Ergot The Genus Claviceps, 1st Edition

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This edition published in the Taylor & Francis e-Library, 2006.

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Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license

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Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the
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 CONTENTS

Preface to the Series vii

Preface ix
Names of Ergot in Various Countries of the World xi
Contributors xiii

1 The History of Ergot 1

Anacleto Minghetti and Nicoletta Crespi-Perellino

2 Biology and Life Strategy of the Ergot Fungi 25

Klaus B.Tenberge

3 The Taxonomy and Phylogeny of Claviceps 57

Sylvie Pazoutová and Douglas P.Parbery

4 Genetics of Claviceps purpurea 79

Paul Tudzynski

5 Biosynthesis of Ergot Alkaloids 95

Ullrich Keller

6 Physiological Regulation of Ergot Alkaloid Production and

Special Cultivation Techniques 165
Vladimír Køen

7 Ergot Alkaloids and Other Metabolites of the Genus Claviceps 173

Martin Buchta and Ladislav Cvak

8 Chemical Modifications of Ergot Alkaloids 201

Petr Bulej and Ladislav Cvak

9 Biotransformations of Ergot Alkaloids 229

Vladimír Køen

10 Analytical Chemistry of Ergot Alkaloids 267

Alexandr Jegorov

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vi CONTENTS

11 Parasitic Production of Ergot Alkaloids 303

Éva Németh

12 Saprophytic Cultivation of Claviceps 321

Zdenìk Malinka

13 Industrial Production of Ergot Alkaloids 373

Ladislav Cvak

14 Ergot Alkaloids and their Derivatives as Ligands for

Serotoninergic, Dopaminergic, and Adrenergic Receptors 411
Heinz Pertz and Eckart Eich

15 Antimicrobial and Antitumor Effects of Ergot Alkaloids

and their Derivatives 441
Eckart Eich and Heinz Pertz

16 Role of Ergot Alkaloids in the Immune System 451

Auna Fišerová and Miloslav Pospíšil

17 Toxicology of Ergot Alkaloids in Agriculture 469

Richard A.Shelby

18 Producers of Ergot Alkaloids out of Claviceps Genus 479

Anatoly G.Kozlovsky

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 PREFACE TO THE SERIES

There is increasing interest in industry, academia and the health sciences in

medicinal and aromatic plants. In passing from plant production to the eventual
product used by the public, many sciences are involved. This series brings together
information which is currently scattered through an ever increasing number of
journals. Each volume gives an in-depth look at one plant genus, about which an
area specialist has assembled information ranging from the production of the
plant to market trends and quality control.
Many industries are involved such as forestry, agriculture, chemical, food,
flavour, beverage, pharmaceutical, cosmetic and fragrance. The plant raw
materials are roots, rhizomes, bulbs, leaves, stems, barks, wood, flowers, fruits
and seeds. These yield gums, resins, essential (volatile) oils, fixed oils, waxes,
juices, extracts and spices for medicinal and aromatic purposes. All these
commodities are traded world-wide. A dealer’s market report for an item may
say “Drought in the country of origin has forced up prices”.
Natural products do not mean safe products and account of this has to be
taken by the above industries, which are subject to regulation. For example, a
number of plants which are approved for use in medicine must not be used in
cosmetic products.
The assessment of safe to use starts with the harvested plant material which
has to comply with an official monograph. This may require absence of, or
prescribed limits of, radioactive material, heavy metals, aflatoxin, pesticide
residue, as well as the required level of active principle. This analytical control is
costly and tends to exclude small batches of plant material. Large scale contracted
mechanised cultivation with designated seed or plantlets is now preferable.
Today, plant selection is not only for the yield of active principle, but for the
plant’s ability to overcome disease, climatic stress and the hazards caused by
mankind. Such methods as in vitro fertilisation, meristem cultures and somatic
embryogenesis are used. The transfer of sections of DNA is giving rise to
controversy in the case of some end-uses of the plant material.
Some suppliers of plant raw material are now able to certify that they are
supplying organically-farmed medicinal plants, herbs and spices. The Economic
Union directive (CVO/EU No. 2092/91) details the specifications for the
obligatory quality controls to be carried out at all stages of production and
processing of organic products.
Fascinating plant folklore and ethnopharmacology leads to medicinal
potential. Examples are the muscle relaxants based on the arrow poison, curare,
from species of Chondrodendron, and the antimalarials derived from species of
Cinchona and Artemisia. The methods of detection of pharmacological activity
have become increasingly reliable and specific, frequently involving enzymes in
bioassays and avoiding the use of laboratory animals. By using bioassay linked
fractionation of crude plant juices or extracts, compounds can be specifically

vii
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viii PREFACE TO THE SERIES

targeted which, for example, inhibit blood platelet aggregation, or have

antitumour, or antiviral, or any other required activity. With the assistance of
robotic devices, all the members of a genus may be readily screened. However,
the plant material must be fully authenticated by a specialist.
The medicinal traditions of ancient civilisations such as those of China and
India have a large armamentaria of plants in their pharmacopoeias which are
used throughout South East Asia. A similar situation exists in Africa and South
America. Thus, a very high percentage of the World’s population relies on
medicinal and aromatic plants for their medicine. Western medicine is also
responding. Already in Germany all medical practitioners have to pass an
examination in phytotherapy before being allowed to practise. It is noticeable
that throughout Europe and the USA, medical, pharmacy and health related
schools are increasingly offering training in phytotherapy.
Multinational pharmaceutical companies have become less enamoured of the
single compound magic bullet cure. The high costs of such ventures and the
endless competition from me too compounds from rival companies often
discourage the attempt. Independent phytomedicine companies have been very
strong in Germany. However, by the end of 1995, eleven (almost all) had been
acquired by the multina-tional pharmaceutical firms, acknowledging the lay
public’s growing demand for phytomedicines in the Western World.
The business of dietary supplements in the Western World has expanded from
the Health Store to the pharmacy. Alternative medicine includes plant based
products. Appropriate measures to ensure the quality, safety and efficacy of these
either already exist or are being answered by greater legislative control by such
bodies as the Food and Drug Administration of the USA and the recently created
European Agency for the Evaluation of Medicinal Products, based in London.
In the USