Alkaloids: Chemistry, Biology, Ecology, and Applications [2 ed.] 0444594337, 9780444594334

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Alkaloids

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Alkaloids

Chemistry, Biology, Ecology, and Applications

Second edition

By

Dr. Tadeusz Aniszewski Helsinki, Finland

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK 225 Wyman Street, Waltham, MA 02451, USA Second edition 2015 # 2015 Tadeusz Aniszewski. Published by Elsevier B.V. All rights reserved. First edition copyright : Copyright # 2007 Elsevier B.V. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress For information on all Elsevier publications visit our website at http://store.elsevier.com/ This book has been manufactured using Print On Demand technology. Each copy is produced to order and is limited to black ink. The online version of this book will show color figures where appropriate. ISBN: 978-0-444-59433-4

Dedicated to the Present and Future Generations

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Contents

List of figures....................................................................................................................xi List of tables .................................................................................................................... xv Preface............................................................................................................................xvii

CHAPTER 1 Definition, typology, and occurrence of alkaloids .................................................. 1 1.1 Definition ................................................................................................ 2 1.2 Typology of alkaloids............................................................................. 7 1.2.1 Bioecological classification of alkaloids ................................... 7 1.2.2 Chemotechnological classification of alkaloids ........................ 7 1.2.3 Chemo-molecular classification of alkaloids ............................ 8 1.2.4 Biosynthetic shape-classification of alkaloids........................... 8 1.3 Occurrence in nature ............................................................................ 15 1.3.1 The Dogbane botanical family (Apocynaceae) ....................... 17 1.3.2 The Aster botanical family (Asteraceae) ................................. 22 1.3.3 The Logan botanical family (Loganiaceae)............................. 24 1.3.4 The Poppy botanical family (Papaveraceae) ........................... 26 1.3.5 The Citrus botanical family (Rutaceae)................................... 29 1.3.6 The Nightshade botanical family (Solanaceae)....................... 32 1.3.7 The Coca botanical family (Erythroxylaceae)......................... 35 1.3.8 The Borage botanical family (Boraginaceae).......................... 35 1.3.9 The Legume botanical family (Fabaceae) ............................... 37 1.3.10 The Monseed botanical family (Menispermaceae) ................. 53 1.3.11 The Berberry botanical family (Berberidaceae) ...................... 55 1.3.12 The Buttercup botanical family (Ranunculaceae) ................... 56 1.3.13 The Lily botanical family (Liliaceae)...................................... 58 1.3.14 The Coffee botanical family (Rubiaceae)................................ 60 1.3.15 The Amaryllis botanical family (Amaryllidaceae).................. 63 1.3.16 The Oleaster botanical family (Elaeagnaceae)........................ 64 1.3.17 The Caltrop botanical family (Zygophyllaceae) ..................... 65 1.3.18 Mushroom................................................................................. 65 1.3.19 Moss.......................................................................................... 67 1.3.20 Fungus and bacter .................................................................... 68 1.3.21 Animals..................................................................................... 70 References.................................................................................................... 73

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Contents

CHAPTER 2 Alkaloid chemistry ........................................................................................................... 99 2.1 Alkaloids as secondary metabolism molecules.................................. 100 2.2 Synthesis and metabolism .................................................................. 106 2.2.1 Skeleton diversity................................................................... 108 2.2.2 Ornithine-derived alkaloids.................................................... 113 2.2.3 Tyrosine-derived alkaloids..................................................... 116 2.2.4 Tryptophan-derived alkaloids ................................................ 119 2.2.5 Nicotinic acid–derived alkaloids............................................ 125 2.2.6 Lysine-derived alkaloids ........................................................ 127 2.2.7 Methods of analysis ............................................................... 130 2.2.8 Biogenesis of alkaloids .......................................................... 162 2.2.9 Methods of alkaloid analysis ................................................. 172 References.................................................................................................. 184

CHAPTER 3 Biology of alkaloids ...................................................................................................... 195 3.1 Alkaloids in biology ........................................................................... 196 3.1.1 From stimulators to inhibitors and destroyers of growth...... 198 3.1.2 The effects of stress and endogenous security mechanisms ............................................................................ 201 3.2 Bioactivity........................................................................................... 204 3.2.1 Secrets of life ......................................................................... 204 3.2.2 Life regulation through high and low cytotoxicity ............... 206 3.2.3 Hemoglobinization of leukemia cells .................................... 209 3.2.4 Estrogenic effects ................................................................... 211 3.2.5 Antimicrobial properties ........................................................ 212 3.2.6 Antiparasitic activity .............................................................. 215 3.3 Biotoxicity .......................................................................................... 216 3.3.1 Research evidence .................................................................. 218 3.3.2 Influence on DNA .................................................................. 219 3.3.3 Selective effectors of death.................................................... 220 3.3.4 Nontoxic to self but deformer for others............................... 221 3.3.5 Degenerators of cells.............................................................. 223 3.3.6 Aberrations in cells ................................................................ 224 3.3.7 Causers of locoism ................................................................. 226 3.4 Bionarcotics ........................................................................................ 226 3.5 Alkaloids in the immune system........................................................ 230 3.6 Genetic approach to alkaloids ............................................................ 233 3.7 Evolutionary influences on alkaloid biology ..................................... 236 References.................................................................................................. 239

Contents ix

CHAPTER 4 Ecology of alkaloids ...................................................................................................... 259 4.1 4.2 4.3 4.4

Molecular basis of ecology ................................................................ 262 Alkaloids in interaction between life units ........................................ 263 Biological communities and alkaloids ............................................... 264 Alkaloids in different ecosystems ...................................................... 266 4.4.1 Alkaloids in aquatic ecosystems ............................................ 267 4.4.2 Alkaloids in terrestrial ecosystems ........................................ 270 4.4.3 Climate and alkaloids............................................................. 270 4.4.4 Alkaloids and invasive species .............................................. 272 4.4.5 Ecological role of alkaloids ................................................... 274 References.................................................................................................. 283

CHAPTER 5 Evolution of alkaloids and alkaloids in evolution .............................................. 291 5.1 5.2 5.3 5.4

Alkaloids, biodiversity, and endemism.............................................. 291 Alkaloids in evolutionary processes................................................... 296 Alkaloids in the diversification of life ............................................... 298 Alkaloids in reinforcement, hybrid zones, and new species through hybridization ......................................................................... 318 5.5 Alkaloids in microevolution ............................................................... 320 5.6 Alkaloids in life form and function ................................................... 330 References.................................................................................................. 334

CHAPTER 6 Applied potential and current applications of alkaloids .................................. 345 6.1 Introduction......................................................................................... 346 6.2 Chemistry applications ....................................................................... 349 6.3 Medicinal applications........................................................................ 350 6.3.1 Regulation of Na+ ions and channels .................................... 350 6.3.2 Regulation of mescarinic cholinergic receptors (MChRs) .... 353 6.3.3 Regulation of acetylcholine esterase (AChE) and choline oxidase (CO) ............................................................. 354 6.3.4 Regulation of opioid and opiate (OO) and cannabinoid (CB) receptors ........................................................................ 355 6.3.5 Regulation of glycine receptors (GlyRs) ............................... 356 6.3.6 Regulation of 5-HT3 receptors (5-HT3Rs)..................................356 6.3.7 Regulations of other receptors ............................................... 357 6.3.8 Regulation of microtubules of the spindle apparatus............ 358 6.3.9 Alteration of mitochondria..................................................... 358 6.3.10 Regulation of microbial activity ............................................ 359 6.3.11 Regulation of stimulation....................................................... 359 6.3.12 Regulation of schizonticide activity ...................................... 361

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6.3.13 6.3.14 6.3.15 6.3.16 6.3.17 6.3.18

Applications in immunotherapy............................................. 362 Regulation of antiproliferative activity.................................. 363 Targeting tubulin polymerization .......................................... 363 Inhibition of panel of kinases ................................................ 364 Inhibiting topoisomerases ...................................................... 364 Inhibiting of ICCM (isoprenylcysteine carboxyl methyltransferase) .................................................................. 365 6.3.19 Induction of apoptosis............................................................ 365 6.3.20 Deregulating of CP and CCC ................................................ 365 6.3.21 Targeting angiogenesis........................................................... 366 6.4 Alkaloids as drugs .............................................................................. 366 6.5 Agricultural applications .................................................................... 370 6.5.1 Alkaloids in food.................................................................... 372 6.5.2 Alkaloids as biological fertilizers .......................................... 376 6.5.3 Alkaloids in plant protection ................................................. 379 6.6 Biotechnology ..................................................................................... 380 6.6.1 Cell cultures............................................................................ 383 6.6.2 Root cultures .......................................................................... 390 6.6.3 Transformation of living tissues ............................................ 391 6.6.4 Fermentation........................................................................... 392 6.6.5 Bioreactors.............................................................................. 392 6.6.6 Blue biotech............................................................................ 394 6.6.7 White biotech ......................................................................... 396 6.6.8 Fungal biotechnology............................................................. 397 References.................................................................................................. 397

CHAPTER 7 Problems of alkaloids in nature and human activity ....................................... 421 7.1 Alkaloid circulation among species ................................................... 422 7.2 Alkaloids in herbivory, carnivory, and omnivory ............................. 423 7.3 Endogenicity and exogenicity of alkaloids ........................................ 426 7.4 Novelization of alkaloids.................................................................... 430 7.5 Narcosis in nature and human behavior............................................. 432 7.6 Additions and their molecular basis................................................... 434 References.................................................................................................. 435 Appendix: Alkaloid extraction protocols ...................................................................... 439 Alkaloid Index ............................................................................................................... 447 Taxonomic Index ........................................................................................................... 461 Author Index .................................................................................................................. 473

List of figures FIGURE 1.1 FIGURE 1.2 FIGURE FIGURE FIGURE FIGURE FIGURE

1.3 1.4 1.5 1.6 1.7

FIGURE 1.8 FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE

1.9 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 1.19 1.20 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 2.20 2.21

Contemporary scheme of morphine. Some alkaloids isolated by pharmaceutical researchers Pierre Joseph Pelletier and Joseph Beinamé Caventou during 1817–1821. Schemes of taxol, vinblastine, vincristine, and vincamine. An example of a true alkaloid. An example of a protoalkaloid. An example of a pseudoalkaloid. Raw extracts of quinolizidine alkaloids from different lupine species in the Research and Teaching Laboratory of Applied Botany of the University of Joensuu. L-tryptophan, with its aromatic side chain, is a precursor of indole, terpenoid indole, quinoline, pyrroloindole, and ergot alkaloids. The devil’s pepper genus contsains L-tryptophan-derived alkaloids. L-phenylalanine is a precursor of alkaloids in the Skythantus species belonging to the Dogbane family. L-ornithine is an important precursor of pyrrolidine, tropane, and pyrrolizidine alkaloids. L-tyrosine, with its aromatic side chain, is a precursor of phenylethylamino- and isoquinoline alkaloids. L-anthranilic acid is a precursor of quinazoline, quinoline, and acridine alkaloids. L-histidine is a precursor of imidazole alkaloids. L-ornithine and L-nicotinic acids are precursors of some alkaloids in the Nightshade family. L-lysine is a precursor of piperidine, quinolizidine, and indolizidine alkaloids Structure of the seed tests of the Washington lupine (Lupinus polyphyllus Lindl.). The structures of jervine, cyclopavine, and protoveratrine. Basic alkaloids of mushrooms. Ergotamine and LSD. Secondary metabolism blocks and amino acid derivation. Pyruvate derivation and acetyl CoA synthesis. General scheme of alkaloid synthesis. L-lysine-derived nuclei. Nuclei and skeletons of izidine alkaloids. The source and forms of the pyrrolidine ring. L-histidine and the nuclei of imidazole and manzamine alkaloids. The nuclei produced by anthranilic acid in alkaloids. The nucleus of alkaloids derived from nicotinic acid. L-phenylanine-derived nuclei in alkaloid biosynthesis. Nuclei supplied to alkaloids by L-tyrosine in the synthesizing process. The L-tryptophan-supplied nucleus during synthesis. Synthesis of alkaloids from ornithine. Synthesis pathway of the pyrrolizidine alkaloids from L-ornithine or L-arginine. Synthesis of hordeine and mescaline. Synthesis pathway of kreysigine and colchicine. Emetine and cephaeline synthesis pathway. Galanthamine synthesis pathway. Psilocybin and serotonin synthesis pathway. Scheme of elaeagnine, harman, and harmine synthesis pathway. Pattern of the ajmalicine, tabersonine, and catharanthine pathway.

3 3 6 14 14 16 16 19 19 22 23 24 30 30 33 39 40 59 67 68 104 105 107 108 109 110 110 111 112 112 113 114 115 116 117 118 118 119 120 121 122

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List of figures

FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE

2.22 2.23 2.24 2.25 2.26 2.27 2.28 2.29 2.30 2.31 2.32 2.33 2.34 2.35 2.36 2.37 2.38 2.39 2.40 2.41 2.42 2.43 2.44 2.45 2.46 2.47 2.48 2.49 2.50 2.51 2.52 2.53 2.54 2.55 2.56 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9

FIGURE FIGURE FIGURE FIGURE

3.10 3.11 3.12 3.13

Diagram of the vindoline, vinblastine, and vincristine pathway. Diagram of the strychnine and brucine pathway. Diagram of the quinine, quinidine, and cinchonine synthesis pathway. Diagram of the eserine synthesis pathway. Diagram of the ergotamine synthesis pathway. Scheme of nicotine and nornicotine synthesis pathway. Diagram of anatabine, anabasine, and ricinine synthesis pathway. Diagram of the pelletierine, lobelanine, and piperine synthesis pathway. Diagram of the swansonine and castanospermine synthesis pathway. Diagram of the lupinine, sparteine, lupanine, and cytisine synthesis pathway. Structural development of piperidine alkaloids. Structural development of indolizidine alkaloids. Structural development of quinolizidine alkaloids. Structural development of pyrrolizidine alkaloids. Structural development of pyrrolidine alkaloids. Structural development of tropane alkaloids. Structural development of imidazole alkaloids. Structural development of quinazoline alkaloid vasicine. Structural development of acridone alkaloids. Structural development of pyridine alkaloids. Structural development of sesquiterpene pyridine alkaloids. Structural development of phenyl and phenylpropyl alkaloids. Structural development of simple indole alkaloids. Structural development of carboline alkaloids. Structural development of corynanthe alkaloids. Structural development of iboga alkaloids. Structural development of aspidosperma alkaloids. Structural development of quinoline alkaloids. Structural development of pyrroloindole alkaloids. Structural development of ergot alkaloids. Structural development of manzamine alkaloids. Chemical explanation for alkaloid biogenesis in organisms (c ¼ catalysers). Chemical model of indole alkaloid formation in Catharanthus roseus. The biochemical model for indole alkaloid formation in Catharanthus roseus. Molecular biology model of Claviceps purpurea alkaloids. Three basic hypotheses on the biological nature of alkaloids. Rutaecarpine, an alkaloid from Evodia rulaecarpa. Effects of foliar application of lupine extracts. Mechanism of regulation of alkaloid content in plants. Alkaloids in the acetylcholine receptor. Fagaronine, an alkaloid from Fagara zanthoxyloides Lam. Model of hemoglobin. Estrogenic activity of the alkaloids. Activity of some alkaloids on Gram-positive (Staphylococcus aureus, 1) and Gram-negative (Klebsiella pneumonia, 2) bacteria. Some alkaloids from Strychnos species. Acute toxicity of berberine and thebaine on mice in relation to form administration. Acute toxicity (LD50) of some quinolizidine alkaloids in mice. Acute toxicity (LD50) of some pyrrolizidine alkaloids in male mice.

122 123 124 124 125 126 126 128 128 130 138 139 140 144 146 147 148 149 150 151 153 154 155 156 157 159 160 161 162 163 164 166 167 169 170 198 200 201 203 207 209 210 211 213 217 220 222 224

List of figures xiii

FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE

3.14 3.15 4.1 4.2 4.3 4.4 4.5 5.1 5.2 5.3 5.4

FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE

5.5 5.6 5.7 5.8 5.9 5.10 5.11

FIGURE 5.12 FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE

5.13 5.14 5.15 5.16 5.17 5.18 5.19 5.20 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 7.1

FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE

7.2 7.3 7.4 7.5 7.6 7.7

Some narcotics and their derivatives. Model of evolutionary interaction between alkaloids and insects. Biological life as a production of energy. Alkaloids as chemical instruments in the biological community. The accumulation of pyrrolizidine alkaloids in some insect species during various developmental stages. The copulation of butterflies. The butterfly's interaction with alkaloidal plants. General scheme of alkaloid appearance in biotaxons. General scheme of global and local circulation of energy, carbon, nitrogen, oxygen, and hydrogen. Estimated total number of species and total number of species described up to the present. Alkaloid-containing species on the globe. 1 ¼ total number; 2 ¼ insects; 3 ¼ fungi; 4 ¼ bacteria; 5 ¼ birds; 6 ¼ mammals. Alkaloids in evolutionary processes of organisms. Alkaloids in life tree. Mass of filamentous algae at the bottom of a lake. General scheme of alkaloid groups in evolutionary processes in nature. Rf of QA(+) individuals in Astragalus spp, 1999–2003. MEC of Astragalus species. Rf of QA(+) individuals in Coronilla varia, Cytisus scoparius, Lotus corniculatus, Lupinus poplyphyllus, Meliotus officinalis, Ononis repens, Ornithopus perpusillus, and Oxytropis campestris. MEC of Coronilla varia, Cytisus scoparius, Lotus corniculatus, Lupinus poplyphyllus, Meliotus officinalis, Ononis repens, Ornithopus perpusillus, and Oxytropis campestris spp. Rf of QA(+) individuals in Lathyrus spp, 1999–2003. MEC of Lathyrus species. Rf of QA(+) individuals in Medicago spp, 1999–2003. MEC of Medicago sativa and Medicago lupulina. Rf of QA(+) individuals in Trifolium spp, 1999–2003. MEC of Trifolium species. Rf of QA(+) individuals in Vicia spp, 1999–2003. MEC of Vicia species. General diagram of alkaloid applications in chemistry. General diagram of alkaloid applications in medicine. Sanguinarine, an alkaloid from Sanguinaria canadiensis. Nitrogen content in different soils after 1 year from sample preparations. General diagram of biotechnology as a large area for alkaloid applications. General diagram of biotechnological production of alkaloids. Diagram of alkaloid production by cell culture. Cell-culture techniques in the organogenesis stage. Stirrer bioreactor with continuous culture of cell suspension. Production of somatic embryos in the small bioreactors in the laboratory. (a) Translocation of alkaloids by physiological pressure coded in the genes to the rhizosphere. (b) Some translocations of alkaloids by ecological behavior. Enkephalin in C terminal, mix of two peptides: Leu- and Met-enkephalin. A structure of enkephalins (Met and Leu). Antipain activity of opioid peptides (nM of IC50 ,GPI) in comparison to morphine. Structure of alkaloid activity and receptor regulation in the nervous system. Novelization of amphetamine into chlorfentermine. Different stages of morphinism.

227 238 261 265 279 281 282 292 293 294 295 297 299 302 321 322 323 324 325 326 327 328 329 330 331 332 333 349 352 352 378 381 382 384 385 393 394 422 426 427 429 430 432 433

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List of tables TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 2.1 2.2 2.3 3.1 4.1 4.2 6.1 6.2 6.3 7.1

Main types of alkaloids and their chemical groups General botanical characteristics of the Dogbane family General botanical characteristics of the Aster family General botanical characteristics of the Logan family General botanical characteristics of the Poppy family General botanical characteristics of the Citrus family General botanical characteristics of the Nightshade family General botanical characteristics of the Borage family General botanical characteristics of the Legume family Main types of alkaloids and their chemical groups General botanical characteristics of the Monseed family General botanical characteristics of the Berberry family General botanical characteristics of the Buttercup family General botanical characteristics of the Lily family General botanical characteristics of the Coffee family General botanical characteristics of the Amaryllis family General botanical characteristics of the Oleaster family General botanical characteristics of the Caltrop family Amino acids and their participation in alkaloid synthesis Some well-known enzymes and coenzymes active in alkaloid biogenesis General characteristics of the methods and techniques of quinolizidine alkaloid analysis Enzymes specifically involved in alkaloid biosynthesis Some new alkaloids from marine environments Selective Toxicity Coefficients (STC) of some alkaloids and selective toxicity in the ecosystem The most important alkaloids used in modern medicine Potential usage of alkaloid-rich and alkaloid-poor Washington lupine (Lupinus polyphyllus Lindl.) in agriculture Some alkaloids produced by cell cultures Fragmentation of pro-enkephalin A, a precursor of human enkephalin

9 18 22 25 27 29 33 36 38 41 54 55 57 58 60 63 65 66 101 168 181 234 267 276 351 371 386 428

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Preface Recent progress in molecular biology leads to a better understanding of nature and natural life processes at the global and local scopes and opens new horizons for chemical and biological technology. Alkaloids, the subject of this book, are a group of very interesting and complex chemical and biological molecules having special tasks in the life machinery of our planet. They are also a good example of how chemical structures occurring in nature can be studied, developed, and used in practical human life throughout history and nowadays. Moreover, these chemical structures are a part of normal physiological activity of thousands species and all environments. They protect cellular and unicellular life but they also have the ability to kill or weaken life in other cells and units. Alkaloids are an active part in the global battle between populations and in the maintenance of strong biological and ecological order in nature. Production and sequestering of alkaloids are two ways and life strategies in service to this battle and order in nature. Moreover, biomolecules, including alkaloids, can also effect different changes in physical materials. It is known presently that these molecules can reduce metal ions to nanoparticles or be used in the synthesis of some metal nanoparticles. Since ancient times, alkaloids were the first medicines, the first poisons, and the first protection agents. This book intends to be a reader-friendly presentation of these compounds in all their polymorphism and in both their intricate and complex reasons for existing and chemico-bio-ecoevoactivity. It is a text for chemists, biologists, ecologists and evolutionists alike. However, the intended audience of this book is not limited to scientists, teachers, and other present and future specialists. My purpose was to compose a beneficial text for an academic and professional audience that could also serve as source of knowledge for anyone who is interested in the fascinating subject of alkaloids. The first edition, published in 2007, has been received by readers with genuine interest and positive feedback. This means that this book was needed and expected. The present, second, edition is extended by presenting new knowledge and new research results in the field of alkaloids. Alkaloids existing in a deep marine environment produced by microorganisms, and aspects of these molecules not researched or unclear are also highlighted in this book. Moreover, the biological, ecological, and evolutionary scope of these compounds is presented. New findings and achievements in the field of alkaloid applications in different areas are also shown. Finally, the problems of alkaloids in nature and human activity, including narcotic use and the danger of these compounds, are demonstrated. Alkaloids: Chemistry, Biology, Ecology, and Applications, second edition, is therefore, a large book presenting the interesting subject with an innovative approach. The reader can find from this book not only answers to many questions connected with alkaloids but also questions still without scientific answers. Therefore, this book probably also will be benefit to active researcher in the field and industry. The literature on alkaloids is growing rapidly. As I stated in the Preface to the first edition, researchers are persistently attempting to decode the many secrets surrounding alkaloids. It is very challenging and difficult work. Full translation of these secrets from code symbols

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into an ordinary understanding of life is still on the way. For this reason, the science, knowledge, and biological life of alkaloids are interesting points of human interest. Each new human generation makes its own contribution to this process. The Web of Science (WoS, all database) mentions (1 January 2015) 156,393 papers containing the keyword alkaloid, and 66,776 of these papers are graded as research papers belonging to the world’s leading scholarly literature (WLSL). This means that the research in the field is active, dynamic, and very productive. From the January 2014 to January 2015, 8190 new papers with the search word alkaloid have been published around the globe. 3406 is graded as research papers belonging to the WLSL. Alkaloids are one of the central issues of the global science today. Alkaloids: Chemistry, Biology, Ecology, and Applications, second edition, provides a clear and up-to-date presentation of alkaloids from an interdisciplinary point of view. Not only do I present the subject, I also approach unresearched areas and several problems that persist in this fascinating field. The book consists of seven chapters, the first of which presents recent knowledge of alkaloid distribution among species and organism kingdoms. The second chapter discusses alkaloid chemistry in biosynthesis, models, and other methodological considerations and basic techniques used. The biology of alkaloids is presented in Chapter 3 and the ecology of alkaloids in Chapter 4. The evolution of alkaloids and alkaloids in evolution are outlined in Chapter 5. The data concerning recent applications of alkaloids is presented in Chapter 6; finally, Chapter 7 discusses problems connected with alkaloids in nature and human behavior. Each chapter features an outline. The last portion of this book includes an appendix listing of alkaloids, plants containing alkaloids, and some examples of basic protocols of alkaloid extraction. Moreover, the attention is directed into the point that a totally perfect and unique protocol still does not exist. Therefore, a process of alkaloid extraction is still important and scientifically difficult, if deeply analyzed by bioorganic chemistry. However, there are many achievements in individual alkaloid extraction, isolation, purification, and novelization. My appreciation and thanks are due to several people around the world. Experienced and young scientists and students are appreciated for their fruitful discussions on the subject. Many of them have visited my Research and Teaching Laboratory of Applied Botany, Biological Interactions, and Ecological Engineering at the University of Eastern Finland (former University of Joensuu) in Joensuu. I thank many universities and research institutions around the world, on all continents for their cooperation during last decades. Collaboration with the editorial staff of the Elsevier Limited was also pleasant and fruitful. The grant of the Association of nonfiction writers of Finland (Suomen tietokirjailijat ry) is appreciated. I extend my sincere thanks to everyone for fruitful university years, cooperation, and life. Dr. Tadeusz Aniszewski Helsinki, Finland

Chapter

1

Definition, typology, and occurrence of alkaloids CHAPTER OUTLINE

1.1 Definition 2 1.2 Typology of alkaloids 1.2.1 1.2.2 1.2.3 1.2.4

7

Bioecological classification of alkaloids 7 Chemotechnological classification of alkaloids 7 Chemo-molecular classification of alkaloids 8 Biosynthetic shape-classification of alkaloids 8 1.2.4.1 True alkaloids 8 1.2.4.2 Protoalkaloids 14 1.2.4.3 Pseudoalkaloids 15

1.3 Occurrence in nature

15

1.3.1 The Dogbane botanical family (Apocynaceae) 17 1.3.2 The Aster botanical family (Asteraceae) 22 1.3.3 The Logan botanical family (Loganiaceae) 24 1.3.4 The Poppy botanical family (Papaveraceae) 26 1.3.5 The Citrus botanical family (Rutaceae) 29 1.3.6 The Nightshade botanical family (Solanaceae) 32 1.3.7 The Coca botanical family (Erythroxylaceae) 35 1.3.8 The Borage botanical family (Boraginaceae) 35 1.3.9 The Legume botanical family (Fabaceae) 37 1.3.10 The Monseed botanical family (Menispermaceae) 53 1.3.11 The Berberry botanical family (Berberidaceae) 55 1.3.12 The Buttercup botanical family (Ranunculaceae) 56 1.3.13 The Lily botanical family (Liliaceae) 58 1.3.14 The Coffee botanical family (Rubiaceae) 60 1.3.15 The Amaryllis botanical family (Amaryllidaceae) 63 1.3.16 The Oleaster botanical family (Elaeagnaceae) 64 1.3.17 The Caltrop botanical family (Zygophyllaceae) 65 1.3.18 Mushroom 65

Alkaloids # 2015 Tadeusz Aniszewski. Published by Elsevier B.V. All rights reserved.

1

2 CHAPTER 1 Definition, typology, and occurrence of alkaloids

1.3.19 Moss 67 1.3.20 Fungus and bacter 68 1.3.21 Animals 70

References

73

Docendo discimus. Seneca

1.1 DEFINITION The definition of the term alkaloid is not simple and, in many cases, is a source of academic controversy. This controversy was especially active at the beginning of 20th century, when the progress in chemical research on alkaloids occurred. Many new achievements in the field have been opposed and very critically discussed. Moreover, scientific discussions around the alkaloids continue.8,23 Alkaloids were fascinating subject in the past, and nowadays, they are an object of strong scientific and economic interest, especially in medicine and the pharmaceutical industry. Difficulties with the definition of such a group of secondary, natural molecules as alkaloids stem, also nowadays, from the similarities between alkaloids and other secondary compounds, despite their diversity and occurrence in complexes with different moieties, sometimes classified as different biochemical types, such as peptides, proteins, and sugars. Moreover, alkaloids were used for long time before their discovery as chemical molecules with a unique nomenclature. For example, the alkaloid quinine was use in herbal form by native people of America many hundred years before it was given that name. Awareness of the possibility of using this herbal medicine in the cure of malaria came in the year 1638, when some aristocrats were officially cured of malaria by this mean.87 Attempts to define the term alkaloid originated at the time of the discovery of these compounds. Friedrich Sert€ urner, an apothecary’s assistant from Westphalia, first isolated morphine (Figure 1.1), one of the most important alkaloids in the applied sense.43 This was in 1805 and proved a significant step forward in chemistry and pharmacology.10,70,366 Using the method developed by Friedrich Sert€ urner, the pharmaceutical researchers Pierre Joseph Pelletier and Joseph Benaime´ Caventou isolated, from 1817 to 1821, a remarkable range of other alkaloids (Figure 1.2), such as brucine (a close relative of strychnine), febrifuge, quinine, caffeine, and veratrine.42–44 After that, the first attempts to discover alkaloids by synthetic means took place in the middle of 1800s. One of them was an attempt to produce synthetic quinine in the laboratory. However, many other chemical compounds have been made synthetically before quinine, which had been done only in 1900s. Nowadays, the synthetic production of some important alkaloid is more common than their isolation from

HO Two condensed Tyr rings lead to the pathway for Morphine O

O

H

H

O

NCH3 N

O

N

O

HO n FIGURE 1.1 Contemporary scheme of morphine. Friederich Surt€urner, who first isolated this alkaloid in an impure form in 1805, knew that it was converted from the pathway of Tyrosine, Tyr. The correct morphine structure was determined by Gulland and Robinson in 1923. Moreover, even 200 years after Surt€urner’s isolation, scientists are still discussing this alkaloid from a molecular point of view. This is a good example of the scientific evolution of knowledge of alkaloids.

N O

H H3CO

H 3C

H H N

H3CO

CH3 N

N

O

H

O

H

N

N

CH3 O Brucine

Caffeine H

H

H HO

N H

H3CO

N

Quinine N H N

H H N

N

O

H H

O O

Febrifuge

Strychnine

n FIGURE 1.2 Some alkaloids isolated by pharmaceutical researchers Pierre Joseph Pelletier and Joseph Beinamé Caventou during 1817–1821. They did not know the exact structures. The compounds thus isolated are combinations of alkaloids rather than one pure alkaloid.

4 CHAPTER 1 Definition, typology, and occurrence of alkaloids

natural sources. The progress in both theoretical and applied chemistry led to the synthesis such new compounds, which do not exist in the nature and can only be manufactured. The term alkaloid was first mentioned in 1819 by W. Meißner, an apothecary from Halle. He observed that these compounds appeared “like alkali” and so named them alkaloids.67 For the biologist, the alkaloid is a pure and perfect natural product. From the biological point of view, the alkaloid is any biologically active and heterocyclic chemical compound that contains nitrogen and may have some pharmacological activity and, in many cases, medicinal or ecological use.11 This definition, as a relatively wide one based on application, can be criticized as inexact. However, it presents a general picture of what kinds of compound are under consideration. The biological and chemical nature of this group of compounds leads to the conclusion that each definition of alkaloids is either too broad or too narrow. A short exact definition is not possible without a long list of exceptions.27,71,138–141,189,206,292–294,307,363,365,381,410,428 Sometimes, to avoid presenting this list of exceptions, the basic characteristics of alkaloids are given in the definition. Winterstein and Tier428 stressed that these compounds had such characteristics as (1) greater or lesser toxicity, which acts primarily on the central nervous system (CNS); (2) the basic character of a chemical construction; (3) heterocyclic nitrogen as an ingredient; (4) a synthesis from amino acids or their immediate derivatives; and (5) a limited distribution in nature. In another definition, Waller and Nowacki410 mentioned many characteristics of alkaloids. They especially drew attention to the fact that alkaloids have nitrogen in the molecule and are connected to at least two carbon atoms. Moreover, this compound has at least one ring in the molecule, and its ring is not necessarily heterocyclic. The authors also stated that alkaloids could not be structural units of macromolecular cellular substances, vitamins, or hormones. More early, Sengbush344 simply stressed that alkaloids are a group of nitrogen-containing bases and most of them are drugs. The most important points for the biologist are that alkaloids are a special group of chemicals that are active at different cellular levels of organisms and they take part in the biological processes of plants, animals, and microorganisms living in different environments. Alkaloids are compounds that form typical molecules for concrete living species. Although their content fluctuates from individual to individual, they are characteristic components of the species (sometimes also of a larger taxon) and can be considered a chemical identification factor, because a species with a genetic ability to synthesize alkaloids cannot exist without alkaloids in its body. For the medical scientist, the term alkaloids means any group of nitrogenous substances of vegetable origin, often of complex structure and high

1.1 Definition 5

molecular mass.246 Moreover, it is important that alkaloids are often heterocyclic and may have primary, secondary, or tertiary bases or may contain quaternary ammonium groups. Certainly, the fact that alkaloids are only slightly soluble in water but soluble in ethanol, benzene, ether, and chloroform is also extremely important and highlighted in the medical definition. This long definition also notes that alkaloids exhibit some general characteristics that are revealed by the coloration or precipitation in alkaloid reagents. Finally, medicine draws attention to the fact that alkaloids create intense physiological action, and they are widely used in the medical fields as curative drugs. Some alkaloids can also be highly toxic, even in very small doses.246 In the database of the National Library of Medicine, it is possible to find the definition of alkaloids, according to which these compounds are nitrogenous bases and occur in the animal and vegetable kingdoms, while some of them have been synthesized.280 Another electronic database also provides a definition of alkaloids, stating that an alkaloid is a nitrogenous organic compound with pharmacological effects on humans and other animals and whose name is derived from the world alkaline.425 As can be seen, the definition of alkaloids in the field of medicine also offers parameters of “may be,” “often,” “slightly,” and “highly,” which are not exact. This is typical of the scientific and practical fields, where alkaloids are well known and used in the bettering of human health but where the term remains relatively difficult to define exactly and concisely. Chemistry provides a definition of alkaloids in purely chemical terms. Chemists stress that alkaloids are any group of complex heterocyclic nitrogen compounds, which have strong physiological activity, are often toxic, and retain their own basic chemical properties. It is also stated that there are a few exceptions to this definition.133 Another chemical definition states only that alkaloids are nitrogen-containing compounds derived from plants and animals.147 Later, chemists stressed that alkaloids were biogenic, nitrogen-containing, and mostly N-heterocyclic compounds. This definition also states that amino acids, peptides, nucleosides, amino sugars, and antibiotics are not considered as to be alkaloids.177 In spite of differences between the research fields of biology, medicine, and chemistry and the fact that there remain some differences of accentuation in alkaloid definitions, such definitions are very similar, indeed almost identical. Scientists are recognizing the vital importance of these products for biology, medicine. and chemistry. What has been learned about alkaloids from the last over 200 years of studies? It is fascinating that alkaloids are just a product of nature, and a very small unit of global nature in both a material sense and the processes as they occur. They are just a product of living cells, for other living cells. The alkaloid is a product of chemical molecules for the production of other molecules. It is synthesized, playing its own role in the metabolism after that.

6 CHAPTER 1 Definition, typology, and occurrence of alkaloids

O O

O O

N

OH NH O

OH HO

N H

HO O

O

O

O

O

N

H3C-O2C

H

O

O H3CO

N

H

OH CO2CH3

CH3

Taxol

Vinblastine

N

OH N H H3C-O2C

N H H

N

O N

H3CO

N

H

CHO

Vincristine

OH CO2CH3

HO CO2CH3

Vincamine

n FIGURE 1.3 Schemes

of taxol, vinblastine, vincristine, and vincamine.

The alkaloid represents perfection in much the same way as perfection appears in life and nature. This is the reason why alkaloids were and are a fascinating subject of study. This is also the reason why definitions of these groups of molecules, provided by scientists of biology, medicine, and chemistry, are acceptably imperfect. However, alkaloids are recognized as a large group of compounds with biological, pharmacological, or physiological and chemical activity. Without alkaloids, stupendous achievements in the battle against malaria, leukaemia, and cancer as well as Parkinson disease would be not possible. Alkaloids will also play an important role in the future research and the development of potential new applications protecting human health and welfare. Some new promising results are presented.154–156,158,165,166 The pharmaceutical industry has succeeded in the use of natural plant alkaloids for the development of antimalarial agents (quinine and chloroquinine), anticancer agents (taxol, vinblastine, and vincristine), and agents promoting blood circulation in the brain (vincamine) (Figure 1.3). Many alkaloids can influence an animal’s nervous system, providing possible changes in the functionality of the organism. The activity of

1.2 Typology of alkaloids 7

alkaloid molecules on a psychomental level (opium latex, papaverine, morphine, cocaine) is one of natural phenomena in the process of species selfprotection and the interactions between producers (plants) and consumers (herbivores). It is also a good example of natural selection mechanisms and results. Nowadays, more than 10,000 (estimation varies 8,000 – 14,000) natural compounds and their derivatives are recognized as alkaloids. Each year, scientists around the globe discover a new molecules. They frequently occur as acid salts, but some also occur in combination with sugars whereas others appear as amides or esters. Alkaloids can also be quaternary salts or tertiary amine oxides.295

1.2 TYPOLOGY OF ALKALOIDS The purpose of any classification of bioactive chemical compounds is to box the molecules into similar groups according their physical and biological characteristics and to help their morphological recognition and possible use for both scientific and applied uses. Alkaloids can be classified in the terms of their (1) biological and ecological activity, (2) relation to chemical and technological innovations, (3) chemical structure, and (4) biosynthetic pathway.

1.2.1 Bioecological classification of alkaloids From the point of view of biological activity, it is possible to divide alkaloids into (1) neutral or weakly basic molecules (e.g., lactams such as ricinine, certain N-oxides such as indicine), (2) animal-derived alkaloids (e.g., anuran, mammalian and arthropod alkaloids), (3) marine alkaloids, (4) moss alkaloids, (5) fungal and bacterial alkaloids, and (6) nonnatural alkaloids (structurally modified or analogues). Nowadays, the group of compounds mentioned as nonnatural alkaloids is growing especially rapidly as a result of bio-organic and stereochemistry research. Pharmacological research and the drug industry rapidly advance and promote the most promising new molecules for possible production applications. This is necessary, since the sources of infections (microorganisms) are constantly changing their species and infection ability, becoming resistant to medicines and antibiotics.

1.2.2 Chemotechnological classification of alkaloids Alkaloids can be divided into three large groups on the basis of their relation to the innovations in the fields of both chemistry and technology: (1) natural alkaloids, (2) biomimic and bionic alkaloids, and (3) synthetic alkaloids. This division serves the scientific and practical needs well.

8 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Natural alkaloids are all alkaloids existing in nature, presently known or still unknown by the science. They are molecules naturally synthesized and novelized in time by living organisms as a result of the evolution of life on Earth. Biomimic alkaloids are natural alkaloids copied artificially by chemists in the laboratories.They are identical in structure to natural alkaloids. Bionic alkaloids are those biomimic molecules being novelized by the chemists and engineers using natural models and high-level technology. Bionic alkaloids are not identical analogues to natural alkaloids. Synthetic alkaloids are molecules totally modeled by chemists and engineers using high-level technology, planned models, and artificial synthesis. Synthetic alkaloids are not produced naturally by the living organisms.

1.2.3 Chemo-molecular classification of alkaloids Alkaloids can be divided into different types according their pure chemical structures295 pointing first at the alkaloid base, a basic chemical nucleus. The following are basic types of alkaloids: acridones, aromatics, carbolines, ephedras, ergots, imidazoles, indoles, bisindoles, indolizidines, manzamines, oxindoles, quinolines, quinozolines. quinolizidines, phenylisoquinolines, phenylethylamines, piperidines, purines, pyrolidines, pyrrolizidines, pyrroloindoles, pyrydines, sesquiterpenes, simple tetrahydroisoquinolines, stereoids, tropanes, terpenoids, diterpenes, and triterpenes. The structural characteristics of some of these groups of alkaloids are presented in Chapter 2.

1.2.4 Biosynthetic shape-classification of alkaloids Alkaloids are generally classified by their common molecular precursors, based on the biological pathway used to construct the molecule. From a structural point of view, alkaloids are divided according to shape, structure, and precursors. There are three main types of alkaloids: (1) true alkaloids, (2) protoalkaloids, and (3) pseudoalkaloids. True alkaloids and protoalkaloids are derived from amino acids, whereas pseudoalkaloids are not derived from these compounds (Table 1.1).

1.2.4.1 True alkaloids True alkaloids derive from amino acid and share a heterocyclic ring with nitrogen. These alkaloids are highly reactive substances with biological activity even in low doses. All true alkaloids have a bitter taste and appear as a white solid, with the exception of nicotine, which is a brown liquid. True alkaloids

1.2 Typology of alkaloids 9

Table 1.1 Main types of alkaloids and their chemical groups Alkaloid type

Precursor Compound

Chemical Group of Alkaloids Parent Compounds

Examples

True alkaloids

L-ornithine

Pyrroline alkaloids

Pyrrolidine

Tropane alkaloids

Tropane

Pyrrolizidine alkaloids

Pyrrolizidine

Piperidine alkaloids

Piperidine

Quinolizidine alkaloids

Quinolizidine

Indolizine alkaloids

Indolizidine

Phenylethylamino alkaloids

Phenylethylamine

Simple tetrahydroisoquinoline alkaloids

Benzyltetrahydroisoquinoline

Cuscohygrine Hygrine Atropine Cocaine Hyoscyamine Scopolamine/hyoscine Acetyl-lycopsamine Acetyl-intermedine Europine Homospermidine Ilamine Indicine-N-oxide Meteloidine Retronecine Anaferine Lobelanine Lobeline N-methylpelletierine Pelletierine Piperidine Piperine Pseudopelletierine Sedamine Cytisine Lupanine Sparteine Castanospermine Swansonine Adrenaline Anhalamine Dopamine Noradrealine Tyramine Codeine Morphine Norcoclaurine Papaverine Tetrandrine Thebaine Tubocurarine

L-lysine

L-tyrosine

Continued

10 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.1

Main types of alkaloids and their chemical groups Continued

Alkaloid type

Precursor Compound

Chemical Group of Alkaloids Parent Compounds

Examples

L-phenylalanine

Phenethylisoquinoline alkaloids

Amaryllidaceae alkaloids

Autumnaline Crinine Floramultine Galanthamine Galanthine Haemanthamine Lycorine Lycorenine Maritidine Oxomaritidine Vittatine

L-tryptophan

Indole alkaloids

Indole Simple indole alkaloids

L-tyrosine

or

Simple β-carboline alkaloids Terpenoid indole alkaloids

Quinoline alkaloids

Quinoline

Pyrroloindole alkaloids

Indole

Ergot alkaloids

Arundacine Arundamine Psilocin Serotonin Tryptamine Zolmitriptan Elaeagnine Harmine Ajmalicine Catharanthine Secologanin Tabersonine Chloroquinine Cinchonidine Quinine Quinidine A-yohimbine Chimonantheine Chimonantheine Corynantheine Corynantheidine Dihydrocorynantheine Corynanthine Ergobine Ergotamine Ergocomine Ergocornine Ergocristine

1.2 Typology of alkaloids 11

Table 1.1 Main types of alkaloids and their chemical groups Continued Alkaloid type

Precursor Compound

L-histidine

Chemical Group of Alkaloids Parent Compounds

Imidazole alkaloids

Imidazole

Manzamine alkaloids

Xestomanzamine

L-arginine

Marine alkaloids

β-carboline

Anthranilic acid

Quinazoline alkaloids Quinoline alkaloids

Quinazoline Quinoline

Examples Ergocryptine α-ergocryptine β-ergocryptine Ergometrine Ergonovine Ergosine Ergostine Fumigaclavine D Fumigaclavine E Fumigaclavine F Fumigaclavine G Fumigaclavine H Histamine Pilocarpine Pilosine Xestomanzamine A Xestomanzamine B Saxitoxin Tetrodotoxin 1-acetyl-β-carboline 7-bromo-1-ethyl-βcarboline Eudistomidin B Eudistomidin G Eudistomidin H Eudistomidin I Eudistomidin J Eudistomidin K Marinacarboline A Marinacarboline B Marinacarboline C Marinacarboline D Seragadine A Veriabine A Veriabine B Peganine Acetylfolidine Acutine Bucharine Dictamnine Dubunidine γ-fagarine Flindersine

Continued

12 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.1

Main types of alkaloids and their chemical groups Continued

Alkaloid type

Protoalkaloids

Precursor Compound

Chemical Group of Alkaloids Parent Compounds

Acridone alkaloids

Acridine

Nicotinic acid

Pyridine alkaloids

Pyridine/pyrrolidine

L-tyrosine

Phenylethylamino alkaloids

Phenylethylamine

L-tryptophan

Terpenoid indole alkaloids Pyrrolizidine alkaloids

Indole Pyrrolizidine

Piperidine alkaloids

Piperidine

Sesquiterpene alkaloids

Sesquiterpene

L-ornithine

Pseudoalkaloids Acetate

Examples Foliosidine Glycoperine Haplophyllidine Haplopine Helietidine Kokusaginine Maculosine Perfamine Perforine Polifidine Skimmianine Acronycine Rutacridone Anabasine Cassinine Celapanin Evoline Evonoline Evorine Maymyrsine Nicotine Regelidine Wilforine Hordenine Mescaline Yohimbine 4-hydroxy-stachydrine Stachydrine Coniine Coniceine Pinidine Cassinine Celapanin Evonine Evonoline Evorine Maymyrsine Regelidine Wilforine

1.2 Typology of alkaloids 13

Table 1.1 Main types of alkaloids and their chemical groups Continued Alkaloid type

Precursor Compound

Chemical Group of Alkaloids Parent Compounds

Examples

Pyruvic acid

Ephedra alkaloids

Phenyl C

Ferulic acid Geraniol

Aromatic alkaloids Terpenoid alkaloids

Phenyl Terpenoid

Saponins

Steroid alkaloids

Adenine/guanine

Purine alkaloids

Cathine Cathinone Ephedrine Norephedrine Capsaicin Aconitine Actinidine Atisine Gentianine β-skytanthine Cholestane Conessine Cyclopamine Jervine Pregnenolone Protoveratrine A Protoveratrine B Solanidine Solasodine Squalamine Tomatidine Caffeine Theacrine Theobromine Theophylline

Purine

Source: Refs 11, 23, 28, 63, 72, 77, 90, 104, 112, 135, 144, 145, 148, 153, 164, 162, 168, 177, 207, 216, 217, 220–222, 230, 233, 245, 268, 270, 272, 279, 283, 284, 295, 309, 321, 322, 332, 362, 371, 379, 384, 422, 425, 432

form water-soluble salts. Moreover, most of them are well-defined crystalline substances that unite with acids to form salts. True alkaloids may occur in plants (1) in the free state, (2) as salts, and (3) as N-oxides. These alkaloids occur in a limited number of species and families and are those compounds in which decarboxylated amino acids are condensed with a nonnitrogenous structural moiety. The primary precursors of true alkaloids are such amino acids as L-ornithine, L-lysine, L-phenylalanine/L-tyrosine, L-tryptophan, and 90,295 L-histidine. Examples of true alkaloids include such biologically active alkaloids as cocaine, quinine, dopamine, morphine and usambarensine (Figure 1.4). A fuller list of examples appears in Table 1.1. True alkaloids can be as natural, biomimic, bionic and synthetic alkaloids.

14 CHAPTER 1 Definition, typology, and occurrence of alkaloids

N N H H H H N N

Usambarensine n FIGURE 1.4 An example of a true alkaloid. The L-tyrosine-derived

usambarensine has strong antimalarial potential. Usambarensine was extracted from the root bark of African Strychnosis usamkbarensis, a small tree in East and South Africa and a bush in West Africa.

1.2.4.2 Protoalkaloids Protoalkaloids are compounds, in which the N atom derived from an amino acid is not a part of the heterocyclic bond.177 Such alkaloid include compounds derived from L-tyrosine and L-tryptophan (see Table 1.1). Protoalkaloids are those with a closed ring, being perfect but structurally simple alkaloids. They form a minority of all alkaloids. Hordenine, mescaline (Figure 1.5), and yohimbine are good examples of these kinds of alkaloid. Chini et al.63 found new alkaloids, stachydrine and 4-hydroxystachydrine, derived from Boscia angustifolia, a plant belonging

CH3O CH3O

CH3O

CH2

CH2

NH2

Mescaline n FIGURE 1.5 An example of a protoalkaloid. Mescaline is the

alkaloid derived from L-tyrosine and extracted from the Peyote cactus (Lophophora williamsii) belonging to the Cactus family (Cactaceae). Mesacaline has strong psychoactive and hallucinogenic properties. Peyote cactus grows in the desert areas of northern Mexico and the southwestern parts of the United States. This plant was used in Pre-Columbian America in the shamanic practice of local tribes.

1.3 Occurrence in nature 15

to the Capparidacea family. These alkaloids have a pyrroline nucleus and are basic alkaloids in the genus Boscia. The species from this genus have been used in folk medicine in East and South Africa. Boscia angustifolia is used for the treatment of mental illness and occasionally to combat pain and neuralgia. Protoalkaloids can be as natural, biomimic, bionic, and synthetic alkaloids.

1.2.4.3 Pseudoalkaloids Pseudoalkaloids are compounds, the basic carbon skeletons of which are not derived from amino acids.177 In reality, pseudoalkaloids are connected with amino acid pathways. They are derived from the precursors or postcursors (derivatives of the indegradation process) of amino acids. They can also result from the amination and transamination reactions90 of the different pathways connected with precursors or postcursors of amino acids. These alkaloids can also be derived from nonaminoacid precursors. The N atom is inserted into the molecule at a relatively late stage, for example, in the case of steroidal or terpenoid skeletons. Certainly, the N atom can also be donated by an amino acid source across a transamination reaction, if there is a suitable aldehyde or ketone. Pseudoalkaloids can be acetate and phenylalanine-derived or terpenoid, as well as steroidal alkaloids. Examples of pseudoalkaloids include such compounds as coniine, capsaicin, ephedrine, solanidine, caffeine, theobromine, and pinidine (Figure 1.6). More examples appear in Table 1.1. Pseudoalkaloids can be as natural, biomimic, bionic, and synthetic alkaloids.

1.3 OCCURRENCE IN NATURE Alkaloids are substances very well known for their biological activity at the beginning of world civilization. They were used in shamanism, in traditional herbal medicine for the cure of diseases, and in weapons as toxins during tribal wars and hunting. They also had, and still have, sociocultural and personal significance in ethnobotany.76,159 Moreover, they have been and continue to be the object of human interest concerning new possibilities for their safe utilization and ensuing health benefits. Of all secondary compounds, historically and contemporaneously, only alkaloids are molecules of natural origin with highly important benefits and diagnostic uses. They can be characterized as the most useful and also the most dangerous products of nature. They can be extracted and purified (Figure 1.7).

16 CHAPTER 1 Definition, typology, and occurrence of alkaloids

N H Pinidine

n FIGURE 1.6 An example of a pseudoalkaloid. The acetate-

derived pinidine is extracted from the Pinus species, for example, from Pinus penderosa. Puinidine has antimicrobial activity. Photo: T Aniszewski.

n FIGURE 1.7 Raw extracts of quinolizidine alkaloids from different lupine species in the Research and Teaching Laboratory of Applied Botany of the University of Joensuu. Observe the different colors of the raw extracts, which signify different concentrations of alkaloids in different species. Photo: T Aniszewski.

1.3 Occurrence in nature 17

Alkaloids are most abundant in higher plants. At least 25% of higher plants contain these molecules. In effect, this means that, on average, at least one in fourth plants contains some alkaloids. In reality, it is not impossible that alkaloids occur more commonly. Using the latest equipment and technology, such slight traces of alkaloids may be detected (e.g., less than 10 gigagrams per kg of plant mass) that these have no real influence on biological receptors and activity. Generally, these species are not considered alkaloid species. Hegnauer138,139,140,141 defines alkaloid plants as those species that contain more than 0.01% of alkaloids. This is right from the point of view of the classification. From the genetic point of view and the genetic mechanism of alkaloid synthesis, it is a real limitation. Paying attention to slight traces of alkaloids in plants, we see the members of the plant family that are relatives. They have a genetically determined alkaloid mechanism with a species expression. Moreover, this expression is also on the hybrid level.282

1.3.1 The Dogbane botanical family (Apocynaceae) Some plant families are especially rich in alkaloids. The Dogbane botanical family (Apocynaceae Lindl., Juss.) is a good example (Table 1.2). This family is distributed worldwide, especially in tropical and subtropical areas. The Dogbane family is a large botanical taxa containing at least 150 genera and 1700 species. Alkaloids are especially abundant in the following genera: devil’s-pepper (Rauvolfia L.), periwinkle (Catharanthus G. Don), milkwood (Tabernaemontana L.), strophanthus (Strophanthus DC.), voacanga (Voacanga U.), and alstonia (Alstonia R. Br.). The species belonging to these genera contain L-tryptophan-derived alkaloids (Figure 1.8). Indian snakeroot (Rauvolfia serpentina) (Figure 1.9) contains reserpine and rescinnamine, the quinine tree (Rauwolfia capra) yields quinine, and iboga milkwood (Tabernaemontana iboga) produces iboganine. Deserpine has been isolated from the roots of Rauwolfia canescens,407 This alkaloid differs from reserpine only by the absence of a metoxy group but shows an interesting profile of biological activity. It has been employed in clinical practice for the treatment of hypertension and as a tranquilizer and also as a controller of other cardiac disorders. Deserpine is a compound with limited availability from natural sources. According to Varchi et al.407, reserpine usually occurs at about 0.10–0.16% of natural extracts and deserpine in only 0.04%. Furthermore, five new indole alkaloids (Nb-methylajmaline, Nb-methylisoajmaline, 3-hydroxysarpagine, yohimbic acid, and isorauhimbic acid) were isolated from the dried roots of Rauwolfia serpentina.175 Srivastava, Singh, and Kulshreshtha370 reported on alkaloids isolated from heynana milkwood (Tabernaemontana heyneana Wall.). They discovered ervatine, tabersonine, coronaridine, heyneanine,

18 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.2 General botanical characteristics of the Dogbane family Botanical Forms and Parts Botanical forms

Some typical genera

Special characteristics Leaves Flowers

Characteristics Trees Shrubs Lianas Herbs Vines Sometimes succulents or cactuslike Alstonia Amsonia Angadenia Apocynum Asclepias Catharanthus Ceropegia Cynanchum Echites Gonolobus Hoya Macrosiphonia Mandevilla Matelea Morrenia Pentalinon Rhabdadenia Rauvolfia Secamone Sarcostemma Skythantus Strophanthus Tabernaemontana Vallesia Voacanga Milky juice or latex, hair Opposite or verticillate with reduced stipules Pinnateveined Regular, radial Calyx with five sepals Tubular corolla Pollen grains usually tricolporate (dicolporate rarely) Two carpels

1.3 Occurrence in nature 19

Table 1.2 General botanical characteristics of the Dogbane family Continued Botanical Forms and Parts

Characteristics

Fruit

Ovary Follicles Sometimes berrylike or drupelike Compressed with tufts of long hair Albumen

Seeds

Source: Refs 25a, 31a, 70a, 101a, 101b, 101c, 186a, 326a, 402a

COO– +

H3N

C

H

CH2 C

CO2

CH NH

NH2 N H L-tryptophan

n FIGURE 1.8 L-tryptophan, with its aromatic side

chain, is a precursor of indole, terpenoid indole, quinoline, pyrroloindole, and ergot alkaloids.

n FIGURE 1.9 The devil’s pepper genus contains L-tryptophan-derived alkaloids. Rauwolfia serpentine appears on the flowers. Photo: T Aniszewski.

20 CHAPTER 1 Definition, typology, and occurrence of alkaloids

voacristine, voacristine hydroxyindolenine, hydroxyibogamine, and coronaridine hydroxyindolenine. These alkaloids show both bioimpact and uterotrophic activity. Moreover, Heijden et al.142 describe the isolation of indole alkaloids from Tabernaemontana elegans, a species that occurs in southern part of Africa and is used in traditional medicine in Zimbabwe, Mozambique, and South Africa. These alkaloids are apparicine, 16-S-hydroxy-16, 22-dihydroapparicine, tubotaiwine, vobasine, vobasinol, tabernaemontaninol, tabernaemontanine, isovoacangine, dregamine, dregaminol, dregaminolmethylether, 3-R/S-hydroxytabernaelegantine B, 3-methoxy-tabernaelegantine C, 3-R/Shydroxy-conodurine, tabernaelegantine A, B, C, and D.142 Alstonia plants produce menilamine, which is known as a new antimalarial alkaloid isolated from alstonia trees growing in the Philippines, where this plant is common.250 These plants are known as prospective medicinal plants, and they are well distributed throughout tropical America, India, and Malaysia as evergreen trees and shrubs. Many prospective liana plants from this family grow particularly in Amazonian America, tropical Africa, and Madagascar. Genus Alstonia is rich in bioactive indole and bisindole alkaloids.17,46,188,189,191,192,250,251,429 Only one species, Alstonia angustiloba Miq., contains, in its all organs (leaves, roots and stems), at least 50 alkaloids. The alkaloids from the stem bark of this plant are especially biologically active. Such alkaloids as andranginine, andransinine, angustilobine, angustilodine, corynanthean, isolucine, 16R, 19E-isositsirikineuleine, strychnan, vallesamine, and vincamine are typical for this species.209 The stem bark of Alstonia angustifolia is also rich in bisindole alkaloids.386 Alstonia scholaris synthetizes alkaloids bearing the angustilobine skeleton, such as scholaricine, tubotaiwine, 19,20-(E)-vallesamine, and N4-methyl angustilobine.250 From Alstonia macrophylla Wall. Ex G. Don growing in Thailand, talcarpine, pleiocarpamine, alstoumerine, 20-Epiantirhine, alstonerine, alstophylline, macralstonine, villalstonine, alstomacroline, and macrocarpamine were isolated.190 All these alkaloids display strong bioactivity and are considered to be of potential use in medicine. Moreover, two other Thai Alstonia species, Alstonia glaucescens and Alstonia scholaris, were also found to be identical or similar to alkaloids such as O-methylmacralstonine.190,249 It should be noted that more than 220 biologically active alkaloids have been isolated from the genus Alstonia. This makes this genus one of the most important in terms of potential alkaloid use. The Alstonia, devil’s pepper and milkwood genera are endemic only in Asia and Australia, but they are distributed around the globe in the tropics and subtropics. Ajmalicine, catharanthine, leurosine, vindoline, vindolinine, vinblastine, vincristine, vindesine, and alioline are present in the periwinkle (e.g., Catharanthus roseus and Vinca spp.). From the leaves of Vinca difformis Pourr, vincamajine, vincamedine, vincadifformine, akuammidine, vellosimine, vincadiffine,

1.3 Occurrence in nature 21

difforlemenine, difforine, and normacusine have been isolated.121 From Aspidosperma megalocarpon M€ ull. Arg. growing in Colombia, three alkaloids were extracted: fendlerine, aspidoalbine, and aspidolimidine.269 All display bioactivity and the potential for applications in medicine. Moreover, Leuconotis spp. contain leuconicines A, B, C, D, E, F, G, and (-)-ebumamaline.115 From this genus (Leuconotis), especially from the stem bark of the trees Leuconotis griffithii and Leuconotis eugenifolius, such alkaloids as leucofoline, leuconolam, leuconoline, leuconoxine, and rhazinilam with their derivatives were found.116,117 It is suggested that, from this genus growing in Malaysia and Indonesia, it may be possible to find alkaloids biotoxic against pathological cells that are resistant to other drugs. However, leucofoline and leuconoline are weak in such activity. Other alkaloids are in the Dogbane family. A new one is ervahainine A (cyano-substituted oxindole alkaloid), found in Ervatamia hainanensis. This alkaloid is considered as biologically active, having potential inhibitory effects on some human cancer cells.242 Twelve terpenoid indole alkaloids and four ervachinines were found in Ervatamia chinensis. Ervachinines A, B, C, and D are of the vobasinyl-ibogan type of bisindoles.126 Ervatamia yunnanensis contains coronaridine, coronaridine hydroxyindolenine, ervataine, heineanine, ibogaine, and voacangine hydroxyindolenine.182 Quinoline alkaloids (14, 15-β-epoxiscandine, meloscandonine, meloscine, meloscine N-oxide, polyneuridine, scandine, scandine N-oxide, tubotaiwine) occur in the genus Melodinus.443 Melodinus henryi growing in China contains melodinoxanine and N-β-methylnortetraphyllicine.201 New indole alkaloids with plumeran skeleton (N-benzoyl-12demethoxycylindrocarine and N-cinnamoyl-12-demethoxycylindrocarine) were found in the bark of Aspidosperma cylindrocarpon.124 They are biologically active and are considered to be used with their antiplasmodial activity. Another Aspidosperma species A. vargasii and A. desmanthum contain ellipticine, N-methyltetrahydroellipticine, and aspidocarpine.143 Pyridocarbazole alkaloids were found in the Peschiera affinis.336 Jokela and Lounasmaa183 presented 1H and 13C-NMR exact spectral data for seven types of ajmaline-type alkaloids from various species of the Dogbane family. These alkaloids are as follows: ajmaline, 17-O-acetylajmaline, isoajmaline, isosandwichine, rauflorine, vincamajine, and vincamedine. Four steroidal alkaloids (conessine, holadysenterine, isoconessimine, and kurchessine) were found in the stem bark of Holarrhena antidysenterica.210 Eleven indole alkaloids were isolated from the stem bark of Kopsia hainanensis Tsiang, which is a species of Kopsia, endemic in China.450 They are ( )-kopsinine, ( )-kopsinnic acid, ( )-kopsinoline, kopsinilam, kopsanome, (+)-5,22-dioxokopsane, eburnamenine, (+)-eburnamine, ( )-isoeburnamine, (+)-tubotaiwine, and (+)-kopsoffine. Moreover, from

22 CHAPTER 1 Definition, typology, and occurrence of alkaloids

COO– +

H3N

C

H

CH2 CO2H NH2 L-phenylalanine

n FIGURE 1.10 L-phenylalanine is a precursor of alkaloids in the Skythantus species belonging to the Dogbane family.

this species, new kopsihainins A, B, and C were found and their antitussive effects recorded.387 Kopsia officinalis Tsiang seems to be very similar with respect to alkaloid content. In both species, ( )-kopsinine is the principal alkaloid.450 Moreover, in the Dogbane plant family also has phenylalanine-derived alkaloids, such as β-skytanthine in the Skythantus species (Figure 1.10, Table 1.2 and, later, Table 1.10). All alkaloids from the Dogbane family have a relatively strong biological and medicinal effect. Many of them are used in cancer chemotherapy.

1.3.2 The Aster botanical family (Asteraceae) The Aster (syn. Daisy) botanical family (Asteraceae Dum.) is very large, containing over 900 genera and more than 20,000 species (Table 1.3).

Table 1.3 General botanical characteristics of the Aster family Botanical Forms and Parts

Characteristics

Botanical form

Herbs Shrubs Trees (rarely) Ambrosia Antennaria Artemisia Aster Baccharis Bidens Centaurea Chrysothamnus Cirsium Coreopsis Cousinia Elephanthopus Erigeron Eupatorium Gallardia Gamochaeta Gnaphalium Haplopappus Helianthus Helichrysum Hieracium Jurinea

Some typical genera

1.3 Occurrence in nature 23

Table 1.3 General botanical characteristics of the Aster family Continued Botanical Forms and Parts

Special characteristics Leaves Flowers

Fruit Seeds

Characteristics Liatris Mikania Rudbeckia Sussurea Senecio Solidago Verbensia Vermonia Milky juice, hair Alternate, opposite, or whorled exstipulate Regular or irregular Bisexual or unisexual Sometimes sterile calyx reduced Corolla tubular or flattened Achene Pappus Exalbuminous

Source: Refs 25a, 31a, 70a, 72a, 178a, 186a, 326a, 402a

Their distribution is worldwide, and species belonging to this family are found everywhere. The Aster plant family contains species yielded in ways similar to some natural alkaloids. The genus Ragwort (Senecio L.) is especially rich in L-ornithine- (Figure 1.11) derived alkaloids (senecionine, senecivernine, seneciphylline, spartioidine, intergerrimine, jacobine, jacozine, sekirkine, jacoline, dehydrosenkirkine, erucifoline, jaconine, adonifoline, neosenkirkine, dehydrojaconine, usaramine, otosenine, eruciflorine, acetylerucifoline, sennecicannabine, deacetyldoronine, florosenine, floridamine, doronine),186,296,396 and the genus Knapweed (Centaurea L.) in alkaloids derived from L-tryptophan, for example, afzelin and apigenin. The content of pyrrolizidine alkaloids (especially jacobine and its derivatives) in Jacobea vulgaris is genotype dependent.186 Agregatum convzoides contains lycopsamine, dihydrolycopsamine, and acetyl-lycopsamine.35 The genus Verbesina produces guanidine alkaloids,271 and genus Artemisia diterpenoid alkaloids such as artekorine and its derivatives.349 Alkaloid-containing species are distributed worldwide throughout the temperate areas. The Ragwort genus is endemic to the Mediterranean and West

NH2

CO2H NH2

L-ornithine

n FIGURE 1.11 L-ornithine is an important

precursor of pyrrolidine, tropane, and pyrrolizidine alkaloids.

24 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Asian regions. Such species of this genus as Senecio carpathicus, Senecio erucifolius, Senecio othonnae, Senecio paludosus, Senecio rupestris, Senecio subalpinus, and Senecio wagneri contain a lot of pyrrolizidine alkaloids.253 Other alkaloids were extracted from Senecio triangularis. They are 9-O-acetyl-7-O-angelyl-retronecine, 7-O-angelyl-, 9-O-angelyl-, and 7-O-angelyl-9-O-sarracinylretronecine. Senecio pseudaureus and Senecio streptanthifolios yield only retrorsine and senecionine.22 However, a phytochemical investigation of Senecio divarigata L. (syn. Gynura divaricata DC.) has shown such alkaloids as intergerrimine and usaramine.325 Senecio bonariensis Hook and Arn. contains in its leaves sesquiterpene alkaloids (β-caryophyllene, β-caryophylene oxide, and germacrene D).84 In Switzerland, the alkaloids of Petasites hybridus, found growing in many places, have been studied.426 Petasin, senecionine, and intergerrimine were detected. Cheng and R€ oder61 isolated two pyrrolizidine alkaloids (senkirkine and doronine) from Emilia sonchifolia. The roots of Chromolaena pulchella contain (–)-supinidine, (–)-supinidine triviridiflorate, and (–)-supinidinediviridiflorate.114

1.3.3 The Logan botanical family (Loganiaceae) The Logan plant family (Loganiaceae Lindl.) is abundant in species containing L-tyrosine- (Figure 1.12) derived alkaloids (Table 1.4). Thirty genera and more than 500 species belong to this family, although new systematic research has proposed that Loganiaceae should be divided into several families. The Logan plant genus (Strychnos L.) is especially rich in many of alkaloids such as strychnine, brucine, and curare. From the genus Strychnos L., which contains 190 species, more than 300 alkaloids have been isolated. This genus provides alkaloids that have important biological activities COO– +

H3N

C

H

CH2

O

O

N

OH L-tyrosine

n FIGURE 1.12 L-tyrosine, with its aromatic side

chain, is a precursor of phenylethylamino- and isoquinoline alkaloids.

1.3 Occurrence in nature 25

Table 1.4 General botanical characteristics of the Logan family Botanical Forms and Parts

Characteristics

Botanical form

Herbs Shrubs Trees Logania Mitreola Mitrasacme Strychnos Spigelia Opposite Simple Regular in cymes or panicles Calyx Corolla 2 carpels Capsule Rarely berrylike or drupe Albuminous Sometimes winged

Some typical genera

Leaves Flowers

Fruit Seeds

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

and strong medicinal impact. Species containing strychnine are as follows: Strychnos nux-vomica L., Strychnos ignatii P. Bergius, and Strychnos wallichiana Steud ex DC. These are found throughout Asia, while Strychnos lucida R. Br. is located in Australia. Strychnos icaja Baillon and Strychnos tienningsi grow in Africa, and Strychnos panamensis L. in South America. Curare alkaloid exists in S. usambarensis, the species distributed throughout tropical Africa and Strychnos guianensis, the species found in the South American Amazonian region. Lansiaux et al.225 report on sungucine and isosungucine, isolated from S. icaja Baillon, and their strong bioactivity. Sungucine and isosungucine interact with DNA, inhibit the synthesis of nucleic acids, and induce apoptosis in HL-60 leukemia cells. Fre´de´rich et al.110 reported on the isolation and biological testing of isostrychnopentamine, an alkaloid in the leaves of S. usambarensis with strong antiplasmodial activity. The leaves of S. usambarensis contain also another antiplasmodial agent, a tertiary indolic alkaloid (17-O-acetyl, 10-hydroxycorynantheol 1).49 Dolichantoside, strictoside, and palicoside are in the stem bark of Strychnos mellodora, a tree found growing in the mountainous rain forests of east Africa, particularly in Tanzania and

26 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Zimbabwe.397 Brucine and strychnine have been extracted from S. nux-vomica.109 The root bark of this species also contains strychnoflavine and strychnoflavine,180 and the seed of this species has at least loganin, strychnine, brucine, and strychnine N-oxide.390 Other new seed alkaloids of this species are 4-N-hydroxymethyl strychnidin-17-acetic acid and 10,11-dimethoxy-4-N-hydroxymethyl strychnidin-17-acetic acid.435 Observations on traditional medicine in Brazil suggest that Strychnos pseudo quina A. St.-Hil. also contains quinine. However, experimental evidence of this is still missing.73 The stem bark of another species, Strychnos malacoclados, contains 3-hydroxylongicaudatine, bisnordihydrotoxiferine, divarine, longicaudatine, and longicaudatine Y and F.394 Strychnos moandaensis De Wild. contains moandaensine.409 Indeed, it is known that the genus Gelsemium from the family Loganiaceae is rich in different types of alkaloids. New alkaloids from this genus are β-carboline compounds.445 Surprisingly, Gelsemiun elegans Benth. contains also gelsochalotine. This indole ring–degraded monoterpenoid indole alkaloid generally is not typical for the family Loganiaceae and recently was first found by spectroscopic and crystal x-ray diffraction studies.232 The roots of Gelsemium elegans contain oxindole alkaloids, such as gelegamines A, B, C, D, E.444 The stem bark of Fagraea racemosa Jack ex Wall. in Indonesia stores fagraeoside. This alkaloid is derived from the natural condensation of secologanin and L-asparagine. Its amino acid component is not aromatic, which is a rare in terpene alkaloids. Fagraeoside is biologically active.374

1.3.4 The Poppy botanical family (Papaveraceae) The Poppy botanical family (Papaveraceae) contains L-tyrosine(Figure 1.12) derived alkaloids, such as morphine, codeine, thebanine, papaverine, narcotine, narceine, isoboldine, and salsolinol. The Poppy family is relatively large, comprising at least 26 genera and about 250 species. The family is distributed in the subtropical and temperate regions of the northern hemisphere (Table 1.5). The opium poppy (Papaver somniferum L.) is a known source of opium from its latex. The Poppy family alkaloids have strong biological and medicinal impact. They are also strong narcotics. A lot of new alkaloids have been reported on within this family. Six alkaloids (norsanguinarine, sanguinarine, dihydrosaneuinarine, oxysanguinarine, lincangenine, and cryptopine) were reported from Papaver coreanum Nakai.212 Papaver nudicale, growing in Mongolia, produces (+)-amuronine, pseudoprotopine, allocryptopine, (–)-dihydroamuronine, (–)-amurensine N-oxide, (–)-amurensine N-oxide, and promorphinane alkaloid (–)-8,14-dihydroflavinantine. Pseudotropine was not reported before as an alkaloid presents in the family Papaveraceae. The same refers to (–)-dihydroamuronine and

1.3 Occurrence in nature 27

Table 1.5 General botanical characteristics of the Poppy family Botanical Forms and Parts

Characteristics

Botanical form Some typical genera

Herbs Adlumia Arctomecon Argemone Canbya Chelidonium Corydalis Dendromecon Dicentra Eschscholzia Fumaria Hesperomecon Meconella Papaver Platystemon Roemaria Romneya Sanquinaria Stylophorum Milky juice Stem with vascular bundles Usually lobed or dissected Bisexual Regular Red Violet Yellow White Two sepals Capsules Dark seed in the capsule

Special characteristics Leaves Flowers

Fruit Seeds

Source: Refs 25a, 30a, 31a, 51a, 70a, 186a, 326a, 402a

both (–)-amurensine N-oxide A and B, which previously considered alkaloids, do not occur in the genus Papaver.171,306 Moreover, Papaver trinifolium Boiss. contains miltanthoridine and miltanthoridinone.338 From greater celandine (Chelidonium majus), widespread in Central Europe, such alkaloids as sanguinarine, cholidonine, hydrastine, berberine, and chelerythine have been isolated.408 Phytochemical investigation of

28 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Glaucium leiocarpum Boiss. revealed 11 isolated alkaloids: (+)-glaucine, 6,6adehydronorglaucine, oxoglaucine, (+)-N-methylglaucine, (+)lastourviline, (+)-predicentrine, (+)-dihydropontevedrine, secoglaucine, ( )N-methylcoclaurine, allocryptopine, and protopine.337 Glaucium paucilobum contains stylopine, protopine, α-allocryptopine, bulbocapnine, corydine, isocorydine, crabbine, and arosine 347 and Glaucium flavum such alkaloids as protopine, glaucine, and bocconoline. All three alkaloids occur in the roots of this species. The bocconoline do not exist in the aerial parts.36,37 Glaucine is a very bioactive compound used as antitussive and also a recreational drug of abuse.267 Protopine is considered as promising agent in cancer theraphy.37 Twenty-three isoquinoline alkaloids have been isolated from Corydalis bulleyana Diels. Hao and Qicheng134 report on such alkaloids as protopine, (+)-consperine, (+)-acetylcorynoline, dihydrosangunarine, (+)-acetylisocorynoline, ()-stylopine, (+)-corynoline, (+)-corynoloxine, (+)-isocorynoline, ( )-chelanthifoline, corycavanine, (+)-scoulerine, (+)isoboldine, acetylcorydamine, allocryptopine, corydamine, bulleyamine, (+)-6-acetonylcorynoline, (+)-12-formyloxycorynoline, (+)-6-oxoacetylcorynoline, (+)-12-hydroxycorynoline, (+)-bulleyanaline, and (+)norjuziphine. Corydalis bulleyana Diels is used in traditional medicine as a febrifuge, antidote, and analgesic. Moreover, other species of this genus, such as Corydalis amabilis Migo, Corydalis yanhusao W. T. Wang, Corydalis ambigua Cham and Schlecht, Corydalis bungeana Turcz., and Corydalis incisa Thunb. are also used in folk medicine in China. They contain identical or similar alkaloids as C. bulleyana Diels.134 From Corydalis humosa, two new alkaloids (1,1-dimethyl-6-methoxy-7-hydroxyl1,2,3,4-tetrahydroisoquinoline and (1R)-(4-hydroxybenzyl)-7-hydroxyl-8O-d-glucopyranosyl-1,2,3,4-tetrahydroisoquinoline) were found.448 Jain, Tripathi, and Pandey176 report on ()-cheilanthifoline and hunnemanine from Eschscholzia californica Cham. L-tyrosine- (Figure 1.12) derived alkaloids, such as bicuculline and metiodine, occur in the genera Bleeding heart (Corydalis L.) and Dutchman’s breeches (Dicentra L.). From the species Corydalis flabellata Edgew, many alkaloids have been isolated: sibiricine, severzinine,348 6 (2-hydroxyethyl)5-6-dihydrosanguinarine, 6-acetonyl-5,6-dihydro sangui narine, 6-acetonyl-5,6-dihydrosanguinarine, N-methyl-2,3,7,8-tetramethoxy-6-oxo-5,6 dihydrobenzophenanthridine, oxosanguinarine, spallidamine, 6-acetonyl5,6 dihydrochelerythrine, 6-oxochelerythrine, and sanguidimerine.204 These alkaloids are well known for their biological activity. For example, spallidamine has been found to display fungitoxic activity.247 Fumaria bracteosa Pomel is characterized by the presence of (+)-adlumidine, (+)-α-hydrastine, (+)-bicucullidine, and protopine.131 The genera Eschschohzia and

1.3 Occurrence in nature 29

Argemone contain at least 23 alkaloids from different alkaloid groups (pavinane, protopine, benzylisoquinoline, benzophenanthridine, aporphine, and protoberberine).45 Argemone mexicana accumulates biologically active alkaloids such as sanguinarine, chelerythine, berberine, protopine, and allocryptopine.329 Tatsis, Bohm, and Schneider392 present interesting results on nudicaulins. These are alkaloids with a unique pentacyclic skeleton composed of an indole ring and a polyphenolic moiety. An interesting point is that nudicaulins confers the intense color of Papaver nudicale and Papaver alpinum flowers, and they are accompanied by other compounds, such as anthocyanins and flavonols. This is a fresh and prospective result for future chemical and taxonomical studies.

1.3.5 The Citrus botanical family (Rutaceae) The Citrus (syn. Rue) botanical family (Rutaceae Juss.) contains more than 150 genera and over 900 species (Table 1.6). These species are distributed worldwide across tropical and subtropical areas. Many species contain both

Table 1.6 General botanical characteristics of the Citrus family Botanical Forms and Parts

Characteristics

Botanical form

Shrubs Shrublets Trees Herbs Agothosma Amyris Citrus Clausena Cneoridium Fagara Glycosmis Haplophyllum Helietta Poncirus Ptelea Pilocarpus Ruta Spathelia Zanthoxylum Usually aromatic with resinous tissues Alternate

Some typical genera

Special characteristics Leaves

Continued

30 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.6 General botanical characteristics of the Citrus family Continued Botanical Forms and Parts

Characteristics Exstipulate Bisexual or unisexual Small Regular or irregular Three to five petals Ovary superior, usually syncarpous Capsule Drupe Samara or berry

Flowers

Fruit

Source: Refs 17a, 25a, 31a, 70a, 186a, 280a, 326a, 402a

CO2H

NH2 L-anthranilic

acid

n FIGURE 1.13 L-anthranilic acid is a precursor of quinazoline, quinoline, and acridine alkaloids.

anthranilic acid– (Figure 1.13) and L-histidine- (Figure 1.14) derived alkaloids. Anthranilic acid–derived alkaloids are dictamnine, skimmianine (in such species as Dictamnus albus, Dictamnus dasycarpus Turcz.412 Dictamnus angustifolius,378 Euxylophora paraensis,170 and Skimmia japonica), acronycine in Acronychia baueri, melicopicine in Melicope fareana, and rutacridone in Ruta graveolens. In the genus Haplophyllum A. Juss., a lot of alkaloids with potential estrogenic activity are reported.279 These are acutine, acetylfolifidine, bucharidine, dubinidine, dubinine, glycoperine, evoxine, y-fagarine, folifidine, foliosidine, haplophyline, haplopine, perfamine, and skimmianine. Moura et al.272 report on alkaloids from Helietta longifoliata Britt., a Rutaceae family plant that grows in South America and is used in Brazilian folk medicine. Helietidine, y-fagarine, flindrsine, kokusaginine, and maculasine have been isolated, and their

COO– +

H3N

C

H

CH2 C

NH

H N

CH C H

N

N

CO2 NH2

L-histidine

n FIGURE 1.14 L-histidine is a precursor of imidazole

alkaloids.

1.3 Occurrence in nature 31

antibacterial activity demonstrated. Alkaloids derived from L-histidine include, for example, pilocarpine and pilosine, in such species as Pilocarpus microphyllus and Pilocarpus jaborandi. Fagaronine, the alkaloid extracted from Fagara zanthoxyloides Lam. has been described. There is evidence that this alkaloid induces erythroleukemic cell differentiation by gene activation.95 From Zanthoxylum integrifolium Merr., an evergreen tree that grows in the northern Philippines and Taiwan, three new alkaloids were isolated: 7,8-dehydro-1-methoxyrutaecarpine, isodecarpine, and 8-demethyloxychelerythine.57 In earlier studies, 1-hydroxyrutaecarpine, rutaecarpine, and 1-methoxyrutaecarpine were isolated from this plant.350 In Zanthoxylum hyemaline St. Hill, two quinoline alkaloids ( )-R-geilbalansine and hyemaline were isolated.272 Bioassay-guided fractionation led to the isolation of three indolopyridoquinazoline alkaloids, 1-hydroxy rutaecarpine, rutaecarpine, and 1-metoxyrutaecarpine, from the fruit of Z. integrifolium.350 Zanthoxoaporphines A–C are in Zanthoxylum paracanthum,335 and L-proline-derived alkaloids such as monophyllidin in Zanthoxylum monophyllum.290 From the bark of this species, thalifoline, berberine, and jathrorrhizine also were isolated. Moreover, Melicope semecarpifolia produces melicarpine and samecarpine.56 The genera Toddalia, Dictamus, Pelea, and Stauranthus are also present in these furoguinoline alkaloids.55,58,134,149,399,400 Eighteen alkaloids are reported from Toddalia asiatica, such as 8-methoxynorchelerythrine, 11-demethylrhoifoline, 8-methoxynitidine, 8-acetylnorchelerythrine, 8,9,10,12-tetramethoxynorchelethrine, isointegriamide, 1-dimethyldicentrinone, and 11hydroxy-10-methoxy-(2,3)-methylenedioxytetrahydroprotoberberine.161,163 Galipea officinalis Hancock, a shrub growing in tropical America and used in folk medicine as an antispasmodic, antipyretic, astringent, and tonic,31,53,123 yields nine quinoline alkaloids, of which galipine, cusparine, cuspareine, demethoxycusparine, and galipinine are the most important.316 The fruit of Evodia officinalis, which have traditionally been used as a folk medicine in Korea for the treatment of gastrointestinal disorders, postpartum hemorrhage, and amenorrhea, contain six quinoline alkaloids: (2-hydroxy-4-methoxy)-3-(3´-methyl-2´-butenyl) quinoline, evocarpine, dihydroevocarpine, evodiamine, rutaecarpine, and 1-methyl-2-[(Z)-6-undecenyl]-4(1H)-quinolone. In addition, the fruit of the similar species Evodia rutaecarpa contain four quinolone alkaloids: 1-methyl-2-tetradecyl-4(1H)quinolone, evocarpine, 1-methyl-2-[(4Z,7Z)-4,7 decadienyl]-4(1H)quinolone and 1-methyl-2-[(6Z,9Z)-6,9-pentadecadienyl]- 4(1H)-quinolone.203 Alkaloids occurring in E. rutaecarpa show various bioactivities, including angiotensin II antagonistic effects, an inhibitory effect on Helicobacter pyroli growth, and DGAT inhibition. Moreover, Rahmani et al.315

32 CHAPTER 1 Definition, typology, and occurrence of alkaloids

reported on the carbazole alkaloid 7-methoxy-glycomaurin, discovered in Glycosmis rupestris Ridely. Rahman and Gray304 reported on carbazole alkaloids from Murraya koenigii (L.) Spreng., a small tree with dark gray bark that grows in Asia. Mahanimbine has been reported to possess insecticidal and antimicrobial properties.314,317 The isolation and identification of six 2-alkyl-4(1H)-quinolone alkaloids from the leaves of previously uninvestigated Spathelia excelsa (K. Krause) has been described by Lima et al.236 These data have chemosystematic significance in clarifying the relationships of this species and the Rutaceae plant family. Moreover, a new carbazole alkaloid, named clausine Z, has been isolated from stems and leaves of Clausena excavata Burm. by Potterat et al.308 A clausine structure was established by spectroscopic methods, and its bioactivity was determined. According to Potterat et al.,308 this compound exhibits inhibitory activity against cyclin-dependent kinase 5 (CDK5) and shows protective effects on cerebellar granule neurons in vitro. The roots of Clausena excavata contain 12 carbazole alkaloids,369 including clausine and its derivatives.393 Later, from this species, other new bioactive carbazole alkaloids, excavatines A–C, also were found.299 Carbazole alkaloids, clausenawallines G–K, are produced by Clausena wallichii,254 harmandianamines A–C by Clausena harmandiana,255 and mafaicheenamines D and E by Clausena lansium.256 The alkaloids glycosmisacridone and glycosmisindole were found in twigs of Glycosmis cochinchinensis. These alkaloids show antibacterial activity.368 From Geijera parviflora, an endemic species in Australia, parvifloranines A and B were found and reported by Shou et al.356 Alkaloids also are found in the genus Conchocarpus. Conchocarpus marginatus and Conchocarpus inipinatus produces acridone alkaloids, such as arborinine, methylarborinine, xanthoxoline, toddaliopsin C, and inopinatin. These species also have quinoline alkaloids such as dictamine.26 Acridone alkaloids were reported from the Citropsis articulata. In the stem bark of this species, 14 alkaloids are present, including citropsine A.266 Orirenierines A, B, and C are alkaloids found in Oricia renieri and reported by Wansi et al.420 These alkaloids are strongly bioactive. Carbazole alkaloids, such as murrayakoenimol, mahanimbine, koenimbine, murrayazoline, and girimbine, were found in Murraya koenigii (Linn) Spreng.54 Moreover, this species contains also another carbazole alkaloid, kurryam,252 and mahanine, pyrayafoline D, and murrafoline I.174 Antimicrobial alkaloids (oriciacridone A and B) were found in the stem bark of Oriciopsis glaberrina.421

1.3.6 The Nightshade botanical family (Solanaceae) The Nightshade plant family (Solanaceae Pers.), containing 90 genera and more than 2000 species distributed in all continents, is particularly abundant in alkaloids (Table 1.7). The plant species belonging to this family grow

1.3 Occurrence in nature 33

Table 1.7 General botanical characteristics of the Nightshade family Botanical Forms and Parts

Characteristics

Botanical form

Herbs Shrubs Small trees Vines Atropa Capsicum Cestrum Datura Deboisia Hyoscyamus Lycianthes Lycium Mandragora Nicotiana Petunia Physalis Solanum Sometimes climbing Hair Alternate Exstipulate Regular or slightly irregular with tabular calyx Corola rotate Hermaphrodite Bisexual Berry or capsule Many seeded Albuminous Embryo straight or curved

Some typical genera

Special characteristics Leaves Flowers

Fruit Seeds

Source: Refs 25a, 31a, 70a, 186a, 285a, 326a, 402a

especially in the tropics and subtropics. However, the majority of the species occur in Central and South America. The L-ornithine- (Figures 1.11 and 1.15) derived alkaloids occur in many species of this family. Hyoscyamine, scopolamine, littorine, anisodamine, and cuscohygrine are reported as typical alkaloids for Solanaceae.178 Hyoscyamine, hyoscine, and cuscohygrine are in the genus Nightshade (Atropa L.). This genus is distributed in large areas from the Mediterranean to central Asia and the Himalayas. Deadly nightshade (Atropa belladonna L.) is a typical species containing tropan

NH2

CO2H

CO2H NH2

L-ornithine

N L-nicotinic

acid

n FIGURE 1.15 L-ornithine and L-nicotinic acids are precursors of some alkaloids in the Nightshade family.

34 CHAPTER 1 Definition, typology, and occurrence of alkaloids

alkaloids.437 Moreover, the genus Jimsweed (otherwise known as Thornapple; Datura L.), from tropical and warm temperate regions, and the genus Pitura plants (Deboisia L.), native to Australia and New Caledonia, also contain these compounds. The Jimsweed genus is especially rich in alkaloids. Fifty-three tropane alkaloids are reported from only one species, Datura innoxia, growing in Morocco. Hyoscyamine is a main alkaloid in the roots of this species and scopolamine in its aerial parts.99,100 From another species, Datura stramonium, over 60 tropane alkaloids are reported to be isolated and identified by many scientists.28,30,101,305 Further, also rich in the above-mentioned L-ornithine-derived alkaloids are the genus of Henbane plants (Hyoscyamus L.) occurring in Europe and North America, as well as in the large area from northern Africa to central Asia. The black henbane (Hyoscyamus niger L.) is a good example of this alkaloid-containing genus, but many more genera have the ability to yield these alkaloids. The white henbane (Hyoscyamus albus L.) contains 34 tropane alkaloids, including hygrine and tropane derivatives, phygrine, and aponorscopolamine.99 The genera of Mandrake plants (Mandragora L.) and Scopolia plants (Scopolia L.) may be mentioned in this context. However, the Nightshade plant family (Solanaceae) also contains other alkaloids, such as the compounds derived from Nicotinic acid (Figure 1.15). The Tobacco plant genus (Nicotiana L.), with approximately 45 species native to the North and South Americas and 21 species native to Australia and Polynesia, contains such alkaloids as nicotine and anabasine. Moreover, phenylalanine-derived alkaloids are also characteristic of the Nightshade plant family (Solanaceae). Capsaicin is a typical alkaloid of the paprika plant genus (Capsicum L.), which has approximately 50 species native to Central and South America. Steroidial alkaloids, such as solanidine, are very common in the potato plant genus (Solanum L.), with more than 1500 species distributed throughout the tropical, subtropical, and temperate zones of the globe. Certainly, the plant species belonging to the genus Solanum L. are endemic only in South America. Solanum lycocarpum St. Hill is an invasive and native shrub in Brazilian savanna. It is well known that this plant contains solamargine and solasodine, present in the unripe fruit.342 Especially, steroid alkaloid solasodine may penetrate animal bodies (experiments with rats), the placental and hematoencephalical barriers, and affect the fetus. According to Schwarz et al.,342 S. lycocarpum fruit may act as phytohormones, perhaps promoting some neural alterations that at adult age may impair the sexual behavior of the experimental female without impairing fertility and sexual hormone synthesis. Eight solanidane alkaloids were found in Solanum campaniforme. Their bioactivity is reported to be limited and focused on inhibition of the main toxic action of Bothrops pauloensis venom.398 Another steroid is solanopubamine, isolated from Solanum schimperianum.9 Such

1.3 Occurrence in nature 35

alkaloids as demissidine, dihydrosolacongestidine, solasodine, and tomatodine were found in aerial parts of Solanum leucocarpum Dunal growing in Colombia.281 Tomatine is a characteristic alkaloid for the Tomato plant genus (Lycopersicon L.), with seven species and native to the Pacific coast of South America.

1.3.7 The Coca botanical family (Erythroxylaceae) Alkaloids also occur in many other plant families. It is relevant to mention the Coca plant family (Erythroxylaceae L.), distributed in the tropics and endemic to South America, especially in the regions of Peru and Bolivia, where the coca bush (Erythroxylum coca) has been known for at least 5000 years.15 Typical characteristics of this family are elliptic, light green leaves (4–7  3–4 cm), small, white flowers, and small, reddish-orange drupes. Nowadays, it is distributed in the Andean region, the African tropics, and in southern Asia. Many L-ornithine-derived alkaloids are found in this plant family, from which three species, the aforementioned E. coca, Erythroxylum truxilense, and Erythroxylum novagranatense, contain cocaine, ecgonine, cinnamylcocaine, α-truxilline, truxilline, methylecgonine, tropine, hygrine, hygroline, and cuscohygrine. These strong alkaloids are commonly used as drugs in mainstream medicine and, at times, are the object of pathological or criminal activity—the source of many personal human tragedies. Zanolari et al.439 reported on new alkaloids from Erythroxylum vacciniifolium Mart., a Brazilian endemic plant used in traditional medicine. From the bark of this plant, nine tropane alkaloids (catuabines H–I, three of their hydroxy derivatives, and vaccinines A and B) have been isolated. These tropane alkaloids are interesting for their ester moieties. In the bark of Erythroxylum zambesiacum, 27 alkaloids were found.65 Ribeiro et al.323 found 7-β-acetoxy-3-β-6-β-dibenzoyloxytropane in the leaves of the species Erythroxylum rimosus O.E. Schultz. This alkaloid is considered a new tropane alkaloid in the genus Erythroxylum. This genus has some 250 species and, apart from the cocaine-producing species, has not been examined systematically by modern analytical methods.

1.3.8 The Borage botanical family (Boraginaceae) The Borage plant (syn. Forget-me-not) family (Boraginaceae Lindl.) contains L-ornithine- (Figures 1.11 and 1.15) derived alkaloids, such as indicine-N-oxide in the heliotrope (Heliotropium indicum) and southern hound’s tongue (Cynoglosum creticum) species (Table 1.8). Farsam et al.104 reported new alkaloids from another heliotrope species, Heliotropium crassifolium: europine and ilamine and their N-oxides. These alkaloids have strong toxic effects.

36 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.8 General botanical characteristics of the Borage family Botanical Forms and Parts

Characteristics

Botanical form

Herbs Rarely shrubs or trees Lianas (rarely) Amsinckia Anchusa Bourreria Cordia Cryptantha Cynoglosum Ehretia Hackelia Heliotropium Lappula Lithospermum Mertensia Myosotis Onosma Onosmodium Pulmonaria Tournefortia Plagiobothrys Symphytum Stiff and bristly hair Alternate Simple Usually rough-hairy Exstipulate Regular Five-parted calyx Regular corola (five-lobed) Blue or white A drupe Rarely berrylike Straight or curved embryo Scant albumen

Some typical genera

Special characteristics Leaves

Flowers

Fruits Seeds

Source: Refs 25a, 31a, 70a, 90a, 186a, 326a, 402a

1.3 Occurrence in nature 37

Moreover, six pyrrolizidine alkaloids were detected in Anchusa strigosa Banks and Sol38 and Heliotrium esfandiarii europine N-oxide.436 Alkaloids of both species have bioimpact. Anchusa strigosa is a plant widely distributed in the Mediterranean region. It is used in local folk medicine as a diuretic, analgesic sedative, sudorific remedy, and for treatment of stomach ulcers and externally for skin diseases.6,331 Siciliano et al.360 analyzed the qualitative and quantitative composition of alkaloids in flowers, leaves, and roots of A. strigosa. This phytochemical study led to the isolation of nine pyrrolizidine alkaloids, from which three have been unidentified. Pyrrolizidine alkaloids (echimidine and its derivatives) are in Echium species: Echium hypertropicum Webb and Echium stenosiphon Webb,52 Echium glomeratum,4 and viridinatine in Onosma erecta81,82. Many pyrrolizidine alkaloids have been isolated from the leaves, roots, and rhizomes of the lung-wort species (Pulmonaria spp.). In both Pulmonaria officinalis and Pulmonaria obscura, such alkaloids as intermedine, lycopsamine, and symphitine have been detected. This means that P. officinalis is not an exception among Boraginaceae in not having pyrrolizidine alkaloids, as had been previously claimed.326 Haberer et al.130 present the evidence for this. Thus, they have advanced the theory of the botanical family base for alkaloid distribution. Acetyl-intermedine and acetyl-lycopsamine are alkaloids yielded in common comfrey (Symphytum officinale L.). Many species belonging to the Borage plant family are native to the Mediterranean area.

1.3.9 The Legume botanical family (Fabaceae) Alkaloids derived from L-ornithine, L-lysine, and L-trypthophan occur in the Legume plant family (Fabaceae Juss.) (Table 1.9). This plant family is the third largest botanical family, with 650 genera and 18,000 species in the humid tropics, subtropics, temperate, and subarctic zones around the globe.12–14,364 L-ornithine-derived alkaloids such as senecionine are present in the genus Crota (Crotalaria L.). The most typical alkaloids for this botanical family are L-lysine- (Figure 1.16) derived alkaloids, such as lupinine, sparteine, lupanine, angustifoline, epilupinine, and anagyrine. Lysine alkaloids occur in many species belonging to the legume family. They are quinolizdine alkaloids occurring in the large and very diverse genus Lupine (Lupinus L.) (Figure 1.17) and the genus of Broom plants (Cytisus L.). Such alkaloids occur also in Sophora species.205 Sophora nuttalliana contains lot of quinolizidine alkaloids, including anagyrine. Moreover, this species contains also swainsonine.213 Chronanthus orientalis, an endemic Fabaceae species of Turkey, contains typical quinolizidine alkaloids (sparteine, lupanine, oxosparteine, etc.). This species also is able to synthesize lidocaine.125 Goat’s rue (Galega officinalis L.) produces a toxic

38 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.9 General botanical characteristics of the Legume family Botanical Forms and Parts

Characteristics

Botanical form

Herbs Shrubs Lianes Vines Trees Acacia Adesmia Aeschynomene Albizia Arachis Astragalus Baptisia Bauhinia Caesalpinia Calliandra Cassia Cercis Chamaecrista Crotalaria Dalbergia Dalea Delonix Desmodium Erythrina Gleditsia Glycine Indigofera Inga Lathyrus Leucaena Lonchocarpus Lotus Lupinus Meliotus Milletia Mimosa Parkia Parkinsonia Phaseolus Pisum Pithecellobium

Some typical genera

1.3 Occurrence in nature 39

Table 1.9 General botanical characteristics of the Legume family Continued Botanical Forms and Parts

Special characteristics Leaves Flowers Fruit Seeds

Characteristics Robinia Rhynchosia Senna Swartzia Tamarindus Tephrosia Trifolium Vicia Wisteria Often twining or climbing Hair Alternate Stipulate Regular or irregular Usually bisexual A legume Big or small seed

Source: Refs 12, 13, 14, 25a, 31a, 70a, 186a, 326a, 364, 402a

alkaloid, galegine.285 Both the genus Swainsona (Swainsona L.) and the genus of Blackbean plants (Castanospermum L.) contain swainsonine and castanospermine. Przybylak et al.310 detected 46 compounds from six Mexican lupin species (Lupinus rotundiflorus, Lupinus montanus, Lupinus mexicanus, Lupinus elegans, Lupinus madrensis, Lupinus exaltatus). It was possible to identify unambiguously 24 detected compounds from the 46 of them. Most of the identified alkaloids are from the lupanine group: sparteine, ammodendrine, epiaphyllidine, epiaphylline, tetrahydrorhombifoline, 17oxosperteine, 5,6-dehydro-α-isolupanine, angustifoline, α-isolupanine, aphyllidine, 5,6-dehydrolupanine, lupanine, aphylline, 11,12dehydrolupanine, dehydrooxosparteine, 3β-hydroxylupanine, multiflorine, 17-oxolupanine, 13αhydroxylupanine, 3β, 13α-dihydroxylupanine, 13αangeloylolupanine, 13αtigloyloxylupanine, and 4β-tigloyloxylupanine. Matrine has been isolated from Sophora subprostata Chun et T. Chen64. Genistein and lupinalbin A were reported from Macroptilium lathyroides.88 Moreover, in plants belonging to the Legume family (Fabaceae L.), alkaloids derived from L-tryptophan also occur. Eserine, eseramine, physovenine, and geneserine are all examples of these kind of alkaloids, which occur, for

COO– +

H3N

C

H

CH2 CO2H

CH2

NH2

CH2

NH2

CH2 +

NH3 L-lysine

n FIGURE 1.16 L-lysine is a precursor of piperidine, quinolizidine, and indolizidine alkaloids.

40 CHAPTER 1 Definition, typology, and occurrence of alkaloids

(a)

(b) n FIGURE 1.17 (a) Structure of the seed tests of the Washington lupine (Lupinus

polyphyllus Lindl.). Transmission electron microscopy (TEM) proved wide structural diversity in both the genus Lupinus L. and the species. The figure shows exotesta (exo), mesotesta (meso), endotesta (endo), and cotyledon (C), the parts that differ in the species and varieties Lupinus spp. In the testa and parts of the storage cells, alkaloids are present. (b) Alkaloidal Lupinus polyphyllus Lindl. in its flowering stage.

1.3 Occurrence in nature 41

example, in the Calabar bean (Physostigma venenosum L.). Erysovine and wrythraline are high toxic alkaloids in Erythrina lysistemon. Lou et al.244 isolated two alkaloids, 2-methoxyl-3-(3-indolyl) propionic acid and 2-hydroxyl3-[3-(1-N-methyl)-indolyl] propionic acid, in peanut skins (Arachis hypogaea L.). These alkaloids had not previously been found in natural sources.244 Moreover, Wanjala et al.419 isolated and identified several new alkaloids in Erythrina latissima, widespread in Botswana, Zimbabwe, and South Africa. One alkaloid, (+)-erysotrine, shows bioimpact as an antimicrobial agent. Moreover, Kajimoto et al.187 reported on isolation of purine alkaloid (locustoside A) from Gleditsia japonica, and Jones et al.184 on pyrrolizidine alkaloid, 1-epilexine, isolated from Australian legume tree Castanospermum australe. Tanaka et al.388 reported on a new alkaloid in Erythrina poeppigiana, a plant found in central and South America. This alkaloid, 8-oxo-α-erythroidine epoxine, is similar to other alkaloids previously found in this species, such as erysodine, erysovine, α-erythroidine, 8-erythroidine, and dihydro-,8-erythroidine.355 Recently, N-[(3R, 7R)-( )-jasmonoyl]-(S)-dopa and N[(3R,7R)-( )-jasmonoyl]-dopamine were isolated from the flowers of broad beans (Vicia faba L.) by Kramell et al.207 These alkaloids are tyrosine-derived compounds. All alkaloids occurring in Fabaceae have both biological and ecological significance. They also have been considered to be taxonomical markers.263 The occurrence of some important alkaloids in nature is shown in Table 1.10.

Table 1.10 Main types of alkaloids and their chemical groups Occurrence in Nature

Precursor Compound of Alkaloid Derivation

Family

Species

Alkaloids

L-ornithine-derived

Solanaceae

Atropa belladonna

()-hyoscyamine ()-hyoscine Cuscohygrine As Atropa As Atropa As Atropa As Atropa As Atropa As Atropa As Atropa As Atropa

alkaloids

Datura innoxia Datura stramonium Datura metel Datura sanguine Duboisia myoporoides Hyoscyamus niger Hyoscyamus muticus Mandragora officinarum

Continued

42 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alkaloids

Erythroxylaceae

Scopolia carniolica Withana somnifera Erythroxylum coca

As Atropa Withasomnine (-)-cocaine (-)-ecgonine Cinnamylcocaine α-truxilline Truxilline Methylecgonine Tropine Hygrine Hygroline Cuscohygrine Indicine-N-oxide Indicine-N-oxide Acetyl-intermedine Acetyl-lycopsamine Senecionine Senecionine Senecionine Stachydrine 4hydroxystachydrine Pelletierine Pseudopelletierine Methylpelletierine Anaferine Obeline Lobelanine Sedamine Piperine Piperidine Anagyrine Cytisine Sparteine Thermopsine Albine Angustifoline Lupanine Sparteine Lupinine Sparteine

Erythroxylum truxilense

L-lysine-derived

alkaloids

Boraginaceae

Heliotropium indicum Cynoglossum spp. Symphytum officinale

Asteraceae Fabaceae Capparidaceae

Senecio vulgaris Senecio jacobaea Crotalaria spp. Boscia angustifolia

Punicaceae

Punica granatum

Crassulaceae

Lobelia inflata

Piperaceae

Sedum acre Piper nigrum

Fabaceae

Baptisia alba

Lupinus albus

Lupinus luteus

1.3 Occurrence in nature 43

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

L-tyrosine-derived

alkaloids

Occurrence in Nature Family

Species

Alkaloids

Lupinus polyphyllus Lupinus angustifolius Lupinus hispanicus

Lupanine Angustifoline Lupinine Epilupinine Anagiryne Sparteine Swainsonine Castanospermine Hordenine Tyramine Mescaline Anhalamine Anhalonine Anhalonidine Salsolinol Curare Tubocurarine Fangchinoline Fangchinoline Fangchinoline N-methylliriodendronine 2-O-N-dimethylliriodendronine Dicentrinone Corydine Aloe-emodin Tetrandrine Stephanine Fangchinoline Fangchinoline Fangchinoline Tubocurarine Morphine Codeine Thebaine Papaverine Narcotine Narceine Isoboldine

Graminae

Lupinus latifolius Cytisus scoparius Swainsona canescens Castanospermum australe Hordeum vulgare

Cactaceae

Lophophora williamsii

Papaveraceae Menispermaceae

Corydalis spp. Chondrodendron tomentosum Cissampelos pereira Cyclea barbata Cyclea peltata Stephania dinklagei

Stephania tetrandra

Loganiaceae Papaveraceae

Stephania harnandifolica Triclisia subcordata Strychnos toxifera Papaver somniferum

Continued

44 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alkaloids

Berberidaceae

Berberis spp.

Ranunculaceae

Mahonia spp. Nandina domestica Hydrastis canadiensis

Berberine Berbamine Hydroxyacanthin Glaucine Berberine (+)-nantenine Berberine Hydrastine Fangchinoline Fuzitine Bicuculline Metiodine Spallidamine Sanguidimerine Oxosanguinarine Bicuculline Metiodine Autumnaline Floramultine Kreysigine Colchicine Emetine Cephaeline Secologanin Ipecoside Mitragynine Lycorine Homolycorine 2-O-acetyllycorine Leucovernine Acetylleucoverine N-demethylgalanthamine Hippeasterine 9-O-demethylhomolycorine 5α-hydroxyhomolycorine 11-hydroxyvittatine Lycorine Galanthamine

Thalictrum orientale Fumariaceae

Corydalis spp. Corydalis flabellate

Dicentra spp. Liliaceae

Kreysigia multiflora

Rubiaceae

Colchicum autumnale Cephaelis ipecacuanha

Amaryllidaceae

Mitragyna speciosa Leucojum vernum

Lycorus radiata Galanthus spp.

1.3 Occurrence in nature 45

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alkaloids

Galanthus plicatus ssp. Pancratium sickenbergeri

Galanthindole Hippadine Trispheridine Pseudolycorine Haemanthidine Norgalanthamine Haemanthamine Vittatine Pancracine Lycorine Galanthine Haemanthamine Lycorine Lycorenine Oxomaritidine Martidine Vittatine Psilocin Psylocybin Psilocin Psilocybin Psilocin Psylocybin Psilocin Psilocybin Gramine Ergotamine

Zephyranthes citrina

L-tryptophan-derived

alkaloids

Agaricacea

Psilocybe semilanceata Cynocybe spp. Panaeolus spp. Stropharia spp.

Graminae

Elaeagnaceae Zygophyllaceae Rubiaceae Apocynaceae

Hordeum vulgare Secale cereale (with C. purpurea) Triticum aestivum (with C. purpurea) Triticale (with C. purpurea) Elaeagnus angustifolia Peganum harmala Pausinystalia yohimbe Ophiorrhiza mungos Alstonia macrophylla

Ergotamine Ergotamine Elaeagnine Harman, harmine Yohimbine Camptothecin Talcarpine Pleiocarpamine Alstoumerine 2-O-epiantirhine

Continued

46 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alstonia scholaris Aspidosperma megalocarpon Rauvolia capra Rauvolfia serpentina

Rauvolfia canescens

Rauvolfia vormitoria Catharanthus roseus

Loganiaceae

Ochrosia elliptica Ervatmia heyneana Strychnos icaja Strychnos nux-vomica Strychnos toxifera

Strychnos usambarensis

Alkaloids Alstonerine Alstophyline Macralstonine Alstomacrophyline Villalstonine Alstomacroline Macrocarpamine Menilamine Fendlerine Aspidoalbine Aspidolimidin Quinine Reserpine Rescinnamine Ajmalicine Reserpine Rescinnamine Deserpine Reserpine Rescinnamine Ajmalicine Catharanthine Vindoline Vinblastine Vincristine Vindesine Alioline Ellipticine Camptothecin Sungucine Isosungucine Strychnine Brucine Curare C-toxiferine Alcuronium Isostrychnopentamine

1.3 Occurrence in nature 47

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alkaloids

Poaceae

Arundo donax

Rubiaceae

Cinchona officinalis

Arundamine Arundacine Quinine Quininidine Cinchonine Cinchonidine Quinine Cinchonidine Quinidine Quinine Cinchonidine Quinidine Corynanthine α-yohimbine Dihydrocorynantheine Corynantheine Corynantheidin Camptothecin Waltherione-A Camptothecin Camptothecin Camptothecin

Cinchona succirubra

Cinchona calisaya

Corynanthe pachyceras

Sterculiaceae Nyssaceae Icacinaceae

Fabaceae

Ophiorrhiza mungos Waltheria douradinha Camptotheca acuminata Nothapodytes foetida Merilliodendron megacarpum Pyrrenacantha klaineana Arachis hypogaea

Physostigma venenosum

Erythrina lysistemon Erythrina latissima Erythrina poeppigiana

Camptothecin 2-methoxyl-3-(3-indolyl)propionic acid 2-hydroxyl-3-[3-(1-N-methyl)indolyl]propionic acid Eserine Eseramine Physovenine Geneserine Erysovine Erythraline (+)-erysotrine 8-oxo-α-erythroidine-epoxine Erysovine α-erythroidine β-erythroidine Dihydro-β-erythroidine

Continued

48 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alkaloids

Convolvulaceae

Ipomea violacea

Ergoline Ergotamine Ergine Ergoline Ergotamine Ergine Afzelin Apigenin Arctigenin Astragalin Schischkinin Dolichotheline Pilocarpine Pilosine Pilocarpine Pilosine Harmin Vaccine Dictamnine Skimmianine Maculosine γ-fagarine Helietidine Flindersine Kokusaginine Dictamnine Skimmianine Acronycine Melicopicine Melicarpine Semecarpine Rutacridone (-)-nicotine Anabasine Discorine Ricinine Arecoline Pyridine

Rivea corymbosa

L-histidine-derived

alkaloids

Asteraceae

Centaurea schischkinii

Cactaceae Rutaceae

Dolichothele sphaerica Pilocarpus microphyllus Pilocarpus jaborandi

Anthranilic acid–derived alkaloids

Zygophyllaceae Acanthaceae Rutaceae

Peganum harmala Justicia adhatoda Dictamus albus Helietta longifoliata

Skimmia japonica Acronychia baueri Melicope fareana Melicope semecarpifolia Ruta graveolens Nicotinic acid–derived alkaloids

Solanaceae

Nicotiana tabacum

Discoreaceae Euphorbiaceae Palmae

Discorea dregeana Ricinus communis Areca catechu

1.3 Occurrence in nature 49

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation Acetate-derived alkaloids

Occurrence in Nature Family

Species

Alkaloids

Umbelliferae Pinaceae Zygophyllaceae

Conium maculatum Pinus spp. Nitraria komarovii

Coniine Pinidine Komavine Acetylkomavine Dihydroschoberine Nitrabirine N-oxide Ephedrine Cathinone Cathine Ephedrine Cathinone Cathine Ephedrine Cathinone Cathine Norpseudoephedrine Capsaicin β-skytanthin Actinidine Dihydroagarofuran Gentiopicroside Arcutin Acorone Accoridine Corypidine Coryphine Tangutisine 12-acetylnepelline Cammaconine Karacoline Karakanine Nepelline Songorine Aconitine Sinomontanine Aconitine Methyllycaconitine Barbine

Nitraria sibirica Phenylalanine-derived alkaloids

Ephedraceae

Ephedra intermedia

Ephedra geriardiana

Ephedra major

Terpenoid alkaloids

Celastraceae Solanaceae Apocynaceae Actinidiaceae Celastraceae Gentianaceae Ranunculaceae

Catha edulis Capsicum annuum Skytanthus acutus Actinida polygama Tripterygium wilfordii Gentiana lutea Aconitum arcuatum Aconitum coreanum

Aconitum karacolicum

Aconitum napellus Aconitum sinomontanum Aconitum vulparia Delphinium berbeyi

Continued

50 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alkaloids

Delphinium corymbosum

Delcorine Delsonine Methyllycaconitine Barbine Norditerpenoid Methyllycaconitine Barbine Ajacine Anthranoyllycoctonine Candelphine Delphyrine Delpoline Delsonine Karacoline Lycococtine Solanidine Tomatine Jervine Cyclopamine Cycloposine Protoveratrine A Protoveratrine B O-acetylfervine Jervine Cyclopamin Cycloposine Protoveratrine A Protoveratrine B Jervine Cyclopamin Cycloposine Protoveratrine A Protoveratrine B Holaphyllamin Conessine

Delphinium occidentale Delphinium nuttalianum Delphinium glaucescens Delphinium poltoratskii

Steroid alkaloids

Solanaceae Liliaceae

Solanum tuberosum Lycopersicon esculentum Veratrum album

Veratrum lobelianum Veratrum californicum

Veratrum viride

Apocynaceae

Purine alkaloids

Rubiaceae

Holarrhena floribunda Holarrhena antidysenterica Coffea arabica

Caffeine Theophylline Theobromine

1.3 Occurrence in nature 51

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Species

Alkaloids

Coffea canephora

Caffeine Theophylline Theobromine Caffeine Theophylline Theobromine Caffeine Theophylline Theobromine Caffeine Theophylline Theobromine Caffeine Theophylline Theobromine Caffeine Theophylline Theobromine Guaranine Theophylline Theobromine Caffeine Theobromine Caffeine Theobromine Caffeine Theobromine Annotinine Lycodine Lycopodine Cernuine Huperzine A Huperzine J Huperzine K Huperzine L Phlegmariurine

Coffea liberica

Theaceae

Camellia sinensis

Aquifoliaceae

Ilex paraguensis

Ilex cassine

Ilex vormitoria

Sapinidaceae

Paullinia cupana

Sterculiaceae

Cola nitida Cola acuminata Theobroma cacao

Moss alkaloids

Lycopodiaceae

Lycopodium annotinum Lycopodium complanatum Lycopodium cernuum Huperzia serrata

Continued

52 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Fungus alkaloids

Alkaloids

Aspergillus terreus

Asterrelenin Terretonin Territrem A Territrem B Chanoclavine Ergoline Ergotamine Tabtoxin Pyocyanine Deformylflustrabromin Lustrabromine Flustramine Saxitoxin Tetrodotoxin Samandarine Samandarone Samandaridine Cycloneosamandaridine Cycloneosamandione Samandenone Cardiotoxin Neurotoxin Batrachotoxin Homobatrachotoxin Histrionicotoxin Dihydroisohistrionicotoxin Gephyrotoxin Pumiliotoxin Pumiolotoxin A Pumiliotoxin B Pseudophrynaminol Castoramine Muscopyridine Cassine Dialkylpyrazine Trialkylpyrazine Norharman

Bryozoa

Rhizopus, Penicillium, Claviceps purpurea (with Secale cereale) Pseudomonas tabaci Pseudomonas aeruginosa Flustra foliacea

Saxidomus

Saxidomus giganteus

Salamandra

Salamandra maculosa

Dendrobatidae

Salamandra samandarine Phyllobates aurotaenia

Bacter alkaloids Animal alkaloids

Species

Dendrobates histrionicum

Dendrobates pumilio

Mammals Mymonoptera

Formicidae

Rhinotermitidae

Pseudophryne coriacea Castor fiberei Moschus moschiferus Solenopsis invicta Dontomachus hastatus Ontomachus brunneus Reticuliterms flavipes

1.3 Occurrence in nature 53

Table 1.10 Main types of alkaloids and their chemical groups Continued Precursor Compound of Alkaloid Derivation

Occurrence in Nature Family

Diploda

Coleoptera

Coccinellidae

Species

Alkaloids

Ticulitermes virginica Glomeris marginata

Norharmane Glomerin Homoglomerin Polyzonimine Adaline Coccinelline Podamine Epilachnene Convergine Podamine Myrrhine Propeleine Propyleine

Polyzonium rosalbum Adalia bipunctata Coccinella septempunctata Pilachna varivestis Podamia convergens Myrrha octodecimguttata

Staphylinidae

Propylea quatuordecimpunctata Chilocorus cacti Paederus fuscipes

Stenusine (+)-pederin Pederone

Sources: Refs 11, 17–19, 21, 28, 35, 37, 51, 57, 61, 63, 68, 72, 81, 83, 90, 94, 98, 101, 103–105, 107, 108, 111–113, 119, 120, 135, 142–146, 148, 153, 156, 162, 163, 164, 166, 168, 177, 185, 194, 195, 208, 211, 213, 216, 217, 220–223, 229, 234, 245, 249, 257–259, 268, 272, 276, 279, 281, 283, 289, 291, 294, 295, 297, 300, 303, 315, 318, 319, 321, 327, 341, 346, 350, 352–354, 358, 361, 362, 370, 371, 375, 377, 406, 411, 413, 422, 423, 425, 427, 434, 438, 440, 444, 447, 450, 451

1.3.10 The Monseed botanical family (Menispermaceae) The Monseed plant family (Menispermaceae) contains L-tyrosine-derived alkaloids (Figure 1.12). Plant species belonging to this family are found throughout the tropics, especially in tropical lowland zones.395 The Monseed botanical family is large, containing about 70 genera and 450 species (Table 1.11). The genus Stephania produces tetrandrine and stephanine, while the genus Curare (Chondrodendron) yields curare and tubocurarine. All are known to be medicinal alkaloids. More than 150 alkaloids have been isolated from plants of the Stephania genus. Camacho47 reported on many of alkaloids found in Stephania dinklagei, a climbing shrub of the deciduous forest of Africa. They were methylliriodendronine, 2-O,N-dimetylliriodendronine, liriodenine, dicentronine, corydine, and aloe-emodin. These alkaloids display strong biological impact with antiprotozal activity. The report by G€oren, Zhou, and Kingston122 notes that these plants also yielded liriodenine, corydine, isocorydine, atherospermidine, stephalagine, and

54 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.11 General botanical characteristics of the Monseed family Botanical Forms and Parts

Characteristics

Botanical form

Trees Lianas Alternate Usually palmately veined Often lobed Regular Small Unisexual Endocarp Curved embryo

Leaves

Flowers

Fruit Seeds Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

dehydrostephalagine. Liriodenine showed strong cytotoxic activity. Corydine and atherospermidine even revealed activity damaging to DNA. Zhang and Yue441 reported on the isolation and structural elucidation of new alkaloids from Stephania longa Lour., a perennial herbaceous liana. They detected stephalonines A–I, norprostephabyssine, isoprostephabyssine, isolonganone, and isostephaboline. Chen et al.60 isolated tetrandrine from the root of a Chinese herb Stephania tetrandra S. Moore. This alkaloid showed the ability to inhibit both culture activation and TGF-β(1)-stimulated activation of quiescent rat hepatic stellate cells (HSCs) in vitro.60 Fangchinoline was isolated from the same species.431 This alkaloid is strongly bioactive inhibiting, for example, the kinase activities. Cepharathine, cepharanoline, isotetrandrine, and berbamine were isolated From Stephania cepharantha Hayata 277 and such alkaloids as cepharantine, tetrahydropalmatine, and xylopinine are reported in Stephania rotunda, a creeper growing commonly in mountains of Cambodia.34 Moreover, in addition to these alkaloids, vireakine, roemerine, and palmatine were isolated and indentified from the same species (Stephania rotunda).21 Aporphine, protoberberine, and morphine are found in Stephania yunnanensis,442 and gindarudine and grabradine in Stephania glabra.345 Cepharanthine is a particularly active component of hair growth. Moreover, the isolation and characterization of alkaloids (cycleanine, cycleanine Noxide, isochondodendrine, cocsoline and quinine) from Epinetrum villosum (Exell) Troupin has also been reported.287 These alkaloids were found to exhibit antimicrobial and antiplasmodial activities. Epinetrum villosum is a twining liana, growing in secondary forests in the coastal areas in Congo

1.3 Occurrence in nature 55

and Angola, and is used in traditional medical for the treatment of fever, malaria, and dysentery.243,287 The genus Cissampelos contains cissampareine, which has potential medicinal uses, but it is also psychoactive. It is a principal alkaloid of dawidjiewortel (Cissampelos capensis), which grows in South Africa. Moreover, this species also contains bulbocapnine, dicentrine, salutaridine, cissacapine, cycleanine, and insularine89; and Cissampelos sympodials Eichl synthesizes a very active biological bisbenzylioquinoline alkaloid, warifteine.74 Two aporphinoid alkaloids, neolitsine and dicentrine, were found by Raoelison et al.313 in the leaves of Spirospermum penduliflorum Thouras from Madagarscar. These alkaloids, especially dicentrine, have relaxant properties. Rhizomes of such Menispermaceae species as Sinomenium acutum contain a lot of alkaloids.62,214 Dauriporphine, 6-O-demethylmenisporphine, bianfungecine, menisporhine, and 6-O-demethyldauriporphine show different bioactivities. The stem bark of the wood Abuta grandifolia (Mart.) contains aporphine alkaloids (e.g., R-nomuciferine) and bisbenzylisoquinoline alkaloids (e.g., (R,S)-2 N-norberbamunine and curine derivatives).69 Subsessiline, oxoaporphine, telixotine, azufluoranthene, isoimerubrine, and tropoloisoquinoline occur in Abuta rufescens.380

1.3.11 The Berberry botanical family (Berberidaceae) L-tyrosine-derived alkaloids also occur in the Berberry botanical family (Berberidaceae Torr., Gray, Juss.) (Table 1.12). Berberine is produced

Table 1.12 General botanical characteristics of the Berberry family Botanical Forms and Parts

Characteristics

Botanical form

Bushes Herbs Trees Small hairs Stems with vascular bundles Generally alternate Simple Regular Olitary or in cymes, racemes, or panicles Bisexual A berry or pod Anatropous with endosperm Small embryo

Special characteristics Leaves Flowers

Fruit Seeds

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

56 CHAPTER 1 Definition, typology, and occurrence of alkaloids

particularly by the Berberry genus (Berberis L.) and the Mahonia genus (Mahonia Nutt.). These alkaloids are found in such species as the common barberry (Berberis vulgaris L.), native to Euroasia; the Japanese barberry (Berberis thunbergii DC.), native to Asia; and the Chita (Berberis aristat DC.) in the Himalayas. It is also present in Holly (Mahonia aquifolium Nutt.), native to Western America, and in the Creeping mahonia (Mahonia repens (Lindl.) G. Don., endemic to the North America. New research reports mention berberine, found in the oblonga barberry (Berberis oblonga Scheid), growing in Kazakhstan but native to Central Asia.194 Moreover, it is also reported that, together with berberine, other alkaloids were detected, such as glaucine, hydroxyacanthin, and berbamine. Orallo286 reported on (+)-nantenine, a natural alkaloid derived from Nandina domestica Thunberg, which was first isolated by Takase and Ohasi in 1926. Subsequently, extracts containing this alkaloid were widely used in Japanese folk medicine for the treatment of whooping cough, asthma, pharynx tumors, uterine bleeding, and diabetes. Berbamine was extracted from Berberis poiretil Echneid, a plant that grows in China.128 This alkaloid shows actions of antiarhythmia, antimyocardial ischemia, and antithrombosis. Berbanine was isolated from Berberis vulgaris in Czech Republic160 and spectroscopically identified. Moreover, the analysis of the aerial parts of Berberis sibirica Pall. from Mongolia showed that they contain 14 isoquinoline alkaloids (derivatives of aporphine, protoberberine, protopine, benzylisoquinoline).173 Another biomolecule, pachycanthine, an isoquinoline alkaloid with significant bioactivity, was found in Berberis pachycantha Koehne.3 Early (–)-tajedine was reported from ssp. australis of the common barberry.373

1.3.12 The Buttercup botanical family (Ranunculaceae) The Buttercup botanical family (Ranunculaceae Juss.) (Table 1.13) yields both L-tyrosine and terpenoid alkaloids. This plant family, which has 50 genera and nearly 2000 species, is situated around the globe in the temperate zones. Tyrosine-derived alkaloids, such as berberine and hydrastine, occur in the Seal genus (Hydrastis L.). Fangcholine and fuzitine have been reported in the genus Thalictrum (Thalictrum orientale), growing in Turkey.102 From the Thalictrum species growing in Bulgaria (Thalictrum flavum L. and Thalictrum aquilegifolium L.), thialigosidine, thialisopidine, columbamine, and corydine are found.172 Both (+)- and (–)-5-hydroxyl-8oxoberberine also are reported in the genus Coptis.411 Terpenoid alkaloids, such as aconitine and sinomontanine, appear in the genus Hood (Aconitum L.). Many other alkaloids have been found in this genus. In

1.3 Occurrence in nature 57

Table 1.13 General botanical characteristics of the Buttercup family Botanical Forms and Parts

Characteristics

Botanical form

Herbs Climbers (rarely) Trees (rarely) Vines (rarely) Medium-sized plants Stems with vascular bundles Alternate with sheathing bases or opposite Regular or irregular Usually bisexual Achene, follicle, or berrylike (rarely) Seeds with endosperm A minute embryo

Special characteristics Leaves Flowers Fruit Seeds

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

Aconitum karacolicum (Rapaics) from Kyrgyzstan, karacoline, karakanine, songorine, nepelline, 12-acetylnepelline, cammaconine, and secokaraconitine were detected.376 In addition, karaconitine was found from the roots of this species.405 In Aconitum arcuatum (Maxim.), a new alkaloid, arcutin with antibacterial and medicinal impact, was located.391 In Aconitum coreanum (Levl.) Rapaics, tangutisine, acorone, acorridine, coryphine, and coryphidine were found, all of which have powerful biological impact.96 Aconitum weixiense contains bioactive diterpenoid alkaloids: weisaconitines A, B, C, and D,446 and the roots of Aconitum carmichaeli Debx. contains bioactive aconitamide.215 Secoaconitine, N-deethyl-3-acetylaconitine, and N-deethyldeoxyaconitine are reported from Aconitum pendulum.417 Moreover, in Aconitum anthora L., typical alkaloids are hetisinone, isotalatizidine, and 10-dehydroxy-8-O-methyltalatizamine107 and trichocarpine in Aconitum tanguticum var. trichocarpum.238 Methyllycaconitine and barbine are typical in the genus Larkspur (Delphinium L.). Delphinium corymbosum contains delcorinine and delsonine,333,334 while Delphinium poltoratskii was found to hold a lot of alkaloids.33 These include methyllycaconitine, lycoctonine, anthranoyllycoctonine, ajacine, karacoline, and delpoline. Delphinium trichophorum contains bioactive trichodelphines A, B, C, D, E, and F.237 Nigeglapine and nigeglaquine are alkaloids characteristic for Nigella glandulifera. They are glycosylated indazole alkaloids with rare distributions in nature.240,241 From this species, deoxynigellamine, nigellamines A and B as well as nigeglamine also have been reported.240,241

58 CHAPTER 1 Definition, typology, and occurrence of alkaloids

1.3.13 The Lily botanical family (Liliaceae) The Lily botanical family (Liliaceae Adans., Juss.) (Table 1.14) is spread worldwide and contains more than 200 genera and around 3500 species. Some genera of this family produce L-tyrosine-derived alkaloids. The genus Kreysigia yields autumnaline, floramultine, and kreysigine. Saffron genus Colchicum (Colchicum L.) produces colchicines. Metacolchicine is in Sandersonia aurantiaca,202 and other colchicines in meadow saffron (Colchicum autumnale L.).273 Stereoidal alkaloids in the Liliaceae family are found in the Hellebore genus (Veratrum Bernch.). Jervine, cyclopamine (Figure 1.18), cycloposine, protoveratrine A, and protoveratrine B yield Veratrum album. Veramadines A and B are reported to be found in Veratratum mackii var. japonicum.389 O-Acetyljervine has been reported in the false hellebore (Veratrum lobelianum Bernch.).207 Steroidal alkaloids

Table 1.14 General botanical characteristics of the Lily family Botanical Forms and Parts

Characteristics

Botanical form

Herbs Shrubs Trees Climbers (rarely) Colchicum Erythronium Fritillaria Gagea Kreysigia Lilium Medeola Tulipa Veratrum Cosmopolitan family Usually bulbs Alternate Parallel veined Regular Solitary or in racemes, panicles, or umbels Dehiscent capsule or a berry Small embryo Albumen

Some typical genera

Special characteristics Leaves Flowers Fruit Seeds

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

1.3 Occurrence in nature 59

CH3

CH3 H H N

O CH3

H O

H

CH3

H

HO Jervine H N

H3C H H3C H CH3 H

HO

H O

H

CH3

H

H

H Cyclopamine CH3

N CH3 H H3C

HO

OH

CH3

O

OH OH

O HO

OCOCHCH2CH3 COOCH3 CH3 O COOCH3 Protoveratrine

n FIGURE 1.18 The structures of jervine, cyclopavine, and protoveratrine.

also occur in such species as Fritillaria anhuiensis, Fritillaria cirrhosa, Fritillaria delavayi, and Fritillaria pallidiflora.51,228,351,357,381 Four new steroid alkaloids (puqienine A, puqienine B, N-demethylpuqietinone, puqietinonoside) have been isolated from Fritillaria species by Jiang et al.180 The bulbs of these plants have been used as an antitussive and expectorant in folk Chinese medicine. All four new alkaloids have been reported to display the antitussive activity in mice.180 The family Liliaceae contains also pyrrolizidine alkaloids. Kim et al.196 reported 10 of these alkaloids from the roots of Liliaceae species Paris

60 CHAPTER 1 Definition, typology, and occurrence of alkaloids

verticulata. According to this research, such alkaloids as verticillatin A., verticillatin B, vertcillatin C, heliovinine N-oxide, and indicine N-oxide were especially bioactive.

1.3.14 The Coffee botanical family (Rubiaceae) The Coffee (syn. Madder) botanical family (Rubiaceae Juss.) (Table 1.15) consists of more than 400 genera and over 6000 species. It grows in the tropics and the subtropics. Plants belonging to this family include trees,

Table 1.15 General botanical characteristics of the Coffee family Botanical Forms and Parts

Characteristics

Botanical form

Trees Shrubs Lianas Herbs Climbers Borreria Casasia Catesbaea Cephalanthus Chiococca Coffea Dioidia Ernodia Erithalis Exostema Galium Gardenia Guettarda Hamelia Hedyotis Ixora Mitchella Morinda Mussaenda Pavetta Pentodon Pickneya Policourea Psychotria Randia

Some typical genera

1.3 Occurrence in nature 61

Table 1.15 General botanical characteristics of the Coffee family Continued Botanical Forms and Parts

Special characteristics

Leaves Flowers

Fruits Seeds

Characteristics Richardia Rondeletia Spermacoce Tarenna Mainly tropical Some genera in temperate zone Lacking internal phloem Hair Opposite or whorled Stipulate Regular Corolla with cylindric tube Usually bisexual A capsule, berry, drupe, or schizocarp Albumen Embryo straight to curved

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

bushes, and liane. The Coffee plant family contains two major purines of adenine-/guanine-derived alkaloids, the so-called purine alkaloids. Purine is a nitrogenous base of nucleotide, which consists of just purine and pentose sugar (D-ribose or 2 deoxy-D-ribose). Typical purine alkaloids are caffeine, theophylline, and theobromine. The same or similar purine alkaloids occur in other plant families as well, such as the Tea plant family (Theaceae), the Guarana plant family (Sapinidaceae), and the Cola plant family (Sterculiaceae). The plants of the Guarana family have one additional alkaloid, guaranine (Table 1.10). Purine alkaloids have a biological and, according to recent clinical results, as well as a positive and prophylactic effect in decreasing the risk of Parkinson’s disease, for example, in the case of caffeine. Coffee consumption is linked into health benefits including possible prevention of several chronic and degenerative diseases. There is evidence that caffeine interact with glutamate receptor gene GRIN2A, which encodes an NMDA-glutamate-receptor subunit as Parkinson’s disease genetic modifier. Caffeine is therefore known as active agent in the prevention of this disease. The biosynthetic pathway of caffeine and related purine alkaloids is relatively well known and their artificial synthesis is developed.18 New methods of caffeine recognition are also established.157 From Waltheria

62 CHAPTER 1 Definition, typology, and occurrence of alkaloids

douradinha St. Hill belonging to the Cola family, walterione A, a tryptophanderived alkaloid, has been discovered.151 This alkaloid has important biological potential. Staerk et al.371 noted five alkaloids isolated from the species Corynanthe pachyceras K. Schum., a member of the Rubiaceae family. Corynantheidine, corynantheine, dihydrocorynantheine, α-yohimbine, and corynanthine were isolated from the bark of this species, and all these alkaloids demonstrate powerful bio- and ecoimpacts (leishmanicidal, antiplasmodial, and cytotoxic activity). Other L-tryptophan-derived alkaloids were found in the stem bark of Cinchona officinalis, belonging to same botanical family (Rubiaceae).321 These are quinine, quinidine, cinchonine, and cinchonidine. Such alkaloids also show bioimpact. Moreover, the latest research focuses on mitragynine, an alkaloid in Mitragyna speciosa, which grows in Thailand. This alkaloid has a powerful, opiumlike effect.152 Mitragynine was also isolated from this plant,262 and the effect of mitragynine on neurogenic contraction of smooth muscle was studied in guinea-pig vas deferens. The alkaloid inhibited the contraction of the vas deferens produced by electrical transmural stimulation. On the other hand, mitragynine failed to affect the responses to norepinephrine and ATP.262 Other alkaloids have also been isolated from Mitragina speciosa.7 They are indole (e.g., 7 β-hydroxy7H-mitraciliatine) and oxindole alkaloids (e.g., isospeciofoleine). Mitragyna diversifolia contains 15 alkaloids, including monoterpene indole alkaloids such as mitradiversifoline, specionoxeine-N(4)-oxide, 7hydroxyisopaynantheine, 3-dehydropaynantheine, and 3-isopaynantheine.50 From the roots and fruit of Mitragyna inermis, naucleactonin has been reported by Donfack et al.93 Mitragyna hirsuta from Thailand contains isomitraphyllinol.200 Moreover, the leaves of Mitragyna parvifolia are known to contain isomitraphylline and mitraphyline and their derivatives.288 From another Rubiaceae species, Nauclea latifolia Smith, indole alkaloids (strictosamide, vincosamide, and pumiloside) were found by Donalisio et al.92 The roots of Nauclea orientalis contain such molecules as naucleficine, naucleactonin, naucleidinal, 19-epi-naucleidinal, and pumiloside,359 which are indole alkaloids.The bark of Nauclea officinalis has such alkaloids as naucline, angustine, angustidine, naulefine, and naucletine.235 β-carboline alkaloids have been reported from Psychoria barbifolia D.C.,86 Galianthe ramosa,85 and Galianthe thalictroides.106 The new indole alkaloids (spirocadambine, dehydraisodihydrocadambine, nitrocadambine A, and nitrocadambine B) have been reported from Neolamarckia cadamba239 and pyrrolidinoalkaloids from Margaritopsis cymuligera (Muell. Arg.39 Quadrigemine A, quadrigemine B, psychotridine, and isopsychotridine C have been isolated from the leaves of Psychotria forsteriana and their cytotoxic activity on cultured rat hepatoma cells (HTC line) reported.328 These alkaloids show a high toxicity on HTC. Moreover, psychotriasine was isolated from the leaves

1.3 Occurrence in nature 63

of Psychotria calocarpa449 and croceaine A and psychollatine from Palicourea crocea from Trinidad278 and from Psychotria umbellata.193 The leaves of Myrioneuron nutants are known to contains antimalarial alkaloids: (–)-myrionidine, myrionamide, and (–)-schoberine.304

1.3.15 The Amaryllis botanical family (Amaryllidaceae) L-tyrosine-derived alkaloids are found in the Amaryllis (syn. Daffodil or Snowdrop) plant family (Amaryllidaceae Hill.), which is distributed throughout the world. This large botanical family (Table 1.16) comprises 50 genera and over 850 species. Lycorine has been detected in the Spider lily genus (Lycorus L.) and galanthamine in the Snowdrop genus (Galanthus L.). Galanthindole was isolated from Galanthus plicatus ssp. byzantinus.83,403,404 From the bulbs of Boophone haemanthoides growing in South Africa, such alkaloids as distichaminol, lycorine, and distichamine

Table 1.16 General botanical characteristics of the Amaryllis family Botanical Forms and Parts Botanical form Some typical genera

Special characteristics

Leaves Flowers

Fruit Seeds

Characteristics Herbs Behria Crinum Cyrtanthus Haemanthus Hippeastrum Hymenocallis Leucojum Narcissus Zephyranthes A medium-sized herbs Bulbs Reduced stems More or less linear from bulbs On a leafless stalk from the bulb or solitary flower (rarely) Corona Bisexual A capsule or a berry Small seeds Testa Embryo curved

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

64 CHAPTER 1 Definition, typology, and occurrence of alkaloids

were isolated.277 Boit, D€ opke, and Stender32 reported isolating four alkaloids from Zephyranthes citrina (Baker) belonging to the Amaryllis plant family: galanthine, haemanthamine, lycorine, and lycorenine. More recently, Herrera et al.145 also isolated oxomaritidine, maritidine, and vittatine from this species. Oxomaritidine was reported for the first time by the authors. Alkaloids from Z. citrina (especially haemanthamine) have a clear bioimpact with inhibitory effects on the growth of HeLa cells and protein synthesis, as well as being a cytotoxic agent against MOLT 4 tumor cells.181,418 Haemanthidine also has a powerful bioimpact as a cytotoxic agent against various human tumor cell lines,16 and galanthine has a high inhibitory capacity with ascorbic acid biosynthesis in the potato.103 Maritidine exhibits antineoplastic activity.5 From Pancratium sickenbergi, hippadine, tris- pheridine, pseudolycorine, haemanthamine, norgalanthamine, haemanthidine, vittatine, 11-hydroxyvittatine, pancracine, lycorine, ent-6α-6β-hydroxybuphasine and ( )-8-demethylmaritidine have been isolated.2 These alkaloids have antiviral, antitumoral, analgesic, and insecticidal effects.2,217–219,260 Three alkaloids, lycorine, homolycorine, and 2-O-acetyllycorine, were isolated from the bulbs of Leucojum vernum by Szla´vik et al.382 and two new alkaloids, leucovernine and acetylleucovernine, by Forgo and Hohmann.108 These alkaloids, like many other new alkaloids from Amaryllidaceae, display antiviral activity. Shihunine and dihydroshihunine exist in Behria tenuiflora Greene. These alkaloids have been shown particularly to be inhibitors of Na+/K+ ATPase in the rat kidney.24,25 Moreover, alkaloids from Crinum stuhlmannii Baker have also been reported. Machocho et al.251 detected eight alkaloids (lycorine, kirkine, 9-O-demethylpluvine, ambelline, crinine, hamayne, crinamin, and amabiline) in this plant. Five alkaloids (lycorine, hamayne, vittatine, ismine, and ungeremine) were isolated from Hippeastrum solandriflorum Herb.24 Pancrimatine A, pancrimatine B, pancrimatine C, norismine, trispheridine, phenanthridine, and some of their derivatives are reported from the species Pancratium maritimum.167 Some of these are biologicaly very active.

1.3.16 The Oleaster botanical family (Elaeagnaceae) The Oleaster botanical family (Elaeagnaceae Lindl.) has three genera and 50 species (Table 1.17). It is distributed around the world, mostly in the temperate climatic zone and especially in the northern hemisphere. It also grows in eastern Australia, as well in some tropical and subtropical areas. The L-tryptophanderived alkaloid elaeagine occurs in the Oleaster genus (Elaeagnus L.), especially in the Russian olive (Elaeagnus angustifolia L.). Elaeagnus multiflora Thunb. and Elaeagnus argentea Pursh. also contain β-carbonyl alkaloids in their bark.1 Alkaloids are found in leaves of sea buckthorn (Hippophae rhamnoides L).29,129 However, deep research of these compounds is still in process.

1.3 Occurrence in nature 65

Table 1.17 General botanical characteristics of the Oleaster family Botanical Forms and Parts

Characteristics

Botanical form

Trees Shrubs Alternate or opposite Exstipule Regular Unisexual Calyx-tube Drupelike With bony testa Ex-albuminous Straight embryo

Leaves Flowers

Fruit Seeds

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

1.3.17 The Caltrop botanical family (Zygophyllaceae) The L-tryptophan-derived alkaloid known as harman and the Anthranilic acid– derived alkaloid known as harmine occur in the Caltrop plant family (Zygophyllaceae R. Brown) (Table 1.18). The family contains near 30 genera and more than 230 species and grows worldwide, especially in the tropics, subtropics, warm temperate zones, and dry areas. Harman and harmine occur in harmala pegan (Peganum harmala L.), the species belonging to the Pegan genus (Peganum L.). From Peganum nigellastrum comes such alkaloids as luotonins C and D, harmine, 3-phenylquinoline, 3-(4-hydroxyphenyl)quinoline, and 3-(1H-indol-3-yl)quinoline.248 Alkaloids derived from acetate, dihydroschoberine, and nitrabirine N-oxide have been found in the genus Nitraria (Nitraria Pall.) from the Siberian nitraria (Nitraria sibirica Pall.).392,401 In Nitraria komarovii (I. et. L.), komavine and acetylkomavine have been detected.402 Alkaloids are found in Fagonia olivieri.320 In Tribulus terrestris, terrestribisamide and tribulusterine are found.

1.3.18 Mushroom Alkaloids occur in many other botanical families. Moreover, these alkaloids, derived from L-tryptophan, occur in mushrooms genera: Hericium mushrooms (Hericium erinaceum), Psilocybe mushrooms (Psilocybe), Conocybe mushrooms (Conocybe), Haymaker’s mushrooms (Panaeolus), and Stoparia mushrooms (Stoparia). Hericirine, serotonin, psilocin, and psilocybin are basic alkaloids derived from these mushrooms (Figure 1.19). They

66 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Table 1.18 General botanical characteristics of the Caltrop family Botanical Forms and Parts Botanical form

Some typical genera

Special characteristics

Leaves

Flowers Fruit Seeds

Characteristics Shrubs Some trees and subshrubs Annual herbs (rarely) Balanites Guaiacum Fagonia Kallstroemia Larrea Porlieria Tribulus Zygophyllum Mainly in warm and arid regions Stems often sympodial Stems joined at the nodes Xylem with vessels Tracheids and fibres Hair Opposite Two ranked Stipulate Regular with four or five free sepals and petals Bisexual Capsules Berries (rarely) One seeded Endosperm present

Source: Refs 25a, 31a, 70a, 186a, 326a, 402a

are powerful psychoactive and neurotransmitter compounds. Recreational use of hallucinogenic mushrooms has been reported in several European countries, including England, Norway, Finland, the Netherlands, and Germany.372 Psilocybe semilanceata and Phanaeolus subbalteatus proved to be the only psilocybin containing fungi that can be gathered in middle and northern Europe in sufficient quantities to permit abuse.372 Hallucinogenic mushrooms alkaloids (e.g., psilocin and psylocibin) are similar in their activity to symphathomimetic alkaloids (e.g., cathionone, cathine) produced by khat tree (Catha edulis FORSK).226 The pyriferines A–C were isolated from the mushroom Pseudobaeospora pyrifera in Germany.311

1.3 Occurrence in nature 67

Norsesquiterpene alkaloid has been reported in the solid culture of mushroom-forming fungus Flammulina velutipes in China.433 Lion’s mane mushroom (Hericium erinaceum) produces isohericenone,198 and Macrolepiota neomastoidea produces the indole alkaloids macrolepiotin199 and lepiotin C.197 Alkaloids are also found in a cultivated Nigerian edible mushroom, Psathyrella atroumbonata Pegler.20 In the fruiting bodies of the mushroom Mycena sanquinoleta, sanquinones A and B together with sanquinolentaquinone are found.302 Mycena rosea produces mycenarubins A and B.302 Ling zhi (Ganoderma lucidum W. Curt.: Fr. P. Karst.), belonging to the Aphyllophoromycetideae, contains a lot of compounds including alkaloids.224 Mushroom alkaloids of the genus Ganoderma are still not researched in detail, although future research seems to be very prospective.

HO

NH2 N H Serotonin

OH N-CH3-CH3 N H Psilocin HO

1.3.19 Moss The moss alkaloids annotinine, lycopodine, cernuine, lyconadins, and complanadine169 occur in the genus Lycopodium L.. Gao et al.119 reported on the isolation of new alkaloids from the Lycopodiaceae family. Researchers working on Huperzia serrata (Thunb.), Trev. detected huperzine J, huperzine K, and huperzine L. These alkaloids have potential effects on Alzheimer’s disease. They occur not only in H. serrata, but also in other species belonging to the genus Huperzia. There are other similar alkaloids, such as huperzine A and its derivatives.19 Moreover, Tan et al.385 reported on several new alkaloids isolated from H. serrata (Thunb.): 11α-hydroxy-phlegmariurine B, 7α-hydroxyphlegmariurine B, and 7α-11α-dihydroxyphlegmariurine. Phlegmariurine was also reported in this species. Hirasawa et al.150 reported on Lycopodium alkaloids, lycobelines A–C, isolated from Huperzia goebelii. Lycopodium japonicum Thunb. constains five fawcettimine-related alkaloids, including 6-hydroxyl-6,7-dehydrolycoflexine, and 6-hydroxyl-6,7dehydro-8-deoxy-13-dehydroserratinine416 as well as 9 lycopodine-related alkaloids.414 Lycopodine-type new alkaloids are recently reported by He et al.136 They have isolated a lot of this type of alkaloids including 4α-hydroxyanhydrolycodoline, 4-α-6-α-dihydroxyanhydrolycodoline, and 6-epi-8-β-acetoxylycoclavine from Lycopodium japonica. Moreover, from Lycopodium obscurum L. such new alkaloids as obscurumine F, and obscurumine G were isolated by Chen et al.59 They have also found in this species acetylacrifoline, acetylannofoline, acetyldihydrolycopodine, acetylfawcettiine, des-N-methyl-α-obscurine, fawcettiine, flabelliformine, lycodoline, lycopodine, lycoflexine, and obscurumine B. Such new alkaloid as serralongamine A is reported to be isolated from Lycopodium serratum var. longipetiolatum by scientists from Taiwan and Japan.179

HO

P

O

O N

CH3 N H Psilocybin n FIGURE 1.19 Basic alkaloids

of mushrooms.

CH3

68 CHAPTER 1 Definition, typology, and occurrence of alkaloids

Lots of bioactive alkaloids were isolated from Icelandic club moss Diphasiastrum alpinum, such as 8S-O-acetylepiclavolonine, 5R, 8R-O-acetylfawcettiine, 5R, 8S-O-acetyllofoline, lycodoline, lycopodine, clavolonine, and anhydrolycodoline.132

1.3.20 Fungus and bacter The fungi Aspergillus, Rhizopus, Penicillium, and Claviceps produce parasitic ergoline and ergotamine alkaloids. The ergot alkaloids derived from L-tryptophan in the fungus Claviceps purpurea, growing on grain in the ears of rye (Secale cereale), wheat (Triticum aestivum), or triticosecale (Triticale), are highly toxic (Figure 1.20). They have been used in the development of lysergic acid diethylamine (LSD), which is hallucinogenic and, in small doses, used in the treatment of schizophrenia. Li et al.227 reported

N

HO H

O

OH

O

N

O

HN

NCH3 H

O O

NCH3 H

N H (+)-lysergic acid N H Ergotamine

N O

N-CH3 H

N H Lysergic acid diethylamide LSD n FIGURE 1.20 Ergotamine and LSD.

1.3 Occurrence in nature 69

isolating a new alkaloid, asterrelenin, from Aspergillus terreus. Moreover, from this fungi species, terretonin, territrem A and territrem B also have been isolated. A lot of alkaloids are isolated from the soil fungus Aspergillus sp. PSU-RSPG185. They are indole-benzodiazepine-2,5-dione derivatives and prenylated indole alkaloids.330 Indeed, indole alkaloids are produced by fungus Neosartorya fischeri (KUFC 6344) as well. Similar properties are found marine-derived fungi Neosartorya laciniosa (KUFC 7896) and Neosartorya tsunodane (KUFC 9213).97 DFFSCS013 benzodiazepine and indole alkaloids were found in deepsea-derived fungus Aspergillus westerdijakiae.298 Moreover, Aspergillus fumigates produces ergot alkaloids during a conidia stage.274 Two new diastereomeric quinolinone alkaloids have been identified from fungus Penicillium janczewskii obtained from a marine sample.137 These compounds show a low to moderate general toxicity. Dalsgaard et al.78 reported the isolation of communesins G and communesins H from the new species Penicillium rivulum Frisvad. The compounds were isolated by high-speed counter-current chromatography and preparative HPLC using UV-guided fractionation and subjected to antiviral, antimicrobial, and anticancer activity tests. In contrast to all other known communesins, communesins G and H were found inactive in these activities studied.78 Sasaki et al.339 isolated perinadine A from the cultured broth of the fungus Penicillium citrinum, which was separated from the gastrointestinal track of a marine fish. Citrinadin A, a pentacyclic indolinole alkaloid, has been isolated from the cultured broth of this fungus, which was also separated from a marine red alga.275 The endophytic fungus Penicilium citrinum produces a lot of secondary compounds, including other alkaloids.223 Marine fungus Penicillium oxalicum produces 2-(4-hydroxybenzol)quinazolin-4(3H)-one, a biologically active alkaloid having potential in reducing of plant viruses, especially TMV.340 Deepsea-derived fungus Penicillium sp. F23-2 produces indole alkaloids,127 and marine-derived fungus Penicillium sp. JF-72 is a source of diketopiperazine alkaloids.312 Endophytes of many plant species (e.g., Fescue species) are know to produce alkaloids. Loline is produced by the mutualist fungal endophytes Neotyphodium sp. and Epichloe sp.324 Endophytic fungus Mortierella alpina, living on the moss Schistidium antarctici in Antarctica, produces pyrrolopyrazine alkaloids.265 Endophytic fungi (Fomitopsis sp., Alternaria sp., and Phomposis sp.) isolated from fruit and seed of Miquelia dentata are known to produce a quinoline alkaloid camptothecine, which is known as anticancer agent.340 An interesting point is the capability of some endophytic fungi to synthetize alkaloids, which is considered a future perspective for the development of methods of highscale, intensive alkaloid production. Fusarium proliferatum is one of these. Empirical evidence exists that isolating BLH51 of this fungus from Mackleaya cordata can produce sanguinarine having antibacterial,

70 CHAPTER 1 Definition, typology, and occurrence of alkaloids

anthelmintic, and anti-inflammatory activities.415 Another endophytic fungus is Phoma sp. NRRL 46751, isolated from the Saurauia scaberrinae and producing a lot antitubercular phomapyrrolidone alkaloids.424 Production of alkaloids by bacteria is not common in nature. The bacteria Pseudomonas spp. are known to produce tabtoxin and pyocyanine, alkaloids with a relatively powerful biological activity. Marine bacterium Pantoea agglomerans P20-14 produces indole alkaloids.118 Endophytic bacterium Pseudomonas brassicaceareum ssp. neoaurantiaca isolated from Salvia miltiorrhiza produces 11 alkaloids,231 and endophytic bacteria isolated from Miquelia dentate Bedd., like some endophytic fungi, produce anticancer alkaloid camptothecine.340 Moreover, the marine bacterium Bacillus pumilus, isolated from the black coral Anthipathes sp., produces bioactive, antitrypanosomal alkaloids.261

1.3.21 Animals Alkaloids are also found in the animal kingdom, especially in millipedes, salamanders, toads, frogs, fish, and mammals. They occur particularly in the genera Saxidomus, Salamandra, Phyllobates, Dendrobates, Castor, and Moschus. Moreover, alkaloid molecules are found in such genera as Solenopsis, Odontomachus, Glomeris, and Polyzonium. Many alkaloids have been isolated from the marine environment, especially from the sponges.113 The discovery of ptilomycalin A from the sponges Ptilocaulis spiculifer and Hemimycale spp. preceded the isolation of several analogues from other sponges, such as Crambe crambe, Monanchora arbuscula, Monanchora ungiculata as well as from the some starfishes such as Fromia monilis and Celerina heffernani. From the Caribbean sponge Monanchora unguifera, the guanidine alkaloids (batzelladine J, ptilomycalin A, ptilocaulin, and isoptilocaulin) have been isolated.113 Many of guanidine alkaloids display ichthyotoxicity and antibacterial, antifungal, and antiviral activity. Antiviral activity has been exhibited against Herpes Simplex virus (HSV-1) and in inhibiting the HIV virus and cytotoxicity against murine leukemia cell lines (L1210) and human colon carcinoma cells (HCT-16). Segraves and Crews343 reported on the isolation of six new brominated tryptophanderived alkaloids from two Thorectidae sponges: Thorectandra and Smenospongia. These alkaloids also have wide ranging biological activities and are attractive compounds for potential applications. Alkaloids occur in amphibians. These vertebrate animals are reliant on water for their reproduction. Some species live both in and out of water and others are exclusively aquatic species. There are three orders of amphibians: the Anura (syn. Salientia) with more than 4500 species of frogs and

1.3 Occurrence in nature 71

toads, the Urodela (syn. Caudata) with 450 species of newts and salamanders, and the Apoda (syn. Gymnophiona) with more than 160 species of wormlike organisms. The skin of amphibians contains alkaloids. Costa et al.75 reported on bufetonin from the Anura species. This tryptamine alkaloid is widely spread as a component of chemical defense system in these species. Bufetonin acts as a potent hallucinogenic factor, showing activity similar to LSD on interaction with the 5HT2 human receptor.75 This compound has been isolated from the skin of three arboreal amphibian species, Osteocephalus taurinus, Osteocephalus oophagus, and Osteocephalus langsdorfii, from the Amazon and Atlantic rain forests. Moreover, it is known that toads belonging to the genus Melanophryniscus contain toxic alkaloids in their skin.264Alkaloids of the pumiliotoxin (PTX) group and indolizidines were isolated from Melanophryniscus montevidensis. The ladybird (Coccinellidae) and other beetles also contain alkaloids. Examples are mentioned in Table 1.10. Conversely, some moths, such as the arctiid moth (Utethesia ornatrix), depend on alkaloids for defense. Utethesia ornatrix, for example, sequesters pyrrolizidine alkaloids as a larva from the food plants of Crotalaria, belonging to the Fabaceae family.48 Longitarsus lateripunctatus (Coleoptera, Chrysomelidae, Alticidae), a leaf beetle feeding on Pulmonaria abscura leaves, contain readily traceable quantities of pyrrolizidine alkaloids.91 On the other hand, some poisonous frogs (Mantella) are known to digest alkaloids in their food. The ants Anochetum grandidieri and Tetramorium electrum, containing pyrrolizidine alkaloids, have been found in the stomachs of Mantella frogs.66,79 The strawberry poison frog (Dendrobates pumilio) contains dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing anthropods distributed in the habitat.383 Some pyrroloindole alkaloids, such as pseudophrynaminol, were found in the Australian frog (Pseudophryne coriacea).367 However, it is known that a diverse array of over 800 biologically active alkaloids have been discovered in amphibian skin,80 With the exception of the samandarines and pseudophrynamines, all alkaloids appear to be derived from dietary sources. It has been discovered that the beetles are sources for batrachotoxins and coccinellinelike tricyclics and ants and mites for pumiliotoxins. Moreover, ants are sources for decahydroquinolines, izidines, pyrrolidines, and piperidines. They are likely sources for histrionicotoxins, lehmizidines, and tricyclic gephyrotoxins.80 From North Sea Bryozoan (Flustra foliacea) several, brominated indole alkaloids have been isolated.301 These include deformylflustramine and flustramine. Deformylfrustrabromine A and deformylfrustrabromine B

72 CHAPTER 1 Definition, typology, and occurrence of alkaloids

have been shown to have affinities in the lower micromolar range with the neuronal nicotinic acetylcholine receptor (nAChR). As early as 1973, it was reported that erythrinan alkaloids (β-erythroidine and dihydro-βerythroidine) with neuromuscular transition blocking activity resembling the effects of curare had been found in the milk of goats (Capra) that grazed on the leaves of Erythrinia poeppigiana.355 The spectrum of alkaloids in mammals41 ranges from isoquinoline derivatives, via 8-carbolines, through to thiazolidines, arising from vitamin B6, and chloral and glyoxylic acid. For a long time, tetrahydroisoquinoline alkaloids were considered to be exclusively of plant origin. Bringmann et al.40 suspected that the formation of such endogenous alkaloids occur naturally in humans and mammals. The spontaneous formation of mammalian alkaloids, their further metabolic fate, and their biological and medicinal roles are a key not only to a better understanding of metabolic diseases but also to novel therapeutic concepts. In the case of animal species, it is necessary to check whether alkaloid molecules detected are endogenous or derived from exochemicals of dietary origin. One example of this problem, which could be mentioned, occurs in the important alkaloid as morphine. The biosynthesis of this alkaloid by plants from the Poppy family (Papaveraceae) is practically resolved, and there are not many research problems. However, the opposite situation occurs in the case of animals. It was reported, and biochemical data was presented to prove, that this alkaloid can occur in animals and humans, in considerable quantities. The only question remaining concerns the origin of this alkaloid in the animal and human body: Is it endogenous? If yes, moreover, the evidence of existing enzymes needed for the biosynthesis of the alkaloid in animals should be presented and biosynthetical activity should be documented. Only after that can the occurrence of alkaloids in the animal species finally be accepted as an endogenous characteristic without any conditions. On the other hand, there is evidence that animal and human bodies can produce endogenous alkaloids.41,430 Mammalian alkaloids derive from L-tryptophan via biogenic amines, such as dopamine, tryptamine, and sorotonine. Small amounts of alkaloids are normal in mammals. When disease strikes, alkaloid levels rise steeply. The common mammalian alkaloids are harman, norharman, tetrahydroharman, harmalan, 6-metoxyharman, salsolinol, norlaudanosoline (THP), dideoxynorlaudanosoline 1-carboxylic acid, and spinaceamines. Newly detected alkaloids are L-histamine derivatives.41,430 Although it is generally accepted or strongly suggested that alkaloids occur in animal species, even as a common matter,40,41,430 the genetic origin of these compounds as purely animal is still under discussion. Many research groups are working on this problem. Certainly, alkaloid chemical and biological research is both very challenging and prospectively fascinating. Alkaloids in nature are a part of production and consumer (feeding) chains.

1.3 Occurrence in nature 73

Moreover, they contribute to species growth, pleasure, and pathology. They are key to the processes of aggression and defense by the species. Alkaloids are used in nature for many purposes and by many species. Homo sapiens is just one of them.

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Chapter

2

Alkaloid chemistry CHAPTER OUTLINE

2.1 Alkaloids as secondary metabolism molecules 2.2 Synthesis and metabolism 106

100

2.2.1 Skeleton diversity 108 2.2.2 Ornithine-derived alkaloids 113 2.2.3 Tyrosine-derived alkaloids 116 2.2.3.1 2.2.3.2 2.2.3.3 2.2.3.4

Mescaline pathway 117 Kreysigine and colchicine pathway 117 Dopamine—the cephaeline pathway 117 Galanthamine pathway 118

2.2.4 Tryptophan-derived alkaloids 2.2.4.1 2.2.4.2 2.2.4.3 2.2.4.4 2.2.4.5 2.2.4.6 2.2.4.7 2.2.4.8

119

Psilocybin pathway 119 Elaeagnine, Harman and harmine pathway 120 Ajmalicine, tabersonine and catharanthine pathway 120 Vindoline, vinblastine, and vincristine pathway 121 Strychnine and brucine pathway 123 Quinine, quinidine, and cinchonine synthesis pathway 123 Eserine synthesis pathway 124 Ergotamine synthesis pathway 125

2.2.5 Nicotinic acid–derived alkaloids 125 2.2.6 Lysine-derived alkaloids 127 2.2.6.1 Pelletierine, lobelanine, and piperine synthesis pathway 127 2.2.6.2 Swansonine and castanospermine synthesis pathway 127 2.2.6.3 Lupinine, lupanine, sparteine, and cytisine synthesis pathway 129

2.2.7 Methods of analysis

130

2.2.7.1 Methodological considerations 132 2.2.7.2 Structural approach 136

2.2.8 Biogenesis of alkaloids 2.2.8.1 2.2.8.2 2.2.8.3 2.2.8.4

162

Chemistry models 165 Biochemistry models 167 Molecular biology models 169 Analytical dilemmas 171

2.2.9 Methods of alkaloid analysis

172

2.2.9.1 Methods in history 172 2.2.9.2 Basic methods and instruments 172

Alkaloids # 2015 Tadeusz Aniszewski. Published by Elsevier B.V. All rights reserved.

99

100 CHAPTER 2 Alkaloid chemistry

2.2.9.3 From iodine to enzyme 173 2.2.9.4 Choice of method and confidence 181 2.2.9.5 Chemical modification of alkaloids 182

References

184

Naturam mutare difficile. Seneca

2.1 ALKALOIDS AS SECONDARY METABOLISM MOLECULES Classification of alkaloids on the basis of bioecological, chemotechnological, chemomolecular, and biosynthetic shape is important for the maintenance of systematic order of these compounds. From a chemical point of view, the most important are the chemotechnological, chemomolecular, and especially chemomolecular and biosynthetic shape (structural) classifications. Both chemomolecular and biosynthetic shape classifications are linked together by the alkaloid structural nucleus and the position among other chemical molecules and structures. Exact determination of a chemical structure in a particular time is impossible without a clear examination of the biosynthetic pathway of this compound. Therefore, the chemical nucleus (base) and precursors are bases for the exact and deep determination of the alkaloid and its´ position among other such compounds. Each compound in organic chemistry can be read chemically according to its carbon and moiety, but the chemical name alone is not sufficient for a clear typology of an alkaloid, as alkaloids have not only a chemical but also a biological nature. The molecules belonging to bioorganic chemistry can be classified not only according to the general chemical classification but also according to the nucleus and precursors. Origin oneself is not sufficient, more information is needed on compound creation form, which is more important in practical use than scientific classification of the alkaloids. The precursors (and postcursors) of true alkaloids and protoalkaloids are amino acids, while transamination reactions precede pseudoalkaloids (Tables 1.1 and 1.10). It is not difficult to see that, from all amino acids, only a small portion of which are known to be alkaloid precursors (Table 2.1). Both true and protoalkaloids are synthesized mainly from the aromatic amino acids, phenylalanine, tyrosine (isoquinoline alkaloids), and tryptophan (indole alkaloids). Lysine is the precursor for piperidine and quinolizidine alkaloid, and ornithine for pyrrolidine, pyrrolizidine, and tropane alkaloids. Pseudoalkaloids are synthesized from other compounds, for example, acetate in the case of piperidine alkaloids (coniine or pinidine). Alkaloids are derived from the amino acid in L-configuration (protein amino acids) and from nonprotein

2.1 Alkaloids as secondary metabolism molecules 101

Table 2.1 Amino acids and their participation in alkaloid synthesis Group of Amino Acids/Amino acids

Alkaloid Type

Participation in Alkaloid Synthesis

Arginine-derived alkaloids

True alkaloids Marine alkaloids

Histidine-derived alkaloids

True alkaloids Imidazole alkaloids Manzamine alkaloids

Lysine-derived alkaloids

True alkaloids Piperidine alkaloids Quinolizidine alkaloids Indolizidine alkaloids

Phenylalanine-derived alkaloids

True alkaloids Phenylethylamino alkaloids Phenylisoquinoline alkaloids Amaryllidaceae alkaloids

L-tryptophan

Tryptophan-derived alkaloids

L-tyrosine

Tyrosine-derived alkaloids

True alkaloids Indole alkaloids Quinoline alkaloids β-carboline alkaloids Pyrroloindole alkaloids Ergot alkaloids Iboga alkaloids Corynanthe alkaloids Aspidosperma alkaloids Protoalkaloids Terpenoid indole alkaloids True alkaloids Phenylethylamino alkaloids Simple tetrahydroiso quinoline alkaloids Phenethylisoquinoline alkaloids Amaryllidaceae alkaloids Protoalkaloids Phenylethylamino alkaloids

Protein amino acids L-alanine L-arginine L-asparagine L-aspartic

acid

L-cysteine L-glutamine L-glutamic

acid

L-glycine L-histidine

L-isoleucine L-leucine L-lysine

L-methionine L-phenylalanine

L-proline L-serine L-threonine

L-valine

Continued

102 CHAPTER 2 Alkaloid chemistry

Table 2.1

Amino acids and their participation in alkaloid synthesis Continued

Group of Amino Acids/Amino acids

Alkaloid Type

Participation in Alkaloid Synthesis

L-ornithine

Ornithine-derived alkaloids

Anthranilic acid

Anthranilic acid-derived alkaloids

Nicotinic acid

Nicotinic acid–derived alkaloids

True alkaloids Pyrrolidine alkaloids Tropane alkaloids Pyrrolizidine alkaloids True alkaloids Quinazoline alkaloids Quinoline alkaloids Acridine alkaloids True alkaloids Pyridine alkaloids Sesquiterpene pyridine alkaloids

Non-protein aminoacids

amino acids, such as ornithine. However, it is important to note that alkaloids should be derived directly from the precursors of amino acids, as in the case of anthranilic acid (the precursor of tryptophan from the shikimate pathway) or acetate (the precursor of lysine via α-ketoadipic acid and transamination in some algae and fungi), for example. The precursors’ substrata for alkaloids can also derive directly from the degradated parts of amino acids, as in the case of nicotinic acid (niacin or vitamin B3), which is the precursor and a key part of coenzymes NAD+ and NADP+ in the degradation process of tryptophan, for example. Alkaloid chemistry is clearly directly connected to the protein amino acids, their precursors, or postcursors in different pathways.30,31 It is difficult to find exceptions to this rule. Although ornithine is a nonprotein amino acid, in reality its precursor is L-glutamate (in plants) and L-arginine (in animals). The importance of ornithine as the precursor of alkaloids is not that this amino acid is not protein but that it is a postcursor of the protein amino acid (L-glutamate). Although the pathways of alkaloids are at present relatively well understood from the point of view of organic chemistry, many questions remain relating to the biological nature of alkaloid synthesis. Mahler and Cordes82 considered and discussed three general examples of the synthesis of alkaloids from amino acids: (1) synthesis of the pyrroline ring and derived alkaloids from ornithine, (2) synthesis of the piperidine ring and derived alkaloids from lysine, and (3) synthesis of isoquinolizidine alkaloids from tyrosine. Nowadays, a lot of new data are available on chemical alkaloid research, but the above-mentioned three classic examples are still important in the understanding of alkaloid synthesis. Certainly, the present trend in alkaloid

2.1 Alkaloids as secondary metabolism molecules 103

chemistry is to underline the significance of the blocks, pathways, and transamination reactions in alkaloid synthesis.32 However, a presentation of chemical pathways and the synthesis of true alkaloids, protoalkaloids, and pseudoalkaloids is impossible in many cases without the characterization of their precursors determined on genetic level. This is especially important in the case of some alkaloids produced by microorganisms. In the case of filamentous fungus Aspergillus flavus, cytochrome P450, DtpC, is responsible for pyrroloindole ring formation and dimerization of N-methylphenylalanyltryptophanyl diketopiperazine.111 The similar vital role of cytochrome P450 in rearrangements of skeletal structure is also found in the case of terpenoid compounds produced by some medicinal plants.2,164 Some precursor compounds can be the same for lot of alkaloids. One example is that alkaloids from the marine sponge Aaptos suberitoides (aaptamine, aaptoline A, isoaaptamine, and demethylaaptamine) have common precursors, L-DOPA and β-alanine aldehyde.74 As already stated, protein amino acids, with their precursors and postcursors in different pathways, with or without transamination reactions, are generally substrates for alkaloids. This concept is very important because it highlights the probable role of alkaloids in metabolism and underlines the significance of protein amino acids, their synthesis, and degradation. Alkaloids exist in some kind of balance between distribution and degradation within amino acid production in the organisms producing them. This claim may provoke some controversy, but the connection between the amino acid pathway and alkaloid synthesis is so evident that it cannot be omitted. The similarity of the alkaloid to each molecule from the secondary metabolism is a consequence of the derivation process in the constructed active block. There are only four basic active blocks for the secondary compounds. Acetyl coenzyme A (acetyl CoA) is used in the acetate pathway, and shikimic acid in the shikimate pathway. The third block, mevalonic acid, is active in the mevalonate pathway; and the last, 1-deoxyxylulose 5-phosphate, key to the deoxyxylulose phosphate pathway (Figures 2.1 and 2.2). The theory of secondary compound synthesizing blocks is one of the most important in chemistry, as well as being interesting from a biological perspective. The establishment of the block needs the energy, and the primary metabolism is the source of it. On the other hand, the building blocks for the secondary metabolism are strongly regulated, and this regulation seems to be genetically determined. The building blocks link the primary and secondary metabolisms. Acetyl CoA is derived from pyruvic acid (the product of a glycolytic pathway) and used in the acetate pathway (Figure 2.1). Pyruvate is derived primarily from glucose 6-phosphate. Another source of this threecarbon α-keto acid are the conversion reactions of oxaloacetate, lactate, and

104 CHAPTER 2 Alkaloid chemistry

Photosynthesis

Glycolysis pyruvic acid

L-phenylalanine

Shikimic acid

Pentose phosphate cycle

CoA Krebs cycle

L-tyrosine L-tryptophan

Deoxyxylulose phosphate L-glutamic

Mevalonic acid

acid

L-ornithine L-arginine L-aspartic

acid

L-lysine

n FIGURE 2.1 Secondary metabolism blocks and amino acid derivation. Note that shikimic acid can be derived directly from photosynthesis and glycolysis through the pentose phosphate cycle or, alternatively, as a pyruvic acid postcursor.

alanine (Figure 2.2). Acetyl CoA is associated with genomewide active mechanism of expression69 and is synthesized by the oxidative decarboxylation of pyruvate and the β-oxidation of fatty acids, as well as from ketogenic amino acids. A part of the acetyl CoA can be exported to the cytosol in the form of citrate, thus participating in fatty acid synthesis. However, in mammals, acetyl CoA cannot be converted back into pyruvate. What is most important regarding alkaloid synthesis is that the pyruvate metabolism is a base for its alkaloid pathway precursors (Figure 2.2). As a group of specific molecules, part of the alkaloids is synthesized in the shikimic pathway. However, alkaloids are not the main product of this pathway, from which many phenols and lignans are also derived. Moreover, the shikimic pathway is a source for only aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan. These amino acids are known to be the precursors of some alkaloids. Other amino acids are alkaloidal precursors from the different pathways. Ornithine is the postcursor of L-glutamic acid and L-lysine is

2.1 Alkaloids as secondary metabolism molecules 105

Glucose

Glucose 6-phosphate

Glucose 1-phosphate

Oxaloacetate

Fructose 6-phosphate

6-phosphate Gluconate

Pyruvate

Alanine

Lactate

Acetyl CoA

n FIGURE 2.2 Pyruvate derivation and acetyl CoA synthesis. Observe that pyruvate, and subsequently the acetyl CoA pathway, has chain roots in the primary metabolism. Pyruvate can also be synthesized by conversion reactions. The secondary acetyl CoA is constructed as a building block on the pyruvate and glycolysis.

postcursor of L-aspartic acid. Both glutamic and aspartic acids originate from the Krebs cycle. However, the shikimic and acetate pathways are very important as the original chain of alkaloids. Shikimic acid is nowadays a promising building block for the synthesis of bioactive compounds produced traditionally and industrially. A lot of microbes (e.g., Citrobacter freundii GR-21, KC 466031) can produce shikimic acid in a fermentation process. This acid itself is bioactive, as it inhibits the hydrolysis of triglycerides by 55–60% and pancreatic lipase activity by 66%.101 This aspect of

106 CHAPTER 2 Alkaloid chemistry

shikimic acid is considered to have application potential, although its basic importance in bioorganic chemistry is just in a block for the synthesis of other bioactive compounds, especially some alkaloids. Certainly, steroid alkaloids originate from the activity of mevalonate and deoxyxylulose phosphate pathways. This means that different alkaloids may derive from different secondary metabolism blocks and pathways. Alkaloid chemistry is, therefore, a part of the total secondary metabolism and has its roots in the primary metabolism, photosynthesis, and the Krebs cycle. The CoA and shikimic acid remain very important blocks for the alkaloids and their chemistry.

2.2 SYNTHESIS AND METABOLISM Each biomolecule of a chemical nature in living organisms has its own synthesizing, transformational, and interconverting processes. Therefore, the formation of the ring of the alkaloid molecule and the flow of the nitrogen atom into this molecule are the basic points for understanding alkaloid synthesis and its metabolism. Alkaloid biosynthesis needs the substrate. Substrates are derivatives of the secondary metabolism building blocks: the acetyl coenzyme A (acetylCoA), shikimic acid, mevalonic acid, and 1-deoxyxylulose 5-phosphate (Figure 2.1). The synthesis of alkaloids starts from the acetate, shikimate, mevalonate, and deoxyxylulose pathways. The acetyl coenzyme A pathway (acetate pathway) is the source of some alkaloids and their precursors (e.g., piperidine alkaloids or anthranilic acid as aromatized CoA ester, anthraniloyl-CoA). Shikimic acid is a product of the glycolytic and pentose phosphate pathways, a construction facilitated by parts of phosphoenolpyruvate and erythrose 4-phosphate (Figure 2.1). The shikimic acid pathway is the source of such alkaloids as quinazoline, quinoline, and acridine. The mevalonate pathway is based on mevalonic acid (three molecules of acetyl-CoA), which is closely related to the acetate pathway, while the deoxyxylulose phosphate pathway is based on a combination of pyruvic acid and glyceraldehyde 3-phosphate (both from the glycolytic pathway). Together, mevalonate and deoxyxylulose phosphate pathways produce terpenoid and steroid compounds. However, it is important to note that the Krebs cycle pathway is also key to many precursors of alkaloids. Ornithine, a postcursor of L-arginine in animals and of L-glutamate in plants, and L-lysine, a principal protein amino acid, deriving from the Krebs cycle pathway compound, are useful examples of the role of the Krebs cycle for alkaloid precursors (Figure 2.1). Moreover, there are other sources of alkaloid substrates, particularly in purine alkaloids. Figure 2.3 represents the general scope of

2.2 Synthesis and metabolism 107

SECONDARY Terpenoid indole alkaloids

Pyridine alkaloids Phenylethylaminoalkaloids Simple tetraIndole hydroisoquinoline PRIMARY alkaloids alkaloids Quinazoline L -Tyrosine Quinoline alkaloids Nicotinic Glucose alkaloids Quinoline glycolysis acid Ergot NAD ADP alkaloids alkaloids ATP NADH2 L -Tryptophan CH3 Acridine Tryptophan Anthranilic C = O Indolizine alkaloids Tyrosine acid COOH Leucine alkaloids Ephedra Pyruvic acid Lysine CO2 Piperidine NAD alkaloids Isoleucine alkaloids L-Lysine Tropane NADH2 Aspartic acid Oxaloacetic Mevalonic CoA alkaloids Asparagine acid acid Fatty acids C = O Pyrrolidine COOH Quinolizidine NADH2 alkaloids CH3 C=O alkaloids CoA Pyrroloindole alkaloids

NAD COOH

lan

ts

HCOH CH2COOH Tyrosine L-Tyrosine Malic acid CH2 Citric acid CH COOH Adenine 2 COOH Phenylalanine Guanine C - COOH KREBS H COOH HC - COOH C Saponins cis-Aconitic acid C COOH H CH2COOH CYCLE FADH2 FAD C - COOH L-Ornithine CO2 HOC - COOH Iso citric acid CH2 COOH CoA CO CH2 COOH H CoA 2 NAD Succinic acid NADH2 L-Glutamate CH2 COOH CH2COOH GDP CH 2 HCH Glutamic acid i L-Phenylalanine GTP O = C - S - CoA NAD O = C - COOH Succinyl CoA ATP

Arginine Histidine Proline L-Histidine

Ketoglutaric acid NADH2

Pyrrolizidine alkaloids

Purine alkaloids Terpenoid alkaloids

imals

an

n

Simple tetrahydroisoquinoline alkaloids

Geraniol

in p

Phenylethylaminoalkaloids

Acetyl coenzyme A CH2 CH2COOH COOH HOC COOH

Steroidal alkaloids

L-Arginine

Phenethylisoquinoline alkaloids Amaryllidaceae alkaloids

Imidazole alkaloids METABOLISM Other alkaloids

Marine alkaloids

METABOLISM

n FIGURE 2.3 General

alkaloid synthesis in the metabolic system of organisms and their energy production. Enzymatic activity is very important in the primary metabolism of glycolysis and the Krebs cycle. Pyruvic acid and CoA are key compounds in the synthesis of alkaloid precursors. Moreover, these precursors (amino acids) can be derived from different points in the glycolysis and Krebs cycles. Consequently, the synthesis of alkaloids as a secondary metabolic activity is a very challenging research subject. Generally, in the literature, alkaloid metabolism in animals and especially in mammals is recognized to be closely related to that of plants.16,160 However, some exceptions exist. Figure 2.3 shows two means of L-ornithine synthesis.

scheme of alkaloid synthesis.

108 CHAPTER 2 Alkaloid chemistry

In plants, this nonprotein amino acid is derived from L-glutamate and in animals from L-arginine. Moreover, Figure 2.3 demonstrates that synthesis of alkaloids is complicated by the ability of the same amino acid to synthesize many different alkaloids.

2.2.1 Skeleton diversity The skeleton nucleus of the alkaloid is the main criterion for determining the alkaloid precursor. Many skeletons are produced in the process of alkaloid synthesis. A good example is indole fragment synthesized in the case of indole alkaloid dragmacidin E.135 Figure 2.4 illustrates some nuclei and skeletons supplied in the synthesis. Alkaloid relates only to the reactions of this stage of the synthesis. Moreover, skeletons can change their form during the synthesis. A case in point is the synthesis of quinine, where the indole nucleus is reconstructed to form the quinoline nucleus. Moreover, nowadays, nuclei can be synthesized totally in the laboratory. A practical method for the synthesis of pyrrolizidine, indolizidine, and pyrroloazepinolizidine nucleus with

CO2H NH2

NH2

Piperidine nucleus

N H

C5N skeleton

N C5NC3 skeleton

Indolizine nucleus

N C5NC4 skeleton

Quinolizidine nucleus

N Pyridon nucleus

n FIGURE 2.4 L-lysine-derived nuclei.

C5NC4 skeleton

2.2 Synthesis and metabolism 109

good yield has been developed.33,56,98 Piperidine nucleus can also be synthetisized.70 Moreover, other examples are the Schiff´s base derivatives with nitroimidazole and quinoline nuclei.83 Nuclei also can be significantly or discretely modified. Modifications of acridine nucleus, for example, are suggested to develop novel compounds with antiprion activity.131 Moreover, skeleton modifications can sometimes be also spontaneous.42 During biosynthetic processes, L-lysine can produce at least four alkaloid skeletons with different alkaloid nuclei: piperidine nucleus (C5N skeleton), indolizine nucleus (C5NC3 skeleton), quinolizidine nucleus (C5NC4 skeleton), and pyridon nucleus (with the variated quinolizidine nucleus and a C5NC4 skeleton). The ability of L-lysine to provide different alkaloid nuclei is related to the role of this DNA amino acid in plant and animal organisms. In plants, this amino acid is an endogenous compound synthesis that is used in both primary and secondary metabolisms. In the animal kingdom, lysine is principally an exogenous amino acid, mainly of dietary origin. Consequently, L-lysine-derived alkaloids with different types of skeletons have very different biological effects on the organisms. Piperidine, indolizine, quinolizidine, and pyridon alkaloids have different effects on the digestive and nervous systems of herbivores. Their acute toxicity and ability to temporarily or permanently change cell numbers or the functional metabolism differ markedly. Moreover, skeleton structure also influences taste. In this regard, the position of the N atom is important. In all the skeletons derived from L-lysine, the position of N is the same as in the substrate. In the case of the izidine alkaloids (Figure 2.5) the position of the nitrogen atom is the same, but the number of C atoms is different. The difference lies in the rings of these alkaloids. They represent different structural groups of alkaloids, although they have two rings and are two-cyclic compounds. This structural point is key to their biological activity. Pyrrol-, indol-, and quinolizidine rings display structural similarities but diversity in both their origin and, what is very important, bioimpact. Even small differences in nucleus can effect huge changes in the alkaloid activity.159 L-ornithine (Figure 2.6) produces the pyrrolidine nucleus (C4N skeleton). This nucleus is also constructed within tropane alkaloids (C4N skeleton +) (Figure 2.6). Alkaloids that contain the pyrrolidine and tropane nuclei are very

N

N Pyrrolizidine

Indolizidine

n FIGURE 2.5 Nuclei and skeletons of izidine alkaloids.

N Quinolizidine

110 CHAPTER 2 Alkaloid chemistry

CO2H NH2

N H

NH2

C4N skeleton

Pyrrolidine nucleus

C4N skeleton +

N

Me

n FIGURE 2.6 The source and forms of the pyrrolidine ring.

vigorous in their biological activity. Common pyrrolidine nucleus alkaloids include hygrine, hyoscyamine, cocaine, and cuscohygrine. The best-known plant alkaloids with pyrrolidine nuclei are henbane (Hyoscyamus niger), deadly nightshade (Atropa belladonna), and Jamestown weed (Datura stramonium). The imidazole nucleus (Figure 2.7) is supplied during alkaloid biosynthesis by L-histamine. Typical alkaloids with the imidazole nucleus include histamine, histidine, procarpine, and pilosine. They are found as basic alkaloids in two principal families, Cactaceae and Rutaceae. The basic alkaloid in Pilocarpus jaborandi (Rutaceae) is pilocarpine, a molecule that contains an imidazole nucleus and is also used as a clinical drug. During alkaloid synthesis, L-histidine can produce the manzamine nucleus (Figure 2.7). These alkaloids are quite widespread, although they

H N N

CO2H N NH2 Imidazole nucleus

N C9N2 skeleton

N NH Manzamine nucleus C9N4 skeleton

N NCH3

n FIGURE 2.7 L-histidine and the nuclei of imidazole and manzamine alkaloids.

2.2 Synthesis and metabolism 111

were first isolated in the late 1980s in marine sponges.21,57,99,100,149,161 They have an unusual polycyclic system and a very broad range of bioactivities. Common alkaloids with this nucleus include manzamine A, manzamine B, manzamine X, manzamine Y, and sextomanzamine A. In the case of C6C2N skeletons (Figure 2.8) converted from the anthranilic acid into the alkaloids quinazoline, quinoline, and acridine, nuclei are constructed inside the cyclic system. Only this part is derived from the precursor, while the rest of the ring system comes from other sources.32 Alkaloids with the C6C2N skeleton occur in many species, such as Peganum harmala, Dictamus albus, Skimmia japonica, and Ruta graveolens. The best-known alkaloids containing these nuclei are peganine (vasicine), dictamine, skimmianine, melicopicine, acronycine, and rutacridone. All alkaloids with the C6C2N skeleton are bioactive; since they constitute a very large group of compounds, they display different properties. As already stated, anthranilic acid provides these alkaloids with a nucleus (Figure 2.8), but the rest of the skeleton comes from other donors. Simply, this can have an influence on the characteristic activities of alkaloids. Nicotinic acid (Figure 2.9) provides alkaloids with the pyridine nucleus in the synthesizing process. This nucleus appears in such alkaloids as anabasine, anatabine, nicotine, nornicotine, ricine, and arecoline. Moreover, many alkaloids contain the pyridine nucleus as part of their total skeleton. For example, anabasine is derived from nicotinic acid and lysine.120 Alkaloids with the pyridine nucleus occur in such plants as tobacco (Nicotiana

CO2H N NH2

N Quinazoline nucleus

Quinoline nucleus

Acridine ring

N

N

n FIGURE 2.8 The nuclei produced by anthranilic acid in alkaloids.

C6C2N skeleton

C6C2N skeleton

C6C2N skeleton

112 CHAPTER 2 Alkaloid chemistry

Nicotinic acid = Niacin = Vitamin B3

CO2H

Pyridine nucleus

(Niacin + Acetate) n FIGURE 2.9 The nucleus of alkaloids derived from nicotinic acid.

N

C6N skeleton

Sesquiterpene pyridine nucleus

tabacum), castor (Ricinus communis), and betel nuts (Areca catechu). The sesquiterpene pyridine nucleus derives partly from nicotinic acid, and partly from the acetate pathway. more than 300 alkaloids in this group are known.29,34,38,61,66,78–80,110,118,165,166 In the alkaloid synthesis, L-phenylalanine (Figure 2.10) provides the phenyl or phenylpropyl nucleus to an alkaloid. These kinds of nuclei occur in cathionine, cathine, ephedrine, pseudoephedrine, and norpseudoephedrine. Such alkaloids are found especially in many species of Ephedra. Natural alkaloid molecules from these plants have similar properties to synthetic compounds used as narcotics (e.g., amphetamine).

CO2H NH2

N Phenyl nucleus

Phenylpropyl nucleus n FIGURE 2.10 L-phenylaninederived nuclei in alkaloid biosynthesis.

C6C2N skeleton

C6C3 skeleton

2.2 Synthesis and metabolism 113

CO2H HO

NH2 Phenyl nucleus

N C6C2N skeleton

Phenylpropyl nucleus

C6C3 skeleton

L-tyrosine

n FIGURE 2.11 Nuclei

L-phenylalanine)

supplied to alkaloids by in the synthesizing process.

(Figure 2.11) is an aromatic amino acid (similar in compound to that also provides phenyl (Figures 2.10 and 2.11) and phenylpropyl (Figures 2.10 and 2.11) nuclei for alkaloids. Molecules containing nuclei from L-tyrosine include, for example, mescaline, anhalamine, papaverine, curare, and morphine. They are biologically very strong natural compounds and occur relatively widely in the plant kingdom (Table 1.10). Another aromatic amino acid, L-tryptophan (Figure 2.12), contains the indole nucleus. This nucleus is synthesized in a large number of alkaloids, such as psilocin, psilocybin, harmine, catharanthine, reserpine, ajmalicine, vindoline, vincristine, strychnine, quinine, ergotamine, and other ergot alkaloids. The alkaloid nucleus as a fragment of the precursor structure given to the new molecule during its synthesis is very interesting and relatively unknown. The evident original donor of each carbon in the alkaloid ring is still not exactly comprehended. However, along the alkaloid pathways from the secondary building blocks to the synthesis of alkaloids is a long chain of reactions. The nucleus translocation into the alkaloid molecule is the most important step in this alkaloid synthesis. The indole nucleus can change during the synthesizing reaction into a quinoline nucleus (Figure 2.12). Moreover, L-tryptophan, the precursor, provides both β-carboline and pyrroloindole nuclei. Iboga, Corynanthe, and Aspidosperma nuclei also originate from L-tryptophan (Figure 2.12). Alkaloids with nuclei derived from this amino acid tend to be very active compounds with a relatively widespread provenance in nature (Table 1.10).

2.2.2 Ornithine-derived alkaloids Ornithine is a metabolically quite active amino acid and the important precursor of pyrrolidine nucleus, which is found in pyrrolizidine alkaloids. Ornithine itself is a nonprotein amino acid formed mainly from L-glumate in plants and synthesized from the urea cycle in animals as a result of the reaction catalyzed by enzymes in arginine.

L-tyrosine

114 CHAPTER 2 Alkaloid chemistry

CO2H

NH2 N H

N Indole nucleus

Quinoline nucleus after transformation from indole

N H

N

Indole C2N skeleton

Quinoline C9N skeleton

N

b-carboline nucleus

N

Carboline C8NC3N skeleton

NH Pyrroloindole nucleus

N H Pyrroloindole C10N2 skeleton

Iboga nucleus Iboga C9 skeleton

Corynanthe nucleus Corynanthe C9 skeleton

Aspidosperma nucleus

Aspidosperma C9 skeleton n FIGURE 2.12 The L-tryptophan-supplied

nucleus during synthesis.

2.2 Synthesis and metabolism 115

NH2

CO2H NH2

ODI/PDI

Pyridoxal phosphate PLP

Putrescine

S -adenosylmethionine SAMe

L-ornithine

N -methylputrescine

Diamine oxidase

N -methyl-Δ1-pyrrolinium cation

Hygrine and other pyrrolidine and tropane alkaloids

The synthesis of alkaloids from L-ornithine starts with decarboxylation by pyridoxal phosphate (PLP) to putrescine (Figure 2.13) and putrescine methylation by S-adenosylmethionine (SAMe) to N-methylputrescine. SAMe is a naturally occurring reaction, when the departing groups convert L-methionine to S-adenosylmethionine. In this process, a positively charged sulfur is produced and facilitates the nucleophilic reaction. By the activity of diamine oxidase, the N-methyl-Δ1-pyrrolinium cation is formed and, after that, the first alkaloid, hygrine. From hygrine, by way of acetyl CoA, hydrolysis and intramolecular Mannich reactions, other pyrrolidine and tropane alkaloids are synthesized: cuscohygrine, hyoscyamine or tropinone, tropine, and cocaine. The Mannich reaction involves the combination of an amine, an aldehyde, or a ketone with a nucleophilic carbon. This reaction is typical in alkaloid synthesis and can be written as follows: R I I N+ = C I I H

I C I

I N I

I C I

I C I

n FIGURE 2.13 Synthesis of alkaloids from ornithine. Alkaloids are derived via putrescine or glutamic semialdehyde. At least two enzymes, ODL (ornithine decarboxylase) or PDL (pyrroline decarboxylase), are needed.

116 CHAPTER 2 Alkaloid chemistry

NAD+ Imine

L-arginine

or L-ornithine Putrescine x Putrescine

Homospermidine and other pyrrolizidine alkaloids n FIGURE 2.14 Synthesis pathway of the pyrrolizidine alkaloids from L-ornithine

or L-arginine.

The synthesis of tropine from tropinione requires dehydrogenase NADPH+. Similarly, the synthesis of cocaine requires the Mannich reaction, SAMe, and NADPH+. Putrescine is a biogenic amine. Other biogenic amines also participate in alkaloid synthesis, for example, cadaverine in the case of lysine alkaloids. Aniszewski, Ciesiołka, and Gulewicz8 drew attention to the fact that the various biogenic amines that actively participate in the biosynthetic process of alkaloids play a role in the equilibrium between basic nitrogen compounds. Moreover, enzyme participation in pyrrolidine and tropane alkaloid synthesis has also been noted. From the L-ornithine, and alternatively also from L-arginine, pyrrolizidine alkaloids are synthesized (Figure 2.14). The L-arginine alternative pyrrolizidine precursor is based on its ability to change into L-ornithine, and alternatively into putrescine, via coenzyme pyridoxal phosphate and agmatine. In the synthetic pathway to homospermidine, which is the first pyrrolizidine alkaloid in this synthesis chain, two molecules of putrescine are condensed by the enzyme NAD+ into imine before NADH converts it to homospermidine. From homospermidine, the synthesis chain continues across oxidative and base formation and the Mannich reactions to synthesize other alkaloids, such as retronecine and its diester senecionine. This synthesis pathway is also characteristic of heliotridine, laburine, lycopsamine, and indicine-N-oxide. All these alkaloids contain the pyrrolidine nucleus, which is derived from ornithine or its precursors and postcursors.

2.2.3 Tyrosine-derived alkaloids Tyrosine is an important precursor of alkaloids with the phenyl and phenylpropyl nuclei. There are four basic alkaloid pathways.

2.2 Synthesis and metabolism 117

CO2H

PLP

NH2

HO

Tyramine

L-tyrosine

SAMe

Tetrahydrobiopterin

Hordeine

L-dopa

Mescaline

PLP

Dopamine

n FIGURE 2.15 Synthesis of hordeine and mescaline.

2.2.3.1 Mescaline pathway This alkaloid pathway starts with PLP decarboxylation to tyramine and subsequently, via SAMe, dimethylation synthesizes hordeine (Figure 2.15). The second synthesis pathway from L-tyrosine is to dopamine across hydroxylation patterns and PLP activity. Dopamine is a very important compound in the synthesis of alkaloids, especially in animals. Only dopamine can be converted to another alkaloid, for example, mescaline. Anhalamine, anhalonine, and anhalonidine can also be synthesized in this way. Like mescaline, they are typical of simple tetrahydroisoquinoline alkaloids.

2.2.3.2 Kreysigine and colchicine pathway From L-tyrosine, and alternatively also from L-phenylalanine, kreysigine synthesis begins with dopamine (Figure 2.16). S-autumnaline is derived via a Mannichlike reaction. S-autumnaline is converted into floramultine by the oxidative coupling. Subsequently, the kreysigine is synthesized through the activities of SAMe. From S-autumnaline, other alkaloids can also be derived. The destination of this pathway is colchicine (Figure 2.16).

2.2.3.3 Dopamine—the cephaeline pathway The pathway of tetrahydroisoquinoline alkaloids, such as emetine and cephaeline (Figure 2.17), also begin from dopamine. Dopamine and secologanin undergo a Mannichlike reaction to produce N-deacetylisoipecoside

118 CHAPTER 2 Alkaloid chemistry

Colchicine L-tyrosine Dopamine L-phenylalanine

S -autumnaline Floramultine

Kreysigine n FIGURE 2.16 Synthesis pathway of kreysigine and colchicine.

N-deacetylisoipecoside Ipecoside

Dopamine

Emetine

Cephaeline n FIGURE 2.17 Emetine and cephaeline synthesis pathway.

and ipecoside, and after hydrolysis and transformation, this is converted to emetine and cephaeline.

2.2.3.4 Galanthamine pathway From L-tyrosine, or alternatively from L-phenylalanine, there is one further alkaloid biosynthesis pathway. This is the galanthamine pathway (Figure 2.18). Galanthamine synthesizes with tyramine, norbelladine, lycorine, crinine, N-demethylnarwedine, and N-demethylgalanthamine. After formation of a Schiff base and a reduction reaction, oxidative coupling and enzyme NADPH and SAMe activity occur in this pathway. Schiff base is a reaction for the elimination of water in formation with the C
N bonds process. From norbelladine, through the activity of the SAMe, the 4’-0methylnorbelladine synthesizes and again is transformed to lycorine, crinine, and by oxidative coupling, to N-demethylarwedine, which is the object of enzyme NADPH activity. Galanthamine is synthesized by transformation trough the activity of the SAMe from N-demethylgalanthamine.

2.2 Synthesis and metabolism 119

L-tyrosine

Tyramine L-phenylalanine

Norbelladine

SAMe

4′-0-Methylnorbelladine

Lycorine Crinine N-demethylarwedine

N-demethylgalanthamine

Galanthamine n FIGURE 2.18 Galanthamine synthesis pathway.

2.2.4 Tryptophan-derived alkaloids Alkaloids derived from L-tryptophan hold the indole nucleus in a ring system. The ring system originates in the shikimate secondary compound’s building block and the anthranilic acid pathway. The shikimate block, in general, and anthranilic acid, in particular, are known to be precursors to many indole alkaloids. However, many rearrangement reactions can convert the indole ring system into a quinoline ring.

2.2.4.1 Psilocybin pathway In this pathway, L-tryptophan is enzymatically transferred to tryptamine and subsequently, through the activity of SAMe, to psilocin. By the reaction of phosphorylation, psilocin is converted into psilocybin (Figure 2.19). Psilocybin and psilocin are psychoactive hallucinogenous alkaloids synthesized from the small mushroom genus Psilocybe spp. On average,

120 CHAPTER 2 Alkaloid chemistry

SAMe L-tryptophan

Tryptamine

Psilocin SAMe

Serotonin

Psilocybin

n FIGURE 2.19 Psilocybin and serotonin synthesis pathway.

the concentration of these alkaloids is 300 g
3 in 100 g of mushroom mass. Structurally, these alkaloids are neurotransmitters 5-HT. From L-tryptophan, the serotonin synthesis pathway also begins. Serotonin is 5-hydroxytryptamine. It is derived from L-tryptophan, which at first is simply hydroxylated to 5-hydroxy-L-tryptophan and subsequently to the serotonin (Figure 2.19). Structurally, serotonin is also a 5-HT monoamine neurotransmitter. It is found in many cellular complexes, such as the central nervous system, the peripheral nervous system, and the cardiovascular system, but it also appears in blood cells.

2.2.4.2 Elaeagnine, harman and harmine pathway From tryptamine (derived from L-tryptophan, Figure 2.19), the synthesis pathway of harman and harmine, which are alkaloids based on a β-carboline ring, also starts. Using the Schiff base formation and Mannichlike reaction, the carboline ring is synthesized. Then, by a Mannichlike reaction using keto acid and oxidative decarboxylation, harmaline is synthesized. Harmaline is converted to harmine and tetrahydroharmine. Certainly, following the above-mentioned Mannich reaction and oxidative decarboxylation, a reduction reaction can ensue, and this leads to the synthesizing of elaeagnine (Figure 2.20). Elaeagnine is synthesized in Elaeagnus angustifolia (Elaeagnaceae). Harmine and harman are alkaloids having fully aromatic β-carboline structures. They have been detected in Peganum harmala (Zygophyllaceae). These alkaloids have psychoactive properties. Harman is a very important mammalian alkaloid.16,55,160

2.2.4.3 Ajmalicine, tabersonine and catharanthine pathway Over 3000 alkaloids are synthesized in this pathway. These are terpenoid indole alkaloids, one of the principal groups of alkaloids in the plant kingdom. Some of the most important alkaloids used widely in medicine belong to this group. As stated in Chapter 1, these alkaloids belong mainly to eight botanical families, of which Apocynaceae, Loganiaceae, and Rubiaceae are the most important from the perspective of existing applications. Ajmalicine

2.2 Synthesis and metabolism 121

Harmaline Tryptamine

Schiff base formation Mannichlike reaction Mannichlike reaction using keto acid Oxidative decarboxylation Harmine

Reduction reaction Harman Elaeagnine Tetrahydroharmine

n FIGURE 2.20 Scheme of elaeagnine, harman, and harmine synthesis pathway.

and akuammicine are typical alkaloids containing the Corynanthe nucleus, and tabersonine is typical for the Aspidosperma nucleus. The Iboga type of these alkaloids may be clearly seen in catharanthine and iboganine. All these alkaloids have C9 and C10 fragments in their structure, which derive from terpenoid. Molecules from these alkaloids originate partly from terpenoid, in combination with tryptamine. The synthetic pathway starts with geraniol and, via iridodial and iridotrial, is synthesized as loganin. Subsequently, through oxidation and formation of alkene and ring cleavage, loganin is converted to secologanin, which crosses tryptamine to form the Corynanthetype nucleus. From this ring, akummicine or ajmalicine is synthesized in turn. Ajmalicine is derived from tryptamine (partly from geraniol) via secologanin, strictosidine, and cathenamine. Reduction of cathenamine to ajmalicine is facilitated by enzyme NADPH activity. Again by transformation, the Aspidosperma-type tabersonine or Iboga-type catharanthine is synthesized (Figure 2.21). Yohimbine is a carbocyclic variant of ajmalicine. It has been found in species belonging to the Apocynaceae and Rubiaceae families. Yohimbine can be converted to reserpine and rescinnamine (trimethoxybenzoyl esters). Rescinnamine is a trimethoxycinnamoyl ester.

2.2.4.4 Vindoline, vinblastine, and vincristine pathway Vincamine, vinblastine, and vincristine are very important clinic alkaloids. They are produced naturally by plants: vincamine by Vinca minor, and vinblascine and vincristine by Madagascar periwinkle (Catharanthus roseus). The vindoline synthesis pathway starts with strictosidine and, via dehydrogeissoschizine, preakuammicine, stemmadenine, and tabersonine, is converted to vindoline and vincristine (Figure 2.22). Conversion from vindoline to vinblastine is based on the NADH enzyme activity. Vinblastine

122 CHAPTER 2 Alkaloid chemistry

Iridodial Iridotrial Deoxyloganin

Geraniol

Loganin

Tryptamine

Deoxyloganin

Strictosidine Akuammicine

Secologanin

Cathenamine Ajmalicine NADPH

Tabersonine Catharanthine Yohimbine

Reserpine Rescinnamine Serpentine

n FIGURE 2.21 Pattern of the ajmalicine, tabersonine, and catharanthine pathway.

Dehydrogeissoschizine Strictosidine Preakuammicine

Stemmadenine

Tabersonine Vindoline Vinblastine

NADH

Vincristine n FIGURE 2.22 Diagram of the vindoline, vinblastine, and vincristine pathway.

2.2 Synthesis and metabolism 123

and vincristine are very similar alkaloids. The difference is that vincristine has CHO connected to N, whereas vinblastine in the same situation has only CO3. This synthetic structural differences influence their activity. Vinblastine is used to treat Hodgkin’s disease (a form of lymphoid cancer), while vincristine is used clinically in the treatment of children’s leukemia. Vincristine is more neurotoxic than vinblastine.

2.2.4.5 Strychnine and brucine pathway The synthetic pathway starts with the preakuammicine structure (Figure 2.22) by hydrolysis, decarboxylation, and condensation reactions to aldehyde (WielandGumlich), and subsequently reacts with acetyl-CoA to make a hemiacetal form of aldehyde (Wieland-Gumlich) and strychnine (Figure 2.23). Strychnine and brucine are extremely toxic alkaloids. Strychnine binds itself to receptor sites in the spinal cord and accommodates glycine. Brucine is a dimethoxy form of strychnine and is less toxic.

2.2.4.6 Quinine, quinidine, and cinchonine synthesis pathway As noted in Chapter 1, quinine, quinidine, cinchonidine, and cinchonine alkaloids are found particularly in the genus Cinchona from the botanical family Rubiaceae. They have a powerful bioimpact and are important antimalarial drugs. Quinidine is also used to treat cardiac arrhythmias because it inhibits fibrillation, where there is no coordinated contraction of muscle fibers in the heart. During the synthesis of these alkaloids, a change occurs in the nucleus. The indole nucleus is transformed into the quinoline nucleus. This is one reason why these compounds are known as quinoline alkaloids. The synthesis pathway starts with strictosidine, which is transformed by hydrolysis and the decarboxylation reaction into corynantheal (Figure 2.24). At this point, the indole nucleus and cinchoamine are synthesized. Cinchonamine is an indole derivative and an intermediate compound

Preakuammicine

Wieland-Gumlich aldehyde

Aldol reaction with CoA

Strychnine

Brucine

n FIGURE 2.23 Diagram of the strychnine and brucine pathway.

124 CHAPTER 2 Alkaloid chemistry

Cinchoamine Strictoside

Corynantheal

Cinchoninone Cinchonidine

NADPH NAD

Quinine Cinchonine Quinidine n FIGURE 2.24 Diagram of the quinine, quinidine, and cinchonine synthesis pathway.

in the quinine pathway. At the next stage of the synthesis, the transformation of the nucleus occurs and the resultant intermediate cinchoninone no longer contains the indole nucleus. By the enzymatic reaction of NADPH, cinchonidine is synthesized from cinchoninone and subsequently changes to quinine. Cinchonidine and quinine are similar alkaloids. The difference is only that cinchonidine has H while quinine has OCH3 in the same position. By epimerization and NADPH activity, cinchonine and quinine are synthesized from cinchoninone. The difference between cinchonine and quinidine is very similar to that between cinchonidine and quinine.

2.2.4.7 Eserine synthesis pathway Eserine (physostigmine) has a pyrroloindole skeleton. This alkaloid is used as an anticholinesterase drug, which is fairly important in the treatment of Alzheimer’s disease. Eserine is synthesized in Physostigma venenosum and stored in the seeds of this leguminous plant. The synthesis pathway starts with tryptamine, which is transformed into eserine (Figure 2.25).

Tryptamine

C-alkylation Ionization

Eserine n FIGURE 2.25 Diagram of the eserine synthesis pathway.

2.2 Synthesis and metabolism 125

2.2.4.8 Ergotamine synthesis pathway Ergotamine is one of the ergot alkaloids produced by the fungus genus Claviceps, which lives on cereal kernels and grass seeds. The toxicity of ergot kernels and grass seeds is extreme. The ergotamine synthesis pathway starts from L-tryptophan and, continuing via chanoclavine-I and chanoclavine-2, then agroclavine, elymoclavine, and pispalic acid, and is converted into D-(+)-lysergic acid. This compound is very important in ergotamine synthesis. By powerful enzymatic activity (ATP and SH) and hydroxylation, ergotamine is synthesized (Figure 2.26). Ergotamine also contains a peptide fragment in its structure.

2.2.5 Nicotinic acid–derived alkaloids Alkaloids derived from nicotinic acid contain a pyridine nucleus. Nicotinic acid itself is synthesized from L-tryptophan via N-formylkynurenine, L-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and quinolinic acid.

L-tryptophan

Chanoclavine-1 Chanoclavine-2

Agroclavine

Elymoclavine

Pispalic acid

D-(+)-lysergic

acid

ATP SH

Ergotamine n FIGURE 2.26 Diagram of the ergotamine synthesis pathway.

126 CHAPTER 2 Alkaloid chemistry

Nicotinic acid

NADPH

Dihydronicotinic acid

NADP+

1,2-dihydropyridine

Nornicotine Nicotine

n FIGURE 2.27 Scheme of nicotine and nornicotine synthesis pathway.

The pyridine nucleus is passed to alkaloids via dihydronicotinic acid, moving from dihydropyridine to nicotine and nornicotine (Figure 2.27). Dihydronicotinic acid is synthesized by the enzymatic activity of NADP and subsequently becomes 1,2-dihydropyridine by a reduction reaction. At the next stage, nicotine is synthesized by the reactions of ionization and enzyme NADP+. Nornicotine synthesis is achieved by hydroxylation, NADPH activity, and in its final stage, by nonenzymatic decomposition. Interestingly, during the ionization reaction, the residue cation from putrescine (a derivate of L-ornithine) appears. The synthesis of other alkaloids derived from nicotinic acid is illustrated in Figure 2.28. Anabasine is synthesized from nicotinic acid using the Δ1-piperidinium cation (from L-lysine), and anatabine from nicotinic acid (as with nicotine) via dihydronicotinic acid and 1,2–dihydropyridine. However, the synthesis of ricine from nicotinic acid is accomplished through nicotinamide. The other alkaloids derived from nicotinic acid, with pyridine nucleus, such as arecoline, arecaidine, and guvacoline, are tetrahydronicotinic acid (guvacine) derivatives.

L-lysine

Nicotinamide

Ricinine

Nicotinic acid Δ1-piperidinium cation

Anabasine

Dihydronicotinic acid Anatabine 1,2-dihydropyridine n FIGURE 2.28 Diagram of anatabine, anabasine, and ricinine synthesis pathway.

2.2 Synthesis and metabolism 127

Nicotinic acid also forms part of the sesquiterpene pyridine nucleus. As is well known, aliphatic and aromatic acids esterify hydroxyl groups of fundamental sesquiterpene. Some sesquiterpene forms bridge with pyridine rings of nicotine acid derivatives, then the sesquiterpene pyridine nucleus appears. More than 300 sesquiterpene pyridine nucleus alkaloids have been documented.34,38,79,80,118,166 Cathedulin alkaloids, isoevoniate, hydroxyisoevoniate, epimeric, norevoniate, wilfordate, hydroxywilfordate, isowilfordate, benzoyloxy- and furanoyloxywolfordate, edulinate, and cassinate constitute the groups of these reported alkaloids. In nature, sesquiterpene pyridine alkaloid formation involves a mixed biosynthetic route. The sesquiterpene moiety originates from acetate metabolism via the mevalonic acid pathway. Sesquiterpene pyridine alkaloids are synthesized when nicotinic acid and (3S)-isoleucine form evoninic acid, which is configured with the sesquiterpene moiety.

2.2.6 Lysine-derived alkaloids L-lysine furnishes alkaloids with at least four different nuclei. It is a protein amino acid, one of the most important alkaloid precursors. L-lysine-derived alkaloids have a basic skeleton with C5N (the piperidine nucleus) and C5N +. . . (indolizidine, quinolizidine, and pyridon nuclei).

2.2.6.1 Pelletierine, lobelanine, and piperine synthesis pathway Alkaloids with the piperidine nucleus, such as pelletierine (Punica granatum), lobelanine (Lobelia inflata), and piperine (Piper nigrum), have a typical biosynthesis pathway. It starts with L-lysine and continues via cadaverine (biogenic amine), Δ1-piperideine and Δ1-piperidinium cations, and lobelanine to be synthesized as lobeline. Piperine is synthesized from Δ1piperideine via piperidine (Figure 2.29). For the transformation from Δ1piperideine to Δ1-piperideine cation, the residue from acetyl-CoA is needed, together with SAMe activity in the transformation to lobelanine. Piperine is synthesized from piperidine through the formation of amide.

2.2.6.2 Swansonine and castanospermine synthesis pathway Swansonine and castanospermine synthesis starts with the α-aminoacid, γ-semialdehyde, and, via piperidine-6-carboxylic acid synthetases, L-pipecolic acid. This compound is a substrate to HSCoA and acetyl-CoA. As a result of this activity, the second ring is established. Subsequently, it changes to 1-indolizidinone and, by an oxidation reaction, produces castanospermine or swansonine (Figure 2.30).

128 CHAPTER 2 Alkaloid chemistry

Co2H NH2

Cadaverine

NH2

L-lysine

Δ1-piperideine

Δ1-piperideine cation

Pelletierine

CoA, SAMe

Lobelanine

Piperine

Piperidine

n FIGURE 2.29 Diagram of the pelletierine, lobelanine, and piperine synthesis pathway.

Co2H NH2 NH2

a-aminoadipic acid d-semialdehyde

L-lysine

Piperideine-6-carboxylic acid

L-pipecolic

acid

HScoA, Acetyl-coA

1-indolizidinone

Castanospermine

Swansonine

n FIGURE 2.30 Diagram of the swansonine and castanospermine synthesis pathway.

2.2 Synthesis and metabolism 129

Both castanospermine and swansonine occur in some legume plants, such as Castanospermum australe and Swainsona canescens, respectively. They are hybrid molecules compounded of pyrrolizidine and quinolizidine alkaloids and have shown some resistance to the AIDS virus. Certainly, the abovementioned alkaloids are also toxic for animals.

2.2.6.3 Lupinine, lupanine, sparteine, and cytisine synthesis pathway L-lysine is a very important precursor for alkaloids with the quinolizidine nucleus. This group of alkaloids can be divided according to their construction into three subgroups as follows: bicyclic alkaloids (first subgroup), tricyclic (second subgroup), and tetracyclic alkaloids (third subgroup).7 In the older studies, a division of quinolizidine alkaloids was established according to alkaloid type. Kinghorn and Balandrin60,71,102,102a divided these alkaloids into (1) quinolizidines with simple substituents, (2) the leontidine type, (3) the sparteine/lupanine type, (4) the esters of sparteine/lupanine type, (5) the tricyclic degradation products of sparteine/lupanine-type, (6) the pyridine bases type, (7) the matrine type, (8) the Ormosia type, and (9) quinolizidine alkaloids having miscellaneous structures. The synthesis pathway of quinolizidine alkaloids is based on lysine conversion by enzymatic activity to cadaverine in exactly the same way as in the case of piperidine alkaloids. Certainly, in the relatively rich literature that attempts to explain quinolizidine alkaloid synthesis,32,40,49,89,146,147 there are different experimental variants of this conversion. According to experimental data,32 the conversion is achieved by coenzyme PLP activity, when the lysine is CO2 reduced. From cadeverine, via the activity of the diamine oxidase, Schiff-base formation and four minor reactions (Aldol-type reaction, hydrolysis of imine to aldehyde/amine, oxidative reaction, and again Schiff-base formation), the pathway is divided in two directions. The subway synthesizes (
)-lupinine by two reductive steps, and the main synthesis stream goes via the Schiff-base formation and coupling to the compound substrate, from which again the synthetic pathway divides to form (+)-lupanine synthesis and (
)-sparteine synthesis. From (
)-sparteine, the route by conversion to (+)-cytisine synthesis is open (Figure 2.31). Cytisine is an alkaloid with the pyridone nucleus.

This pathway clearly proves that the first quinolizidine alkaloid to be synthesized is (
)lupinine (two cycling alkaloids) and subsequently both (+)lupanine and (
)-sparteine. This is a new approach to the synthesis of this type of alkaloids, because in the older literature, just four cycling alkaloids (lupanine and sparteine) are mentioned as the first synthesized molecules.40,89,146 In the cadaverine conversion, the participation of diamine oxidase is more reliable than the oxosparteine synthase mentioned by some older studies.49,146

130 CHAPTER 2 Alkaloid chemistry

Diamine oxidase CO2H

NH2 NH2

PLP

Cadaverine

L-lysine

Aldol-type reaction Schiff base Hydrolysis of imine to amine Schiff base Oxidative deamination

(–)-Lupinine

Schiff base coupling

(–)-Sparteine C (+)-Cytisine

(+)-Lupanine

n FIGURE 2.31 Diagram of the lupinine, sparteine, lupanine, and cytisine synthesis pathway. Abbreviations: PLP ¼ coenzyme pyridoxal phosphate; C ¼ cleavage of C4 unit.

2.2.7 Methods of analysis This presentation of basic alkaloid synthesis pathways clearly reveals the diversity and complicity of this process in nature. Moreover, the large number of pathways and synthesis routes proves the status of alkaloids as a phenomenon of the metabolic activity of organisms. Here, we have seen only the basic pathways and routes. In reality, each alkaloid has its own synthesis route. It is possible to find or to place it on one of the basic pathways. Certainly, experimental data is required for this, the obtaining of which necessitates deep research into molecular structure. Although the technical level of research in the leading laboratories is very high, deep structural and synthesis research is not easy. It is a very expensive and complex job. Pure chemical structure analysis does not suffice today to explain the nature of alkaloid behavioral synthesis in depth. Reactions require a lot of the energy derived from the Krebs cycle and, generally, enzymatic activity. Alkaloid studies use chemical methods of analysis to clarify the constructional and taxonomical nature of these compounds, together with biological and semi-biological methods to describe the role and behavior of these

2.2 Synthesis and metabolism 131

molecules in life processes. It is well known that natural product molecules are biosynthesized by a chain of reactions that, with very few exceptions, are catalyzed by enzymes.32,134 This is especially important in the case of alkaloids—biologically very active secondary compounds, which have genetic background and environmental oscillators.6 A discussion on the synthesis of alkaloids derived from different substrates in the metabolic system of a living organism should cover the form and construction blocks of substrate and the changes occurring in the synthesis. The most important step is to provide the answer to the question of origin and the link to the different compounds in the synthesis reactions. An alkaloid metabolism is concerned with the formation of new molecular substances or the degradation of synthesized molecules. This metabolism is, in reality, connected with the active transport of metabolites, inorganic ions, and organic atoms. Moreover, the converting of energy in biosynthetic and degradative processes is also a base for the reaction chains. The metabolic system of alkaloid pathways is clearly regulated. Preiss and Kosuge96 emphasize that enzyme synthesis or degradation and enzyme activity are integrated to produce a more efficient modulation. Simpkins119 notes that there must be a positive correlation between the rate of overall physiological processes and the kinetic and regulatory properties of key enzymes. Moreover, he contends that the specific activity of key enzymes involved in a metabolic pathway would be expected to be high. Therefore, alkaloid synthesis is regulated by a mechanism linked to enzymatic strategy. This strategy seems to be one of the fundamental blocks in alkaloid analysis. It is generally known that enzymes are proteins with catalytic activity in the life system and metabolic processes. They were discovered by Sumner, who first isolated the enzyme urase in crystalline form from jack bean meal in 1926. This historic event was very important for both chemistry and biology. However, from today’s perspective it is difficult to understand why the first isolation and crystallization of enzymes met with such strong criticism, and even derision, among large groups of leading scientists. Only the methodological development and subsequent works with crystallization, and the identification of many new enzymes, published more than 20 years later by Northrup, Kunitz, and Herriot92 confirmed Sumner’s discovery and finally established that the enzyme was a protein. The enzyme as a catalytic unit has a direct relation to alkaloid synthesis research. Even nowadays, not all metabolic enzymes have been isolated and named in alkaloid synthesis pathways. The development of enzymatic methods for the detection of the steps of the synthesis and degradation of alkaloid molecules seems to be very challenging for contemporary research. Mahler and Cordes82 show that enzymes catalyze reactions in

132 CHAPTER 2 Alkaloid chemistry

four directions: (1) increasing reaction efficiency and speed, (2) increasing of the utilization of the substrate, (3) broadening the spectrum of reactions, and (4) controlling cellular metabolism. All these catalyzing purposes are applicable to alkaloid synthesis and degradation reactions. Methods of alkaloid analysis are therefore focused on the molecular-level biology inside and outside the organism. On the other hand, any analysis of alkaloids is impossible without pure classical chemistry and bioorganic chemistry methods.

2.2.7.1 Methodological considerations Alkaloids generally, and especially investigations into them throughout history, retain some kind of mystery. This is connected with their strong biological activity, which was observed and used even in ancient times but without any explanation. Even in the middle of the 20th century, the scientific explanation of alkaloids was based on the opinion that these compounds were the products of waste organic metabolic processes. Only in 1960s and 1970s did alkaloids begin to be seen as evolutionary and biogenetic markers of living organisms.128 Despite the fact that alkaloids are very different compounds, they can be classified and harmonized according to morphology and metabolism. As stated earlier, it is evident that an alkaloid pathway consists of a series of reactions and compounds as well as enzymes.5,6,133 The sequence of all reactions leading to any alkaloid synthesis can be presented as follows:

χ1…….n

α

β

ϕ

A

P

χ

A

P

w

A

P

A chance compounds and chemical elements

2.2 Synthesis and metabolism 133

α ¼ precursor β ¼ basic intermedia φ ¼ obligatory intermedia χ ¼ second obligatory intermedia χ1. . ..n ¼ in some cases one or multiple secondary intermedia ω ¼ final product of metabolism A ¼ alkaloid synthesis P ¼ postcursor of alkaloid. The alkaloid is not the final product of a secondary metabolism. This new conception explains why alkaloids were traditionally considered to be unnecessary and undesirable compounds in organisms. Although the “waste” theory is no longer seriously entertained, many questions remain unanswered. It is not quite clear why plants, animals, and particularly microorganisms produce alkaloids. Certainly, there are many hypotheses and theories regarding this problem, with compelling arguments but also with points open to criticism. A similar question is related to biosynthesis: Why does an alkaloid need its intermediate molecule in the synthesis process and why is it not derived directly from the precursor? In natural processes, there is a tendency to cut corners in developing links and pathways. However, in the alkaloid synthesis pathway, just the opposite occurs, although this is also a process occurring in nature. Take, for example, the synthesis of quinolizidine alkaloids. From a theoretical point of view, the structural transformation of L-lysine into (
)lupinine should be simpler; but according to empirical studies into ring and carbon spectra, a direct reaction does not exist.32 This transformation occurs only by intermedia (cadaverine) reactions.

2.2.7.1.1 The precursor The alkaloid precursor is a substrate for the alkaloid molecule as well as being the source of the alkaloid’s nitrogen and skeleton. Precursors of alkaloids are very diverse in their structure, type, and metabolic function. They also can be fragments of the molecules.91 Generally, alkaloid precursors are nonprotein amino acids (e.g., ornithine, nicotinic acid, anthranilic acid), protein amino acids (e.g., lysine, tyrosine, tryptophan, histidine, phenylalanine), and various compounds (e.g., acetate and malonate), L-phenylalanine in the case of providing nitrogen from amination (geraniol, cholesterol, adenine, guanine in transamination reactions). In some cases, there may be more than one precursor, when alkaloids can be synthesized via alternative pathways. This is typical, for example, of L-tyrosine and L-phenylalanine. Both are protein amino acids with aromatic side chains, but L-phenylalanine is not so frequently used in alkaloid synthesis. Moreover, L-phenylalanine generally contributes only carbon atom units (e.g., C6C3, C6C2, C6C1) to

134 CHAPTER 2 Alkaloid chemistry

the alkaloid skeletons without providing a nitrogen atom. Phenethylisoquinoline alkaloids, for example autumnaline, floramultine, kreysigine, and colchicine, are derived from both these amino acids. Although L-phenylalanine is a protein amino acid and is known as a protein acid type of alkaloid precursor, its real role in biosynthesis (providing C and N atoms) relates to only carbon atoms. L-phenylalanine is a part of “magic 20” (a term deployed by Crick in his discussion of the genetic code) and just for this reason should also be listed as a protein amino acid type of alkaloid precursor, although its duty in alkaloid synthesis is not the same as other protein amino acids. However, in relation to the “magic 20,” it is necessary to observe that only part of these amino acids are well-known alkaloid precursors. They are formed from only two amino acid families: Histidine and Aromatic17,90 and the Aspartate family.17

2.2.7.1.2 The basic intermedia The basic intermedia is a compound formed from the precursor in each alkaloid synthesis pathway. In the case of nonprotein amino acids as precursors of alkaloids, the intermedia is generally a biogenic amine, for example, putrescine in hygrine and other pyrrolidine and tropane alkaloid pathway (Figure 2.13), dihydronicotinic acid in the nicotine pathway (Figure 2.27) or nicotinamide in the ricinine pathway (Figure 2.28), and the amide formation compound in peganine or the dictamnine pathway. Generally speaking, the transformation of a precursor to an intermedia is done by an enzyme (e.g., PLP in the hygrine pathway and NADPH in the nicotine pathway) or by CoA with part of another substrate (e.g., anthraniloyl-CoA with part of L-ornithine in the peganine pathway). If differences in the formation of intermedia are sought, the intermedia derived from anthranilic acid should be found in this group of precursors. As stated previously, for intermedia formation, the CoA and part of the other precursor is needed. In many other cases, the metabolism of anthranilic acid as an alkaloid precursor is slightly different from standards of nonprotein amino acids. It is related to the origin of this precursor. Anthranilic acid can be an intermediate compound (precursor) in tryptophan biosynthesis and also the postcursor of the tryptophan during the degradation process. In the case of protein amino acids as precursors of alkaloids, the intermedia is biogenic amine, for example, L-dopa in the mescaline pathway or tyramine in the hordeine pathway (Figure 2.15), putrescine in the homospermidine pathway (Figure 2.14), dopamine in the kreysigine and colchicine pathways (Figure 2.16), tyramine in the galanthamine pathway (Figure 2.18), and tryptamine in the psilocybin pathway (Figure 2.19). Each DNA amino acid as a precursor of alkaloids has a clearly determined

2.2 Synthesis and metabolism 135

intermedia. The transformation of a precursor to an intermedia is achieved by PLP or DC enzymes. Other precursors of alkaloids form intermedia as acids (e.g., capric acid in the coniine pathway, 26-hydroxycholesterol in the solasodine pathway, and piperidine in the jervine pathway). Moreover, in the case of purine as an alkaloid precursor, the intermedia is inosine monophosphate (IMP). One of the characteristics of intermedia is that, in many cases, it is not a stable compound (e.g., cadaverine). Intermedia is a compound that can be the final product of any pathway. However, an alkaloid can convert from an intermedia (e.g., norbelladine from tyramine in the galanthamine pathway), although this process is restricted. Generally, the synthesis pathway continues to establish the next compound, the obligatory intermedia.

2.2.7.1.3 Obligatory intermedia An obligatory intermedia is a compound that follows the basic intermedia in the synthesis process of the alkaloid and metabolism pathway. The synthesis of this generally not stable compound is obligatory or alternative for alkaloid formation. In the case of nonprotein and protein amino acids as precursors of alkaloids, the obligatory intermedia is derived, in most instances, from biogenic amine (intermedia) by SAMe-dependent N-methylation (e.g., the conversion from putrescine to N-methylputrescine in the hygrine pathway), enzyme NAD+ in the conversion of putrescine to imine in the homospermidine pathway, or enzyme DAO in the conversion of cadaverine to Δ1-piperideine in the quinolizidine alkaloids pathway. In the case of nonamino acid precursors, the conversion from intermedia to obligatory intermedia occurs by a coupling reaction, for example, from piperidine to protoverine in the jervine pathway or from IMP to XMP in purine alkaloids. The basic characteristic of obligatory intermedia synthesis is that there cannot be an alkaloid between intermedia and obligatory intermedia, but in some cases, the obligatory intermedia can be an obligatory alkaloid needed for synthesis of other alkaloids, for example, protoverine in the jervine pathway. In these cases, the obligatory intermedia has biological activity as, for example, with protoverine. After the obligatory intermedia, the alkaloid can be synthesized or the obligatory intermedia converts to the second obligatory intermedia.

2.2.7.1.4 Second obligatory intermedia In the case of protein amino acid–derived alkaloids, the second obligatory intermedia are synthesized from the obligatory intermedia by chemical reactions. In the pelletierine synthesis pathway started with L-lysine, the second obligatory intermedia is A1-piperidinium cation. It is formed by a Mannich

136 CHAPTER 2 Alkaloid chemistry

reaction from A1-piperidine (obligatory intermedia) and COSCoA. The second obligatory intermedia, by hydrolysis decarboxylation, produces pelletierine. In the case of nonprotein amino acid–derived alkaloids, the second obligatory intermedia is derived from the obligatory intermedia enzymatically and by the Schiff-base formation as, for example, in the hygrine pathway. The second obligatory intermedia is, in this case, the N-methyl-Δ1-pyrrolinium cation. The general characteristic of the second obligatory intermedia is that this compound is not stable. It is poison and biologically active. Subsequently, the second intermedia alkaloid is synthesized.

2.2.7.1.5 One or multiple secondary intermedia In some cases, the metabolic pathway leads to the establishing of one or even a lot of additional compounds in chain from the second obligatory intermedia. Alkaloids can also be synthesized from this chain, directly from the additional secondary intermedia or cross molecule or even its fragment of the second obligatory intermedia. The nature of this process is connected with the unstable feature of the molecules and the unexpected, random reactions.42,122 Intermedia molecules can be biologically active, even very active.

2.2.7.1.6 Final product In many cases, alkaloid synthesis is not the end of the metabolic pathway as part of the secondary metabolism block. An alkaloid is generally the subproduct of this metabolism and, in many cases, can be used by living cells in neurophysiological activity. Part of the alkaloid substrate can again be metabolized to the alkaloid postcursors in the metabolic subpathway in the form of synthesis or degradation. In the case of a primary metabolism, it is relatively simple to show the final product. In the case of a secondary metabolism, it is not possible in every case to predict the final product, because there are many possibilities of stopping or prolonging the reaction chain according to physiological needs and signaling. Although alkaloid pathways exist, they are parts of the more general secondary metabolism. Alkaloids are not the final products of this metabolism, and their nature is to be a part of the metabolic chains.

2.2.7.2 Structural approach Alkaloids as nonfinal products of the secondary metabolism are very different in their structure and life functions in organisms. Many groups of

2.2 Synthesis and metabolism 137

alkaloids are known. As mentioned, they have different precursors and rings. They also have different subpathways and intermedia.

2.2.7.2.1 Piperidine alkaloids Piperidine alkaloids contain the piperidine nucleus. The structural development of this group of alkaloids in synthesis is presented in Figure 2.32. Here α is L-lysine and β is cadaverine. The basic ring of β is the same as in α, although the activity of PLP reduces carbon dioxide. The β is a biogenic amine, neither a stable nor a poisonous compound. By oxidative deamination, in which diamine oxidase (DAO) is active together with Schiff-base formation, the β converts into φ. The φ is Δ1piperideine, which cannot be substituted by other compounds, although several hypothetical obligatory intermedia, such as glutardialdehyde and 5-aminopentanal, have been proposed.128 In the case of piperidine alkaloids, these hypothetical substitutes of φ are ruled out. The φ has slowly changed the basic ring, but it is no longer piperidine. The deeper change in the ring occurs in the next reaction, the Mannich reaction (MR), the result of which is χ. For this purpose, the nucleophilic acetoacetyl-CoA with (
) is used for (+), closing the ring. In this case, the intermolecular Mannich reaction is under question. Subsequently, χ, which is a Δ1-piperidinium cation, has a ring similar to piperidine, although not identical. Only after hydrolysis and decarboxylation will the compound be A (pelletierine) with the piperidine nucleus. By the activity of SAMe, A is converted to P, which is N-methylpelletierine. P can be converted by the intramolecular Mannich reaction to the next postcursor, P1, which is pseudopelletierine. Other postcursors of pelletierine are possible, for example anaferine, in the intermolecular Mannich reaction. True piperidine alkaloids have one-cycle compounds with the C5N nucleus.

2.2.7.2.2 Indolizidine alkaloids Indolizidine alkaloids contain the indolizidine nucleus with two different cycles. The structural development of this kind of alkaloid is presented in Figure 2.33. The α is L-lysine, as in the case of piperidine, but the β is different. The β is α-aminoadipic acid δ-semialdehyde. The φ is L-pipecolic acid, which is synthesized in plants from piperideine-6-carboxylic acid. In the case of many other organisms, the obligatory intermedia (φ) is derived from the β. The φ retains one ring structure. The indolizidine nucleus is formed only in the synthesis of the χ. The deep structural change occurs when φ is transformed by a chain of reactions: the formation of CoA ester (CoAe), the Claisen reaction with acetyl or malonyl CoA (Cra/mCoA) and the ring closure process (by amide or imine) to 1-indolizidinone, which

138 CHAPTER 2 Alkaloid chemistry

PLP

DAO

j

CO2H NH2

NH2

–CO2

NH2

NH2

N

SBF

Cadaverine

L-lysine

a

b

H+

COSCoA

c Δ1-piperidinium cation

+ NH

O Acetoacetyl-CoA

P

MR O N H

A

O N H

N H

Pelletierine

Anaferine SAMe

P

O N CH3

N - methylpelletierine

MR

H3C

P1

N O

Pseudopelletierine

n FIGURE 2.32 Structural development of piperidine alkaloids.

is the χ. The second obligatory intermedia (χ) has only the indolizidine nucleus. The χ is transformed by hydroxylation to A, which is castanospermine. The A is a typical subpath product. The main pathway is transformed to the χ by hydroxylation and the ring fusion to another A, which is swansonine.

2.2 Synthesis and metabolism 139

a

b

CO2H NH2

CO2H NH2

O

NH2

a-aminoadipic d-semialdehyde

L-lysine

HSCo

CoAe Cra/m Co

Acetyl-CoA

CO2H NH L-pipecolic

H

j acid

O

c

N

1-indolizidinone

OH H

OH

OH H

OH

HO OH N

N

Castanospermine

Swansonine

HO

A

A

n FIGURE 2.33 Structural development of indolizidine alkaloids.

Both alkaloids (castanospermine and swansonine) have the ability to inhibit glycosidase enzymes (GEs), the activity of which is necessary in glycoprotein biosynthesis.

2.2.7.2.3 Quinolizidine alkaloids The third structural group of alkaloids, from the same α, are quinolizidine alkaloids (QAs). It is a large group of compounds with very different abilities.6,7,12,23,24,36,45,46,49,60,71,72,97,102,102a,112,133,137,138,146,147,148,151,159,162 The structural development of quinolizidine alkaloids is presented in Figure 2.34. The α (L-lysine) provides the basic components of the quinolizidine nucleus and skeleton. The β is cadaverine and is synthesized in the same way as piperidine alkaloids (by the activity of PLP). The transformation from β to φ also occurs through the activity of diamine oxidase (DAO). The φ is Δ1-piperideine, which develops by the Schiff-base formation, the

140 CHAPTER 2 Alkaloid chemistry

DAO

a

b

NH2

CO2H NH2

NH2

NH2

Cadaverine

L-lysine

SBF OH

A

H

H

N

N +

CHO

j (–)-lupinine SBF SBF + N

H

c

N +

O H

OH

N N

P

H

A

13a-hydroxylupanine

O

H

H

N

O

N N

N

N

H

N

H Sparteine

Lupanine

A CCU

H

Angustifoline

O H

O N N

N

P

H

HN (+)-cytisine

P

a-isolupanine

N

P1

N O

n FIGURE 2.34 Structural development of quinolizidine alkaloids.

Anagyrine

aldol-type reaction between enamine and iminium, the hydrolysis of imine to aldehyde, oxidative deamination, and again, the Schiff-base formation. During this stage, the quinolizidine nucleus is formed. From φ as a subpath product the (
)-lupinine is synthesized, which is a two-cycle quinolizidine

P

2.2 Synthesis and metabolism 141

alkaloid. The first A is therefore a two-cycle quinolizidine alkaloid,32 although in previous studies four-ring quinolizidine alkaloids have been said to form first.40, 89,146,147 The main way that the product is formed is by the step reaction of the Schiff-base formation, for which the molecule of the β or the φ is again needed. The four-cycle quinolizidine skeleton is formed in this stage by the molecule coupling with cationed nitrogen in the χ (second obligatory intermedia), which is simply two transformed molecules of the φ with cationed nitrogen. In reality, this χ is two molecules of (
)-lupinine connected together in an opposite molecule order with H atom reduction. This strongly suggests that there is also an alternative way for four-cycle quinolizidine alkaloids synthesis from (
)-lupinine. The χ is transformed in two directions: (
)-sparteine and (+)-lupanine, the two basic quinolizidine alkaloids that occur in nature and play an important role in the ecosystem. The (
)-sparteine is transformed by the cleavage of the C4 unit to (+)-cytisine, a three-cycle quinolizidine alkaloid with a pyridon nucleus, and from this step to the other pyridon quinolizidine alkaloids (P, P1). The (+)-lupanine converts to the lupanine derivatives, angustifoline, α-isolupanine and 13α-hydroxylupanine (P) (Figure 2.34).

2.2.7.2.3.1 Bicyclic quinolizidine alkaloids Bicyclic quinolizidine alkaloids have the simplest chemical structure, based only on the quinolizidine nucleus.6 Typical representatives of this type of alkaloids are lupinine, epilupinine, and lusitanine.6,9,25 Lusitanine is an alkaloid derived from Genista lusitanica L.,18 Lupinus excubitus, and Lupinus holosericeus.86 In their absolute configurations, the melting point of Lusitanine is 184–186 ° C, of dihydro 96–100 ° C, and of epidyhydrolusitanine 140–144 ° C.18 Lupinine and epilupinine are typical bicyclic quinolizidine alkaloids in lupines, especially in Lupinus luteus L., Lupinus hispanicus L., Lupinus hirsutus L. They have also been found in Anabasis aphyla. In absolute configuration, lupinine is in its (
)- form, which is nonstable thermally and easily epimerized to epilupinine, which is a stable (+)- form of lupinine.9,18 The melting point of (
)-lupinine is 70–71 ° C, of mixed (+ and
)-lupinine 63–64 ° C, and of (+)-lupinine (synthetic) 167–168 ° C. Lupinine and epilupinine contain esters, which have been found in L. luteus seedlings.6,9,18 2.2.7.2.3.2 Tricyclic quinolizidine alkaloids Tricyclic quinolizidine alkaloids occur in lupines. Angustifoline, with its derivatives, and albine are examples of this structural group of alkaloids. Angustifoline is identical with jamaicensine, an alkaloid isolated from Ormosia jamaicensis. Angustifoline is a compound found in L. angustifolius L., L. polyphyllus Lindl., and Lupinus albus L.15 Angustifoline is in the (
)- form in absolute configuration, with a melting point of 79–80 ° C.18 From L. albus L. and viable seeds of Lupinus termis L., (+)-angustifoline as a diastrereoisomer of

142 CHAPTER 2 Alkaloid chemistry

(
)-angustifoline has been isolated by Wysocka and Przybył157,158 in the Alkaloid Chemistry Laboratory in Poland. Other derivatives of angustifoline are dihydroangustifoline, with a melting point of 82–83 ° C,18 and isoangustifoline, with a melting point of 96–97 ° C.3,4,7 Albine has been found in L. albus141 and structurally reinvestigated by Wysocka and Brukwicki,153,154 Wysocka et al.,155,156 and Wysocka and Przybył.158

2.2.7.2.3.3 Tetracyclic quinolizidine alkaloids Tetracyclic quinolizidine alkaloids can be divided into two types, according to both chemical structure and, especially, biological activity. These are tetracyclic alkaloids that contain a quinolizidine nucleus and others with a pyridone nucleus. Here, the first type of alkaloids (with a quinolizidine nucleus) are discussed. The second type are considered in the next subsection as pyridone alkaloids. Sparteine is one of the basic, and probably most important, tetracyclic alkaloids with a quinolizidine nucleus. In absolute configuration, sparteine occurs as (
)-sparteine, which is lupinidine. Lupinidine, with a melting point of 181 ° C, occurs in all lupine species, although in different concentrations. L. luteus sparteine (lupinidine) is a major alkaloid, and consequently, this yellow lupine has been described as a “typical” sparteine species. However, sparteine is also found in Lupinus mutabilis,145 L. polyphyllus,3,4,7 and Genista tinctoria.87 The (+)-sparteine has been detected in Lupinus pussilus, Cytisus caucasicus, and many other plants.76,77 This alkaloid was discovered in Hovea linearis,75 Maackia amurensis,109 Termopsis mongolica,26 Lygos raetam,1 and in L. albus.152 The melting point of (+)-sparteine is 173–174 ° C, and it is also known as pachycarpine.18 Sparteine is also familiar in the form of ()-parteine. Its melting point is 231 ° C. According to the literature, this alkaloid form does not occur in the lupin species.18 Lupinus sericeus contains (
)-7-hydroxy-β-isosparteine and 10, 17-dioxoβ-isosparteine, which are also sparteines: tetracyclic quinolizidine alkaloids with a quinolizidine nucleus. Moreover, other alkaloids from this group include epiaphylline and aphylline, alkaloids from L. latifolius, and (
)-lindenianine, an alkaloid from Lupinus lindenianus and Lupinus verbasciformis.9,85 Nuttalline (4β-hydroxy-2-oxosparteine) is a tetracyclic quinolizidine alkaloid from Lupinus nuttalli.9 An alkaloid, sparteine can be converted to α-isosparteine or β-isosparteine, which occurs particularly in Cytisophyllum sessilifolium.15,87,140 In contrast to aphylline, 17oxosparteine is known to be synthesized only under energetic conditions.15 One of the most important tetracyclic quinolizidine alkaloids with a quinolizidine nucleus is lupanine, which is in fact 2-oxo-11α-sparteine. In absolute configuration, lupanine is (+)-lupanine with a molecular weight of 248 and melting point of 127 ° C.6,18 Lupanine occurs in L. polyphyllus,

2.2 Synthesis and metabolism 143

L. albus, and L. angustifolius and is, like sparteine, probably found in all lupine species in different concentrations, from main compounds to mere traces. Lupanine is the main alkaloid in the seeds of Lupinus rotundiflorus, Lupinus exaltatus, and Lupinus mexicanus. It has been discovered in considerable amounts in Lupinus montanus and Lupinus madrensis, but only traces were noted in Lupinus elegans.119 Moreover, lupanine occurs in Cytisus scoparius and Leontice eversmannii.18 The (
)-lupanine is hydrorhombinine and has been isolated from L. pussilus and Lupinus macouni as well as other species, such as Baptisia versicolor and Podalyria calyptrata. The melting point of (
)-lupanine is 190 ° C.18 In such species as L. albus and L. termis, lupanine occurs as ()-lupanine with melting points of 127–128 ° C and 250–252 ° C.18 In L. polyphyllus Lindl., L. angustifolius L., and L. albus L., 17-oxolupanine has also been detected.15,87,158 Hydroxylupanine and their esters occur in Lupinus bicolor, Lupinus densiflorus, L. latifolius, Lupinus polycarpus, Lupinus ruber, Lupinus burkei,145 L. rotundiflorus, L. montanus, L. exaltatus, L. mexicanus, L. madrensis,97 and L. polyphyllus.3,4,7

2.2.7.2.3.4 Pyridone alkaloids This group of alkaloids has a pyridone nucleus and generally takes the tetracyclic or tricyclic form. The α for pyridone alkaloids is L-lysine, while the β, φ, and χ are the same as for other quinolizidine alkaloids. Quinolizidine alkaloids containing the pyridone nucleus are the P from the (
)-sparteine by cleavage of the C4 unit.32 The first quinolizidine alkaloid with the pyridone nucleus is tricyclic cytisine, which converts to four cyclic alkaloids. In this synthesis, the anagyrine, the most poisonous quinolizidine alkaloid with a pyridone nucleus, has its own synthesis pathway. Anagyrine has a molecular weight of 244 and a melting point of 264 ° C. It only takes a ()-form.18 This alkaloid occurs in L. latifolius,85 Lupinus arboreus, Lupinus caudatus, L. densiflorus, L. sericeus, Lupinus argenteus, Lupinus leucophyllus.86 Anagyrine was found in neither bitter nor sweet L. polyphyllus Lindl., which grows in Finland.3,4,7

2.2.7.2.4 Pyrrolizidine alkaloids The pyrrolizidine nucleus is characteristic of this group of alkaloids. The α is either L-ornithine or L-arginine, and the β is a biogenic amine, the putrescine. Oxidative deamination by the enzyme NAD+ converts two molecules of putrescine into the imine (φ). By the activity of NADH, imine is reduced to homospermidine (χ). Then, the pyrrolizidine nucleus is formed via a chain of reactions such as oxidative deamination, Schift-base formation, oxidative reaction again, and the intramolecular Mannich reaction.

144 CHAPTER 2 Alkaloid chemistry

Retronecine (A), which is a simple pyrrolizidine alkaloid, commonly occurs in nature. The formation of the pyrrolizidine structure is presented in Figure 2.35. Retronecine and its P, the senecionine, necine, heliotrine, indicine-N-oxide, malaxine, monocrotaline and absulin, are typical representatives of this group of alkaloids.32,103–105,137 Pyrrolizidine alkaloids are widely dispersed throughout the natural world. According to Robins,105 these alkaloids have been found in 15 families, although three plant families

L-ornithine

NH2

CO2H NH2

NH2 NH2 NH2

or NH N2 H

H2N Putrescine

Putrescine CO2H

N H

OD

NH2

NAD+

L-arginine

NH2

NH2

j N (Imine) NADH

A

Retronecine HO

OH

H

OD

CO2H N

NH2

NH2

NH

NH2

MR Homospermidine

CO2H NH2

HO O

O

O

O H

N Senecionine

n FIGURE 2.35 Structural development of pyrrolizidine alkaloids.

P

c

b

2.2 Synthesis and metabolism 145

(Boraginaceae, Asteraceae, and Fabaceae) are the most important sources of these compounds.32 In Fabaceae, the genus Crotalaria is particularly representative of pyrrolizidine alkaloids, and in Asteraceae, the genus Senecio.137 The characteristics of these alkaloids are as follows: (1) they are accumulated in plants as N-oxides; (2) they are poisons; (3) some of them have a bioimpact (e.g., indicine-N-oxide).

2.2.7.2.5 Izidine alkaloids Izidine alkaloids cover compounds that contain one of the izidine skeletons. There are three different skeletons in this group: pyrrolizidine, indolizidine, and quinolizidine. Izidine alkaloids are, therefore, compounds with a bicyclic nucleus, which have different α, β, φ, and χ. Izidines present structural similarities but organic and functional differences. These alkaloids include more than 800 compounds. Several of them show interesting physiological and pharmacological behavior. Izidine alkaloids with the pyrrolizidine nucleus (e.g., hellosupine, senecionine, retronecine, acetyl-intermedine, acetyl-lycopsamine, and indicine-N-oxide) are toxic and known to affect the liver.24 Izidine alkaloids containing the indolizidine nucleus (e.g., slaframine, elaeocanine, securitinine, tylophorine, swansonine, and castanospermine) play an important role as actual and potential drugs in the fight against viruses, including AIDS. Some of these compounds, for example pumiliotoxin B, have an ecological function in nature. Izidine alkaloids with the quinolizidine nucleus (e.g., lupinine, lusitanine, lamprolobine, angustifoline, lindenianine, sparteine, lupanine, nuttalline, aphylline, sophoridine, isomatrine, albertidine, aloperine, and nitraramine) contain toxic compounds with strong selective ecological impact.6

2.2.7.2.6 Pyrrolidine alkaloids Pyrrolidine alkaloids have a pyrrolidine (C4N skeleton) nucleus. The structural α of these alkaloids is L-ornithine (in plants) and L-arginine (in animals). The pyrroline skeleton is synthesized after β (putrescine) and φ (N-methylputrescine), when DAO activity and Schiff-base reaction forms χ, which is N-methyl-Δ1-pyrrolinium cation. Subsequently, A (hygrine) is formed. Typical pyrroline alkaloids are (
)- and (+)-hygrines (Figure 2.36).

2.2.7.2.7 Tropane alkaloids Tropane alkaloids have a tropane (C4N skeleton +) nucleus. Structurally, these alkaloids synthesize as postcursors of pyrrolines (Figure 2.37). The α, β, φ, and χ in the tropane pathway are the same as in pyrrolines. Typical tropane alkaloids (e.g., atropine, hyoscyamine, cocaine, tropinone, tropine, littorine, and cuscohygrine) have a strong biological activity, especially as neurotransmitters.50–53

146 CHAPTER 2 Alkaloid chemistry

NH

Urea cycle (animals)

a NH2

CO2H NH2

CO2H

N H

N2H

a

NH2

ARG L-arginine

L-ornithine

– CO2

PLP

Urea NH N H

H2N

NH2 Agmatine

Hydrolysis of imine functionality in guanidine system

O Plants N2H

N H NH2

N-carbamoylputrescine – CO2

Hydrolysis of urea

PLP H2N NH2

Putrescine

N-methylation

SAMe

O

SBF

DAO

+ SCoA

N

NH

CH3

CH3

N-methyl-Δ1-pyrrolinium cation

O

O

A CH3

n FIGURE 2.36 Structural development of pyrrolidine alkaloids.

N R (–)-hygrine

O

NH

NH2

CH3 N -methylputrescine

A N S

b

CH3 (+)-hygrine

j

2.2 Synthesis and metabolism 147

A (–)

N-methyl-Δ¹-pyrrolinium cation

A (+)

O

P1

P1

O

N IMR

H3C O Tropinone

N H3C

N Cuscohygrine

CH3

SAMe NADPH

P2

NADPH

L-phenylalanine

N H3C

L-phenylalanine

OH Phenyl-lactic acid Tropine N OH

H3C

P3

O L-phenylalanine

O

(–)-hyoscyamine CO2-CH3 N

O2 2-oxyglutamate

OH

H3C

CH3

Methylecgonine

N

P1

H

HO 6b-hydroxyhyoscyamine

N H3C

O

P2

Cocaine

CH2-OH

O

CO2-CH3

P4

O

O

O2 2-oxyglutamate

OFER CH3 N H

O O (–)-hyoscine (scopolamine)

2.2.7.2.8 Imidazole alkaloids This group of alkaloids is an exception in the transformation process of structures, because the imidazole nucleus is already made at the stage of the precursor. The α of these alkaloids is L-histidine, and the first A is developed in a decarboxylation process by histidine decarboxylase (HDC). The histamine is a product of this reaction (Figure 2.38). Other alkaloids from this group include, for example, dolichotheline, pilocarpine, and pilosine.

CH2-OH

P5

O

n FIGURE 2.37 Structural development of tropane alkaloids.

148 CHAPTER 2 Alkaloid chemistry

L-leusine

a

H N

CO2H NH2

N

L-histidine

HDC

– CO2

H N N

H N

H N O Dolichotheline

A NH2

N

Histamine

? ? ? CH3 CH3

N O

N O N

O Pilocarpine

HO CH3

N

O Isopilocarpine

A

A

? = no experimental evidence

H

N O N

A

O Pilosine

n FIGURE 2.38 Structural development of imidazole alkaloids.

2.2.7.2.9 Quinazoline alkaloids Quinazoline alkaloids contain more than 100 compounds. They have been isolated from animal and plant sources. The plant family Rutaceae is especially rich in these alkaloids. Typical quinazoline alkaloids include arborine, glomerin, homoglomerin, glycorine, glycosminine, febrifugine, and vasicine (Figure 2.39). The α of quinazoline alkaloids is anthranilic acid, although there are in some cases alternative α, such as phenylalanine in the case of arborine or ornithine. The β is anthranoylphenylalanine.

A

2.2 Synthesis and metabolism 149

L-ornithine

a

CO2H

NH2

COSCoA

+

HN

b

NH2 ••

Anthranilic acid

Nucleophilic attack of amine to the iminium

Anthraniloyl-CoA

CO2H

b

O

O SCoA •• HN

j

N N -acetyl-anthranilic acid

N H Amide formation

CO2H H2N

N

CO2H H

L-asparagine

A N OH Vasicine (pegamine)

n FIGURE 2.39 Structural

development of quinazoline alkaloid vasicine.

Quinazoline alkaloids are known as biologically active compounds. Arborine inhibits the peripheral action of acetylcholine and induces a fall in blood pressure. Febrifugine is an antimalarial agent, and vasicine acts as a uterine stimulant. Glomerin and homoglomerin are alkaloids of the defensive system in some organisms (e.g., in the glomerid millipede).

2.2.7.2.10 Acridone alkaloids Fewer than 200 acridone alkaloids are known. Typical compounds include atalaphylline, acronycine, and preacronycine. Acridone alkaloids occur in plants and animals. They are especially characteristic of the Rutaceae plant family. The α is anthranilic acid, the β is N-methyl anthraniloyl-CoA, and

150 CHAPTER 2 Alkaloid chemistry

3 * malonyl-CoA

a

b

CO2H

COSCoA

NH2

NH-CH3 N -methyl anthraniloyl-CoA

Anthramilic acid

Claisen reaction neophilic addition dehydration and enolization

j

O

OH

OH

N CH3

1,3-dihydroxy-N-methylacridone

Melicope fareana

Ruta graveolens O

OH

Acronychia baueri O

O

O-CH3

O-CH3 N

O-CH3

A O

CH3 O-CH3

N

A

O-CH3

CH3 Melicopicine

O

N

Rutacridone

CH3 Acronycine

A n FIGURE 2.40 Structural development of acridone alkaloids.

the φ is 1,3-dihydroxy-N-methylacridone (Figure 2.40). Acridone alkaloids are biologically active. Acronycine is also known for its antitumor activity.

2.2.7.2.11 Pyridine alkaloids Pyridine alkaloids are compounds with a pyridine nucleus and a pyrrolidine or piperidine unit. The pyrrolidine ring appears in nicotine and the piperidine ring in anabasine. Typical alkaloids from this group are nornicotine and anatabine. The α of pyridine alkaloids is nicotinic acid, the β is dihydronicotinic acid, the φ is 1,2-dihydropyridine (Figure 2.41). The A is nicotine and its P is nornicotine.32

2.2 Synthesis and metabolism 151

L-ornithine

+ NADPH

b

H

H

O

H NH2

CO2H NADPH H

Putrescine

N

H

N

a

O

H

-

NH2

+ H

Nicotinic acid Dihydronicotinic acid

Aldol-type reaction

H

A H N

N-CH3

NADP+

Nicotine

..

H H

N H

j

1,2-dihydropyridine NADPH

Non-enzymic decomposition

H N

N H

P

Nornicotine

2.2.7.2.12 Sesquiterpene pyridine alkaloids Compounds belonging to this group of alkaloids are sourced from the Celastraceae and Hippocrateaceae families and contain the sesquiterpene nucleus. More than 300 alkaloids are known in this group.38,78–80,118,166 Sesquiterpene pyridine alkaloids can be divided into several types, such as evoninate, wilfordate, edulinate, cassinate, lower-molecular-weight sesquiterpene pyridine, and noncelastraceous sesquiterpene pyridine alkaloids.38,78–80,118,166 Evoninate sesquiterpene alkaloids can again be divided into evoninate, isoevoninate, hydroxyisoevoninate, epimeric evoninate, and norevoninate compounds. Evoninate sesquiterpene alkaloids are compounds in which evonic acid esterifies the sesquiterpene nucleus. Typical alkaloids of this subgroup are evonimol derivatives (e.g., evonine), euonyminol derivatives

n FIGURE 2.41 Structural development of pyridine alkaloids.

152 CHAPTER 2 Alkaloid chemistry

(e.g., euonymine and hippocrateine), isoeuonyminol derivatives (e.g., emarginatine), 4-deoxysesquiterpene cores (e.g., chuchuhuanine), cathedulin alkaloids (e.g., cathedulin), and dimacrocyclic sesquiterpene pyridine alkaloids (e.g., triptonine). A typical isoevoninate sesquiterpene alkaloid is hippocrateine, and a typical hydroxyisoevoninate one is hypoglaunine. Acanthothamine is a representative compound of epimeric evoninate alkaloid. Norevoninate alkaloid has been isolated from only Hippocratea excels.38,78–80,118,166 Wilfordate alkaloids are another type of sesquiterpene pyridine compound. They are sesquiterpenes, macrocyclic compounds esterified by wildorfic acid. Wilforine is an example of this kind of alkaloid. Edulinate sesquiterpene pyridine alkaloids are sesquiterpene compounds esterified by edulinic acid. Cathedulin is one example. The cassinate group of sesquiterpene alkaloids contains orthosphenine and cassinine. These alkaloids have dihydroagarofuran sesquiterpenes esterified by cassinic acid. Lower-molecular-weight sesquiterpene pyridine alkaloids have been isolated only from plants of the Celastraceae family and are characterized by the absence of a macrocyclic ring. They have a sesquiterpene core and a nitrogenous base through esterification. Lower-molecular-weight sesquiterpene pyridine alkaloids are also known as nocotinoyl sesquiterpene alkaloids.38,78–80,118,166 Noncelastraceous sesquiterpene pyridine alkaloids are those compounds isolated from other plants not belonging to the Celestraceae family. Rotundine, for example, has been isolated from Cyperus rotundus (Cypraceae). This is a structurally interesting alkaloid because it has a sesquiterpene skeleton containing a cyclopentane ring attached to the pyridine ring.38,78–80,118,166 Natural sesquiterpene pyridine alkaloid formation needs two precursors, one for the pyridinium moiety and another for the sesquiterpene moiety. The α for formation of the pyridinium moiety is nicotinic acid, which reacts with isoleucine and, by oxidative reaction, produces evoninic acid, wilfordic acid, or edulinic acids. The α for the sesquiterpene moiety is still open to question, but E, E-famesyl cation has been suggested as one possibility and hedycarylol as another. This moiety is dihydroagarofuran. Therefore, the α for the sesquiterpene pyridine alkaloids is nicotinic acid and E, E-famesyl cation and, controversially, hedycaryol. The β is amacrocycling ring formation substance (two moieties), from which the alkaloid forms (Figure 2.42). The sesquiterpene pyridine alkaloids have antifeedant and insecticidal activities. Some alkaloids, such as triptonine B, hypoglaunine B, hyponine B, and wilfortrine, have antivirus activity potential. Others, such as emarginatines A–B, E–G. and emarginatine, have cytotoxic activity. Ebenifoline and cangorinine have immunosuppressive activity.

2.2 Synthesis and metabolism 153

a

CO2H

a

a

NH2

N

+

Nicotinic acid

CO2H

OH

Isoleucine E,E-farnesyl cation

CO2H

N

Hedycaryol

CO2H

CO2H

CO2H

N

Wilfordic acid

b

Evoninic acid

b

CO2H

O Dihydroagarofuran

N

Edulinic acid

O Dihydroagarofuran

CO2H

OAc OAc

OAc

O

Macroajelic ring formation

OH ••

O OAc

O

SCoA

OH

O

SCoA

j

OAc

OH ••

O

O O

O N

O

A N Evonine

2.2.7.2.13 Phenyl and phenylpropyl alkaloids These alkaloids have a phenyl or phenylpropyl nucleus. The group includes simple phenyl amine (tyramine, hordenine), catecholamine (dopamine, noradrenaline, adrenaline), simple tetrahydroisoquinoline (mescaline, anhalamine, anhalonine, anhalonidine), benzylisoquinoline (e.g., papaverine),

n FIGURE 2.42 Structural development of sesquiterpene pyridine alkaloids.

154 CHAPTER 2 Alkaloid chemistry

phthalideisoquinoline (e.g., noscapine), phenethylisoquinoline (autumnaline, floramultine and kreysigine), tetrahydroisoquinoline (emetine and cephaeline), and terpenoid tetrahydroisoquinoline (secologanin and ipecoside) alkaloids. The α for this group of alkaloids is L-tyrosine (in some cases, also phenylalanine), and the β is L-dihydroxyphenylalanine (L-DOPA). Simple phenylamines, such as tyramine and hordenine, are derived from α by PLP (Figure 2.43). The A is tyramine and by activity of the SAMe its P is hordenine. Phenyl and phenylpropyl alkaloids form a very large and diverse group. Many of the compounds belonging to them have very important commercial implications and are used in pharmacology and medicine. The alkaloids from Amaryllidaceae (e.g., norbelladine, lycorine, crinine, and galanthamine) are also considered to be a part of this large group, although they are structurally very different. However, the pattern of biosynthesis, especially for α, is the same as for other alkaloids in this large group.32 The term isoquinoline alkaloids was previously used for the group of phenyl and phenylpropyl alkaloids.120 a

HO

CO2H NH2

HO

CO2H N

HO

b

L-DOPA

L-tyrosine

PLP

PLP OH

PLP

A

A NH2

HO

HO

SAMe OH

SAMe HO SAMe

N-(CH3)2

HO

Hordenine

P2 NH-CH3 Adrenaline

H3C-O NH2

H3C-O O-CH3

P1

n FIGURE 2.43 Structural development of phenyl and phenylpropyl alkaloids.

Noradrenaline

SAMe

P

NH2

HO

Dopamine

SAMe

HO

NH2

HO

Tyramine

P1

HO

Mescaline

2.2 Synthesis and metabolism 155

2.2.7.2.14 Indole alkaloids This structural group of indole alkaloids covers simple indole alkaloids (e.g., tryptamine, serotonin, psilocin, and psilocybin), β-carboline alkaloids (e.g., harmine), terpenoid indole (e.g., ajmalicine, catharanthine, and tabersonine), quinoline alkaloids (e.g., quinine, quinidine, and cinchonidine), pyrroloindole alkaloids (e.g., eserine), and ergot alkaloids (e.g., ergotamine). Indole alkaloids form a very important group from the perspective of their application.

2.2.7.2.14.1 Simple indole alkaloids The α for structural development of serotonin (a simple indole alkaloid) is L-tryptophan, and the β is 5-hydroxyL-tryptophan (Figure 2.44). Serotonin is a monoamine. It is a bioactive CO2H

CO2H NH2

a

NH2

HO

N H 5-hydroxy-L-tryptophan

N H L-tryptophan

–CO2

NH2

b

NH2

HO

N H

N H

Tryptamine

Serotonin SAMe SAMe

OH N-(CH3)2

A

N-(CH3)2 HO

N H

N H

Psilocin

Gramine

OP N-(CH3)2

P N Psilocybin

n FIGURE 2.44 Structural development of simple indole alkaloids.

A

156 CHAPTER 2 Alkaloid chemistry

alkaloid known as a neurotransmitter. It has been found in the cardiovascular system, in blood cells, and the peripheral and CNS. The α for the structural development of psilocin and psilocybin is L-tryptophan, and the β is tryptamine. Psilocin is A and psilocybin is P. A and P are the main alkaloids in hallucinogenic mushrooms belonging to the genus Psilocybe.

2.2.7.2.14.2 Carboline alkaloids The α of carboline alkaloids is L–tryptophan, and the β is tryptamine, while the φ is dihydro-β-carboline. The carboline nucleus is formed at the stage of the φ. The A is elaeagnine, harman, and harmaline, and the P is tetrahydroharmine or harmine (Figure 2.45). Carboline alkaloids, and especially β-carbolines, are common in mammals.108

2.2.7.2.14.3 Terpenoid indole alkaloids This group of alkaloids is very large and contains more than 3000 compounds. Three types of nucleus occur here: the corynanthe, iboga, and aspidosperma:

CO2H

a

NH2 N H

L-tryptophan

NH2

b

SBF

N

MR

N H

b-carboline

N H

Tryptamine

A

j

N

N

P N H

H3C-O

Dihydro-β-carboline

NH

N H

RS N H

H3C-O

Harmaline

Tetrahydroharmine

A NH RS N H

N

N

A N H

H3C-O

N H Harmine

Elaeagnine

Harman

n FIGURE 2.45 Structural development of carboline alkaloids.

P

2.2 Synthesis and metabolism 157

1. Corynanthe alkaloids. The α of structural development is L-tryptophan, and the β is tryptamine. The strictosidine is the φ. Moreover, the χ is dehydrogeissoschizine, and the A is cathenamine. Ajmalicine is the P of cathenamine (Figure 2.46). CO2H

a

NH2 H

N H

OGlc

OHC

L-tryptophan

O H CO2-CH3

b

Secologanin

NH2 N H

Tryptamine

+

j

NH N H Strictosidine

H

H

N

OGlc O

N H

•• OH

H H

H

c

Dehydrogeissoschizine CO2-CH3

CO2-CH3

N N H

H

O H

Cathenamine

A CO2-CH3

N N H Ajmalicine

H

H

O H

P CO2-CH3

n FIGURE 2.46 Structural development of corynanthe alkaloids.

158 CHAPTER 2 Alkaloid chemistry

2. Iboga alkaloids. The common monomeric iboga alkaloids are ibogamine, ibogaine, coronaridine, voacangine, and catharanthine. Ibogamine and catharanthine are prototypical structures. The α for cathenamine is L-tryptophan, and the β is strictosidine, as in the case of corynanthe alkaloids. Ibogaine is the P of cathenamine (Figure 2.47). The iboga-type nucleus is derived from the corynanthe type.32 3. Aspidosperma alkaloids. These alkaloids have an aspidosperma-type nucleus. The α and β are the same as in corynanthe type. Catharantine is the P4 from cathenamine (Figure 2.48).

2.2.7.2.14.4 Quinoline alkaloids This group of alkaloids has two structurally different forms of α. The α of alkaloids found in the genus Cinchona (Rubiaceae), such as quinine, quinidine, cinchonidine, and cinchonine, is L-tryptophan. The β is tryptamine, and the φ is strictosidine. The corynantheal is the χ. A is cinchonamine and cinchoninone. Cinchonine, quinidine, quinine, and cinchonidine are the forms of P of cinchoninone (Figure 2.49). The second type of quinoline alkaloid, especially found in the Rutaceae family, has α as anthranilic acid. Typical compounds from this group are edulitine, halfordamine, folifidine, folinine, casimiroin, foliosidine, and swietenidine. The β is 3-carboxyquinoline, and the A is graveoline in the case of those alkaloids from Ruta angustifolia. In the case of S. japonica, the A is eduline, formed directly from α in the decarboxylation process. In the case of microorganisms, for example Pseudomonas aeruginosa, the α is incorporated directly to the A, which is 2-heptyl-4-hydroxyquinoline. Quinoline alkaloids from the Rutaceae family (α ¼ L-anthranilic acid) show antimicrobial activity. Moreover, quinoline alkaloids of the Haplophyllum species are known for their powerful biological properties. For example, skimmianine has sedative, hypothermic, and antidiuretic uses. Haplophyllidine is a strong depressant of the CNS.

2.2.7.2.14.5 Pyrroloindole alkaloids The α of this group of alkaloids is 121 L-tryptophan, the β is tryptamine, and the φ is methyltryptamine (Figure 2.50). The best-known alkaloids belonging to this group are eserine (A), chimonanthine (P), eseramine, physovenine, rivastigmine, eptastigmine, neostigmine, pyridostigmine, and distigmine. 2.2.7.2.14.6 Ergot alkaloids The α for ergot alkaloids is L-tryptophan, and the β is a four-step alkaloid reaction chain. The φ is paspalic acid, which converts to the D-(+)-lysergic acid (Figure 2.51). Ergotamine and ergometrine are the best-known members of this group.

2.2 Synthesis and metabolism 159

CO2H NH2

a

N H

L-tryptophan

b N H

NH H

H OGlc H O

H3C-O2C

Strictosidine

N H

j

N H H

H3C-O2C

OH 20,21-dehydrocorynantheine aldehyde

A N H

N H CH3 H O

H3C-O2C

Cathenamine

N

P

H3C-O

N H

Ibogaine

n FIGURE 2.47 Structural development of iboga

alkaloids.

160 CHAPTER 2 Alkaloid chemistry

CO2H NH2

a

N H

L-tryptophan

N H

NH H

H OGlc H O

b

H3C-O2C Strictosidine

N H

N

20,21-dehydrocorynantheinealdehyde

H H

H3C-O2C

j

OH

P2 N

N H

N

N H CH3

Cathenamine H O

H3C-O2C

N

H3C-O2C

N H H3C-O2C

H CH2-OH

P1

Preakuammicine

H CH2-OH

Stemmadenine

A

N

P1

N N H H3C-O2C

P1 n FIGURE 2.48 Structural development of aspidosperma alkaloids.

Catharanthine

N H H3C-O2C Dehydrosecodine

2.2 Synthesis and metabolism 161

CO2H

a

NH2

A O

N H

N L-tryptophan

N O

Camptothecin

OH O NH2

b

A

O

A

N H

O O

N

Tryptamine

N

H O

H

N H

H

j

H

NH

OGlc

Pumiloside

OGlc

H

O H H

Strictosamide

OGlc

O

H

N H

N H

OH

H H3C-O2C

Strictosidine

N

A c

H

N H

N H

N H H Cinchonamine

CHO

H corynantheal

H HO

NADPH

A

P

N H

H

Cinchonidine

O N H

N NADPH

cinchoninone

H N

HO

N H

H3C-O

NADPH NADPH

N

H

Quinine

P

H

HO

HO

N H

H

N

N H

H3C-O

P

Quinidine N

P

Cinchonine

n FIGURE 2.49 Structural

development of quinoline alkaloids.

162 CHAPTER 2 Alkaloid chemistry

CO2H

a

b

NH2

NH2

N H

N H

Tryptamine

L-tryptophan

j

H3C NH N H

H

Methyltryptamine

H N

H3C-N H3C O

H3C-HN

N-CH3

N-CH3 N H

Chimonanthine

O

H

P

H

Eserine

A

n FIGURE 2.50 Structural development of pyrroloindole alkaloids.

2.2.7.2.15 Manzamine alkaloids Manzamine alkaloids can be isolated from marine sponges. They often contain β-carboline. This group has a diverse range of bioactivities. It also has its own way of establishing its structures. An intramolecular Diels-Alder reaction for manzamines has been proposed. The α is bisdihydropyridine (derived probably from amonia), and the β is an intramolecular cycloaddition in a pentacyclic structure.21,57,99,100,161 The φ is a tetracyclic intermedia, and the A is manzamine A, manzamine B, and manzamine C (Figure 2.52). The best-known manzamines are ent-8-hydroxymanzamine A, manzamines B–M, and X, keramamine, kauluamine.

2.2.8 Biogenesis of alkaloids The synthesis and structural analysis of alkaloids leads to the following basic questions: Why are alkaloids synthesized in an organism and on which mechanism is alkaloid formation and degradation dependent in the life

2.2 Synthesis and metabolism 163

CO2H

a

NH2 N H

L-tryptophan

b HO

OHC

NH-CH3 H

NH-CH3 H

N-CH3 H

HO

N-CH3 H

NADPH H

H

H

N H ChanoclavineI

H

N H

N H ChanoclavineII

N H

Agroclavine

HO2C

HO2C

N-CH3 H

H

Elymoclavine

N-CH3 H H

c N H D-(+)-lysergic

j N H

acid

Paspalic acid

ATP L-alanine L-phenylalanine L-proline

ESH Enzyme

HO

N H

O

O

N HN O

O N-CH3 H

A

Ergotamine

N H

n FIGURE 2.51 Structural

development of ergot alkaloids.

164 CHAPTER 2 Alkaloid chemistry

CHO

CHO

N+

NH3 CHO OHC N

NH3

a CHO

b

OHC

j

C10

C3

N N H

H

N

OH

H

A N

A

Manzamine A

N N H

H

N

N N H

O

H

N NH

A Manzamine B

n FIGURE 2.52 Structural development of manzamine alkaloids.

Manzamine C

cycle? It is known that alkaloids have a genetic nature93 and that alkaloid content is diverse inside and between the species.134 In nature, the same species of plants may have both high and low alkaloid content.3,4 Natural hybridization has been successfully used in plant breeding for the development of

2.2 Synthesis and metabolism 165

the so-called sweet cultivars in crop production. “Sweet cultivars,” however, are not without alkaloids. The total removal of alkaloids is impossible. Sweet cultivars are therefore plants, in useful organs of which alkaloids are present at a very low level, the bioactivity of which is not of any significant or observable level. However, alkaloid decrease by hybridization is an indirect but strong argument for the case that alkaloids have a heredity nature and their presence in plants is of an evolutional character. This is fundamental in answering the first question connected with the biogenesis of alkaloids. Alkaloids have a strong genetic-physiological function and background in the organisms that produce them. The biogenesis of alkaloids is therefore a part of the total genetic-functional strategy of such metabolisms.

2.2.8.1 Chemistry models From 1805, when alkaloid chemical research started, the problem of the biogenesis of alkaloids proved central for chemists. The background to this problem was the fact that chemical compounds are synthesized, used, and degraded by plants. In the middle of the 20th century, it was still difficult to truly ascertain the purpose of alkaloids in plants. Certainly, the use of these compounds in many applications outside of the organisms producing them was well recognized. Their role within the plants, especially in their metabolism, was not known. The general consensus was that alkaloids were the “waste” product of metabolisms and had no active role to play.16 Therefore, chemical chains of alkaloid production were explained as chemical reactions, the “technical” process of life. Later, especially since the late 1970s, the theory of “wastes” was debated and corrected.134 However, chemical research has now extensively proven the existence of new alkaloids, the pathways of their biosynthesis, and structural modification. Three directions in this research have been followed, one purely chemical, the second biochemical, and the third purely biomolecular or in the molbiological direction. The chemical explanation of alkaloid biogenesis is based on the consideration that all reactions are of a chemical nature and that the energy needed for life is produced by chemical reactions. Figure 2.53 shows a diagram of the chemical explanations for alkaloid biogenesis. From this diagram, it is clear that alkaloid is one of the metabolic objects in the system. It has a long chemical chain, which includes chemical synthesis before and chemical degradation after its functional activity in the metabolism. Biogenesis is, therefore, considered by chemistry to be the chain of the reactions between chemical molecules and by chemical means, in which reactions, conditions, and catalyzers are of special importance. Chemistry and organic chemistry consider alkaloid biogenesis to be the transformation of organic material

166 CHAPTER 2 Alkaloid chemistry

Primary metabolism

Precursor Obligatory intermedia

c

Second obligatory intermedia

c

Secondary metabolism blocks

Organisms producing alkaloids

Postcursors

c

Synthesis pathways

ALKALOIDS

Functional chemical receptors in metabolism system

c

c Chemical degradation c c n FIGURE 2.53 Chemical explanation for alkaloid biogenesis in organisms (c ¼ catalysers).

with reaction catalyzers. Different alkaloids have their own biogenesis and they are used, separately or together, with biochemical models in developing the methods for synthetic reactions and the modification of structures. Moreover, these models are also used in biotechnology.106,107 Figure 2.54 presents the chemical model for the synthesis of Catharanthus alkaloids. It shows the primary metabolism as the background for alkaloid formation, although the Catharanthus alkaloids are the yields of a secondary metabolism. The connection between primary and secondary metabolisms is an important area for future studies in chemistry. From the model presented, it is clear that Catharanthus alkaloids are postcursors from three basic compounds: acetate, glucose, and tryptophan. In the Catharanthus alkaloids, three types of ring nucleus are presented. The chemical model describes biogenesis from the point of view of the formation nucleus and skeleton of alkaloids, together with connected chemical molecule reactions in their structural and dynamic changes. Torssell128 used the term “mechanistic approach for the secondary metabolism to describe the chemical approach to this metabolism. Chemical routes, and alternative routes and their options, are very important for chemical models. In particular, the tendency

2.2 Synthesis and metabolism 167

PRIMARY METABOLISM Acetate

Glucose

Secondary metabolism Geraniol

Tryptophan Secondary metabolism Tryptamine

Secologanin

Strictosidine

Aspidosperma nucleus type

Catharantus alkaloids

Iboga necleus type

Corynanthe nucleus type

n FIGURE 2.54 Chemical model of indole alkaloid formation in Catharanthus roseus. The arrows represent the direction of formation, the flux of compounds skeleton construction.

of natural processes and reactions to shorten synthesis pathways is significant. Nowadays, the principles of alkaloid biochemistry and their biosynthetic means are widely recognized by specialists, and chemical or mechanistic approaches to synthesis and biosynthesis, form a basic part of research. Without carbons, nucleus, skeleton, ring, and moiote, the alkaloid does not exist. To research, these structural components of alkaloids’ chemical models of approach are the most effective.

2.2.8.2 Biochemistry models The description of single enzyme activity in chemical reactions, together with the activity of other biomolecules, is typical for biochemical models of alkaloid biogenesis. There is no contradiction between chemical and biochemical, which serve to enrich one another. In many cases, typical chemical and biochemical models are unified in papers today.106,107 Biochemical reactions are basically the same as other chemical organic reactions with their thermodynamic and mechanistic characteristics, but they have the enzyme stage. Laws of thermodynamics, standard energy status, and standard free energy change, reduction-oxidation (redox), and electrochemical potential equations are applicable to these reactions. Enzymes

168 CHAPTER 2 Alkaloid chemistry

catalyze reactions and induce them to be much faster.128,144 Enzymes are classified by international convention into six classes on the basis of the chemical reaction they catalyze. According to Enzyme Commission (EC) rules, the enzyme classes are (1) oxidoreductases (transfer of hydrogen or oxygen atoms and electron forms), (2) transferases (transfer of chemical groups), (3) hydrolases (catalyzing of hydrolytic reactions), (4) lyases (cleaving substrates by reactions other than hydrolysis), (5) isomerases (intramolecular rearrangements), (6) synthases (catalyse covalent bond formation). The best-known enzymes and coenzymes active in alkaloid biogenesis are presented in Table 2.2. The biochemical model contains the pathways of the enzymatic reactions in the synthetic routes. Models can be constructed for each alkaloid. Figure 2.55 presents a biochemistry model of Catharantus alkaloids. Table 2.2 Some well-known enzymes and coenzymes active in alkaloid biogenesis Enzyme Type

Reactions

Decarboxylases (DC) Tryptophan decarboxylase (TDC) Phenylalanine decarboxylase (PDC) Dimerases (DM) Hydroxylases (H) Methylases (MT) Synthases Oxidases (O) Peroxidases (PO) N-methyltransferase (MT) Amine oxidases (AO) Monoamine oxidase (MO) Diamine oxidase (DO) Dehydrogenases (DHG) NAD+(nicotinamide adenine dinucleotide) NADP+(nicotinamide adenine dinucleotide phosphate) Pyridoxal phosphate (PLP) CoA S-adenosylmetionine (SAMe)

Decarboxylation

Dimethylallyl diphosphate (DMAPP) Transaminases (TA) Reductases (RD) Source: Refs 2, 13, 14, 31, 32, 50–54, 58, 103, 121, 142.

Dimerization Hydroxylation +CH3 Synthesis Removing hydrogen from a substrate Using hydrogen peroxide Transfer of methyl group Oxidizing reactions Dehydrogenation to an imine Oxidizing to aldehyde Removes two hydrogen atoms from the substrate Tends to be utilized as hydrogen acceptor Tends to be utilized as hydrogen acceptor Coenzyme in transamination and decarboxylation Involves biological reactions Provides positively charged sulfur and facilitates nucleophilic substitution Nucleophilic substitution Transamination Reduction

2.2 Synthesis and metabolism 169

PRIMARY METABOLISM Acetate

Glucose

Secondary metabolism Geraniol G10H

Secologanin

Tryptophan

TDC

Secondary metabolism Tryptamine

SS

Strictosidine

Aspidosperma nucleus type

NADPH+ P, O, NADH+ Catharantus alkaloids

Iboga nucleus type

Corynanthe nucleus type

n FIGURE 2.55 The biochemical model for indole alkaloid formation in Catharanthus roseus. The arrows represent the direction of the formation and the flux of compounds in skeleton construction. On the diagram, enzymes are shown by a circle.

The most important enzymes on this model are TDC (tryptophan decarboxylase), G10H (geraniol 10-hydroxylase) and SS (strictoside synthase). NADPH+, PO (peroxidase), O (oxidase), and NADH+ are all active in different Catharantus alkaloid formations. The biochemical models are subject to both qualitative and quantitative alkaloid analysis. Not all enzymes participating in alkaloid synthesis and degradation are yet known. Alkaloid enzymatology is, therefore, a growing research area.

2.2.8.3 Molecular biology models Alkaloid research and bioanalysis of central-processing molecules (DNA and RNA) led to the important concept of the heredity nature of alkaloid metabolisms. Recent investigations have proved empirically that alkaloids have a genetic background and that all their biogenesis is genetically determined.44,48,59,117,130 According to Tudzynski et al.130, cpd1 gene coding for

170 CHAPTER 2 Alkaloid chemistry

dimethylallyltryptophan synthase (DMATS) catalyzes the first step in the biosynthesis of ergot alkaloids from Claviceps purpurea. The second gene for ergot alkaloid biosynthesis is cpps1, which encodes for a 356-kDa polypeptide showing significant similarity to fungal modular peptide synthetases. According to Tudzynski and his research group,130 this protein contains three amino acid–activating modules, and in the second module, a sequence is found that matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthase 1 (LPS1) of C. purpurea. The authors proved that cpps1 encodes LPS1. Cpd1 is also involved in ergot alkaloid biogenesis. Cpox1 probably encodes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), while the second putative oxidoreductase gene, cpox2, is closely linked to it in inverse orientation.130 At least some genes of ergot alkaloid biogenesis in C. purpurea were found to be clustered. This means that detailed molecular genetic analysis of the alkaloid pathway is possible.130 These results were confirmed by the research of Haarmann et al.48 Moreover, Huang and Kutchan59 found three genes (cyp80b1, bbe1, and cor1) that encode the enzymes needed for sanguinarine synthesis. Molecular biology models may be constructed for each alkaloid biogenesis. An example of this kind of model is presented in Figure 2.56.

Gene cluster cpd1 cpps cpox1 cpox2

DMATS

LPS1

FAD

a b .... n FIGURE 2.56 Molecular biology model of Claviceps purpurea alkaloids.

2.2 Synthesis and metabolism 171

Molecular biology research on alkaloids is very revealing. Its results can be used in the construction of alkaloid biogenetic models. At present, only a few alkaloid metabolism genes are known.

2.2.8.4 Analytical dilemmas Chemical, biochemical, and biological models of alkaloid biogenesis can be constructed only according to scientific research on the small chains of the synthesis of each alkaloid, enzyme, and gene involved in these chains. Models are constructed from the experimental data on synthesis and degradation of alkaloids. Even though a high technical level of analytical equipment exists and research is exact, the results do not give a direct answer to some questions of biogenesis. For structural (chemical), enzymatic (biochemical), and genetic (molbiology) research, different techniques are deployed. They work under different conditions, and the results received are from these different conditions, although from the same places in the pathway. In model construction, a researcher should unify these results for a logical chain. In many cases, there are theoretical or hypothetical conclusions. Certainly, the common analysis in the same trial of the structural, biochemical, and biological aspects of alkaloids is the best method. Such kinds of supertechnique are still not developed. Therefore, the primary analytical dilemma concerns the question of separate or common analysis.5 On the one hand, this question covers the chemical, biochemical, and molbiology aspects and, on the other, the isolation of alkaloids. The dominant way is separate analysis for separate alkaloids. Separate alkaloid analysis gives exact results in microscale, microplace, and microimportance. In many cases, such results would be compromised in macroscale analysis (the biogenesis model). However, in the extraction of some group of alkaloids, the same method (common in this sense) is accepted in many papers. One example is quinolizidine alkaloids. Although quinolizidine alkaloids are very similar, they also have differences in optimal dissolving (temperature and pH value), purity, stability, and (+) and (
) forms. The same method of extraction for all alkaloids is a great compromise and causes compromised results, which have been regarded as sufficient. Another analytical dilemma is the problem of in vitro and in vivo conditions. Alkaloids should be studied in their physiological conditions in organisms. This is not possible in many cases. In vitro experiments give compromised data. Theoretical conclusions and hypotheses in analysis, although they are in many cases indicators of a new breakthrough, also have some problems and some risks. Analytical dilemmas are one reason why continuous novelization of structural, biochemical, and molbiological results is necessary. These dilemmas

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merit attention nowadays, more than 210 years after the first alkaloid was isolated.

2.2.9 Methods of alkaloid analysis These analytical dilemmas interfere with the methods of alkaloid analysis. Each group of alkaloids has its own methods of extraction, isolation, and crystallization, as well as detection in structure, molecule, and dynamism. Not all these stages are still possible in the majority of alkaloids. In recent years, many techniques have been used in alkaloid detection. There are atomic and molecular electronic spectroscopy, vibration spectroscopy, and electron and nuclear spin orientation in magnetic fields, mass spectroscopy, chromatography, radioisotope, and electrochemical techniques. Although important developments in methodology and methods of alkaloid analysis have occurred over the last 200 years, the most efficient methods are still awaited. The oldest parts of these methods seen to be the extraction and isolation stages.

2.2.9.1 Methods in history The first method of alkaloid analysis was developed in 1805, in the case of morphine. This method of isolation, with minor and major variations, is still used today. By this method, the first quinolizidine alkaloids were also extracted: sparteine in 1851, lupinine in 1865, and lupanine two years later. At the beginning of the 20th century, the extraction and determination of total quinolizidine alkaloids in the same analysis (common) was carried out by Jurkowski,68 Nowotno´wna,94 Trier,129 Ivanov,62,63 Sengbusch,113–116, Łukaszewicz,81 and Wuttke.150 Reifer and Niziołek102 and Wiewio´rowski and Skolik139 initiated research in which the sum of the contents of the different and separate alkaloids is the total alkaloid content. The method of isolation of quinolizidine alkaloids was developed next by Wysocka et al.155,156 and Wysocka and Przybył.157,158

2.2.9.2 Basic methods and instruments The first step in the development of methods was the evidence that molecules synthesized and degraded and that intermediate compounds existed. Initial methods provided molecule isolation and, subsequently, the place in the metabolic chains. The basic methods of alkaloid determination developed historically as follows: iodine, taster, seed color, Dragendorff’s reagent, fluorescence, calorimetry, photometry, electrophotometry, spectrometry, paper chromatography, thin-layer chromatography, highperformance liquid chromatography, gas chromatography, gas liquid chromatography–mass spectrometry, nuclear magnetic resonance, x-ray,

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enzymelike immunosorbent assay, radio immuno assay, and scintillation proximity assay methods.5 The most effective method for establishing a metabolic pathway is the use of isotopes in radiotracing and mass spectrometry methods. The basic instruments that have been developed are photometers, calorimeters, analyzers, spectrometers, chromatographs, and different mass spectrometers. These instruments have subsequently been improved to be more exact and have been through many generations in their development by many different producers.

2.2.9.3 From iodine to enzyme Alkaloid analytical methods were developed by applications based on different hypotheses, from the simple to the very complicated. Subsequently, corresponding instruments were developed. This development can be seen by considering the example of quinolizidine alkaloids.

2.2.9.3.1 Iodine Iodine was discovered by Barnard Courtois in 1811 in France. It has been successfully researched and used in biochemistry and clinical research since 1825. In analytical work, however, it is necessary to draw attention to the fact that iodine can occur in biological material as free iodine or in other forms, such as iodoaminoacids and iodoproteins. Three chemical methods for the quick determination of quinolizidine alkaloids were developed for use in practical breeding in 1927.116 All these methods were based on the use of iodine. In the first of Sengbusch’s methods, the alkaloids were extracted from whole seeds or leaves by means of hot water and precipitated with iodine-quicksilver-potash of iodine. By this method of alkaloid analysis, the first “sweet” plants (without indication of alkaloid content) of L. luteus were found.116 With material of low alkaloid content, the second method could be evolved in which the alkaloids were extracted with hydrochloric acid instead of hot water. According to Sengbusch,116 this method is suitable mainly for the investigation of leaf material, whereas in the testing of seeds, precipitation of nonalkaloid substances apparently also occurs. The hydrochloric acid method with cold water extraction was then developed. In this method, the alkaloids are extracted from the seed with cold water then precipitated with iodine-potash. According to Sengbusch,116 this method permits the testing of seed material without damage to germination, so that the tested seeds can be sown. This is very important, for example, in plant breeding. The use of iodine in determining total alkaloid content has been developed by plant breeders in field conditions up to the present. The basic iodine solution contains 100 g J and 140 g KJ as well as 1000 ml water. Before

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application, the basic iodine solution is diluted by 1:3 or 1:5. The color of leaves without alkaloids is not changed after application of the iodine solution. Following this method, the leaf color changes to red-brown.116,150 Very similar to the method of Sengbusch was that developed by Schwarze, during which the juice of leaves was transferred onto blotting paper. After that, the blotting paper was put into the iodine-potash-iodine solution. The brown color that appeared on the blotting paper was caused by alkaloids, and the color of the blotting paper in which alkaloids were absent was slightly yellow or green,5 A similar solution is also known as the old Dragendorff’s reagent and the KI/I2 test. The use of KI/I2 needs 2–3 hours for sample determination.

2.2.9.3.2 Taster method This is a nonchemical, and probably the first biological, method of determining the presence of alkaloids. It was first used particularly with quinolizidine alkaloids in lupine plants. The tasters were men or animals, even in ancient times. It is based on the fact that quinolizidine alkaloid has a bitter taste. This method is qualitative. Taste is a subjective and individual category, especially in the case of animals. It is known generally that hares and sheep are more tolerant of quinolizidine alkaloids than other animals. This method can be described as a simple attempt to determine bitterness (alkaloid) in lupine plants.

2.2.9.3.3 Seed color method The simple observation that white seeds are sweeter that black seeds was used in the construction of a practical method of judging lupine seeds qualitatively. This method cannot be used with confidence, because, especially in white lupine, even very white seeds can have high alkaloid content. On the other hand, plants from the same species are “sweet.” In some species, for example in the case of L. angustifolius or L. luteus, the tendency of white seeds to be “sweet” is more likely but not absolutely certain.

2.2.9.3.4 Dragendorff reagent Dragendorff’s reagent (DRG) was developed as a reagent for detecting alkaloids, heterocyclic nitrogen compounds, and quaternary amines. It is used particularly in plant drug analysis.132 At least six Dragendorff’s reagents are known. Each one also contains potassium iodine. Dragendorff’s reagent is still often used, and its research value is high, as researchers can check by visualization the existence of the alkaloids.66,67,88 To understand the significant of this reagent, it is necessary to pay attention that lot of studies with alkaloid determination without the use of a control reagent should be

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rejected. Without visualization of an alkaloid occurring, the proof of alkaloid content does not exist.This is a message to many laboratories, scientists, faculties, and publishers on our globe in the year 2015.

2.2.9.3.5 Fluorescence method This method is based on the fluorescence characteristics of lupanine and its derivatives in L. albus. They have a fluorescence capacity to a light of 366 nm.10,11,95 Fluorescence is an emission of light from a molecule that is returning to its normal ground state from the lowest vibrational level of an excited singlet state light.95 Fluorescence is closely related to absorption, because absorption must precede emission of fluorescence. For this method, a UV lamp with light of 366 nm is necessary. Bitter seeds are fluorescent and sweet seeds are not. Generally, the fluorescence method of lupine seed analysis is considered to be qualitative only.10 In reality, this method can, after development, also be quantitative. For this purpose, it is necessary to ensure that (1) the intensity of fluorescence is directly proportional to the molecular absorptivity, (2) the intensity of fluorescence is directly proportional to the concentration of the fluorescent species, and (3) the intensity of fluorescence is directly proportional to the intensity of the incident light.95 This method, with some innovations, is also used currently in the detection of other alkaloids, especially for detection of a 9-acridone moiety in UV of 401, 352, 323, 285, 275, and 269 nm and a xanthone skeleton. The fluorescence method is used in the process of lupine seed qualification. This method is relatively easy. The possible risk of the destruction of seeds does not exist. However, this method is not perfect. The humidity of seeds is a very important factor in their fluorescence. The best results have been obtained with 90–92% dryness of seeds.

2.2.9.3.6 Calorimetry method The idea of calorimetry is based on the chemical reaction characteristic of molecules. The calorimetry method does not allow absolute measurements, as is the case, for example, with volumetric methods. The results given by unknown compounds must be compared with the calibration curve prepared from known amounts of pure standard compounds under the same conditions.95 Practical laboratory work uses very different applications of this method, because there is no general rule for reporting results of calorimetric determinations. A conventional spectrophotometry is used with a calorimeter.95 The limitations of many calometric procedures lie in the chemical reactions on which these procedures are based rather than on the instruments available. This method was first adapted for quinolizidine alkaloid analysis in 1940 by Prudhomme and subsequently used and developed by many authors. In particular, a calorimetric microdetermination of lupine and

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sparteine was developed in 1957.102 The micromethod depends on the reaction between the alkaloid bases and methyl range in chloroform.

2.2.9.3.7 Photometry method The basis of the photometry method is a comparison of the extent of the absorption of radiant energy at a particular wavelength in a solution of the test material with that in series of standard solutions. Filter photometers are suitable for routine methods that do not involve complex spectra. In practical laboratory work, the photometric micromethod was developed for determination of sparteine, lupanine, lupinine, hydroxylupanine, and angustifoline.139 This method, tested on model solutions, is suitable for the determination of alkaloids in vegetal material of very low alkaloid content.

2.2.9.3.8 Electrophotometry method The use of electrophotometry requires a sample preparation with a colored solution. Together with an electrophotometer for alkaloid analysis, constant light intensity, and a filter as well as an electronic installation for measurement must be used. The electrophotometry method is an application of both calorimetry and photometry in the same analysis.

2.2.9.3.9 Paper chromatography Paper chromatography as a method of alkaloid analysis has a long history. It was first proposed in Russia by M. S. Tswett in 1903, after the successful separation of a mixture of plant pigments.27,143,144 The solution containing the alkaloid is transferred onto tissue paper. The color of the tissue paper is very important and can be compared against a standard. This is a qualitative and quantitative method of analysis if the standard is scaled. The paper used is very similar to that for thin-layer chromatography but without the need of special coatings.35 Modern chromatographic methods are based on the principles of this first method, in which the different distribution and behavior of compounds or their parts in the stationary and mobile phases of the solid are fundamental considerations.

2.2.9.3.10 Thin layer (planar) chromatography Thin-layer chromatography (TLC) is widely adopted for the analysis of alkaloids. The basic characteristics of thin-layer chromatography as a method are as follows: qualitative and semi-quantitative analysis, speed of analysis, and a chromatographic fingerprint (Rf values and colors, color photography, densitometry or fluorometry of the chromatogram at certain wavelengths and a photographic alkaloid atlas). At the mobile phase, the mixture moves across the layer from one side to the opposite. This

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movement of the solid transfers analyte placed on the layer at the rate determined by its distribution coefficient (K) between the stationary and mobile phases.144 The movement of the analyte, therefore, can be expressed by factor Rf, which is the relation between the distance moved by the analyte from its origin to the distance moved by the solvent from its origin (Rf ¼ Ka/Ks). The sample applied to the TLC should contain at least 50–100 μg of alkaloids. This method was used for alkaloid metabolite extraction, analysis, and purification.142

2.2.9.3.11 High-performance liquid chromatography High-performance liquid chromatography (HPLC) is a modern application of liquid chromatography. High-performance liquid chromatography guarantees a high sensitivity and, at the same time, this technique has its gas analogue. The principle of HPLC is the same as that of liquid chromatography (LC), liquid- solid chromatography (LSC), and liquid-liquid chromatography (LLC). High-performance liquid chromatography is the most recent technique. The stationary phase may be a solid or liquid on a solid support. The mechanisms responsible for distribution between phases include surface absorption, ion exchange, relative solubilities, and steric affects.35,43,123,144 High-performance liquid chromatography is a useful method for quinolizidine alkaloid analysis, especially when pure standards are available.5 This method was used for alkaloid metabolite extraction and analysis.142,163 A simple reversed-phase liquid chromatographic method has been developed for the simultaneous quantitation of four anticancerous alkaloids, vincristine, vinblastine, and their precursors catharanthine and vindoline, using a specific HPLC column.47,65

2.2.9.3.12 Gas chromatography Gas chromatography is a method similar to HPLC. It provides a quick and easy way of determining alkaloids in a mixture. The only requirement is some degree of stability at the temperature necessary to maintain the substance in the gas state.35 Gas chromatography is divided into two subclasses according to the nature of the stationary phase. One of these is GSC (gas-solid chromatography). The fixed phase consists of a solid material, such as granual silica, alumina, or carbon. Gas-solid chromatography is an important method in the separation of permanent gases and low-boiling hydrocarbons.35 The second subclass, more important for lupine alkaloid analysis, is gas-liquid chromatography (GLC). A gas chromatograph is needed for the analysis. Basically, a gas chromatograph consists of six parts: (1) a supply of carrier gas in a

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high-pressure cylinder with attendant pressure regulators and flow meters, (2) a similar injection system, (3) the separation column, (4) detectors, (5) an electrometer and strip-chart recorder (integrator), and (6) separate thermostated compartments for housing the columns and the detector to regulate their temperature. Helium is the preferred carrier gas. For alkaloid analysis, a nitrogen detector is needed. Gas-liquid chromatography is a qualitative but also quantitative method of alkaloid analysis. It is very sensitive. The only problem concerns the distribution of the alkaloid mixture in the chromatographic process and the identification of alkaloids, which must be achieved by a different technique.3,4 A very positive characteristic is the possibility of totally computerizing this method of alkaloid detection.

2.2.9.3.13 Gas-liquid chromatography–mass spectrometry Capillary gas-liquid chromatography combined with mass spectrometry (MS) has been successfully used for the separation of complex mixtures of alkaloids. The aim of gas-liquid chromatography–mass spectrometry (GLC/MS) is to operate both a gas chromatograph and a mass spectrometer. Gas chromatography is an ideal separator, whereas the mass spectrometer is an identifier.19,28,35,39,41,43,144 The technique of mass spectrometry was discovered in 1912 and developed to become one of the most effective methods for biomolecular research. The mass spectrometer or mass spectrograph, as it is also called, generally consists of four units: (1) an inlet system, (2) an ion source, (3) an electrostatic accelerating system, and (4) a detector and readout system. Different mass spectrometers exist. The mass spectrometer determines the mass spectrum from the alkaloid analysis. The mass spectrum of a compound contains the masses of the ion fragments and the relative abundance of these ions plus, often, the parent ion. A mass spectrometer can be used for electron impact (EI+) or for EI+ and chemical ionization (CI) of compounds. This molecular fragmentation is the basis for alkaloid identification. The basis of mass spectrometry analysis is that, under the same conditions, the molecular fragments of the alkaloid mass must be identical.

2.2.9.3.14 Nuclear magnetic resonance The nuclear magnetic resonance (NMR) method is based on the interaction between matter and electromagnetic forces, and can be observed by subjecting a sample simultaneously to two magnetic fields: one stationary and the other varying at a certain radio frequency. At particular combinations of fields, energy is absorbed by the sample, and this absorption can be observed as a change in the signal developed by a radio frequency detector and

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amplifier.35 Nuclear magnetic resonance spectrometry was discovered in 1946, and became one of the basic methods in organic chemistry. In quinolizidine alkaloids, two techniques of NMR are currently used: 1H-NMR and 13 C-NMR. In the case of 1H-NMR analysis, the basis is that energy absorption can be related to the magnetic dipolar nature of spinning nuclei. Quantum theory is used in this case. In 1H-NMR analysis, the H-nuclei from the alkaloid molecule is very important. In the case of 13C-NMR, the sensitivity of the 13C isotope is used. 13C-spectra show chemical shifts that are more sensitive to details of structure than proton shifts. 13C–1H spin–spin interaction is capable of being tested. For example, non-protonated 13C gives a singlet, 13CH a doublet, 13CH2 a triplet and so on. Nuclear magnetic resonance spectroscopy is today a basic method in the structural studies of alkaloids.

2.2.9.3.15 X-ray method As structure and function are intimately related, X-ray crystallography is the most comprehensive technique, which elucidates the three-dimensional structure of the molecule. X-ray crystallographic study provides an accurate and complete chemical characterization of the compound. This method has been used successfully for the analysis of such opioid alkaloids as morphine and evaluated as very precise and even suitable for the research of novelizations of compounds. The use of this method can also help the estimation of the receptor, because compound structure is important in binding to the receptor. Quinolizidine alkaloid analysis also utilizes the X-ray method, which is based on the absorption of X rays, diffraction of X rays, wavelength, and radiant power measurements of X rays. When an atom is excited by the removal of an electron from an inner shell, it usually returns to its normal state by transferring an electron from some outer shell to the inner with the consequent emission of energy as an X ray. The x-ray method is applied to quinolizidine alkaloids that have a crystalline form. In this sense, it is the same as the R€ontgen (RTG) methods, which can be applied only to crystalline materials. X rays can be absorbed by the material, and this gives rise to x-ray absorption spectra.41 The spectrum provides material for the identification of compounds.

2.2.9.3.16 Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) is a method sometimes used in alkaloid studies.13,14 The application of ELISA to alkaloid study is based on antibody incubations. It differs from classical precipitation-based methods in that specific antigen-antibody interactions are recognized by assaying

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an enzyme label conjugated to one reactant, usually an antibody. Because of the sensitivity with which enzyme markers can be detected by their reaction with an appropriate substrate, ELISA offers several possibilities of greater sensitivity. In this technique, an enzyme is labeled with an alkaloid molecule. The labeled enzyme is then bound by an antialkaloid antibody. In the complex, the enzyme is rendered inactive. When a free alkaloid (e.g., hydroxylupanine) is present, it competes with the enzyme alkaloid for antibody-binding sites again and reacts with the bacterial substrate present in the tube. The enzyme activity is directly related to the concentration of free alkaloid in the sample.127 Enzyme-linked immunosorbent assay is a heterogenous immunoassay. Reactions involve a solid phase to which components are sequentially presented and successively bound. This method is very effective in the determination of the total alkaloid content. The positive characteristics of this method are the use of nontoxic reagents and basic equipment with low cost, a small sample volume, and the ability to measure alkaloids in crude sample extracts. According to the literature, compared with results obtained from GLC, the precision of ELISA for quinolizidine alkaloids is not as high as that of the gas chromatography procedure but is adequate for plant breeding purposes. The use of enzymes in developing the methods of quinolizidine alkaloids analysis looks likely to increase in the future.

2.2.9.3.17 Radioimmunoassay Radioimmunoassay (RIA), like ELISA, is based on the radioactive labeling of the antibody molecules. The labeled antibody reacts with the antigen present in the tube; the amount of radioactivity present in the bound complex is directly proportional to the amount of antigen added to the tube.127

2.2.9.3.18 Scintillation proximity assay Scintillation proximity assay (SPA) is a variety, or part, of RIA. This method is based on the measurement of the scintillation of radioactive molecules. On the basis of the power of the scintillation, it is possible to determine the amount of radioactivity. Then, it is possible to count the alkaloid content. This method was used recently in the biosynthesis of communesin alkaloids.142

2.2.9.3.19 Capillary zone electrophoresis Capillary electrophoresis is suitable to separate a wide spectrum of both large and small biological molecules. This method was used for analysis of opium alkaloids, such as thebanine, codeine, morphine, papaverine, and narcotine.

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2.2.9.4 Choice of method and confidence There exists a long list of different methods of quinolizidine alkaloid analysis (Table 2.3). These methods are of a chemical and biological nature. The development of methods of alkaloid analysis has been a long and difficult process. Each method has its drawbacks. The various methods for the determination of alkaloids are very diverse, and the results of measurements are not comparable. This is a particular problem when plant materials are being compared. A problem of great importance is the isolation of alkaloids. Traditionally, very strong solvents have been used. This presents some difficulties connected with the confidence with which the results can be treated. The isolation of all alkaloids from the sample and the purity of this isolation are also a significant problems. A further problem that must be resolved is the resistance of quinolizidine alkaloids to other amines and nitrogen compounds during the analysis. The general conclusion is that a perfect method of alkaloid analysis does

Table 2.3 General characteristics of the methods and techniques of quinolizidine alkaloid analysis Method of Technique

Nature of Method

Kind of Measurements

Sensitivity

Iodine Taster Seed color DRG Fluorescence Calorimetry Photometry Electrophotometry Spectrophotometry PC GLC GLC/MS HPLC NMR X Ray, RTG ELISA RIA SPA

ch bio bio ch ph, ch ph, ch ph, ch ph, ch ph, ch ph, ch ch ch ch ph, ch ph, ch bio bio, ph bio, ph

qual qual qual qual qual, qual, qual, qual, qual, qual, qual, qual, qual, qual, qual, qual, qual, qual,

y/n, c y/n, nc y/n,nc y/n, nc y/n, nc 1 μg, c 1–50 μg 1–50 μg 1–50 μg y/n, 1 mg 1 μg, c 1 μg, c 1 μg, c 0.017% 0.04% y/n, 0.001% y/n, 0.001% y/n, 0.001%

quant quant quant quant quant quant quant quant quant quant quant quant quant quant

Abbreviations: ch ¼ chemical; bio ¼ biological; phys ¼ physical; qual ¼ qualitative; quant ¼ quantitative; y ¼ yes, alkaloid exists; n ¼ no alkaloid, absent; c ¼ confident; nc ¼ not confident.

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not exist. Therefore, the explanation of results in light of the aforementioned factors is vital in each case. The history of the use and development of methods of analyzing quinolizidine alkaloids shows a move away from the deployment of iodine toward the use of complicated biological processes, such as antialkaloid antibody and enzymatic processes. It seems to be necessary to incorporate biological methods of alkaloid analysis into the system of analytic-chemical monitoring used in modern laboratories.

2.2.9.5 Chemical modification of alkaloids Chemical modification is a process in the change of the structure, skeleton, configuration, moiety groups, biosynthetic ability, or form of a compound. Modification is connected with structural changes, including the changes in bioactivity of alkaloids.8 By chemical modification, many medicines may be developed for the pharmacological market. Modification of alkaloids can be considered in three aspects: chemical, biochemical, and molbiological. Mechanical changes (chemical) cover structural alterations in all possible parts of compounds. Biochemical changes are connected with the modifications of enzyme activity, while molbiological changes cover biofactor manipulation inside the alkaloid. The latter is a new approach to the modification of alkaloids. The modification of an enzyme and its transfer to an alkaloid molecule is currently a growing research area. Such a modification can be achieved by changes in alkaloids directly or by changes in their precursors, postcursors, or connected proteins. These changes remain possible but very challenging in alkaloid research. He et al.54 and Teng et al.124,125 reported on chemical modification of tryptophan enzymes, which also have potential significance for alkaloid research and modifications. Similar studies have also been carried out by Masuda, Ide, and Kitabake84 in Japan and Januszewski et al.64 in the United States. Phenylalanine enzymes have been modified as well.136 Moreover, some solvent-stabilized Pt (2.3–2.8 nm) and Pd (83.7–3.8 nm) nanoparticles can accelerate and modify alkaloids, as in the case of Cinchona alkaloids.22 Krasnov, Kartsev, and Vasilevskii73 reported on chemical modification of plant alkaloids and especially the reaction of cotarnine with bifunctional NH- and CH-acids. In this research, substituted 1,2,3,4-tetrahydroisoquinoline systems were prepared by the reaction of cotarnine with the NH- and CH-acids methyl and acyl derivatives of pyrazole and 1,3-dicarbonyl reagents. According to Krasnov et al.,73 bifunctional pyrazole nucleophiles can deliver substitution products in the N atom, methyl. or acyl group, depending on the structure and reaction conditions.

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2.2.9.5.1 Basic techniques Basic techniques of alkaloid modification are grounded on the following reactions: (1) the Schiff formation and Mannich reaction, (2) the Aldol and Claisen reactions, (3) the Wagner-Meerwein rearrangements, (4) the Michaels reaction, (5) the regioselective intramolecular Friedel Crafts reaction, (6) the modified Bischler-Napieralski reaction, (7) nucleophilic substitution, (8) electrophilic addition, (9) decarboxylation, (10) the transamination reaction, (11) enzymatic reactions (oxidation, reduction, and dehydrogenation), (12) elimination reactions, (13) coupling reactions, (14) reactions with reagents. The Schiff formation is a reaction in the formation of C
N bonds, with a nucleophilic addition followed by the elimination of water and the given imine (Schiff base). The Mannich reaction is also connected with C
N bonds formation. Meanwhile, the protonated form of imine reacts with the nucleophilic addition. The Aldol and Claisen reactions are connected with C
C bond formation. Wagner-Meerwein rearrangements are related to the generation of more stable carbocations. Nucleophilic substitution is connected with SAMe, which produces positively charged sulfur and promotes nucleophilic substitution (SN2). Electrophilic addition is connected with the C5 isoprene unit in the form of dimethylallyl diphosphate DMAPP which can ionize to generate a resonance-stabilized allylic carbocation then react with isopentenyl diphosphate IPP. Decarboxylation is the reduction of carbon, while transamination is the exchange within the amino group of an amino acid to a keto acid (the introduction or removal of nitrogen). Enzymatic reactions change the oxidation and hydroxylation state of the molecule through the activity of enzymes. Elimination reactions are connected to exchange within hydroxyl, amino, or mercapto groups. Coupling reactions are connected with the unification of two or more phenolic systems in a process readily rationalized by means of free radical reactions. The solvent reaction of NH- and CH-acids with alkaloid can produce modification. Some other reagents and some solvent-stabilized Pt and Pd nanoparticles can accelerate and modify alkaloids, as for example in the case of Cinchona alkaloids.22 These reactions are widely used in alkaloid modification. A good example of alkaloid modifications for clinical curation purposes are opioids. Morphine and codeine are natural products of Papaver somniferum. However, codeine is naturally produced in small amounts. This is one reason why it is produced synthetically from morphine by modification. As codeine is the 3-O-methyl ether of morphine, the mono-O-methylation occurs in the acidic phenolic hydroxyl. Pholcodine is obtained by modification of morphine through alkylation with N-(chloroethyl)morpholine. Moreover, dihydrocodeine, hydromorphone, and heroin are also obtained from morphine through modification.

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Modification of alkaloids is very important for their use in medicine.20,37,126 In particular, modification through biological processes and bioengineering may lead to a new generation of compounds for medical applications.

2.2.9.5.2 Chemical achievements Alkaloid chemistry is a small part of chemistry, whose history began in 1805, when the first alkaloid was isolated. Since this time, many famous achievements have occurred in research and product development. A host of excellent scientists have worked successfully in this field. Alkaloid chemistry has saved many millions of lives by producing the knowledge on the bases of which alkaloid-based medicines have been developed against malaria and other diseases. Chemistry has not only investigated alkaloids, their structures, and activity58 but also developed methods for their modifications and structural manipulation. These methods are successfully used in both the pharmaceutical industry and biotechnology.

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139. Wiewio´rowski M, Skolik J. Photometrische Mikrobestimmung der LupinusAlkaloide. Roczniki Chemii 1959;33:461–9. 140. Wiewio´rowski M, Pieczonka G, Skolik J. Futher studies on the stereochemistry of sparteine, its isomers and derivatives. Part 1. Synthesis, structure and properties of 16,17-endo-methylene-lupaninium perchlorate, 17β-methyllupanine and 17β-methyl sparteine. J Mol Struct 1977;40:233. 141. Wiewioro´wski M, Wolinska-Mocydlarz J. Structure of the new lupine alkaloid, dehydroalbine. Bull Pol Acad Sci Chem 1964;12:213–22. 142. Wigley LJ, Mantle PG, Perry DA. Natural and directed biosynthesis of communesin alkaloids. Phytochemistry 2005;67(6):561–9. 143. Wilson K. Biomolecular interactions: I. Enzymes. In: Wilson K, Walker J, editors. Principles and Techniques of Practical Biochemistry. 5th ed. Cambridge, UK: Cambridge University Press; 2000. p. 357–402. 144. Wilson K. Chromatographic techniques. In: Wilson K, Walker J, editors. Principles and Techniques of Practical Biochemistry. 5th ed. Cambridge, UK: Cambridge University Press; 2000. p. 619–86. 145. Wink M. Methoden zum Nachweis von Lupinen-Alkaloide. In: Wink M, editor. Lupinen 1991. Forschung, Anbau und Verwertung. Heidelberg, Germany: University of Heidelberg, IFB; 1992. p. 78–90. 146. Wink M. Quinolizidine alkaloids: Biochemistry, metabolism, and function in plants and cell suspension cultures. Planta Med 1987;53:509–14. 147. Wink M, Hartmann T. Localization of enzymes of quinolizidine alkaloids biosynthesis in leaf chloroplast of Lupinus polyphyllus Lindl. Plant Physiol 1980;70:74–7. 148. Wink M, Meiβner C, Witte L. Patterns of quinolizidine alkaloids in 56 species of the genus Lupinus. Phytochemistry 1995;38(1):139–53. 149. Winkler JD, Londregan AT, Ragains JR, Hamann MT. Synthesis and biological evaluation of manzamine analogues. Org Lett 2006;8(15):3407–9. 150. Wuttke H. Einfache Alkaloiduntersuchungsmethoden von gelben und blaue Lupinen. Z€ ucht 1942;14:83–6. 151. Wyrostkiewicz K, Wawrzyniak M, Barczak T, Aniszewski T, Gulewicz K. Evidence for insectoside activity of some preparations from alkaloid-rich lupin seeds on Colorado potato beetle (Leptinotarsa decemlineata Say), larvae of the large white but terfly (Pieris brassicae L.), black bean aphid (Aphis fabae Scop.) and on their parasitoids (Hymenoptera: Parasitica) populations. Bull Pol Acad Sci Biol Sci 1996;44(1–2):30–9. 152. Wysocka W. (+)-sparteine: A new minor alkaloid from Lupinus albus L. Sci Legum 1995;2:137–40. 153. Wysocka W, Brukwicki T. Lupin alkaloids. I. Reinvestigation of the structure of N-methylalbine. Planta Med 1988;54:522–3. 154. Wysocka W, Brukwicki T. Minor alkaloids of Lupinus albus: 13α-hydroxy-5dehydromultiflorine and 13β-hydroxy-5-dehydromultiflorine. Planta Med 1991;57:579–80. 155. Wysocka W, Brukwicki T, Macioszek E, Wolski W. The influence of the isolation method on the quantitative and qualitative composition of the alkaloids from Lupinus albus seeds. In: Twardowski T, editor. Proceedings 5th international Lupin Conference, July 5–8, 1988. Poznan, Poland: Polish Academy of Sciences, Institute of Bioorganic Chemistry; 1988.

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156. Wysocka W, Brukwicki T, Jałoszynski R, Hoffman K. A new and efficient method of extraction of alkaloids from lupine seeds. Lupin Newsletter 1989;13:59–65. 157. Wysocka W, Przybył A. (+)-Angustifoline: A minor alkaloid from Lupinus albus. Planta Med 1993;59:289. 158. Wysocka W, Przybył A. Alkaloids from Lupinus albus and Lupinus angustifolius L.: an efficient method of extraction. Sci Legum 1994;1:37–50. 159. Wysocki W, Gulewicz P, Aniszewski T, Ciesiołka D, Gulewicz K. Bioactive preparations from alkaloid-rich lupin. Relation between chemical composition and biological activity. Bull Pol Acad Sci Biol Sci 2001;49:9–17. 160. Xe XS, Tadic D, Brzostowska M, Brossi A, Bell M, Creveling C. Mammalian alkaloids—Synthesis and O-methylation of (S)-3’-hydroxycoclaurineand R-3’-hydroxycoclaurine and their N-methylated analogs with S-adenosyl-L[methyl-C-14]methionine in presence of mammalian catechol O-methyltransferase. Helv Chim Acta 1991;74:1399–411. 161. Yousaf M, Hammond NL, Peng JN, Wahyono S, MsIntosh KA, Charman WN, et al. New manzamine alkaloids from an indolo-pacific sponge. Pharmacokinetics, oral availability, and the significant activity of several manzamines against HIV-I, AIDS opportunistic infections, and inflammatory diseases. J Med Chem 2004;47 (14):3512–7. 162. Yovo K, Huguet F, Pothier J, Durand MK, Breteau M, Narcisse G. Comparative pharmacological study of sparteine and its ketonic derivative lupanine from seeds of Lupinus albus L. Planta Med 1984;50:420–4. 163. Zhang P, Cui Z, Liu D, Wang D, Liu N, Yoshikawa M. Quality evaluation of traditional Chinese drug toad venom from different origins through a simultaneous determination of bufogenins and indole alkaloids by HPCL. Chem Pharm Bull 2005;53 (12):1582–6. 164. Zhao YJ, Cheng QQ, Su P, Chen X, Wang XJ, Gao W, et al. Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants. Appl Microbiol Biotechnol 2014;98(6):2371–83. 165. Zhao ZB, Sun JZ, Mao SC, Guo YW. Fasciospyridine, a novel sesquiterpene pyridine alkaloid from Guangxi sponge Fasciospongia sp. J Asian Nat Prod Res 2013;15 (2):198–202. 166. Zhu JB, Wang MG, Wu WJ, Ji ZQ, Hu ZN. Insecticidal sesquiterpene pyridine alkaloids from Euonymus species. Phytochemistry 2002;61(6):699–704.

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Chapter

3

Biology of alkaloids CHAPTER OUTLINE

3.1 Alkaloids in biology

196

3.1.1 From stimulators to inhibitors and destroyers of growth 198 3.1.2 The effects of stress and endogenous security mechanisms 201

3.2 Bioactivity 3.2.1 3.2.2 3.2.3. 3.2.4 3.2.5 3.2.6.

3.3 Biotoxicity 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7

204

Secrets of life 204 Life regulation through high and low cytotoxicity Hemoglobinization of leukemia cells 209 Estrogenic effects 211 Antimicrobial properties 212 Antiparasitic activity 215

206

216

Research evidence 218 Influence on DNA 219 Selective effectors of death 220 Nontoxic to self but deformer for others 221 Degenerators of cells 223 Aberrations in cells 224 Causers of locoism 226

3.4 Bionarcotics 226 3.5 Alkaloids in the immune system 230 3.6 Genetic approach to alkaloids 233 3.7 Evolutionary influences on alkaloid biology References 239

236

Natura non facit saltus. Carl von Linné

Alkaloids play a very important role in organism metabolism and functional activity.19 They are metabolic products in plants, animals, and microorganisms. They occur in both vertebrates and invertebrates as endogenous and exogenous compounds. Many of them have a distributing effect on the Alkaloids # 2015 Tadeusz Aniszewski. Published by Elsevier B.V. All rights reserved.

195

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nervous systems of animals.103,105,164,328 Alkaloids are the oldest successfully used drugs throughout the historical treatment of many diseases,306 and some of them are still used or considered for use in the modern medicine.38,165 All the time, new alkaloid molecules are discovered, and new horizons appear in the field. Nowadays, especially the deepsea bioenvironment is considered a new source of natural alkaloids.225,315,323,324 Moreover, new alkaloids also are discovered by chemical synthesis and modifications.110,233 Synthetic and semi-synthetic alkaloids are coming all the time to be more important in many fields, and in many cases, they are biologically more active than natural alkaloids. Modifications of alkaloids in their amount and structures are arrived at nowadays by genetic (organism transformation, breeding, tissue culture) and transgenic modification of organisms (GMO).82,188 Moreover, scientific achievements have discovered a new, realistic vision, that structurally the same alkaloids in different group of organisms (e.g., plants and animals) are not exactly the same in structural complex and origin and can substantially differ in their functionality. These chemical changes and differentiations (angle of valence of C, N, H, and moiety) of the same alkaloid molecules in different groups of organisms or even individuals open new challenges for both chemical and biological research and can lead to the new box classification of alkaloids according to the organism, such as sparteinep (plant sparteine), sparteinea (animal sparteine), sparteinef (fungal sparteine), and so forth. Micro-level discrete changes in chemical structure (even in electrons inside atoms) can lead into considerable changes in biological activities. Becoming of some disease agents to be resistant to some traditional alkaloid drugs is probably just a result of these changes. Therefore, biological research of alkaloids together with the research of bioorganic chemistry is becoming more important and can lead into significant changes in one’s views on the nature and on life essence on the globe.

3.1 ALKALOIDS IN BIOLOGY For many years, the nature of alkaloids in biology was a mystery. It has been difficult to understand the function of these compounds in plant metabolism. There are many explanations for why plants, animals, and microorganisms produce alkaloids. Nowadays, when genomes, DNA and genes serve as the basis for biological explanations, this issue is of great importance and still open for discussion and deep scientific analysis. Despite the advanced research in the field, a final comprehensive biological explanation of the nature of alkaloids is still on the way. In this sense, the alkaloid mystery continues to exist.

3.1 Alkaloids in biology 197

New compounds are being discovered all the time; however, their biological significance remains unexplained. The slow pace of science and scientific research requires a lot of time to arrive at such explanations. Moreover, the structures of many of these compounds are unexpected, and their bioactivity is surprising. The molecular mechanisms and metabolic roles of newly discovered compounds have remained unclear. Alkaloids from marine, and especially deep-marine, environments and those produced by microorganisms and animal skin are, in particular, objects of current chemical and biological research. Moreover, the nature and role of alkaloids has been based on a multitude of theoretical hypotheses compiled during over the last 200 years. It has even been hypothesized that alkaloids are plant wastes and an end product of metabolism.177 Today, the role of alkaloids can be explained by two factors: the functions of these compounds inside and outside the organism producing them. The external function of alkaloids is presently a particularly strong and growing research area.7,218,273,307,308 This trend in alkaloid research is based on the hypothesis that alkaloids are compounds that solely play a protective or aggressive role (chemical defense or chemical attack) in interaction with other organisms (as some kind of organic bioweapons). This seems to be a rather limited oversimplification of the issue. Although there is strong evidence of this kind of activity256 and even suggested applicable potential,331 it is not entirely clear if it is a basic function of these compounds in the organisms producing them. The idea that this ecologically important role may be only a secondary function and that alkaloids primarily function in connection with the regulation of metabolism as the result of gene expression should not be dismissed. Some new researches suggested just this regulation.107,255 Moreover, it is also known that, in the case of quinolizidine alkaloids, the total removal of these compounds by genetic means leads to the death of the lupine plant.16 This suggests that alkaloids are compounds fundamental for cell activity and gene code realization in the genotype.20 This also means that alkaloids basically function in connection with genes, enzymes, and proteins inside the organism. Moreover, it is also known that quinolizidine alkaloids are able to change their structural chemical configurations under changing cellular pH conditions. This observation and experimentally measured effect, first noted in the 1990s, has unfortunately been given little literary attention by other scientists. Although this self-regulation process is still not understood in detail, many recent studies prove that chemical structural changes influence large changes in the biological activity of chemical compounds.58,65,166,210,321 Alkaloids are nontoxic in vacuoles where they are stored but toxic when they escape from the vacuoles. They have to change their chemical

198 CHAPTER 3 Biology of alkaloids

Alkaloids as metabolic wastes

Alkaloids as biological weapons

Alkaloids as gene code metabolic self-regulators

n FIGURE 3.1 Three basic hypotheses on the biological nature of alkaloids.

configurations and biological activity in different cells and tissues according to pH changes. This means that some alkaloids can have different biological activity in different cell conditions and different receptors. This process has to be genetically regulated (Figure 3.1). Future detailed studies will likely clear up this fascinating and complex matter concerning the biological nature of alkaloids.

3.1.1 From stimulators to inhibitors and destroyers of growth Waller and Nowacki299 distinguished the role of alkaloids in plants as growth stimulators and inhibitors and also as protective agents and reservoirs of nitrogen. More recently, Nguyen et al.212 showed evidence that some alkaloids (e.g., nuciferine) stimulate secretion of other compounds in plants. They observed that a plant, Nelumbo nucifera, uses the alkaloid nuciferine for secretion of insulin and producing the antidiabetic characteristics of the plant. Moreover, some alkaloids are known neurotransmitters in animals and can also be considered part of the signaling system. This system is constructed as a part of cell and metabolic operations controlled by functional mechanisms of biological membranes, channels, receptors, and enzymes. It is known that some alkaloids, for example, purine and steroidal alkaloids, can bind to some compounds presented on cell membranes. As a result of this interactive process, the moiety segment of alkaloids can be

3.1 Alkaloids in biology 199

changed by addition to different parts (e.g., lipophilic, hydrophilic) of the molecule, which assists in binding to the receptor. There are different receptors for different compounds transported in the organism. Alkaloids can promote receptor activity or inhibit it. This is also, in many cases, connected with alkaloid moiety. The steroidal alkaloid gagamine, which has been isolated from the roots of Cynanchum wilfordi Hamsley (Asclepiadaceae), can be mentioned as an example. This alkaloid is known to have an inhibitory effect on the activity of aldehyde oxidase, which metabolizes heterocyclic rings.160 Some other steroidal alkaloids (e.g., tomatine and tomatidine) are known to afford a neuroprotective effect against glutamate-induced toxicity in SH-SY5Y neuroblastoma cells by preserving mitochondrial membrane potential and reducing oxidative stress.278 Alkaloids have their own signaling systems. According to Makhov et al.179 a natural alkaloid of the fruit of long pepper, piperlongumine, has strong impact on Akt/mTOR signaling. Generally, in alkaloid signaling systems, receptors and membranes play an active role. The role of biological membranes is also connected with the action of the specific ion channels of Ca2+, Na+, and K+ and their active pumps (e.g., Ca2+-ATPase). Only alkaloids can promote or inhibit activity of ion channels and their active pumps. Therefore, these channels are important in an alkaloid signaling system. This mechanism is connected directly or indirectly to receptor proteins. Alkaloids such as dopamine, histamine, or serotonin are well-known neurotransmitters with their own receptors. The stimulation of a neurotransmitter system (especially ion channels) is caused by an influx of Na+ ions. This large-scale and rapid influx activates a so-called voltage gate of Na+ and K+ channels, which is essential for alkaloids. Neurotransmission is one of the most important biological characteristics of alkaloids. However, the research data by Pineda et al.230 presents information about the effects of the crude extracts of lupine quinolizidine alkaloids, which were intracerebroventricularly administrated in adult rat brain tissue. These extracts were administrated to the right lateral ventricle of adult rats through a stainless steel cannula for five consecutive days. The researchers stated in their report that, immediately after the administration of quinolizidine alkaloid from Lupinus exaltatus and Lupinus montanus seeds, the rats began grooming and suffered from tachycardia, tachypnea, piloerection, tail erection, muscular contractions, loss of equilibrium, excitation, and an unsteady gait. Moreover, Pineda et al.230 reported that the rats treated with alkaloids had damaged neurons. Although there was no statistical significance, damages were observed and may suggest a histopathological influence on neurons. The most frequent abnormalities observed in this brain tissue were “red neurons” with a shrunken eosinophilic cytoplasm, strongly stained

200 CHAPTER 3 Biology of alkaloids

pyknotic nuclei, neuronal swelling, spongiform neuropil, “ghost cells” (hypochromasia), and abundant neuronophagic figures in numerous brain areas. If these results are proven in the future by no direct administration of alkaloids to the brain, they will serve as evidence of the destructive role of alkaloids in the animal body. Although the research of Pineda et al.230 is interesting in many aspects, the results cannot be considered evidence of such destruction caused by alkaloids. It is evidently known that crude extracts cannot be physiologically transported to the animal brain. The direct administration of crude extracts to the brain tissues from outside the liver affects the influence of all components of these extracts, from which alkaloids are only one part. There is evidence in the literature that alkaloid biology is connected with regulation, stimulation, and induction functions. Tsai, Chang, and Lin282 proved that caffeine levels in the blood, brain, and bile of rats decreased when given a treatment of rutaecarpine, an alkaloid from Evodia rutaecarpa (Figure 3.2). It is known that caffeine has been found to enter the brain by both simple diffusion and saturable carrier-mediated transport.183 The hepatobiliary excretion of caffeine has also been reported in humans,115 rabbits,28 and rats.282 Caffeine is now also known to induce tumor cytotoxicity via the regulation of alternative splicing in subsets of cancerassociated genes.176 A treatment of rutaecarpine causes an increase in renal microsomal enzymes related to CYP1A and enhances the activity and protein levels of CYP1A. It is known that caffeine is a mild stimulant. It is metabolized in the liver by CYP1A2, and it also has been shown to be an inducer of CYP1A2 in rodents on account of the increase in hepatic microsomal CYP1A2.63 Rutaecarpine is an inducer of cytochrome P450(CYP)1A in mouse liver and kidney.285

N N H N

n FIGURE 3.2 Rutaecarpine, an alkaloid from Evodia rulaecarpa.

O

There is evidence that alkaloids influence plant growth, as both stimulators and regulators. A large series of applied studies in Germany and in Poland, started in the 1980s, proved that quinolizidine alkaloids in crude lupine extracts had effects on both yield amount and quality (Figure 3.3). Foliar application of lupine extract on several crops resulted in yield increases of 17–20%135,136 and 15–25%.74 Moreover, these results proved that crude lupine extract with quinolizidine alkaloids influenced the balance of nitrogen compounds in plants. Increases in protein concentration and changes in amino acid contents have been observed. Snap bean (Phaseolus vulgaris L.) seed yield after foliar application of the extract increased by 16.4%, and the biological value of protein measured with essential amino acid coefficients increased by 2.87%.74 The stimulation role of alkaloids can be explained by more intense nitrogen metabolism after application. In the 1950s, a case of

3.1 Alkaloids in biology 201

Evidence of the alkaloidal plant growth stimulation Effects of foliar application of lupine extracts on Yield increase – cereals 17–20% – sugar beets – maize – potato – snap bean

Change of – total aminoacids – essential aminoacids

n FIGURE 3.3 Effects of foliar application of lupine extracts. Sources: Refs. 74,135,136,193.

applying pure lupanine solution to the leaves of alkaloid-poor Lupinus albus L. was shown to have a growth-stimulating effect.299 However, there are also old findings indicating some plants exhibited no effects at all when treated. In some cases, they exhibited growth inhibition or the effects of poisoning. More recent research indicates that carbon and nitrogen nutritional components influence better growth and alkaloid accumulation in plants (Cyrtanthus guthrieae L).211 Recent studies14a in Russia suggest that alkaloids isolated from marine sponges have ability to stimulate growth of seedling roots of some plants. According to Anisimov et al.14a these alkaloids (damirone A, damirone B, makaluvamine G, debromohymenialdisine, dibromoagelaspongin) stimulated growth of barley (Hordeum vulgare L.), buckwheat (Fagopyrum esculentum Moench), corn (Zea mays L.), soybean (Glycine max (L) Men.), and wheat (Triticum aestivum L.) seedling roots. They have selective growth stimulation of these species. Damirone A, makaluvamine G, and debromohymenialdisine stimulate barley. Damirone B, makaluvamine G, and debromohymenialdisine stimulate buckwheat. Wheat roots are stimulated by dibromoagelaspongin. The mechanism of this selectivity is not presently know. Such mechanism should be cleared in the future.

3.1.2 The effects of stress and endogenous security mechanisms In biology today, the basic question concerning alkaloids is connected with the relation between their internal and external roles. It appears that the external role is only secondary, and the endogenous use of alkaloids as genetically coded is the primary function. H€ oft et al.122 have studied the sources of alkaloid formation and changes in Tabernaemontana pachysiphon plants. In this research the endogenous factors were leaf age, plant

202 CHAPTER 3 Biology of alkaloids

age, leaf position in the crown and teratological leaf dwarf growth on leaf alkaloid contents. Environmental factors were soil and other climatic factors controlled in a greenhouse in the case of young plants. In the case of the old trees, environmental factors were measured in natural habitat. H€ oft, Verpoorte, and Beck122 clearly documented that higher leaf alkaloid content is thought to result from higher nitrogen and cation availability. The relationship between nutrients in the soil and changes in alkaloid amount occurring in plants is one of the most important topics in alkaloid biology and furthermore in plant physiology and biochemistry. Alkaloid content in plants, for example, in tobacco (Nicotiana) or lupine (Lupinus), may increase with treatments high in nitrogen. There are, however, many exceptions to this. Amounts of indole, purine, and steroid alkaloids in plants do not change rapidly in response to such treatments. It does seem that alkaloid content is generally related to the nitrogen levels available to plants. Two basic factors seem to influence this relation: (1) the biosynthetic nature of alkaloids themselves and (2) the balance of nitrogen and other nutrients in the soil. The alkaloid biosynthetic pathway is important in the sense that, during synthesis, the nitrogen existing in the precursor can be liberated or additional nitrogen may bind. Some precursors are richer in nitrogen than alkaloids, for example, in the case of morphine, nicotine, and hyoscyamine. In the case of gramine or caffeine, the amount of nitrogen is the same as in their precursors. In alkaloids such as tomatidine or coniine, the amount of nitrogen is higher than in their precursors. This is the reason why some alkaloids are more sensitive to nitrogen availability than others. Moreover, the balance of nitrogen in the soil seems to be very important. High or low concentrations of nitrogen in soil seem to influence alkaloid content in the plant despite the biosynthetic nature of alkaloids (Figure 3.4). In both mentioned cases, the plant suffers from nutritional stress and the production of alkaloids seems to increase. Nutritional stress seems to be the reason for this. It is affected by absences and a high demand for nitrogen during metabolism. Plant stress in this sense can be determined as a force that strengthens alkaloid production for both continuing storage in vacuoles and for continuing their departure from vacuoles for the metabolic regulation of stress. It is necessary to mention that this topic has not yet been the object of larger specialized laboratory studies. Therefore, this explanation remains a strong hypothesis to be investigated in future studies. It is, however, known that nitrate uptake promotes alkaloid accumulation and is preferred over ammonium uptake. Soil acidity and temporary drought stress are also known to block nitrification and may thus contribute to low leaf alkaloid accumulation.24 The aforementioned experiments by H€ oft 122 et al. proved that, in Tabernaemontana pachysiphon, the differences in

3.1 Alkaloids in biology 203

M E T A B O L I C

Stress

Alkaloid production in plants

OH/OL LGF

Storage in vacuoles

RS Genetic code

n FIGURE 3.4 Mechanism of regulation of alkaloid content in plants. Abbreviations: RS, regulation system; OH, overhigh level; OL, overlow level; LGF, life growing factors. Observe that this regulation system is coded in genes. Life growing factors (light, water, CO2, nutrients including nitrogen, temperature, etc.) influence stress, which is also dependent on this system.

alkaloid levels due to endogenous factors such as leaf age or dwarf growth were much more pronounced than any other difference caused by environmental factors. The influence of age or teratological leaf growth differed depending on the alkaloid. Apparicine content was enhanced in very young leaves and equally high contents in dwarf leaves of old trees.122 The different positions of leaves may have been caused either by small differences in leaf age or by a plant’s internal nutrient and water fluxes. According to H€ oft et al.,122 differences in alkaloid levels according to tree age were rather marginal. Although their research does not directly answer the question of the internal and external roles of alkaloids, it does show the factors influencing alkaloid production. These factors also indirectly mean that alkaloids become more needed in a plant when the factors influencing their production are present. When summarizing and generalizing empirical results, it can be stated that stress and stressful situations in plants induce alkaloid production and their needs in regulatory processes of metabolism. The high content of alkaloids in old leaves suggests a metabolism regulation function similar to growth hormones, although it is also known that plant hormones such as cytokinins were found to stimulate the alkaloid synthesis.79 Moreover, this also suggests that the basic biological function of alkaloids is endogenous. Many present research results suggest just this. For example, research by Henriques et al.116 shows that alkaloid production and accumulation in plants of Psychotria leiocarpa (Rubiaceae) increases with plant age and light exposure. The alkaloids in this case are needed for physiological

204 CHAPTER 3 Biology of alkaloids

and metabolic regulation by a plant. Another good example of alkaloid production and accumulation and its function can be observed in the case of βcarboline alkaloids in humans. These alkaloids occur in mammals.242 As neurotransmitters, they play a regulative role in various metabolic processes. The natural concentration of harman, an endogenous inhibitor of monoamine oxidase subtype A with a high affinity in brain and peripheral organs, in the rat brain is reported to be less than 0.5 ng/g tissue. Norharman induces pro-conflict behavior in limbic-hypothalamic structures and alterations of motor activity.242 This also proves that endogenous activity seems to be a basic function of some alkaloids. Alkaloids are therefore some kind of natural endogenous medicine needed for ordering metabolic processes by inhibiting or accelerating other active molecules. In this sense, the external role, especially in growing environments and species interaction, seems to be secondary. Nowadays, knowledge of alkaloid’s biological function is based on empirical results. The most important biological function in plants involves the chemical and biological protection of cells. Alkaloids protect plant bodies from physical stresses like ultraviolet light and heat.326 Other biological functions are protection against pathogens and herbivores, protection of generative reproduction, an acute source of nitrogen, nitrogen storage, and the stimulation of growth and adaptation to the local environment.

3.2 BIOACTIVITY The general characteristics of alkaloids are their chemical flexibility in regard to structure and, as a consequence of this, their biological activity. Individual alkaloids do not play only one role. The same alkaloid in different cell conditions is able to change its structure and thereby its biological activity. This ability makes the alkaloids a special group of secondary compounds.

3.2.1 Secrets of life Alkaloids are structurally very similar to plant growth hormones. Waller and Nowacki299 critically considered the possibility that alkaloids have a hormonal influence on plant growth. This old hypothesis is still open for discussion; examples in literature attempt to both prove and disprove it. The contradictory results derive from the diversity of alkaloids, not to mention plant diversity and that of other organisms producing alkaloids. There are alkaloid-rich and alkaloid-poor plants from the same species. One such plant is Washington lupine (Lupinus polyphyllus Lindl.), which is capable of growing under various climatic conditions in both the Northern and the

3.2 Bioactivity 205

Southern Hemispheres.16,18 The freely growing genotypes of this plant contain 1.74–3.15 mg of alkaloids in 100 mg of seeds, whereas one hybrid contained only 0.0004 mg. The alkaloid content in leaves was about 1.6 mg in natural genotypes and 0.05 mg in hybrids. The content in shoots was 1.7 and 0.1 mg, respectively.18 The Washington lupine also is known by many other common names, such as Blomsterlupin (in Swedish), Dauerlupine (in German), De belle lupine (in French), Komea lupiini (in Finnish), Łubin wieloletni (in Polish), and Mnogoletnii liupin (in Russian). As a wild plant it is originally from North America, where its distribution extends from California to Alaska. This plant was brought to Europe in the 19th century and it distributed rapidly in numerous countries as a decoration and animal fodder in pastures and game animal farming.16,18 As a perennial and crosspollinated species, it has many different geno- and ecotypes with different alkaloid levels. It has been hypothesized that the role of alkaloids in alkaloid-rich and alkaloid-poor geno- and ecotypes differs because their amounts and structure vary. The diversity of alkaloid content in the same species and hybrids is one of the most interesting secrets of life. Chemical diversity generally constitutes an intrinsic property of biosynthesis, which is an inherent property. This diversity-oriented strategy is widespread in biosynthesis. Schwab,257 when discussing the diversity of secondary compounds, concludes that the number of metabolites in one species often exceeds the number of genes involved in their biosynthesis and that increasing compound diversity does not correlate with increasing gene number. It has also been suggested that multifunctional enzymes are ubiquitous in the plant kingdom.257 In the case of alkaloids, the diversity in content among plants is connected to the genetic code. The proof of this is evident in hybridization, where it is possible to noticeably decrease the alkaloid level of the Washington lupine. This has been done over the period 1982–1990 in Finland.18 The mechanism of determining the alkaloid-rich and alkaloidpoor plants is connected with enzymatic activity and production of an alkaloid precursor. In the case of quinolizidine alkaloids, the alkaloid is plant specific and its occurrence in individual plants is connected to the metabolism of lysine. In expanded vegetation, there is a surplus of lysine, which leads to the production of quinolizidine alkaloids through the activity of HMT/HLTase and ECTase.272 In individual plants without such alkaloids, the biosynthetic pathway of the alkaloids with HTM/HLTase is blocked.119 The difficulty of studying the effect of alkaloids as growth regulators is similar to the problem of the alkaloid content variation in plants. Waller and Nowacki299 clearly took up this issue for methodological discussion. The level of alkaloid richness affects further addition of alkaloids to a plant. However, environmental growth factors such as light, moisture,

206 CHAPTER 3 Biology of alkaloids

temperature, and nutrition, and the genetic factors, such as genotype and photosynthesis capacity of a species, influence alkaloid precursors and their derivation to alkaloids. The concentrations of these compounds in plants influence their activity as growth regulators. However, many questions arise in the light of this. Do alkaloid-rich plants grow better and faster than alkaloid-poor plants? What empirical evidence exists that alkaloids also have the effect of growth regulators? Waller and Nowacki299 mention that alkaloids are growth regulators. They mentioned differences in regulator activity and also pointed out exceptions. The answer to the first question is certainly not exactly the same. Research has advanced as the development of techniques and equipment illustrates. According to my studies and observations carried out in experiments in Finland, the answer is just opposite to the one given by Waller and Nowacki.299 The alkaloid-rich plants grow at a higher rate and to higher canopy than alkaloid-poor plants.16,17 However, when ripening periods are compared, alkaloid-rich plants ripen more slowly than alkaloid-poor plants. The growing conditions in the Boreal zone of Finland are generally very favorable for perennial lupines and especially for the Washington lupine. The populations of this species have been large, and this species has had no factors reducing populations (e.g., herbivory or disease). Rapid growth and higher growth rate per day can be considered a result of regulator activity. Empirical studies support this. Lupinus angustifolius cult. Mirela (an alkaloid-rich plant) grows more rapidly than alkaloidpoor species. In chamber experiments, the mean photosynthetic uptake of L. angustifolius cult. Mirela (alkaloid-rich) was 12.71 mg CO2 dm
2 h
1 and that of L. polyphyllus Lindl. (alkaloid-poor) was 10.04 mg CO2 dm
2 h
1 21.

3.2.2 Life regulation through high and low cytotoxicity Alkaloids from the plant family Amaryllidaceae are known to have a wide range of biological activities. They have analgesic, antiviral, antimalarial, antineoplastic properties and display effects on the CNS. Elgorashi, Stafford, and Van Staden86 studied 25 Amaryllidaceae alkaloids for possible inhibitory activity of their acetylcholinesterase enzyme (AChE). This enzyme is biologically very important. According to the cholinergic hypothesis, Alzheimer’s disease symptoms result from AChE activity, which reduces brain acetylcholine activity. Crinine, crinamidine, epivittatine, 6hydroxycrinamine, N-desmethyl-8α-ethoxypretazettine, N-desmethyl-8βethoxypretazettine, lycorine, 1-O-acetyllycorine, 1,2-di-O-acetyllycorine, and cherylline have been shown to inhibit AChE.86 Lycorine-type alkaloids are the most active against AChE.86,173 The action mechanism of these alkaloids on AChE inhibition is still not exactly known, although it has been

3.2 Bioactivity 207

ALKALOIDS Activators Inhibitors

Ca2+ channels AChE

XI AChR

Acetylcholine activity Na+ channels

n FIGURE 3.5 Alkaloids in the acetylcholine receptor. Abbreviations: AChE, acetylcholine enzyme; AChR, acetylcholine receptor; XI, inactivity of AChE.

reported that the crystal structures of the acetylcholinesterase inhibitors, such as galanthamine, huperzine A, tacrine, and edrophonium, demonstrated binding to the active site gorge of AChE. Studies on steroid alkaloids, such as saracocine, saracodine, saracorine and alkaloid-C isolated from Sarcococca saligna101a, suggest that these alkaloids are also calcium antagonists and AChE inhibitors. AChE is known to be located on the acetylcholine receptor (AChR), which is also bound by such alkaloids as anabasine, arecoline, coniine, C-toxiferine, cytisine, hyoscyamine, lobeline, lupanine, muscarine, nicotine, pilocarpine, tubocurarine, scopolamine, sparteine, and so on. These alkaloids can activate AChE or inhibit it by the influence of enzyme AChE (Figure 3.5). As in cases of the Amaryllidaceae alkaloids, AChE can be inhibited. As a result of this, acetylcholine activity increases. Acetylcholine activity is needed for human brain function. It seems that Amaryllidaceae alkaloids have a wide biological regulatory ability. It is known that lycorine, one of the most important Amaryllidaceae alkaloids, is actively antiviral. Pseudolycorine and pretazettine are active against several types of leukemia by the inhibition of protein synthesis and prevention of peptide-bond formation.1 Galanthanine has analgesic, anticholinergic, and anticholinesterase properties. The minor Amaryllidaceae alkaloids studied by Abd El Hafiz et al.1 are also biologically active. The lycorine-type alkaloid (pratorinine) and the crininetype alkaloid (6αhydroxybuphanisine) showed a moderate cytotoxic activity. Moreover, (
)-spectaline, a piperedine alkaloid isolated from the legume Cassia leptophylla Vog.,44 has been proven in studies by Alexandre-Moreira et al.9 to have no significant toxicity effects but rather antinociceptive traits. In these experiments, conducted on mice, (
)-spectaline was able to significantly inhibit abdominal writhing in the mice in comparison to the control animals. It was suggested that this bioactivity of (
)-spectaline was connected to a direct interaction of the binding of the vanilloid system or excitatory amino acid on its receptors.9 This is a promising research direction when considering possible bioapplications of this alkaloid.

208 CHAPTER 3 Biology of alkaloids

Neolitsine, dicentrine, cassythine, and actinodaphine are aporphine alkaloids isolated from Cassytha filiformis. These alkaloids have been studied by Ste´vigny et al.269 for their cytotoxic activities on cancerous and noncancerous cell lines in vitro. Neolitsine was very active against HeLa and 3 T3 cells, and cassythine and actinodaphnine showed activity against Mel-5 and HL-60 cells. Dicentrine was previously reported to be cytotoxic against several tumor cells and has been shown to inhibit DNA and RNA biosynthesis.124 Dicentrine also acts as a topoisomerase II inhibitor.63 Chen et al.64 researched aporphine alkaloids isolated from the trunk bark of Hernandia nymphaeifolia. These alkaloids also showed potent cytotoxicities against P-388, KB16, A549, and HT-29 cell lines. Similar results were previously noted in the case of dimeric aporphine alkaloids isolated from the same species.64 Very interesting results concerning the cytotoxicity of alkaloids isolated from the flowers of ornamental legume plant Senna spectabilis have been noted by Sriphong, Sotanaphun, and Limsirichaikul.265 N,O-diacetylcassine, 3(R)-benzoyloxy-2(R)-methyl6(R)-(11’-oxododecyl)-piperidine, and 5-hydroxy-2-methyl-6-(11’-oxododecyl) pyridine N-oxide exhibited cytotoxicity against KB cell lines. One of the most common biological properties of alkaloids is their cytotoxicity against cells of foreign organisms.120,181 These activities have been widely studied for their potential use in the elimination and reduction of human cancer cell lines. Wu et al.319 studied the cytotoxicity of 53 isoquinoline alkaloids and their N-oxides against A-549, HCT-8, KB, P-388, and L-1210 cells. The isoquinoline alkaloids represent a different structural type of alkaloids. Among all structural types investigated (tetrahydroprotoberbirines, protoberberines, aporphines, morphinadienone, oxoaporphines, phenanthrenes, and their N-oxides), the most active were some of the oxoaporphines. Liriodenine especially showed potent and wide spectrum activity against all the cell lines tested.319 Moreover, it has been evident in this research that human KB cells appear to be the most sensitive in detecting active compounds of different alkaloids. The same result has been noted by Jagetia et al.132 in the case of echitamine, which is a monoterpene indole alkaloid. This research investigated HeLA, HepG2, HL60, KB and MCF-7 cells in vitro and in mice. Jagetia et al.132 reported antitumor properties of echitamine in vitro and in vivo. Moreover, Long and Li noted the antitumorous characteristics of alkaloid extracted from Oxytropis ochrocephala and concluded that the activity is dose dependent. This antitumor effect is associated with the expression of inhibition of proliferating cell nuclear antigen (PCNA) and mutant p53 protein.167

3.2 Bioactivity 209

The cytotoxic activity of phenanthroquinolizidine alkaloids has also been reported.168 Of two studied alkaloids (boehmeriasin A and boehmeriasin B) isolated from Boehmeria siamensis Craib (Utricaceae), only boehmeriasin A possessed cytotoxicity against 12 cell lines from six types of cancer, including lung, colon, breast, prostate, kidney cancer, and leukemia. The antimitotic and cytotoxic activities of guattegaumerine, a bisbenzylisoquinoline alkaloid isolated from the bark of Guatteria gaumeri, was studied by Leclerq et al.169 According to the results, guattegaumerine exerts activity on B16 melanoma, which is a relatively resistant tumor. The cytotoxic activity of 8-OCinnamoylneoline, an alkaloid isolated from flower bud of Aconitum carmichaeli (Ranunculaceae), was studied by Taki et al.274 This alkaloid was detected only in flower buds. These acute toxicity and analgesic activities are connected with the presence of C-8 substituent in its ring. The tubers of the Aconitum species have been known to be biologically very active. In China and Japan, these species are known as herbs with strong bioactive potential. They contain masconitine, hypaconitine, and aconitine that are extremely toxic.274 Taki et al.274 concentrated their attention on the relatively lower toxicity of alkaloids in the flower buds. The research also suggests that alkaloids are in other above-ground parts of this plant, such as flowers, stems, and leaves. Alkaloids from other plant parts may have lower acute toxicities compared to the tubers. Biologically active alkaloids are regulators not only of endogenous life processes in the organisms that produce them but also in the organism that has consumed them.

3.2.3 Hemoglobinization of leukemia cells The biological activity of alkaloids can be demonstrated by fagaronine (Figure 3.6), an alkaloid isolated from Fagara zanthoxyloides Lam. (Rutaceae). This alkaloid alone has been tested by many research groups as a biological agent of the hemoglobinization of human leukemia cells.68,84,191,260 One of the characteristics of leukemia cells is escaping the normal regulatory pathway controlling cell proliferation and differentiation. As early as in 1972, Messmer et al.191 reported on fagaronine antileukemic activity against murine leukemia P388. Four years later, Sethi’s260 OH H3C-O N H3C-O

O-CH3

+

CH3

n FIGURE 3.6 Fagaronine, an alkaloid from Fagara zanthoxyloides Lam.

210 CHAPTER 3 Biology of alkaloids

study was published with evidence indicating that fagaronine inhibits DNA polymerase activity in murine embryos. It was also found that fagaronine inhibits human DNA ligase I275 and reverse transcriptases from RNA viruses.259 This last finding was confirmed by Tan et al.276 in the case of human HIV-1 reverse transcriptase in vitro. In the year 1983, Pezzuto et al.229 reported that fagaronine inhibits nucleic acid and protein synthesis in KB cells. Fagaronine was also reported to induce the hemoglobinization of leukemic K562 cells.68 Later studies pointed to the ability of this alkaloid to intercalate DNA, to interact with the ribosomal system,59 and to inhibit the activities of the DNA topoisomerase I and II.94,157 The research group of Dupont in France examined the effect of fagaronine on erythroid differentiation and growth of leukemic K562 cells.84

H

n FIGURE 3.7 Model of hemoglobin. Explanation: H, heme. Peptides are part of myoglobin.

The results of this deep research can be considered promising in the field, and the studies are in agreement with results obtained in previous studies by Comoe¨ et al.68 Dupont et al.84 observed that fagaronine induces the hemoglobinization of K562 cells and inhibits leukemic cell growth in 80% of cells. Moreover, fagaronine has no acutely toxic effects on the K562 cell line.84 The mechanism of this biological influence was explained by Dupont et al.84 as resulting from the action of several genes. Hemoglobin synthesis mediated by fagaronine treatment was preceded by the increased transcription of several genes known as the markers of erythroid differentiation. They are α- and β-globins, PBGD and EPO-R. Moreover, hemoglobin synthesis in leukemic cells was preceded by an overexpression of GATA-1 and NF-E2 mRNAs as well as by GATA-1 protein accumulation.84 Hemoglobin is a known tetramer of protein subunits with two α and two β subunits, myoglobin, and two glutamic acid residues in β subunits. A heme is an iron-containing porphyrin acting as a prosthetic (Figure 3.7). Moreover, fagaronine has caused increased transcriptional activity of the luciferase gene downstream of the α-globin, EPO-R, and GATA-1 promoters. This study is very interesting and proved that alkaloids have strong biological activities in foreign organisms. In this respect, fagaronine has probably more activity than in F. zanthoxyloides, a plant producing it, although this is only a hypothesis. The biological activity of fagaronine in plant cells is understudied in comparison to that in human cells. Its activity in plant cells should not be dismissed, for it may provide a better understanding of fagaronine activity in human cells. There are presently many other studies on alkaloid bioactivity in human leukemia cells and the role of alkaloids as competitive antagonists of cytotoxic agents.143,190 Sampangine, for example, is an alkaloid extracted from the stem bark of Cananga odorata. Kluza et al.143 show that the treatment of human HL-60 leukemia cells for 48 hours with sampangine induced

3.2 Bioactivity 211

oxidative processes and proved that this alkaloid has anticancer properties. Another alkaloid, voacamine, extracted from Peschiera fuchsiaefolia has been found to be an inhibitor to P-glycoprotein action and a competitive antagonist of cytotoxic agents.190 Pitzalis et al.231 studied the influence and molecular alternation of retrorsine on the rat hepatic cell cycle. Retrorsine has been found to block proliferation of resident cells. Cyclin D1 mRNA and protein levels were found to be elevated in rats treated with this alkaloid. The PCNA was also elevated.231 The conclusion from this study is that such a persistent block outside the resisting phase may contribute to selective replacement of resident cells during liver repopulation. The hemoglobinization of human leukemic cell lines by alkaloids demonstrated through in vitro means that these compounds are biologically very active. Alkaloids are therefore a promising botanical to be used in future applications.

3.2.4 Estrogenic effects Biological activity, although typical for alkaloids, can be very different and dependent on the chemical structure of alkaloid molecules. Quinoline alkaloids extracted from the plant belonging to the genus Haplophyllum A. Juss. (family Rutaceae) have strong biological activity with an estrogenic effect.210 The receptor for estrogenic activity is located in the nucleus (Figure 3.8). Therefore, this activity can be considered initiated by these receptors. C

D N A

N u c l e u s

N u c l e u s m e m b r a n e

y

Quinoline alkaloids t

HSP

o R p l a

s m

n FIGURE 3.8 Estrogenic activity of the alkaloids. Abbreviations:

R, receptor; HSP, heat shock proteins.

212 CHAPTER 3 Biology of alkaloids

Empirical results with 15 quinoline alkaloids have been studied in mature intact rats. It was found that all the alkaloids studied (fagarine, haplopine, skimmianine, glycoperine, evoxine, dubinidine, dubinine, perforine, haplophyllidine, perfamine, bucharidine, folifidine, acetylfolifidine, foliosidine, and acutine) cause the uterus to hydrate. Some alkaloids changed the menstrual cycle of mature intact rats by lengthening the estrus phase. If the average duration of a single menstruation was 1 day, the alkaloids extended it to 1.4 days.210 However, the alkaloids studied differed concerning the intensity of estrogenic activity. According to the results, quinoline alkaloids have the highest estrogenic activity at doses from 50 to 100 mg kg
1. This interesting study also observed that the estrogenic activity of perforine was many times greater than that of haplophyllidine. According to Nazrullaev, Bessonova, and Akhmedkhodzhaeva,210 estrogenic activity depends on the heterocyclic skeleton, N, and the nature of the substituent. Estrogenic activities of alkaloids from Crotalaria palida Ailton are reported currently to be excellent from the point of view of the possibility of a practical hormone replacement in menopause therapy.41 Moreover, recent results from the field of molecular endocrinology show also estrogenic activity of some erythroidine alkaloids. α-erythroidine and β-erythroidine isolated from legume plant Erythrinia poeppigiana are considered as potential estrogenic agents to be use in clinical practice.81a Extracts from Tabernaemontana divaricata flowers have also significant estrogenic activity and even antifertility effects.207 Antifertility effects in rats are also known with the application of the extract from stem bark of Dysoxylum binectariferum.141

3.2.5 Antimicrobial properties It is generally recognized by more than 1500 scientific papers that alkaloids have strong antimicrobial, antibacterial, and antifungal biological properties.6,23,42,51,56,58,70,91,102,109,123,129a,131,139,143,145,147a,148,162,163,170a,200, 205,208,213,214,224,226,237,238,277,292,300a,325,330a Moreover, some studies (more than 130 scientific papers) have found antiparasitic activity in this group of compounds.69,87,214,238a,244 Caron et al.58 investigated 34 quasi-dimeric indole alkaloids for antimicrobial activity using eight test microorganisms. They found that all of the studied alkaloids showed activity against Staphylococcus aureus and Bacillus subtilis, which are Gram-positive bacteria. Caron et al.58 found that 31 alkaloids showed biological activity against microorganisms. The microorganisms tested by Caron et al.58 such like B. subtilis, S. aureus, Mycobacterium smegmatits, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. This study concluded that antimicrobial activity of alkaloids is connected with the stereochemistry of the carbon ring, its aromatic substitution, and oxidation.58

3.2 Bioactivity 213

The antimicrobial activity of pendulamine A, pendulamine B, and penduline isolated from the root extract of Polyalthia longifolia var. pendula was studied by Faizi et al.91 All three alkaloids showed bioactivity, although the most active was found to be pendulamine A. In this study, the following Grampositive organisms were used: B. subtilis, Corynebacterium hoffmanii, S. aureus, Streptococcus faecalis, Streptococcus pyogenes, Streptococcus viridans, and Micrococcus lysodicklycus. The Gram-negative microorganisms were E. coli, K. pneumoniae, P. aeruginosa, Proteus mirabilis, Salmonella paratyphi A., S. paratyphi B. and Salmonella typhi.91 Antibacterial properties of the alkaloids isolated from Zanthoxylum rhifolium have also been noted in the study of Gonzaga et al.102 Significant bioactivity was displayed by 6-acetonyldihydronitidine, 6-acetonyldihydroavicine, and zanthoxyline. Gonzaga et al.102 used the following Gram-positive bacteria in their research: S. aureus, Staphylococcus epidermilis, and Micrococcus luteus. The Gram-negative bacteria were K. pneumoniae, Salmonella setubal, and E. coli. Antifungal, antibacterial, and antimalarial properties are mentioned in the case of sampangine as a result of in vitro studies.143 This alkaloid has also shown other bioactivities, especially a novel opportunity to be used as anticancer agent. Figure 3.9 presents antibacterial activity by some alkaloids. There are clearly different minimum inhibitory amount of alkaloids for this activity. It is also clear that alkaloid antibacterial activity is selective.

Antibacterial activity (minimum inhibitory amount in micrograms plate–1)

60 Scutianine E Condaline A Pendulamine A Penduline Xanthoxyline Berberine Sparteine Scutianine E Condaline A Pendulamine A Xantoxyline

55 50 45 40 35 30 25 20 15 10 5 0 1 Staphylococcus aureus

2 Klebsiella pneumoniae

n FIGURE 3.9 Activity of some alkaloids on Gram-positive (Staphylococcus aureus, 1) and

Gram-negative (Klebsiella pneumoniae, 2) bacteria. Explanations: Observe that the value for Scutianine E and Xanthoxyline is the same (12.5 μg). Alkaloids indicated in 1 but not in 2 have values of more than 100 μg. In the case of Penduline, no data are available. Sources: Refs. 15,91,102,205.

214 CHAPTER 3 Biology of alkaloids

Morel et al.205 reported on the antimicrobial activity of cyclopeptide alkaloids isolated from Scutia buxifolia Reiss (Rhamnaceae). The organisms used in this research were identical to those used in the study by Gonzaga et al.102 Condaline and scutianine have shown the widest range of bioactivity. It is necessary to mention that the antimicrobial activity of alkaloids is one of the many possible biological activities of these molecules. Recent studies provide vast amounts of new information about the status of marine environments as large reservoirs for biologically active alkaloids. The indole alkaloids from marine environments are a promising and active group of molecules. Their biological activity covers the cytotoxic, antiviral, antiparasitic, antiinflammatory, serotonin, and antagonistic realms.106 Another alkaloid group from marine environments consists of the pyridoacridone alkaloids. They have been reported as having a wide range of biological properties.69,162,163,200 These alkaloids have been known as having antitumorous and antifungal ability. Moreover, Sas-Piotrowska, Aniszewski, and Gulewicz250 address the fungistatic effects of the quinolizidine alkaloid fractions from extract of Lupinus spp. on potato pathogens. The alkaloid fractions had the strongest effect on the colonization of certain potato leaf fungi, such as Altenaria solani, Cladosporium herbarum, Colletotrichum coccodes, and Vertricillum albo-atrum. Moreover, this study shows evidence that alkaloids from Lupinus luteus L. strongly inhibit the growth of potato tuber fungi, such as Rhizoctonia solani and Phoma exigua. This study also suggests that alkaloid influence is stronger in the case of facultative fungi than in specialized fungi. However, the clearly different sensitivities of various potato pathogen species observed in the experiments carried out by Sas-Piotrowska et al.250 to alkaloid preparations have been explained by pathogenic cell structures and the chemical structure of the alkaloids. On the other hand, it was also possible that the fungistatic action of the preparation was based on the influence of the different synergetic levels of various compounds found in fungi species. Bringmann et al.49 researched naphthylisoquinoline alkaloids isolated from the tropical lianas Ancistrocladus abbreviatus and Triphyophyllum peltatum. The most prominent naphthylisoquinoline alkaloid is dioncophylline A, which has a variety of bioactivities. It was found that this alkaloid possesses high fungicidal and insecticidal properties. It has proven very active in limiting the fungus Botrytis cinerea and inhibiting the growth of the insect Spodoptera littoralis. Moreover, this alkaloid has antiparasitic bioactivity against Plasmodium falciparum and is larivicidal in the case of Anopheles stephensi. Bringmann et al.49 studied naphthylisoquinoline alkaloids for their molluscicidal activity against the tropical snail Biomphalaria glabrata. This study indicated a strong molluscicidal activity by the alkaloid.

3.2 Bioactivity 215

It is necessary to mention that the antimicrobial activity of alkaloids has been studied relatively extensively even during the 1940s–1980s. These studies have reported nearly 50 steroids,43,95,170,178,194–197,310–313 over 100 isoquinolizidines,14,67,101,114,125–127,152,154,195,196,198,267,287,310,316–318,329,330 and at least 90 terpenoid indole alkaloids to have antimicrobial activity.3–5,11,37,58,77,180,216,227,243,281,288,289,294,295 Research has reported large diversity in antimicrobial activity against bacteria, yeasts, and fungi. In the 1990s, the best known and most widely used alkaloids were berberine and sanguinarine, due to their antimicrobial activity. Berberine has antidiarrhetic and sanguinarine anticarries properties. This research trend continued in the first decade in 2000s. Presently, antimicrobial activity of alkaloids is studied with new compounds, unknown before, predominantly isolated from marine environments148,323 and from traditional and ethnopharmacological medicinal plants predominantly in Asia, Africa, and Latin America.8,123,236

3.2.6 Antiparasitic activity A parasite is an organism living in or on, and metabolically depending on, another organism. Endoparasites live inside an organism, and ectoparasites live on the surface of the host. Parasites can be carnivorous if living with animals or herbivorous if living with plants. Analyses of parasite-host suggest strong evidence of anticarnivorous, antiherbivorous action of alkaloids. A good example is with protozoan parasites (Plasmodium spp.) injected into humans by mosquitoes of the genus Anopheles. The life cycle of this parasite includes a sexual reproductive stage with multiplication (sporogony) occurring in the mosquito gut lumen and an asexual reproductive phase with multiplication (schizogony) occurring in the human host. Symptoms of this protozoan injection to humans and resulting symptoms of its asexual multiplication are known as malaria. Copp et al.69 investigated the antiparasitic potential of alkaloids against this kind of organism. In in vitro studies of antiparasitic activity on Plasmodium falciparum, Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei rhodesiense, evidence arose that connected pyridoacridone alkaloids with antiparasitism. However, these alkaloids also exhibit high cytotoxic activity, which can limit the use of their bioactivity in possible antimalarial product development. Kapil138 studied the bioactivity of piperine on L. donovani promastigotes in vitro and received very promising results. According to this study, piperine exhibited a concentration-dependent inhibition of L. donovani promastigotes. More recently Wright314 analyzed the bioactive possibilities of cryptoleptine, the main alkaloid from Cryptolepis sanguinolenta, as an antimalarial agent. The bioactivity of alkaloids against parasites is becoming

216 CHAPTER 3 Biology of alkaloids

increasingly important, because some parasites (e.g., P. falciparum) are presently resistant to traditional malarial medication. Cryptoleptine was considered by Wright314 as an alkaloid having possibility to be an antimalarial bioagent. However, more research in this direction is very important. As is known, the first alkaloid to be used against malaria was quinine, obtained from the bark of Cinchona. Treatment was later commonly focused on quinoline-based drugs, such as chloroquine, quinine, mefloquine, primaquine, and fansidar. Observations that P. falciparum became resistant to chloroquine, mefloquine, and halofartine2,303 aroused awareness of a problem. This has been studied in connection to indole alkaloids (Figure 3.10) from the Strychnos species (Loganiaceae) by Frederich, Tits, and Angenot.99 Sungucine presented very little activity, but some compounds (strychnogucine B and 18-hydroxyisosungucine) displayed more active qualities against quinine- and chloroquinine-resistant strains of P. falciparum. Antiparasitic alkaloid activity against Leishmania spp. has also been reported in other studies.174,203,301,302 Montenegro et al.203 studied alkaloids (xylopine, nornanteine, cryptodorine, nornuciferine, lysicamine, and laudanosine) from Guatteria amplifolia Triana and Planch (Annonaceae). Their results provide evidence that xylopine, cryptodorine, nornanteine, and nornuciferine have significant bioactive properties against Leishmania mexicana and Leishmania panamensis. Xylopine was the most active compound.203 Moreover, Sari et al.249 studied the bioactivity of alkaloids from Papaver lateritium Koch, a plant endemic to Turkey. The quaternary alkaloid fraction with (
)-mecambridine showed the highest lethality to brine shrimp larvae. Moreover, a study of the bioactivity of Stemona alkaloids provides evidence that these alkaloids have antitussive activity.65 This study also demonstrates a clear structure-bioactivity relationship in such alkaloids. Through substitution of a constituent of the alkaloid ring structure, it is possible to change bioactivity. New research indicates that new or novel alkaloids have a strong antiparasitic effects.50,244,300b

3.3 BIOTOXICITY Many alkaloids are toxic to foreign organisms. Toxicity is a secondary function of the alkaloids, because they are generally nontoxic to the organisms producing them. This is very important for understanding alkaloid nature. Many studies on alkaloid toxins have been published in recent years.13,60,71,73,75,76,83,100,232,253,254,262,291,300,332 The biotoxicity of alkaloids is selective and dependent on different organisms and the chemical structure of alkaloids themselves. Multiple bonds and different bond groups and subgroups especially directly or indirectly influence toxicity mechanisms.

3.3 Biotoxicity 217

N

N

N

N

O

O

O N

N

N

N

O

O

Sungucine

Strychnogucine A N

N OH O

N N H H

N

H H3C

H N N

N O

O

Strychnogucine B

Strychnopentamine N

N N H

HO H3C

N O

N H N H3C

N

N

N

Isostrychnopentamine N

N

Strychnohexamine n FIGURE 3.10 Some alkaloids from Strychnos species.

218 CHAPTER 3 Biology of alkaloids

3.3.1 Research evidence Alkaloids are active bioagents in animal tissues. There is clear scientific evidence of this. Crawford and Kocan71 tested the toxicity of steroidal alkaloids from the potato (Solanum tuberosum), such as α-chaconine, α-solanine, solanidine, and solasodine and the Veratrum alkaloid jervine on fish. The results of Crawford and Kocan’s research proved that rainbow trout exhibited a toxic response to chaconine, solasidine. and solanine, while medaka only did so to chaconine and solanine. Embryo mortality was observed as an effect of toxicity in both species. Many other alkaloids are known to disturb or cause disorder in animal reproductive systems. For example, gossypol from cotton-seed oil is known as a clear reducer of spermatogenesis and premature abortion of the embryo. Schneider et al.253 studied ergot alkaloid toxicity in cattle. The observed symptoms of the toxicity were hyperthermia, loss in milk production, loss of body mass, and reduced fertility. The toxicity symptoms were effected by ergotamine, ergosine, ergocornine, and ergocryptine. These ergot alkaloids caused gangrenous necrosis of extremities in young cattle. Their impact on livestock production is realized in significant financial losses each year.254 Piperidine alkaloids such as coniine and (
)-coniceine are very poisonous. They occur in hemlock (Conium maculatum L.), known as a very toxic plant. One of the characteristics of these piperidine alkaloids is smell. Moreover, they are neurotoxins, which have acute effects, such as chronic toxicity. There are known cases of death by respiratory failure resulting from coniine alkaloids. Pregnant cattle habitually ingesting amounts of plants with these alkaloids, for example, from hay, gave birth to deformed offspring.239 Rabbits have reportedly experienced toxic effects.175 The classic toxic symptoms of coniine alkaloids range from paralysis, muscular tremors, muscle weakness, and respiratory failure preceding death.239 It is not difficult to observe that the bioactivity of coniine alkaloids and especially their symptoms are similar to these of curare239 or of nicotine.45 Piva et al.232 studied the toxicities of pyrrolidine and tropane alkaloids. In this research, a synthetic scopolamine and hyoscyamine mixture in different concentrations was used on test pigs. Toxicity was observed in the gastrointestinal tracts, where the mucous membrane showed lymphocytic infiltration and a loss of epithelium. The villi were necrotic. It was also observed that the high levels of alkaloids increased the blood concentration of total lipids, cholesterol, and increased concentrations of urea and uric acid in the blood. Moreover, some alkaloids can inhibit digestive enzymes. Such kinds of alkaloids are, for example, swansonine or castanospermine. Ergot alkaloids are nowadays studied with their toxic

3.3 Biotoxicity 219

effects on the breeding stallion reproductive system in the University of Georgia, in the United States of America.92 This interesting study proved reduced toxicity of ergot alkaloids. They decreased the gel-free volume of stallions, but statistical significant effects were not found.

3.3.2 Influence on DNA The phenethylisoquinoline alkaloids, present in some members of the Lily family (Liliaceae), are known to be toxic. Wang and Wang300 researched the activity of veratridine on rats. This alkaloid causes persistent opening of the voltage-gate Na+ channel and reduces its single-channel conductance by 75%. However, its toxicity is concentration dependent. The toxicity of isoquinoline alkaloid berberine is low in concentrations of 0.05% for living plant cells.88 In these concentrations, berberine did not kill onion, corn, or broad bean cells, although it did reduce the growth rates of corn and bean. Moreover, in these concentrations, berberine is used as a mobile apoplastic tracer. Sequential application of berberine hemisulphate and potassium thiocyanate to plant tissue affects crystal formation in unmodified walls and in the lumina of dead cells. However, berberine does not affect crystals in lignified and suberized cell walls.88 Berberine was alone tested for possible genotoxicity, mutagenicity, and recombinogenic activities in microorganisms.221 An SOSchromotest with this alkaloid shows that there is no genotoxic activity nor significant cytotoxic, mutagenic, or recombinogenic effects in in vitro (nongrowing) conditions. However, Pasqual et al.221 observed the metabolic activity of this alkaloid in dividing cells. It induced important cytotoxic and cytostatic effects in proficient and repair-deficient Saccharomyces cerevisiae strains. According to Pasqual et al.,221 berberine’s cytotoxicity results from a mutational blockage in the DNA strand-break repair pathway (rad521). The influence of this alkaloid on DNA is evident. Pasqual et al.221 observed the same cytotoxicity in a triple mutant blocked in the excision (rad2-6), in the mutagenic (rad6-1), and in the recombinogenic (rad52-1) repair pathways. Although this toxicity was observed in dividing cells, Pasqual et al.221 concluded, in their discussion of results, that berberine is not a potent mutagenic agent, although one cannot rule out possible implications of DNA topoisomerases in berberine toxicity mechanisms. The results presented by Pasqual et al.221 have sufficiently characterized the nature of alkaloid activity and its potential toxicity. These characteristics are also typical for other alkaloids in general, although there may be some exceptions and reservations. Figure 3.11 presents the acute toxicities of berberine and thebaine. These alkaloids are very selective in their toxicity. There are also strong differences in acute toxicities according to the form in which these alkaloids were administrated to mice.

220 CHAPTER 3 Biology of alkaloids

Berberine, i.p. Berberine, i.v. Berberine, p.o. Thebaine, i.p. Thebaine, i.o Thebaine, p.o.

Acute toxicity (LD50 mg kg–1) of berberine and thebaine in mice

60

50

40

30

20

10 1

2 Alkaloids

n FIGURE 3.11 Acute toxicity of berberine and thebaine on mice in relation to form administration. Abbreviations: 1, berberine; 2, thebaine.; i.p., intraperitoneal; i.v., intravenous; p.o., oral.

Some studies suggest that nicotine influences human carcinogenesis.One such study was carried out by the Kleinsasser research group from the University of Regensburg in Germany.142 To assess the genotoxicity of this alkaloid, researchers tested the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue. The experimental data by Kleinsasser et al.142 evidently indicated that nicotine significantly and directly causes genotoxic effects in human target cells in vitro. However, there were no differences in DNA damage observed in cells from smokers and nonsmokers incubated without nicotine. Kleinsasser et al.142 suggest that the lack of higher DNA damage in smokers compared to nonsmokers is connected only with nicotine dose. New results indicate that nicotine induced mutations and genomic variations, and has implications on carcinogenesis.27a Beta-carboline alkaloids from Galianthe thalictroides (Rubiaceae) are reported to inhibit DNA topoisomerases I and II.93

3.3.3 Selective effectors of death One of the most known toxic alkaloid is strychnine. Vanderkop290 and Sterner et al.268 are examples of those who studied its toxicity, although it is practically rather evident. This alkaloid has been used as a strong

3.3 Biotoxicity 221

rodenticide.280 It is also known for being dangerous to humans. An interesting case example is the death of Alexander the Great. Recent studies suggest that, if he was poisoned, a source of poison was strychnine from Veratum album, which offers a more plausible cause than arsenic.251 One general characteristic of strychnine is its chemical stability. This is some kind of exception in the alkaloids, which are generally flexible heterogeneous compounds. In cases of poisoning, this alkaloid can be detected in exhumed bodies even many years after death. However, in the case of strychnine, some selectivity has been observed. The study of Sterner et al.268 is interesting in the sense that there is clear evidence of the selectivity of strychnine’s subchronic dietary toxicity being species dependent. Sterner et al.268 studied the subchronic toxicity of strychnine on the northern bobwhite quail (Collinus virginianus) and the mallard duck (Anas platyrhynchos). The authors found that strychnine toxicity was much lower in C. virginianus than in A. platyrhynchos. Others have also investigated strychnine. The research of Altememi et al.12 deserves mentioning when considering the species selectivity of strychnine. This study showed evidence that the addition of two acetylenic triazole derivatives increases the potentiation of strychnine toxicity and lethality in mice. Strychnine may also cause convulsions and disorders of the CNS. This is a result of the strychnine activity mechanism. It is known that the strychnine binds to a receptor site in the spinal cord that normally binds with glycine. Schmidt et al.252 proved that strychnine exhibits a fast conformational exchange on the NMR timescale at room temperature. Therefore, the selectivity of strychnine to different species can be considered a new point of view to the consideration of alkaloid toxicity in general. Some of the selectivity also may be present in other very toxic alkaloids. This means that the poisonous nature of alkaloids as immediate death effectors may not hold true for all species and individuals. Therefore, the exotic and colored legends of the use of alkaloids for acute effects of death (murders, executions, weapons, etc.) presented by some scientific books need to be critically checked in the light of present research data. The lethal dose (LD50) should be explained very carefully and critically. Alkaloid toxicity is not absolute; it is dependent on species, individuals, presence of other chemicals, and its own concentration.

3.3.4 Nontoxic to self but deformer for others Quinolizidine alkaloids are nontoxic to the legumes that produce them. On the other hand, the quinolizidine alkaloids can be toxic and in some cases very toxic to other organisms.19 The biotoxicity of alkaloids has for some time been considered to be connected with their bitter taste.159,202 The quinolizidine alkaloids are certainly bitter in taste to humans. However, not all

Acute toxicity (LD50 mg kg–1) of some quinolizidine alkaloids in mice (oral dose)

222 CHAPTER 3 Biology of alkaloids

420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0

1 Sparteine 2 Lupanine 3 13-Hydroxylupanine 4 17-Hydroxylupanine 5 Oxolupanine 6 Cytisine 7 N-Methylcytisine 8 Angustifoline 9 N-Methylangustoline 10 Anagyrine 11 Tinctorine 12 Rhombifoline

1

2

3

4

5

6

7

8

9

10

11

12

Quinolizidine alkaloids n FIGURE 3.12 Acute toxicity (LD50) of some quinolizidine alkaloids in mice. Sources: Refs. 15,52,120,182,304.

alkaloids are. Literature states that some pyrrolizidine and indolizidine alkaloids are not bitter in their pure forms.202 Furthermore, many nonalkaloid compounds, such as flavonoids, are bitter in taste but nontoxic. Therefore, although quinolizidine alkaloids are bitter, the connection between biotoxicity and bitter taste is not absolute. The most toxic quinolizidine alkaloids are tetracyclic with a pyridone nucleus. One of these is anagyrine. One case mentions anagyrine being passed into the human body via milk from goats foraging on Lupinus latifolius.187 The anagyrine caused severe bilateral deformities of the distal thoracic limbs in a baby boy. The literature presents terrible cases of the poisoning of human adults and children by lupine alkaloids.62,104,187,219 According to the results, the acute toxicity of a mixture of quinolizidine alkaloids varies. The lethal dose (LD50) for the extract of L.angustifolius L. is 2279 mg kg
1, and for extract with lupanine 1464 mg kg
1. In other studies the oral LD50 value of sparteine is 220 mg kg
1 and of lupanine 410 mg kg
1. According to some results (Figure 3.12), the LD50 value for sparteine is 60 mg kg
1, lupanine 159 mg kg
1, 13-hydroxylupanine 189 mg kg
1, 17-hydroxylupanine 177 mg kg
1, and oxolupanine 190 mg kg
1.118 The biological effect of the quinolizidine alkaloids is on the nervous system. Tremors, convulsions, and pulmonary arrest have been noted in laboratory animals. Quinolizidine alkaloids cause depression, labored breathing, trembling, convulsions, and respiratory paralysis in sheep.62

3.3 Biotoxicity 223

Yovo et al.327 stated that these alkaloids act via inhibition of ganglionic impulse transmissions of the sympathetic nervous system. It is evident that each alkaloid has its own effect. Anagyrine caused skeletal deformity in fetuses when pregnant cows consumed toxic lupines.62 On the other hand, some quinolizidine alkaloids are used as a drug in folk medicine.22 They probably have chronic toxicity.19 However, adequate knowledge about the chronic toxicity of these alkaloids and especially of chronic toxicity across generations is not available. The premise that quinolizidine alkaloids have not produced hereditary symptoms has not been checked with total reliability.

3.3.5 Degenerators of cells The biotoxicity of pyridine alkaloids is well studied and the toxicity of nicotine is one of the best examples of a very active alkaloid study area. Aydos et al.26 studied 20 rats injected daily with nicotine at doses 0.4 mg 100 g
1 of body weight over three months and made comparisons to a control group of 20 rats. The researchers concluded that ultrastructural alternations in rats exposed to nicotine occurred. Aydos et al.26 underlined the particularly detrimental effects of nicotine on germ cells, peritubular structures, and Sertoli cells. The germ cells were degenerated, and spermatids retained excess cytoplasm and accumulated electron-dense lipid droplets in the cytoplasm. Moreover, the results of Aydos et al.26 proved that the acrosomes in rats exposed to nicotine were irregular and abnormally configured. It is not difficult to interpret these results as evidence of active nicotine toxicity. Moreover, this chronic toxicity is reported also by Sener et al.,258 who studied aqueous garlic extract as an antioxidant. In this research, male Wistar albino rats were injected with nicotine, which led to increased collagen contents in tissues. Although Sener et al.258 reported the aqueous garlic extract was a protector of rat tissues, there is evidence of nicotine-induced oxidative damage. Nicotine toxicity also has been studied in humans.10,25,36,66,78,97,128,129,151,153,192,220,240 None of these studies question the symptoms of acute and chronic toxicity of nicotine. Moreover, the study by D’Alessandro et al.78 points to evidence of the risk of nicotine toxicity for tobacco harvesters. They absorbed approximately 0.8 mg of nicotine daily. Harvesters had higher levels of nicotine in their blood, and urine nicotine levels were also elevated.78 Nicotine toxicity is considered a health risk for agricultural workers on tobacco plantations in India.220 Nicotine toxicity is also connected to pica disease.10 Many symptoms of nicotine toxicity were observed in smokers in numerous studies,25,66,129,153 A dramatic case of nicotine toxicity is presented by Rogers, Denk, and Wax,240 who mention a case of acute ingestion of this alkaloid by a child. Hypoxia and irreversible encephalopathy ensued in this rare and tragic emergency case. Berthier et al.36

224 CHAPTER 3 Biology of alkaloids

1 Acobine

Acute toxicity LD50 (mg kg–1) in male rats (intraperitoneal injection)

2 Echimidine 3 Heliotrine

580 560 540 520 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0

4 Lasiocarpine 5 Lasiocarpine N-oxide 6 Monocrotaline 7 Retrorsine 8 Retrorsine N-oxide 9 Senecionine 10 Seneciphylline 11 Sekirkine 12 Symphitine

1

2

3

4

5

6

7

8

9

10

11

12

Pyrrolizidine alkaloids n FIGURE 3.13 Acute toxicity (LD50) of some pyrrolizidine alkaloids in male mice. Sources: Refs. 52,120,181,304.

reported on a case of a 30-year-old woman with symptoms of acute edematous pancreatitis. The combination of a nicotine patch and tobacco smoking induced an overdose of nicotine in this case.36 Some studies also claim that a chronic administration of high doses of nicotine results in axonal degeneration in the central core.66 Studies of the efficacy of nicotine replacement therapy have produced mixed findings. Moreover, nicotine toxicity is also a topic of clinical and theoretical studies.151,192 The mechanism of this toxicity is still not completely known in detail, but the research in the field is advanced and promising. On the other hand, it is also a difficult research area because of the large industry and large amount of trade involved with tobacco plants as a part of commercial products.

3.3.6 Aberrations in cells Pyrrolizidine alkaloids are toxic to foreign organisms (Figure 3.13). This problem was largely studied in the 1960s–1980s.39,53,72,121,182,228,234,270 Serious livestock poisoning episodes are mentioned in literature from the effects of the pyrrolizidine alkaloid of the Senecio genus, especially Senecio riddellii, Senecio douglasii, and Senecio jacobaea.201 The toxicity of

3.3 Biotoxicity 225

pyrrolizidine alkaloids to livestock was considered coincidental. Johnson and Molyneux133 and Johnson, Molyneux, and Stuart134 stated that experimental feedings of pyrrolizidine alkaloids to cattle empirically proved that the threshold level of ingesting alkaloids must be excessive for toxicity to occur. On the other hand, known cases of animal poisoning from pyrrolizidine alkaloids are found in Cynoglossum officinale (Boraginaceae). Baker et al.27 reported cases of calves being poisoned, and Knight et al.144 connected the deaths of two horses to poisoning by pyrrolizidine alkaloids. The acute toxicity of these alkaloids varies widely; it is recognized by the International Programme on Chemical Safety (IPCS) that for rats the LD50 of most alkaloids is 34–300 mg kg
1.304 Lasiocarpine doses equivalent to 0.2 mg kg
1 body weight per day lead to the development of tumors in rats. For pigs, 1.8 mg kg
1 doses cause chronic liver damage. For humans, the lowest reported intake level causing veno-occlusive disease (VOD) is estimated to be 0.015 mg kg
1 body weight per day.304 Some pyrrolizidine alkaloids are thought to cause lung damage, affect blood pressure and lead to secondary effects of the functioning of the right side of the heart. Moreover, according to the WHO data, pyrrolizidine alkaloids produce chromosomal aberrations in mammalian cells. Some pyrrolizidine alkaloids and their N-oxides are active as tumor inhibitors.270 They also can induce genetic changes and produce cancer in the livers of rats. The toxic effects of these alkaloids can be acute or chronic. Toxicity laboratory trials with retrorsine reported by White, Mattocks, and Butler309 resulted in centrilobular necrosis in rats, mice, and guinea pigs; periportal necrosis in hamsters; and focal necrosis in fowl and monkeys. Even in the 1940s, Wakim, Harris, and Chen296 reported that senecionine produces necrosis in the periportal and midzonal areas of liver lobules. Later, Dueker et al.83 studied monocrotaline metabolism using rat and guinea pig hepatic microsomes. These results suggest that guinea pigs are resistant to pyrrolizidine alkaloid toxicity. Esterase hydrolysis was observed in the metabolism of the guinea pig, and in the case of rats, there was no esterase activity. This explains the guinea pig’s resistance to pyrrolizidine alkaloid toxicity. Monocrotaline was also researched by Vaszar et al.291 Their results showed statistically significant increases in proteases in rats as a result of the activity of this alkaloid toxin. Moreover, Smith et al.262 researched pyrrolizidine alkaloid toxicity in horses. They concluded that these alkaloids led to chronic active hepatitis and, furthermore, chronic heart damage, including right ventricular hypertrophy as a consequence of pyrrolizidine lung damage. However, it is important to note that pyrrolizidine alkaloid toxicity depends on alkaloid structure and its possible reduction. The mechanism of toxic activity of these alkaloids is connected to metabolism in the parenchymal cells, where pyrrolizidine alkaloids change to pyrroles acting on hepatocytes and blood

226 CHAPTER 3 Biology of alkaloids

vessels in the liver or lungs. McLean185 reported that, as a consequence of this, disaggregation of polyribosomes, absence of pyruvate oxidation, and lysosomal activity and necrosis occur. It is important to note that pyrrolizidine alkaloids are inactive as a cell poison by themselves.

3.3.7 Causers of locoism Indolizidine alkaloids are also known as active biotoxins. Swansonine is especially cited in literature as a cause of locoism. This is a neurological lesion, especially in horses, cattle, and sheep.201 According to Elbein and Molyneux,85 swansonine is toxic due to the imbibition of α-mannosidase, an enzyme needed for proper functioning of mammalian cells. It is also known that swansonine inhibits several hydrolases. In addition, Astragalus lentiginous produces lentiginosine, which is an alkaloid related to swansonine.223 It is known as a good inhibitor of several α-glucosidases. This is due to the suppression of digestive enzymes.202 Li et al.,164,171 in a series of experiments, studied swansonine from a new locoweed, Oxytropis serioopetala, and its clinical and pathological features in poisoned rabbits. Application of swansonine to rabbits was in dose 1.5 mg kg–1 of body weight once daily. The rabbits appeared depressed and anorexic after 20th day from the first application. Other symptoms were dull eyes, severely weak limbs, and tremors on movement. The changes in the activities of serum enzymes occurred significantly, and pathomorphological lesions were observed. Moreover, extensive vacuolation occurred in the liver, kidney, and brain of poisoned rabbits. Pathological futures were similar to the symptoms of locoism.

3.4 BIONARCOTICS All alkaloids are neurotransmitters and active agents in the nervous system. Many alkaloids from natural plants and also modified alkaloids can impress euphoric, psychomimetic, and hallucinogenic properties on humans. Some of them can influence narcosis, states of stupor, unconsciousness, or arrested activity. Some of them in moderate doses dull the senses, relieve pain, and induce sleep. In excessive doses, they can cause stupor, coma, or convulsions. They are known as narcotics, a term derived from the narcoticus in Latin, narkotics in Greek, and narcotique in French. In the 1920s, lysergic acid diethylamide (LSD) was developed on the structural basis of ergotamine, the alkaloid produced by the fungus Claviceps purpurea living on rye (Secale cereale L.). Lysergic acid diethylamide has been developed and used primarily for treatment of schizophrenia. This compound is hallucinogenic. In the small doses, it causes psychedelic effects. It is for this reason LSD has been and is used as a narcotic. Narcotics (Figure 3.14) are stimulants that are active on the central nervous system, causing disorders and some temporary or permanent changes in this

3.4 Bionarcotics 227

O

H3C

CO2-CH3

O

N O

H3C

O

O

Cocaine

O O

H3C

HO

N-CH3

H

H

Heroine (diamorphine)

O N-CH3

H

H

N

HO

O

N-CH3 H

Morphine HO

O

N H

NH H

H

LSD

HO

NH-CH3 CH3

Normorphine

Methamphetamine HO

HO

HO H

O N

N-CH3

H

H HO

Apomorphine

Nalmorphine HO

HO

H3C

N H CH3

Phenazocine

H

H

N-CH3

Dextromethorphan

n FIGURE 3.14 Some narcotics and their derivatives.

Continued

228 CHAPTER 3 Biology of alkaloids

H3C-O

HO

O N

H3C

H CH3

N-CH3

H

H HO

Codeine

Pentazocine H3C-O

O N O

O

O H

H

N-CH3

H

HO

H

N-CH3

HO

Dihydrocodeine

Pholcodine

O

O N-CH3

O

N-(CH 3 )2

O

H O-CH3

CO2H O-CH3

O-CH3 O

O O-CH3 O

Narcotine

O-CH3

O-CH3

Narceine

n FIGURE 3.14, CONT'D

system and behavior. The serious negative consequences of narcotics include dependence, a chronic disorder. The best known narcotics are the opium alkaloids, such as morphine, codeine, thebaine, papaverine, noscapine, and their derivatives and modified compounds such as nalmorphine, apomorphine, apomopholcodine, dihydrocodeine, hydromorphone, and heroin, also known as diamorphine. Synthetic narcotics share the structural skeleton of morphine and include dextromethorphan, pentazocine, phenazocine meperidine (pethidine), phentanyl, anfentanil, remifentalin, methadone, dextropropoxyphene, levoproxyphene, dipipanone, dextromoramide, meptazinol, and tramadol. Thebaine derivatives are also modified narcotics and include oxycodone, oxymorphone,

3.4 Bionarcotics 229

etorphine, buprenorphine, nalbuphine, naloxone, and naltrexone. Narcotics can be semi-synthesized or totally synthesized from the morphine and thebaine models. The compounds serve various purposes in clinical practice. The natural source of these narcotics is Papaver somniferum L. and papaveretum, a mixture of purified opium alkaloids. Papaveretum is approximately 85.5% morphine, 8% codeine, and 6.5% papaverine. Only purified alkaloids are considered here, as the total alkaloid content of ripe poppy capsules is only 0.5%. It is recovered from the ripening capsule of papaver when it is in the process of changing color from blue-green to yellow. When the tubs are cut, it is possible to procure the milk. During coagulation, the milk’s color changes to brown. Fresh opium is soft but it hardens during storage. Crude opium has been used in the past as a sleep inducer and in folk medicine for many purposes and smoked for the feeling of pleasure. Lasting use leads to drug dependency and unpleasant withdrawal symptoms. Narcotics are reportedly among the most widely abused substances in the world, particularly the CNS stimulants cocaine and methamphetamine.33,77,150,155,217,235 These narcotics are a very serious problem because they may lead to strong drug dependence. Possible treatments for this dependence are relatively difficult. Common ones are based on the so-called dopamine hypothesis, according to which stimulants have the ability to increase extracellular dopamine, which has an additional narcotic effect.57,140,186,235 Cocaine has similar affinity for the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT).263 Narcotic dependency can be treated with the use of the synthetic compounds, chemically similar to narcotics. Dependence, therefore, is a very serious problem. Although new findings indicate kappa-opioid receptors may be involved in the modulation of some abuse-related effects and dopamine levels, problem-free therapy exists only in an optimistic future. It is known that the administration of cocaine up-regulates kappa-opioid receptors.67a,189,261,283 However, K opioid receptor agonists offer an indirect possibility to modulate some of the abuse-related effects of narcotics. Presently, more research is needed on this subject. Humans have known of and used opium for nearly 5000 years, mainly for medical purposes. Its abuse has been around as long. Government and international laws and licences regulate the production of P. somniferum, and only the clinical use of narcotics is legal and reasonable.

230 CHAPTER 3 Biology of alkaloids

3.5 ALKALOIDS IN THE IMMUNE SYSTEM As has been stated in this book, alkaloids are special secondary compounds. This is due to a general metabolic dependence on the genetic code and their expression through both the genetic code and metabolic scale mediated by growing factors. It is known that alkaloids can interact with DNA or DNA-processing enzymes and can inhibit protein synthesis, as has been mentioned in this book when referring to bioactivity. Moreover, alkaloids can influence electron chains in metabolism and can modulate enzyme activity. As has been presented, these compounds are biologically very active. Another important role alkaloids play is in the immune systems of living organisms. The amount of empirical research on this role is scant. Immunity is here defined as the ability to resist infections. Infections caused by microorganisms can be avoided in many ways. There are external and internal barriers to possible infections. The external barriers are skin (impermeable to many infectious agents), cuticles, skin secretions and pH-value, and washing. The internal barrier to infection is, namely, phagocytosis, a process of killing the infectious microorganisms by special cells. This ability, first discovered by Metchnikov in Russia in the 1880s, is the basis for immune systems in living organisms. Immune systems in animals and plants are quite different. The two types of immune systems in animals are (1) innate, so-called non-specific or passive immunity, and (2) adaptive, so-called specifically acquired, active, or cell-mediated immunity. Innate immunity is based on barriers to infectious agents, and adaptive immunity is based on multiplicative and specific antibody release after contact with an antigen (infectious agent). The so-called memory cells in animals respond to secondary contact with an antigen. Immune systems in plants are based on passive, structural immunity, such as a waxy surface or cuticle, and active immunity exists in the expression of some chemicals. The mechanism of this system is to prevent infectious agents from gaining access to plant cells. Plant immunity may also be protoplasmic. This means that the protoplast in cells is an unfavorable environment for pathogenic development. Plants do not, however, produce antibodies like animals do. The protoplasmic immunity is arranged generally by phytoalexins, nonspecific compounds whose concentrations increase in response to infections. Some alkaloids may act in a similar way to phytoalexins or in the direct chemical prevention of the infectious agent its growth.33,77,217,241 Although there are many differences between immunity systems in plants and animals, there are similarities. Both systems have two kinds of immunity: passive and active. Alkaloids may take part in both systems.

3.5 Alkaloids in the immune system 231

Many recent studies have proved that many alkaloids have antiviral properties. This is directly connected with the immune systems of organisms. It is known that the surfaces of viruses contain hemagglutinin, which helps adhering to cells prior to the infection.33,77,217,241 It is also known that viruses continually change the structure of their surface antigens through processes of antigenic drift and antigenic shift. Drift is a mutation in the viral genome, and the shift process is the change of a virus in the host. These processes lead to alterations in hemagglutinin. Infection can occur only when alterations in hemagglutinin are sufficient to render previous immunity ineffective. The potential role of alkaloids becomes apparent in this stage. These compounds break the alterations in the hemagglutinin. Moreover, alkaloids strengthen antiviral cytotoxic T-cells. Viruses normally work to inhibit these cells by hemagglutinin. Alkaloids seem to benefit the immune system when they decrease hemagglutinin’s ability to alter and, furthermore, when they strengthen and protect T-cells. CD8+T cells have an especially crucial role in an organism’s pathogenic resistance.61 These cells can kill malignant cells. Moreover, it is stated that some critical functions of these CD8+T cells depend on helper activity provided by CD4+T cells. The cooperation of these immunity cell subsets involves recognition of antigens.61 Some alkaloids may weaken antigens and malignant cells. It is also known that NK cells are cytotoxic to cells infected with viruses.33,77,217,241 In the immune system, the interaction between protective cells and chemicals is constant. T-cells also have a very important role in antifungal activity. Fungal infections are very serious problems for many organisms. Fungi try to go across passive immune systems, in which some alkaloids are also important. Many alkaloids have fungistatic properties. In the defense process, the T cells and the NK cells are very important, because they are cytotoxic to fungi. The influence of alkaloids on fungi is evident in the reduction of their growth and in the advancement of T and NK cells. Bacterial infections are problematic for many organisms, both animals and plants, because bacteria physiologically try to avoid phagocytosis by surrounding themselves with capsules. Capsule bacteria excrete exotoxins meant to kill phagocytes and destroy the immune system. Antibody defense neutralizes the toxins. Bacteria growing in intracellular spaces are killed by cell-mediated immunity (CMI) through specific synthesis of T cell helpers, which powerfully activate the formation of nitritic oxide (NO
), reactive oxygen intermolecules (ROIs), and other microbicidal mechanisms. The role of the alkaloids is in the prevention of bacterial growth and replication. Therefore, alkaloids help immune system activity. The biological activity of alkaloids against parasites has been mentioned. Many authors have reported on this kind of activity. A lot of parasites cause

232 CHAPTER 3 Biology of alkaloids

infections and resulting diseases. One group of these parasites is protozoa. Malaria, as previously mentioned, is caused by the protozoan Plasmodium spp. (Plasmodium vivax, P. falciparum, Plasmodium ovale, Plasmodium malariae). Leishmaniasis (Tropical sare, Kala azar, Espundia) is caused by Leishmania spp. (Leishmania tropica, L. donovani, L. brazilensis). Chagas’s disease results from infection by Trypanosama cruzi, and sleeping sickness by Trypanosama rhodesiense and Trypanosama gambiense. Helminths and trematodes (flukes) make up another group of protozoa and cause the schistosomiasis. This disease results from infection by Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. Nematodes (roundworms) make up a third group and cause the diseases trichinosis (Trichinella spiralis), hookworm (Straongyloides duodenale, Necator americanus), and filariasis (Wuchereria bancrofti, Onchocerca volvulus). These three groups of parasites are especially connected to infections in humans and animals. In the case of plants there are many parasites in the form of microorganisms and nematodes. There is large diversity among these parasites. In the steppe grasslands in Eastern Austria, 58 nematode genera were found, including the dominating species Acrobeloides, Anaplectus, Heterocephalobus, Prismatolaimus, Aphelenchoides, Aphelenchus, Tylenchus, and Pratylenchus.333 Moreover, an average of two to six individuals lived in one gram of soil. Although Zolda’s333 study found the plantfeeding nematodes to be third in numeral comparison (after the bacterial and fungal feeding), this group of nematodes is known to stress plants. Moreover, recent studies demonstrate important linkages between dwarf mistletoe infection, host plant vigor, and the ectomycorhizal colonization and fungal community composition of the pinyon pine (Pinus edulis).206 This study demonstrated that high levels of dwarf mistletoe infection were not associated with an increased mortality of infected trees. The infected trees showed only lower shoot growth. This is a good example of both plant adaptation to parasites and also indirectly of the immune system of the trees. Plant immunity seems to be imperfect, because parasites did establish infections in cells. The reduced growth of shoots was a means of preventing cell death. However, it is necessary to mention the numerous amounts of species of microorganisms that have intimate, beneficial, and sometimes essential relationships. Only a small fraction of these organisms are harmful. Therefore, the action of alkaloids as possible part of plant immunity is connected with strong selectivity and specifics. Although parasites live in the host organism and generally impart no benefits to the host, they have little or no harmful effects on the host in some cases, and their presence may be unapparent. However, microorganisms that do damage host organism are pathogens. Pathogens and pests should be

3.6 Genetic approach to alkaloids 233

foremost considered when addressing the potential influence of alkaloids on the immune systems of plants. However, it is known that Cuscuta reflexa and Cuscuta platyloba parasitize Berberis vulgaris but not Mahonia aquifolium. Moreover, Cascuta reflexa parasitized Datura arborea but not Datura stramonium.266 The alkaloid patterns in Berberis and Mahonia are similar but not identical. The same can be stated in the case of D. arborea and D. stramonium. Quinolizidine, pyrrolizidine and indole alkaloids are known to have toxic, repellent, deterrent, neutral, and stimulating activities, depending on the specific aphid-–plant relations.286 The alkaloid gramine taken from Hordeum is recognized as a chemical that disturbs the feeding activities of cereal aphids.

3.6 GENETIC APPROACH TO ALKALOIDS Alkaloid biogenesis in an organism is determined genetically.46,55,108,111,149,245–248,271,299,305,322 This means that many specific genes participate in alkaloid metabolism, and gene participation in metabolism is a very important basis for understanding the alkaloids. As is widely recognized, the gene is a unit of hereditary information encoded in a discrete segment of a DNA molecule, which carries an enormous amount of genetic information. It has been generally estimated that human cells contain from 50,000 to 100,000 genes on 23 chromosomes. The initial results of the Human Genome Project have been published beginning in June 2000 and finally in 2003. As one result of the project, it became clear that the human genome has only 30,000–40,000 genes, which was less than expected in previous estimations.297,298 The mouse (Mus musculus) has about 25,000 genes, the nematode (Caenorhabditis elegans) 19,000 genes, the fruit fly (Drosophila melanogaster) about 13,700 genes, and the common wall cress plant (Arabidopsis thaliana) has 25,500 genes.54 Genetic information connecting to the metabolism of alkaloids signifies that these secondary compounds are more important for the life cycle of organisms, as they are not coded in the genome. Lal and Sharma156 studied alkaloid genetics in P. somniferum. The alkaloids of this plant are determined by dominant and recessive genes. The inheritance of morphine, codeine, and thebaine content from parent plants to the next generation is 21–36%, and that of narcotine only 10.5–14.5%.156 The authors156 also cited previous work of Briza, according to which the heritability of morphine content in P. somniferum ranged from 43% to 68%. When considering narcotine content, some degree of epistasis is reflected.156 The dominant and recessive gene determination of alkaloid content makes alkaloid genetics a very difficult research topic. However, to date, at least more than 30 genes coding for enzymes involved in alkaloid biosynthesis pathways have been isolated and cloned (Table 3.1). Recently,

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Table 3.1 Enzymes specifically involved in alkaloid biosynthesis Alkaloids of Plant Species Purine alkaloids

Pyrrolizidine alkaloids Indole alkaloids

Enzymes

Coded by DNA

Caffeine synthase Xanthosine 7-N-methyltransferase 7-Methylxanthine 3-N-methyltransferase Caffeine xanthinemethyltransferase 1 (CaXMT1) Caffeine methylxanthinemethyltransferase 2 (CaMXMT2) Caffeine Dimethylxanthinemethyltransferase (CaDXMT1) Theobromine 1-N-methyltransferase Homospermidine synthase Tryptophane decarboxylase

Camellia sinensis, Coffea arabica Coffea arabica Coffea arabica Coffea arabica Coffea arabica Coffea arabica Coffea arabica Senecio vernalis, Senecio vulgaris Catharanthus roseus Camptotheca acuminata Catharanthus roseus Catharanthus roseus Rauvolfia serpentina Rauvolfia serpentina Catharanthus roseus Catharanthus roseus Catharanthus roseus Papaver somniferum Arabidopsis thaliana Eschscholtzia californica Coptis japonica Coptis japonica Coptis japonica Coptis japonica Coptis japonica Thalictrum tuberosum Eschscholzia californica Berberis stolonifera Papaver somniferum Papaver somniferum Hyoscyamus niger Atropa belladonna Hyoscyamius niger Datura stramonium Hyoscyamius niger Datura stramonium Solanum tuberosum Ruta graveolens

Secologanin synthase Strictosidine synthase

Isoquinoline alkaloids

Polyneuridine aldehyde esterase Taberosine 16-hydrolase Desacetoxyivindoline acetyltransferase Geraniol/nerol 10-hydroxylase Tyrosine/DOPA decarboxylase

Tropane alkaloids

Berberine bridge enzyme Norcoclaurine 6-O-methyltransferase Coclaurine N-methyltransferase 30 -Hydroxy-N-methylcoclaurine4-O-methyltransferase Scoulerine 9-O-methyltransferase Columbamine O-methyltransferase O-methyltransferases N-methylcoclaurine 30 -hydroxylase Berbamunine synthase Codeinone reductase Salutaridinol 7-O-acetyltransferase Hyoscyamine 6β-hydroxylase Tropinone reductase-I Tropinone reductase-II

Acridone alkaloids

Acridone synthase

Sources: Refs. 48, 64a, 80, 81, 90, 90a, 112, 113, 137, 147, 151a, 180a, 184, 199, 204, 205a, 209, 216a, 222, 247, 273a, 283a, 284, 293.

3.6 Genetic approach to alkaloids 235

acetylajmalan esterase (AAE) was isolated and purified together with a fulllength AAE cDNA clone from Rauvolfia cells.246 This enzyme plays an essential role in the late stages of ajmaline biosynthesis. It is also known as the eighth functional alkaloid gene extracted from this gene, and the sixth identified ajmaline biosynthetic pathway-specific gene. Strictosidine synthase with cDNA and genomic DNA (str1) has been isolated from Rauwolfia serpentina and Rauwolfia mannii.47,48,149 This enzyme coded in cDNA has been isolated also from Catharanthus reseus.184,222 Tryptophan decarbocylase encoded by cDNA has also been isolated from this plant and then described.80 Moreover, Cane et al.55 discovered that the synthesis of the nicotine in the roots of Nicotiana tabacum is strongly influenced by the presence of two nonallelic genes, A and B. Hibi et al.117 reported on putrescine N-methyltransferase isolated from the nicotine biosynthetic pathway coded by cDNA. Recent advances in cell and molecular biology of alkaloid biosynthesis have heightened awareness of the genetic importance. Biosynthetic genes involved in the formation of tropane, benzylisoquinoline, and terepenoid indole alkaloids have been isolated.90 Hyoscyamine 6-β- hydrolase137 and tropinione reductases209 encoded in cDNA and involved with tropane alkaloids have been isolated as well. Moreover, some genes involved in the metabolism of isoquinoline alkaloids and encoded in cDNA are also known. The berberine bridge enzyme from Eschscholtzia californica81 and berbamurine synthase from Berberis stolonifera147 belonging to this group of enzymes are encoded in cDNA. Coclaurine N-methyltransferase and columamine O-methyltransferase involved in the biosynthetic pathways of the isoquinoline alkaloids berberine and palmatine, respectively, have been found and cloned in Coptis japonica.112,113 Salutaridinol 7-O-acetyltransferase forms an immediate precursor of thebaine along the morphine biosynthetic pathway, and its cDNA was obtained from a cell suspension culture of the opium poppy (P. somniferum).112,113 Caffeine is formed from xanthosine through three successive transfers of methyl groups and a single robose removal in coffee plants. The methylation is catalyzed by three N-methyltransferases: xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT), and 3,7dimethyltransferase (DXMT), which participates in the caffeine synthetic pathway.284 There is the evidence that genes involved in alkaloid metabolism can be isolated and engineered to new plants. The biotechnological potential is apparent especially in cytochrome P450 genes isolated from Catharanthus roseus. P450 genes are involved in the 16-hydroxylation of tabersonin in this

236 CHAPTER 3 Biology of alkaloids

plant and establish recombinant system CY71D12 as a tabersonine 16hydroxylase. In Lonicera japonica, P450 genes are involved in the conversion of loganin into secologanin systems as CYP72A1, as a secologanin synthase, and CYP76B6, as a geraniol/nerol 10-hydroxylase. CYP72A1 from higher plants catalyze ring-opening reactions. CYP76B6 and CYP71D12 catalyze alkaloid moiety. In the indole alkaloid biogenesis, P450 genes catalyze a large of number of reactions; for example, P450 genes are important in the formation of parent ring systems of alkaloids. Engineered plant defense and herbicide tolerance is developed by transferring of some 450 genes. Animal enzymes encoded by P450 indicate potential use in plant defense system after their translocation by biotechnological engineering.204 Knowledge of these key genes can be used to enhance alkaloid production in the cell cultures.204,293 The biological importance of alkaloids is connected with the structural, metabolic, functional, and evolutionary role of these compounds in living organisms. The present research on the genes involved in the biosynthesis of alkaloids is advanced and many enzymes have been isolated and cloned. However, the major challenge in the near future is to isolate new genes and new enzymes. Research needs to uncover more information about the regulation of metabolism at different levels, such as genes, enzymes, alkaloid production, and accumulation. A challenge in this research area is to provide more data on genetic information as a means of mapping metabolic networks between these levels. This could help develop better models for alkaloid biosynthesis and production, which will support the metabolic engineering of alkaloids in the future.

3.7 EVOLUTIONARY INFLUENCES ON ALKALOID BIOLOGY Alkaloids have genetic dimensions and hold many secrets of life. They are toxic and many of them can be used as narcotics. As important secondary compounds, they categorically determine much about life. They also play a large role in evolution due to their characteristics. Charles Darwin’s theory of evolution revolutionized biology and has motivated biologists to make empirical studies of evolutionary phenomena in nature and in the laboratory. As a result of this, a fundamental science presently exists based on this theory. A serious biologist must pay heed to Darwin’s statements along with later neo-Darwinistic developments and Mendelism when searching for a deeper understanding of life. Molecular biology, which is presently powering all the biological sciences, is strengthened by Darwin’s and Mendel’s theories and has completely supported them.

3.7 Evolutionary influences on alkaloid biology 237

Many studies consider evolution and coevolutionary interactions between plants and insects.40,96,130,146,161,334 Many of these proved that there is interdependence between plant chemistry and the animals that feed on these plants, especially insects.29–31,34,35,89,98,264,279 Literature is accordant in pointing out the importance of plant and animal chemistry in both evolutionary and coevolutionary processes. Alkaloids are good examples of this chemical role. The classical example is the potato beetle Leptinotarsa decemlineata living on the S. tuberosum and other Solanum species. These species contain solanine, solanidine, and other minor steroid alkaloids. Solanine and solanidine are toxic. However, L. decemlineata tolerates these alkaloids when feeding (on the green mass of potato). Moreover, L. decemlineata does not store these alkaloids in its body, and they are eliminated during metabolism. The study concerning the effects of quinolizidine alkaloids on the potato beetle (Leptinotarsa decemlieata) proved that these alkaloids reduce populations of Leptinotarsa and the development of their larvae.320 Elsewhere, in a case concerning steroid alkaloids (solanine, solanidine), the Colorado beetle has not adapted to the alkaloid lupin. Moreover, in coevolutionary development, some aphids not only feed on alkaloid plants but also sequester the alkaloids and keep them in the own body. Examples of this are the case of Macrosiphum albifrons with quinolizidine alkaloids or the case of Aphis jacobaeae or ladybirds (Coccinella) with pyrrolizidine alkaloids. On the other hand, it is necessary to pay attention to the fact that aphids, ladybirds, and other insects feed on the alkaloid-poor or alkaloid-free forms of the same species. This can be explained as an example of coevolutionary development. Alkaloids are molecules developed in coevolutionary processes with the environment. The evolution of the ability to use some alkaloids by some insects is a consequence of this (Figure 3.15). When food sources change, an organism needs to adapt to the new conditions. This is a basic matter of evolution. Moreover, cytochrome c, one of the basic enzymes, exists largely in plants, animals, fungi, and some bacteria, as for example Rhodospirillum rubrum. Nearly 60% of amino acids of cytochrome c in the homogenic position are identical in wheat and humans and even 30% are identical in R. rubrum and humans. This is only one piece of evidence that genes for cytochrome c have evolved from the first gene of prebacteria in the history of life on the globe. Genes for alkaloids evolved in a similar way. New evidence of evolution is presently available.32,158,215 Evolution of viruses as complex genomes146 and the development of nucleoprotein into RNA and after that to DNA are two hypotheses considered very important for understanding the mechanisms of life.96 It has been stated that alkaloids can influence DNA and RNA as well as protein synthesis in general, because their

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Changes BEHAVIOR

A L K A L O I D S

Ajmalicine Brucine Vincamine

Syntomis

Berberine Codeine Coniine Yohimbine Strychnine

Phormia

Caffeine

Bruchidius

Gramine

Sitobion

Lupinine

Acyrthosiphon

Solanine Solanidine

Leptinotarsa

I N S E C T S

TOXICITY TOLERANCE Changes n FIGURE 3.15 Model of evolutionary interaction between alkaloids and insects.

metabolism is encoded genetically. Even the smallest changes in the gene code influence this mechanism. The starting point for all changes is the cell. The chemical behavior of one organism is affected that in another. Jackson et al.130 described the evolution of antipredator traits in response to a strategy by predators, and Lion, van Baalen, and Wilson172 address the evolution of parasite manipulation of host dispersal behavior. This study reveals that parasites can manipulate their host’s dispersal. The evolution of herbivorehost plant specialization requires low levels of gene flow between populations. Leonardo and Mondor161 show that the facultative bacterial symbiont Candidatus regiella insecticola alters both dispersion and mating in the pea aphid Acyrthosiphon pisum. Changes in dispersal and mating associated with symbionts are likely to play a key role in the initiation of genetic differentiation and in the evolution of pea aphid–host plant specialization. The evidence of the participation of alkaloids in the evolution of organisms is observed in interactions with numerous microorganisms. As has been stated, many alkaloids have antimicrobial activity. However, there are several alkaloids without this characteristic. Some microorganisms as symbionts of Bradyrhizobium spp. can live in both alkaloid-rich and alkaloid-poor plants with the some level of activity. The same can be stated

3.7 Evolutionary influences on alkaloid biology 239

in connection to some fungi, for example. mycorrhiza. Adaptation processes in nature lead to permanent evolution and coevolution between alkaloids as a part of biochemistry and organisms (as a part of the environment). The evolution and ongoing coevolution of alkaloids and organisms is an example that alkaloidal defense of a plant is only a secondary function of these molecules that also changes in the evolutionary process. Just this change of alkaloids in form and activities is biological phenomena, partly unknown in detail.

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283a. Uefuji H, Ogita S, Yamaguchi Y, Koizumi N, Sano H. Molecular cloning and characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in cofee plants. Plant Physiol 2003;132(1):372–80. 284. Uefuji H, Tatsumi Y, Morimoto M, Kaothien-Nakayama P, Ogita S, Sano H. Caffeine production in tobacco plants by simultaneous expression of three coffee N-methyltransferases and its potential as a pest repellant. Plant Mol Biol 2005;59 (2):221–7. 285. Ueng YF, Wang JJ, Lin LC, Park SS, Chen CF. Induction of cytochrome P450dependent monooxygenase in mouse liver and kidney by rutaecarpine, an alkaloid of the herbal drug Evodia rutaecarpa. Life Sci 2001;70:207–17. 286. Urbanska A, Leszczynski B, Matok H. How does gramine affect probing behaviour of grain aphid? Electronic J Pol Agric Univ Biol 2006;9(1):1–7. 287. Vallejos RH, Roveri OA. Alkaloid inhibition of yeast respiration. Prevention by Ca2+. Biochem Pharmacol 1972;21:1160–7. 288. Van Beek TA, de Smidt C, Verpoorte R. Phytochemical investigation of Taberaemontana crassa. J Ethnopharmacol 1985;14:315–8. 289. Van Beek TA, Verpoorte R, Baerheim Svendsen A, Fokkens R. Antimicrobially active alkaloids from Tabernaemontana chippi. J Nat Prod 1985;48:400–23. 290. Vanderkop MA. Strychnine toxicity in livestock. Can Vet J 1993;34:124. 291. Vaszar LT, Nishimura T, Storey JD, Zhao GH, Qiu DM, Faul JL, et al. Longitudinal transcriptional analysis of developing neointimal vascular occlusion and pulmonary hypertension in rats. Physiol Genomics 2004;17:150–6. 292. Verpoorte R, Kodde EW, Van Doorne H, Baerheim Svendsen A. Antimicrobial effect of the alkaloids from Strychnos afzelii. Planta Med 1978;33:237–42. 293. Verpoorte R, Memelink J. Engineering secondary metabolite production in plants. Curr Opin Biotechnol 2002;13:181–7. 294. Verpoorte R, Ruigrok CLM, Baerheim Svendsen A. Medicinal plants of Suriname. Antimicrobial active alkaloids from Aspidosperma marcgravianum. Planta Med 1982;46:149–52. 295. Verpoorte R, Kos-Kuick E, Tsin a Tsoi A, Ruigrok CLM, de Jong G, Baerheim Svendsen A. Medicinal plants of Suriname. III. Antimicrobially active alkaloids from Aspidosperma marcgravianum. Planta Med 1983;48:283–9. 296. Wakim KG, Harris PN, Chen KK. The effects of senocionine on the monkey. J Pharmacol Exp Ther 1946;87:38–45. 297. Walker R. Genes and DNA. London: Kingfisher Publications PLC; 2003. 298. Walker R. Geny i DNA. Warsaw: SAMP; 2004. 299. Waller GR, Nowacki EK. Alkaloid Biology and Metabolism in Plants. New York, London: Plenum Press; 1978. 300. Wang GK, Wang SY. Veratridine block of rat skeletal muscle Nav1.4 sodium channels in the inner vestibule. J Physiol 2003;548:667–75. 300a. Wang JF, Lin XP, Qin C, Liao SR, Wan JT, Zhang TY, et al. Antimicrobial and antiviral sesquiterpenoids from sponge-associated fungus, Aspergillus sydowii ZSDS1F6. J Antibiot 2014;67(8):581–3. 300b. Wang N, Wicht KJ, Shaban E, Ngoc TA, Wang MQ, Hayashi I, et al. Synthesis and evaluation of artesunate-indoloquinoline hybrids as antimalarial drug candidates. Medchemcomm 2014;5(7):927–31.

3.7 Evolutionary influences on alkaloid biology 257

301. Weniger B, Aragon R, Deharo E, Bastida J, Codina C, Lobstein A, et al. Antimalarial constituents from Guatteria amplifolia. Pharmazie 2000;55:867–8. 302. Weniger B, Robledo S, Arango GJ, Deharo E, Aragon R, Munoz V, et al. Antiprotozoal activities of Colombian plants. J Ethnopharmacol 2001;78:93–200. 303. Wernsdorfer WH. Epidemiology of drug resistance in malaria. Acta Tropical 1994;56:143–56. 304. WHO. IPCS. International Programme on Chemical Safety. Health and Safety Guide No. 26. Generva: World Health Organization; 1989. 305. Williams W, Harrison JEM, Jayasekera S. Genetic control of alkaloid production in Lupinus Mutabilis on the effect of a mutant alle mutalisolated following chemical mutagenesis. Euphytica 1984;33:811–7. 306. Wink M. A short history of alkaloids. In: Roberts MF, Wink M, editors. Alkaloids. Biochemistry, Ecology, and Medicinal Applications. New York, London: Plenum Press; 1998. p. 11–44. 307. Wink M. Evolution of secondary metabolities from an ecological and molecular phylogenetic perspective. Phytochemistry 2003;64(1):3–19. 308. Wink M, Mohamed GIA. Evolution of chemical defense traits in the Leguminosae: Mapping of distribution patterns of secondary metabolites on a molecular phylogeny inferred from nucleotide sequences of the rbcL gene. Biochem Syst Ecol 2003;31 (8):897–917. 309. White INH, Mattocks AR, Butler WH. The conversion of the pyrrolizidine alkaloid retrorsine to pyrrolic derivatives in vivo and in vitro and its acute toxicity to various animal species. Chem Biol Interact 1973;6:207–18. 310. Wolters B. Der Anteil der Steroid-saponinen an der antibiotischen Wirkung von Solanum dulcamara. Planta Med 1965;13:189–93. 311. Wolters B. Zur antimikrobiellen Wirksamkeit pflanzlicher Steroide und Triterpene. Planta Med 1966;14:392–401. 312. Wolters B. Zur Verwendung voreschichteter Folien bei der Dunnschichtchromatografischen Untersuchung pflanzlicher Fungistatica. Planta Med 1969;17:42–50. 313. Wolters B. Antimicrobial activity of Veratrum alkaloids. Planta Med 1970;19: 189–93. 314. Wright CW. Plant derived antimalarian agents: New leads and challenges. Phytochemistry Rev 2005;4(1):55–61. 315. Wu GW, Lin AQ, Gu QQ, Zhu TJ, Li DH. Four new chloro-eremophilane sesquiterpenes from an Antarctic deep-sea derived fungus, Penicillium sp p R19N–1. Mar Drugs 2013;11(4):1399–408. 316. Wu WN, Beal JL, Clark GW, Doskotch RW. Antimicrobial agents from higher plants. Additional alkaloids and antimicrobial agents from Thalictrum rugosum. Lloydia 1976;39:65–75. 317. Wu WN, Beal JL, Leu RP, Doskotch RW. Isolation and characterization of alkaloids from roots of Thalictrum podocarpum. Lloydia 1977;40:384–94. 318. Wu WN, Beal JL, Doskotch RW. Alkaloids of Thalictrum. XXII. Isolation of alkaloids with hypotensive and antimicrobial activity from Thalictrum revoltum. Lloydia 1977;40:508–14. 319. Wu Y-C, Liou Y-F, Lu S-T, Chen C-H, Chang J-J. Cytotoxicity of isoquinoline alkaloids and their N-oxides. Planta Med 1989;55:163–5.

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320. Wyrostkiewicz K, Wawrzyniak M, Barczak T, Aniszewski T, Gulewicz K. An evidence for insecticide activity of some preparations from alkaloid-rich lupin seeds on Colorado potato beetle (Leptinotarsa decemlineata Say), larvae of the large white butterfly (Pieris brassicae L.), black bean aphid (Aphis fabae Scop.) and on their parasitoids (Hymenoptera: Parasitica) populations. Bull Pol Acad Sci Biol Sci 1996;44(1–2):30–9. 321. Wysocki W, Gulewicz P, Aniszewski T, Ciesiołka D, Gulewicz K. Bioactive preparations from alkaloid-rich lupin. Relation between chemical composition and biological activity. Bull Pol Acad Sci Biol Sci 2001;49:9–17. 322. Yamazaki M, Saito A, Saito K, Murakoshi I. Molecular phylogeny based on RFLP and its relation with alkaloid patterns in Lupinus plants. Biol Pharmacol Bull 1993;16:1182–4. 323. Yang B, Tao HM, Zhou XF, Lin XP, Liu YH. Two new alkaloids from marine sponge Callyspongia sp. Nat Prod Res 2013;27(4–5):433–7. 324. Yang XW, Zhang GY, Ying JX, Yang B, Zhou XF, Steinmetz A, et al. Isolation, characterization, and bioactivity evaluation of 3-[(6-methylpyrazin-2-yl)methyl]1H-indole, a new alkaloid from a deep-sea-derived actinomycete Serinicoccus profundii sp nov. Mar Drugs 2013;11(1):33–9. 325. Yang Y, Zuo WJ, Zhao YX, Dong WH, Mei WL, Dai HF. Indole alkaloids from Kopsia hainanensis and evaluation of their antimicrobial activity. Planta Med 2012;78(17):1881–4. 326. Yazaki K. ABC transporters involved in the transport of plant secondary metabolities. FEBS Lett 2006;580(4):1183–91. 327. Yovo K, Huguet F, Pothier J, Durand MK, Breteau M, Narcisse G. Comparative pharmacological study of sparteine and its ketonic derivative lupanine from seeds of Lupinus albus L. Planta Med 1984;50:420–4. 328. Zangara A. The psychopharmacology of huperzine A: An alkaloid with cognitive enhancing and neuroprotective properties of interest in the treatment of Alzheimer’s disease. Pharmacol Biochem Behav 2003;75:675–86. 329. Zbierska J, Kowalewski Z. Anticancer and antibiotic properties of chelidonine methyliodide. Herba Polonica 1979;25:209–17. 330. Zbierska J, Kowalewski Z. Anticancer and antibiotic properties of N-methylchelidonine methylsulfate. Herba Polonica 1979;25:311–6. 330a. Zhang L, Hua Z, Song Y, Feng C. Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities. Fitoterapia 2014;97:142–7. 331. Zhang XJ, Kuca K, Dohnal V, Dohnatova L, Wu QH, Wu C. Military potential of biological toxins. J Appl Med 2014;12(2):63–77. 332. Zhang N, Scott V, Al-Samarrai TH, Tan YY, Spiering MJ, McMillan LK, et al. Transformation of the ryegrass endophyte Neotyphodium lolii can alter its in planta mycelial morphology. Mycol Res 2006;10:601–11. 333. Zolda P. Nematode communities of grazed and ungrazed semi-natural steppe grasslands in Eastern Austria. Pedobiologia 2006;50(1):11–22. 334. Zorin NA, Zorina VN, Zorina RM. Evolution of proteins of macroglobulin family. J Evol Biochem Physiol 2006;42(1):112–6.

Chapter

4

Ecology of alkaloids CHAPTER OUTLINE

4.1 4.2 4.3 4.4

Molecular basis of ecology 262 Alkaloids in interaction between life units 263 Biological communities and alkaloids 264 Alkaloids in different ecosystems 266 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5

Alkaloids in aquatic ecosystems 267 Alkaloids in terrestrial ecosystems 270 Climate and alkaloids 270 Alkaloids and invasive species 272 Ecological role of alkaloids 274 4.4.5.1 Sexual behavior 280 4.4.5.2 Feeding attraction and deterrence 282

References

283

Naturam mutare difficile est. Seneca

Alkaloids have long represented a research subject in organic chemistry and pharmacology. The main object of research has been to recognize the profound chemical structure and the physical characteristics of these compounds. Advances in alkaloid chemistry also advanced biological studies of alkaloids, since the chemical structure of these compounds defines their biological activity. Although, today, some areas remain still unclear and underresearched, the knowledge of alkaloids is relatively large and very detailed in many fields. Moreover, this knowledge has been utilized in the development of many contemporary applications important to human life and society. From the beginnings of alkaloid research (from the discovery of morphine) to today, one of the most interesting questions has been and remains to be the function of alkaloids. In particular, the external function of alkaloids has Alkaloids # 2015 Tadeusz Aniszewski. Published by Elsevier B.V. All rights reserved.

259

260 CHAPTER 4 Ecology of alkaloids

been a popular area of the study. However, the spectrum of our knowledge in research will be made right when attention is paid to the fact that some scientists are of the opinion that even the physiological role of alkaloids is not clear and still presents a challenge for researchers.57 However, in spite of some critical scientific pessimism, chemistry and biology together with ecology try in their research to advance the progress in the alkaloid field step by step. Sometimes, it is difficult to clearly define the border between the sciences involved in alkaloid research in a large scope. The ecological scope to alkaloid study is younger than a chemical and biological scope. Ecology itself as an independent science lacks the strong history of development of chemistry or biology. In its present form, ecology is a relatively new field; it has the roots in the second half of 1800s. Oikoslogy (ecology) means the science (-logy) of a place, home (oikos). Early ecology was just the science of the organisms and their relation to their living place, biocenosis, ecosystem, and biome. Ecology can be defined as the science that studies all aspects of organisms in relation to their environment and other organisms, individuals or populations. Ecology, therefore, is a large science, based on natural science with the focus of mutual interactions or one-way relations to other units of life. Ecology is generally considered a part of biology, although in many aspects ecological research and teaching goes beyond the biosciences and can be placed in the fields of chemistry, geography, statistics, behavioral, and applied science. Therefore, ecology is, in reality, an interdisciplinary science based on biological terms of species, their determination, distribution, self-management, and development in the environment. Just natural self-management is very important in ecology, as it is done by interaction of the species. Alkaloid occurrence and the role of natural self-management of the species are major topical processes. The basis for the ecological study of alkaloids is adaptation theory, according to which all living organisms adapt to environmental change.2,7,13,21,28,39,50,56,78,79 Especially, during recent years, the adaptation theory of species is described by a lot of cases involving alkaloids. This ecological research was based on observations, experiments, and laboratory chemical studies. The results, in many cases, are explained by statistical evidence and the hypotheses. Therefore, it is open to the discussion whether ecology, even chemical ecology, is an exact or inexact science. From the point of view of alkaloid studies, it is important that the organisms produced or containing these compounds can be researched on the level of global distribution, on the level of populations and individuals. Individuals seem to be very important, as they are organism units at the population level. The interaction within or between populations is, in reality, the interaction of individuals. It is necessary to remember that all the interactions in biology

CHAPTER 4 Ecology of alkaloids 261

are based on life activity, which depends on energy and molecule behavior. Biological life is a production of energy (Figure 4.1). A cell is the basis of energy production, although bioorganic and chemical reactions play fundamental roles in this production. Energy production is stimulated and lead by both genetic and ecological factors. The principal manager and permanent controller of this production is energy balance, the conduction of which originates in gene impulses and chemical reactions and their density. This is possible only in the active metabolism and molecular changing, growing, evolution, and degradation. Therefore, alkaloids and their metabolisms depend on this energy production. Adaptation and ecological interactions in which alkaloids take part are closely connected with the life as a process of active metabolism. Individual life can be described by many factors, of which metabolic rate is very important. This is the amount of energy that is needed for the maintenance of life during a unit of a time. One of the best known indicators is the basal metabolic rate. This indicator is exceptionally important for the individual, as its normal functionality is determined by the minimum amount of energy with which individual organism can respire and maintain the life functions. The life functionality of the organism is its primary and secondary metabolism and the consequences of energy production and consumption: growth,

Ec

Ee Ea

Ep Biological life

Ch, G

f

Efch n FIGURE 4.1 Biological life as a production of energy. Abbreviations: C, cell; Ch, chemistry; bor, bioorganic reactions; R, reactions; gl, global life; d, density; r, reduction; b, biotransformation; i, individuals; I, interactions; p, populations; e, environment; G, genes; f, form of life; Ec, cell energy; Ep, production of energy; Efch, energy form change; Ee, evolutionary energy; Ea, alternative energy.

262 CHAPTER 4 Ecology of alkaloids

reproduction, and aging changes up to cellular sclerosis and death. Individuals are born, grow, reproduce, becoming older, and die, but their energy transfer moves on to the next generation and into the environment. Without the functional metabolism and energetic processes of organisms, the occurrence of alkaloids is impossible. Functional metabolism is a sum of the pathways of chemical reactions to perform some tasks of functionality, and the energetic process is the result of work carried out, the capacity of to do work or transfer energy, supply heat. Therefore, all the life is based on chemistry although described by biology and ecology. Alkaloids, in a way similar to organisms, go the distance from birth to degradation. Circulation of energy in and outside organisms and the ability to change form is one of the most important life processes on the globe. Alkaloids are in this global process, although the individual organism energy is a product of chemical, biochemical, and molecular primary metabolism. A part of this energy is distributed into and by secondary metabolism. Some organisms, especially plants, have ability to produce secondary compounds with the objective to be in contact with, to be protected by, or to be attractive in relation to the environment. One group of such significant metabolomics is alkaloids.

4.1 MOLECULAR BASIS OF ECOLOGY Ecology is usually divided into sections such as organism, population, community, ecosystem, behavior, and evolution. All these sections are very important at the macro analytical level of the research with the objective to receive the data on organisms’ behavior, interactions, occurrence, diversity, and management. This division of ecology is more structural than vital for understanding life unit activities and their machinery and components. The ecology is presented by many ecologists as a static descriptive field based on mathematical and statistical analysis and large theories and hypotheses. In reality, ecology is based on biological and chemical grounds with strong dynamics at the macro level. Molecules are smallest units of these dynamics. The oikos cannot exist without molecules, life organic and inorganic, grouped to organisms, ecosystems, biomes, and the local, global, and universal environments. Therefore, ecology, in reality, is based on the ordering, changes, and interactions of molecules with other molecules in a local and global oikos and its stability, change over time, and evolution. Scientific characterization of one species (outecology), all communities (ecosystem ecology), and their interactions with other species, ecosystems, and even inorganic materials would not be understood today without strong molecular evidence of the claims. Moreover, interactions of between molecules play an important role, not only in all natural environments and ecology but also in industry. It is known today that biomolecules interact not

4.2 Alkaloids in interaction between life units 263

only with biomaterial but with inorganic surfaces and molecules.25 Researching this, the methods of atomic force and fluorescence microscopy, surface plasmon resonance, quartz crystal microbalance, spectroscopy (X-ray photoelectron, polarization modulation, infrared reflection absorption, sum frequency generation, and time of flight secondary ion mass spectroscopy) should be used to detect small changes of molecular status in a new material. The atomic scale of the molecular interactions, therefore, seems to be the basis for a better understanding of life processes and all ecology. In the literature, however, the process of moving from molecular inventories to functional understanding is recognized to be complex and challenging, and many thousands of dynamic interactions are needed for life or structural phenomena.63 Moreover, molecule-based approaches to the ecological studies reveal a broader diversity than obtained by use of other methods. The evidence of this is found at least in the case of microbial biomes and natural environments.10,11,25,27,31,33,54,63,107 Cells and molecules seems to be the basic activity units in all fields of ecological interaction. In this sense, the fundamentals of ecology are in molecules, both biotic and abiotic. Therefore, structural ecology is and will be in the future strongly molecular as well. Cross-molecular studies increases our vision and knowledge of the species, populations, ecosystems, biomes, and biotic and abiotic space in the global environment and universe, and their interactions will change and be deeply researched in the future. Technological development of molecular analyses will gain importance in all ecological fields, including their bio-physio-chemical diversity.

4.2 ALKALOIDS IN INTERACTION BETWEEN LIFE UNITS One type of molecules produced by living cells of the organisms are alkaloids, and the living cells can be considered units of life, although some times the gene or living protein can be considered the unit of life. As all other molecules, especially metabolomics and foodolomics, the alkaloids can interact with the other molecules inside the same life unit or outside it, with another life-unit molecule. Molecule-molecule interaction, molecule condensation, and dynamic evolution in time are basic characteristics of living organisms in spite of their size or the kingdom to which they belong. Different life units can interact using molecules of natural compounds or the synthetic compounds. Camptothecin, a plant alkaloid, can block both DNA and RNA synthesis in the human body. This is possible because this alkaloid interacts with human topoisomerase I, a molecule known as a monomeric enzyme. This molecule catalyzes the relaxation of supercoiled DNA during replication, transcription, recombination, and chromosome condensation.20 This kind of alkaloid interaction (inhibition) with enzymes

264 CHAPTER 4 Ecology of alkaloids

also is known in the case of some other natural molecules, such as terpenoids, flavonoids, some stilbenes, and fatty acids. Other examples of molecular interaction is the dependence of the alkaloid content of ragwort (Jacobaea vulgaris) on root herbivory;59 of indole alkaloids on mycorrhization in Catharanthus roseus and Nicotiana tabacum plants;3 of benzo[c]phenanthridine alkaloids on double-stranded DNA oligonucleotides of Sanguinaria canadiensis;75 of azoles on excess of vinca alkaloid;65 of biscoclaurine alkaloids, berbamine, isotetrandrine, and cycleanine interaction with heat shock protein 90 alpha;47 and of beta-carboline alkaloids from Peganum harmala L. on bovine serum albumin.66 Some studies have recognized that the molecules of keystone significance are vital in structuring ecological communities.37 Exchange of molecules between life units on the species level can lead to the evolution of functional status of keystone molecules in the new life unit. Chemical defenses of autotrophic or microbial species often originate from the introduction of these molecules. In the case of life units of consumer species, these molecules are used either in chemical defense against higher-order predators or as chemosensory cues that elicit alarm and predatory search. Alkaloids are just molecules of keystone significance that provide critical information to phylogenetically diverse life units on the species level, initiate major trophic cascades, and structure communities within terrestrial and marine habitats. Two guanine alkaloids (tetrodotoxin and saxitoxin), keystone molecules, are reported to have neurobiological and ecological aspects.37 Chemosensory-mediated behavior among life units (cells, individuals, species, etc.) and the ecological function of the molecules in transition seem to be very important in chemical cues and signals in higher levels of biological organizations.109 However, it is necessary to say that these problems have not been researched in detail, and future intensive research is needed.

4.3 BIOLOGICAL COMMUNITIES AND ALKALOIDS A biological community is an assemblage of populations living in the same area and environment and being a living part of the ecosystem. The biological community is dynamic and changes in space and time as a result of community structure, diversity, and development. In the biological community, the many strong processes of symbiosis, mutualism, competition, and antagonism between organisms occurs. These processes, especially their intensity, influence biological community growth or decline over space and time. Metabolomics, especially alkaloids, are chemical instruments of many organisms in a biological community for their vitality, defense, adaptation, competition, and capability in life (Figure 4.2). They are also

4.3 Biological communities and alkaloids 265

gol Chemical instruments in biological community

vdac

M ia A fw

fch

n FIGURE 4.2 Alkaloids as chemical instruments in the biological community. Abbreviations: v, vitality; d, defense; a, adaptation; c, competition; gol, get over in the life; fw, food web; fch, food chains; A, alkaloids; M, metabolomics.

important in the food web of the biological community. The food web is composed of many food chains, and some of these are controlled or attracted by alkaloids. Alkaloids can be produced and used by autotrophs (primary producers such as plants) and by heterotrophs (consumers or decomposers) (Figure 4.2). They can be also translocated by a consumer or a decomposer to another population and even another community. Sometimes, this translocation is a pathway of the activities of herbivores or carnivores, being constructed by historical evolution of the biological communities. A good example is the ladybird beetle (Coccillinedae), which hunts aphids feeding on plant juice containing some alkaloids. Alkaloids are located in ladybird body and used for protection. Moreover, ladybirds, as carnivore insects, have the ability to produce their own alkaloids, typical for the species (endoalkaloid) coccilline, being in parts colored red. An interesting point is that some ladybirds also can be omnivores, organisms that eat both animals and plants. In this case, the alkaloid consumption by food can be considerable. The alkaloid function in a biological community is therefore complex, and the ecological pathway can be long and difficult to study. Some populations are stronger than others, due to their ability to protect themselves. Some studies suggest that endophyte infection in some

266 CHAPTER 4 Ecology of alkaloids

populations can decrease species richness in the associated plant community. Other studies, however, suggest that endophyte (endosymbiont, fungus, or bacterium living with a plant) presence, such as in Festuca arundinacea, is important for all the community and has no negative relationship on community diversity.84 Fungal endophyte-grass symbioses are considered by some authors as a process that can produce dramatic ecological effects in both plant and animal communities and ecosystem processes, such as nutrient cycling. It is known that, within the tall fescue (Schedonorus arundinaceus) and fungal endophyte (Neotyphodium coenophialum), symbiosis with such alkaloids as loline and ergot alkaloids are produced. It is supposed on the basis of empirical experiments that fungally produced alkaloids directly inhibit fescue decomposition80 and, in this way, protect this species in the community. Other research proves that pyrrolizidine alkaloids associated with Senecio jacobea cannot protect this plant against soil fungi.9 On the other hand, it is known that the soil microorganisms can affect the chemistry of plants.19 However, each population in the biological community has its own role in the life dynamics. In the case of a marine environment, some cyanonobacterium Nodularia spumigena strains can produce lot of allelochemicals, especially alkaloids, that have antimicrobial activity.38,77 These phytochemicals are used in the biological marine community as promoters of some populations and antagonists of others. This mechanism is not still known in detail in marine biological communities, although it is a very important in many cases for understanding of the possibility of life itself. Moreover, recent studies on changes in biological communities suggest that the role of allelopathy is important in ecological restoration in terrestrial environments. The case of Cytisus scoparius, a legume in new growing place, proved that allelochemicals, including alkaloids, can influence the biological community immediately, by invasion, and persistently, by the killing or transformation of microbial communities.43 Competition among species and populations in the biological community is done in many cases by alkaloids or other metabolomics. In this sense, the most studied groups of alkaloids are pyrrolizidine and quinolizidine alkaloids. Competition among species in a community may alter or decrease species richness.

4.4 ALKALOIDS IN DIFFERENT ECOSYSTEMS The ecosystem consists of biological community and physical environment of the area, in which community is established and spends its life. In communities and all organisms, the endo- and exometabolomics important for normal functionality depends on the ecosystem conditions. Therefore the appearance of alkaloids is dependent also on the species and biocoenose life factors.

4.4 Alkaloids in different ecosystems 267

4.4.1 Alkaloids in aquatic ecosystems The topical direction of alkaloid research nowadays is the search for and description of new alkaloids from the aquatic, marine, and oceanic environments. These environments are biologically rich ecosystems covering nearly three fourths of the globe and can be divided into freshwater and saline (oceans) environments. Oceans contain 97% of the total water on the Earth and 3% is fresh water. Large numbers of organisms live in the aquatic ecosystems, and those, especially those from the oceans, are less or unknown. Therefore, the profound study of these ecosystems was and remains to be a challenge for ecologists, biologists, chemists, and pharmacologists. There is still hope that the oceanic ecosystem will hold an organism with special alkaloids having new model activity and strong cure for the diseases of civilization, especially for different cancers and the like. This supposition is based on the human experience with terrestrial ecosystems and plant-derived vinca alkaloids having efficacy in some cancers. However, the research of aquatic ecosystems is more difficult and generally not deeply recognized until recently; and the limitations of accessibility to aquatic samples generally have been overcome from the technical point of view only recently. From aquatic environments, many new alkaloids are found in such organisms as marine cyanobacteria, sponges, algae, and tunicates (Table 4.1). Table 4.1 Some new alkaloids from marine environments Alkaloid

Natural source

Aaptoline Acanthomanzamines Agelamadins Anatotoxin-a Aplysinopsins Aprotoxin Aurantiomides A – C Bistellettazines A – C Bistellettazole A Chartelline A Chartellamides A – C Cylindradines A – B Cylindrospermopsin

Aaptos suberitoides Acanthostrogylophora ingens Agelas sp. Cyanobacteria Aplysinopsis sp. Lyngbya majuscule Penicillium aurantiogriseum Stelletta sp. Stelletta sp. Chartella papyracea Chartella papyracea Axinella cylindratus Cyanobacterium Anabaena sp. Cyanobacterium Aphanizomenon ovalisporum Cylindrospermopsis raciborskii Raphidiopsis sp. Umezakia natans

Continued

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Table 4.1

Some new alkaloids from marine environments Continued

Alkaloid

Natural source

Dragmacidine F 7-epicylindrospermopsin Deoxycylindrospermopsin Dolabellin Ergosinine Fascaplysin Hanishin Hectochlorin Hydroxyterezine D Iboganine Lamellarin O Leptoclinidamines Lyngyabellins A - I Marineosins A - B Matemon Meridianins A – G 6-methoxyspriotryprostatin B Mirabilin N-methyldibromoisophakellin Netamine Nostocarboline

Halicortex sp. as cylindrospermopsin as cylindrospermopsin Lyngbya majuscule Pleurobranchus forskalii Hyrtios sp. Agelas ceylonica Lyngbya majuscula Asperigillus sadowi Theoretically active in novel aquatic models Ianthella sp. Leptoclinides durus Lyngbya majuscule Actinomycete Iotrochota purpurea Aphlidium meridianum Aspergillus sydowi Acanthela cavernosa Stylissa caribica Acanthella cavernosa Cyanobacterium Aphanothece Cyanobacterium Calotrix anomala Cyanobacterium Calotrix thermalis Cyanobacterium Cylindrospermum sp. Cyanobacterium Fischerella sp. Cyanobacterium Nostoc sp. Aspergillus sp. Hyrtios sp. Aspergillus sadowi Phorminidium sp. Pelomonas puraquae Psammopemma sp. Cyanobacteria Hyrtios sp. Securiflustra securifrons Penicillium janthinellum, Actinomycete Penicillium janthinellum, Actinomycete Penicillium janthinellum, Actinomycete Aspergillus sp. Aspergillus sp. Monanchora pulchra Kirkpatrickia varialosa Paecilomyces variotii

Notoamides F – K 6-oxofascaplysin 18-Oxotryprostatin A Phormidinines A – B Pelopuradazole Psammopemmins Saxitoxin Secofascaplysin Securamines A - D Shearine A Shearine D Shearine E Shornephine A Speradines Urupocidin A Variolins A – D Varioxepine A

Sources: Refs 8, 14, 17, 24, 36, 46, 48, 62, 64, 70, 83, 88.

4.4 Alkaloids in different ecosystems 269

A metabolomic approach to these organisms seems to be a prospective research area and can produce useful data for a deep understanding the nature of marine alkaloids. From cyanobacteria alone, more than 300 alkaloids were extracted, and they are reported to contain unusual chemical structures with strong biological activity.88,89,92 Cyanobacteria are bloom-forming prokaryotic microorganisms. They produce such toxic alkaloids as anatoxin a, saxitoxin, and cytotoxin cylindrospermopsin.14,46,64 They are very harmful to many animal species. Anatoxin a and cylindrospermopsin both inhibit protein synthesis in various cell types. Cylindrospermopsin, which is hepatotoxic polyketide-derived alkaloid, is presently considered not only as a cytotoxic but also a genotoxic molecule.64 Cyanobactrerium also produces nostocarboline, which is known as a strong inhibitor of acetyicholinosterase and trypsin. Nostocarboline is being considered a metabolite with a large regulatory function in the aquatic ecosystem.8 From the same environment, the fungi Aspergillus, Penicillium, and Actinomycytes species contain a very active alkaloids, such as prostatins, shearinines, and arcyriaflavin. Sponge-derived alkaloids include aaptamine, aldisine, amphimedine, arenosclerins, bastadins, manzamines, and haliclonacyclamies.106,108 A lot of alkaloids are also found in marine tunicates, such alkaloids as alphidine, diplamines, eudistomin, and kottaimides (Table 4.1). The cyabobacterium Lyngbya majuscula is currently reported to be found in the littoral zone and to the depth of 30 m in tropical, subtropical, and temperate regions as an important contributor to the coral reef ecosystem.48,90 This cyanobacterium produces lot of strong alkaloids, such as lyngbyatoxin A and debromoaplysiatoxin, which are highly bioactive and harmful to other animals and humans, with a variety of possible negative dermatological outcomes. Moreover, the natural occurrence of new alkaloids in marine and aquatic systems seems to be constructed as a barrier to the free movement of humans and many mammals in these environments. It is known that harmful cyanobacterium bloom in surface waters is accompanied with the production of lot of toxic alkaloids, having the characteristics of hepatotoxin, neurotoxin, cytotoxin, and dermatotoxin.24 Moreover, the results from some empirical research proves that, in the aquatic ecosystem, alkaloid effects as a neurotransmitter of some aquatic animals is possible.17 An example mentioned is the indole alkaloid ibogaine anxiolyticlike effects on zebra fish. Moreover, in the aquatic ecosystem of Ishigaki Island in Japan, the ergot alkaloid was currently found.95 This finding by Japanese researchers in 2013 is the new, because previously it was generally known that ergot alkaloids are typical and occur only in terrestrial ecosystems. The finding of ergosinine in an aquatic ecosystem leads to the probable finding of other alkaloids and neurotransmitters in this fascinating environment, which seems to be more and more chemically similar to the other ecosystems on the globe.

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The biological activity of these alkaloids is connected basically with their organism protection in the environment. Many of them are produced mostly as potent toxins against natural predators, but some are also used in the research of their diverse range of activities against diseases and possible development on the new drugs. For human society and especially marine and food economy, the occurrence of new alkaloids in aquatic ecosystems is problematic and harmful, although they open new possibilities for potential use in other fields, such as possible pharmacological uses and the development of effective practices for detoxification, such as depuration.44

4.4.2 Alkaloids in terrestrial ecosystems The larger portion of all alkaloids known are found in terrestrial ecosystems, especially in terrestrial plants, animals, and microorganisms. Plants are particularly rich in alkaloid metabolism. Cyanobacterial bloom in surface water are reported to be harmful not only for the aquatic basin animals but also for terrestrial plants, including food crop plants. Corbel, Mougin, and Bouaicha24 studied this problem empirically in France. They introduced cytotoxic alkaloids from cyanobacterial bloom into the soil ecosystem and measured the content of alkaloids in plants growing in this environment. Their results proved that plants from terrestrial ecosystems, including crop plants, can accumulate cyanotoxins originating from the aquatic system. If these results are confirmed, this means that alkaloid circulation from one ecosystem to another is possible also by physiological process through water uptake. Moreover, alkaloids as natural products are found from terrestrial alga Nostoc commune Vauch. Nostocionone, 3-oxo-beta-ionone, and indole alkaloids were found: scytonemin, N-(p-coumaroyl)tryptamine, and N-acetyltryptamine.67 Nostocionone and reduced scytonemin demonstrated string antioxidative activities. Moreover, in terrestrial ecosystems, alkaloids can circulate from one animal to another by sequestration. Maternally derived alkaloids are known to deter predation mechanisms in many species, such as the strawberry poison frog Oophaga pumilio.86

4.4.3 Climate and alkaloids Metabolite production is connected with environmental factors, especially growing factors. Growth and development are important, as they determine not only primary but secondary metabolism. This means that some species can produce different amounts of metabolites under different conditions. Climate change is one factor with strong implications to the alkaloid occurrence and density in their production by many organisms. It is connected with a climate influence into the new distribution of these organisms.

4.4 Alkaloids in different ecosystems 271

One example is aforementioned cyanobacterium Lyngbya majuscula and alkaloids lyngbyatoxin A and debromoaplysiatoxin.90 With an order to check the possible influence of climate change on secondary compounds, Uhlig et al.94 carried out interesting research in Norway. They applied a multianalyte LC-MS/MS method for the semi-quantitative determination of 320 fungal and bacterial metabolites in southern Norwegian cereals. They detected molecules of 46 metabolites of fungal origin in 76 samples of barley, oats, and wheat. It was found that such compounds as deoxynivalenol, HT-toxin, zearalenole, culmorin from Altenaria, Penicillium, Aspergillus species. A lot of other metabolites exist in high prevalence and relatively high concentrations. This information is important for understanding the possible induction of these metabolites in climate change to the wetter summer seasons preferable for fungi invasions, although no any radical catastrophe causing great suffering and destruction is expected, even if climate change is rapid and strong. The results of Uhlig et al.94 were based on samples taken during the unusually wet summer seasons 2010–2013 in southern Norway. Climate change will probably influence secondary metabolites, including alkaloids, more in an evolutionary than revolutionary way in nature. Maybe, after first increasing of fungal and bacterial metabolites affected by climate change, the contents of these molecules will be stabilized and balanced by discrete changes in metabolic systems as results of adaptation processes. However, it is necessary to pay attention to climate change effects in the future regarding changes in the feed of animals in nature and in livestock. Weston, Weston, and Hildebrand99 researched populations of Paterson’s curse (Echium plantagineum) with the objective to monitor the presence of pyrrolizidine alkaloids. Pyrrolizidine alkaloids and related N-oxides were found. It is just suggested that the abundance of secondary metabolites in Echium plantagineum is dependent on climate, and possible climate change might result in greater production of these plant-protective molecules in shoots and roots. Moreover, climate change effects on metabolite production seem to be only partly dependent on climatic conditions. The results of Swedish investigations on loline alkaloid production suggest that endophyte-infected plants produced loline alkaloids in different quantities without the influence of nitrogen fertilization levels. Endophyte-infected plants produced clearly more tillers per plant than plants without the endophyte. However, there was no significant difference in the proportion of reproductive tillers, winter survival, or total biomass between endophyteinfected and noninfected plants.16 Effects of elevated temperature, increased ultraviolet-B (UVB) radiation, and fertilization on the secondary chemistry of one-year-old Norway spruce (Picea abies L. Karst) seedlings was studied by Virjamo, Sutinen, and Julkunen-Titto97 at the University of

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Eastern Finland and Finnish Forest Research Institute in Joensuu. The first results in this subject suggest that, after one growing season, elevated temperature increased the shoot ratio and concentration of needle piperidine alkaloids. All experimental treatments have effected some changes in the chemistry of the seedlings. This suggests the existence of interaction and complex character of possible environmental changes on molecular content of the seedlings. The problem of current climate change research is the short period of empirical study and the construction of final judgements on data extremely short in time and subject. This kind of research, although of large volume, presently seems to have more political and philosophical aspects than pure scientific aspects. Therefore, even empirical results from only one growth period must be interpreted as only one of millions of other possibilities in nature.

4.4.4 Alkaloids and invasive species Species in ecosystem are in dynamic relationships. Competition between species is strong, and as a result of this, some species are able to establish larger populations than others. All the time, to the ecosystem, a new environment is a very important. It has been presently reported that the night blooming invasive weed Datura ferox, containing lot of chemicals including alkaloids, has reproductive success for invasion of in modified ecosystems such as crop fields.93 Soil communities through just their soil biota and interactions with plants determine the success of invasive species.91 For their success, some invasive species develop an adaptation to alkaloids and increase their tolerance. An example is the ladybird Harmonia axyridis, an invasive species occurring in large areas of Europe, Africa, and America. This invasive species is known as a predatory threat to native invertebrates, especially aphid-eating ladybirds. This is interesting because these aphid-eating ladybirds have species-specific alkaloid defenses. Taking aphid-eating ladybirds as food, Harmonia axyridis has developed a tolerance for ladybird prey alkaloids. Sloggett and Davis82 researched this alkaloid fate in Harmonia axyridis larvae after consumption of two other ladybird species with two alkaloids: isopropyleine and adaline. Isopropyleine is an alkaloid occurring historically within the predator’s native range, and adaline is a novel alkaloid in this system. Experimental results by Sloggett and Davis82 open a new direction in understanding the nature of alkaloid sequestering by animals. According to their interpretation, Harmonia axyridis has ability for rapid physiological modification of alkaloids historically exposed, such as isopropyleine, and has no ability to digest the novel, more toxic alkaloid (adaline). Physiological modification of alkaloids less

4.4 Alkaloids in different ecosystems 273

harmful to Harmonia axyridis probably occurs outside of the gut, and novel alkaloids exist for a long time unchanged in the body of Harmonia axyridis. Slogget and Davis82 suggest that metabolic alkaloid specialization as a result of physiological adaptation has made the invasive species Harmonia axyridis a successful predator of other ladybirds. The accumulation in the body of novel alkaloids without an ability to digest them proves just this specialization and its evolutionary origin. The strong competition ability of some invasive plants is based on alkaloids. For example, invasive plants as pale swallow-wort (Vincetoxicum rossicum) and black swallow-wort (Vincetoxicum nigrum) with large, strong expansion populations in the northeastern United States and southeastern Canada.41 The invasive success of these species are connected with the presence of the highly bioactive phenanthroindolizidine alkaloid (-)-antofine in roots, leaves, and seeds. (-)-Antofine has a strong allelopathic influence on the growth of neighboring plants, such as Asclepias tuberosa, Asclepias syriaca, and Apocynum cannabinum. Moreover this alkaloid has also antifungal and antibacterial activity. Allelopatic and bioactivity mechanisms give both pale and black swallow-worts an advantage over more competitive, better adapted, and more aggressive plants in the local ecosystems. Hornoy et al.53 studied the role of quinolizidine alkaloids in the invasive species gorse (Ulex europaeus) and concluded that quinolizidine alkaloid concentrations are traits integrated into the seed predation avoidance strategies of gorse. Some interesting results about the role of alkaloids in invasive species were reported by the researchers from Italy.42 They studied some molecular and cellular effects in the white seabream Diplodus sargus, which consumed in the diet the green alga Caulerim racemosa, an invasive species in the Mediterranean. An interesting finding is that the algal capacity to produce secondary metabolites that modulate the strong competition ability of some invasive plants is based on alkaloids. The level of caulerpin, an indole alkaloid from algae, was used as an indicator of molecular relationships in fish. Gorbi et al.42 concluded that a direct molecular relationship with caulerpin was not established by the empirical study. However, the Caulermin racemosa–enriched diet of the fish can modulate biotransformation and fatty acids metabolism of Diplodus sargus. Future long-term studies will produce the data that definitely clears this problem and will explain the molecular behavior of invasive species and its impact on the fish-controlled bioactive invasive populations. Ecological studies stress the bioactivity of alkaloid in advancing of the invasiveness of the species. In the clinical investigations, alkaloids are considered as just opposite. New alkaloids (e.g., new inone alkaloid derivatives) exhibited potent antiinvasive activity and reduced invasive activity of human MDA-MB-231

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breast cancer cells.35 Diversity of alkaloid bioactive mechanisms in relation to molecular invasiveness and aggressiveness is still open to scientific discussion.

4.4.5 Ecological role of alkaloids Animal responses to secondary compounds, including alkaloids, are as diverse as natural chemicals. In the case of alkaloids produced by plants, animal responses depend on evolutionary and coevolutionary factors. Some animals tolerate alkaloids relatively well, while others have well-developed detoxification systems. Some animals, including mammals, can be harmed or even poisoned by these compounds. There are many known cases of poisoning in cattle by pyrrolizidine alkaloids (senecionine) from the Senecio species.26,50,51,55,100 Anagyrine, from the quinolizidine alkaloid group with pyridine nucleus, has been known to cause skeletal deformities in the fetuses of pregnant cows consuming toxic lupines.4,22 Some animals, including dairy cows, have been shown to selectively feed on only alkaloid-poor green plants.6 Similar results were observed in a field test trial in 1983 at the Central Finland Research station in Torikka (Laukaa), where a lupine green mass of three cultivars, two bitter and one sweet, were offered to outdoor-grazing dairy cows. One cow approached the sweet green mass of one cultivar, tasted it, and continued to consume it for approximately 20 minutes. Afterward, it grazed on grass. Two other cows tried the bitter mass, tasted it, and in both cases spat it out. They were restless during the spitting and did not consume any more lupine mass. The behavioral responses were very important to the researchers. The chemical analysis of the grass matter clearly confirmed field observations and the animals’ consumption behavior. These tests were done later by many farmers in Finland interested in lupine production. The results were identical or a very similar. Their observations and experience lead to the conclusion, that a factor that influences animal behavior is connected not only with alkaloid content but also with the content of fiber of the lupine green mass. Younger, fresh green mass is more acceptable in eating than older mass. Too high a content of a fiber in green mass (high dry matter percentage) leads to the refusal of the proposed lupine feed by cows. However, this additional observation does not change the basic interpretation of the first test results; therefore, this is also a proof that a testing method of alkaloid analysis can be useful in some cases, especially when it is necessary to do so quickly, as in the decision on green mass quality as fodder. This simple test also provides interesting matter for discussion. One might ask what is the cow’s mechanism for recognizing the alkaloids in the green matter. Despite a lack of deep investigation into this question, the mechanism is most likely based on the

4.4 Alkaloids in different ecosystems 275

recognition of bitterness by the animal’s taste receptors. Moreover, the configuration of alkaloid skeletons might not be an adequate fit for the configuration possibilities of the taste receptors, since the lupine juice from green mass matter started the bitter taste reaction mechanism of a cow. The chemical configuration of alkaloid molecules and their suitability for animal taste receptors was an interesting study direction in the research group in the Department of Stereo Chemistry of the Adam Mickiewicz University in Pozna
n (Poland) and in the Department of Natural Product Chemistry of the University of Economy in Pozna
n (Poland) directed by Professor 81 Waleria Wysocka. They tried to find an answer to a simple question: Why is sparteine bitter? The explanation is proposed to be connected with a configuration of the chemical molecule. However, such results also provide evidence of the possibility of the development of low-alkaloid-content cultivars and generally highlight ecological problems of alkaloid consumption by animals. Moreover,the exactly measured taste of the alkaloid probably also is an indicator for the structural judgment in the chemistry of quinolizidine alkaloids. Animal sequestration of alkaloids is connected not only with taste but also with the toxicity of these compounds. It has been stated that the toxicity of alkaloids is very selective. Aniszewski5 reported on data with some LD50 coefficients for some alkaloids and some pesticides and compared their toxicity from a selectivity point of view. There was clear evidence that alkaloids (sparteine and lupanine) are much more toxic for vertebrates than are some pesticides (e.g., malatione, phenitrothione). For, invertebrates, pesticides were clearly more toxic than alkaloids. Selective toxicity coefficients (STCs) were counted by dividing the LD50 for vertebrates by the LD50 for invertebrates. When the STC ¼1.0, there is not selectivity; when STC >1, there in invertebrate selectivity; and when STC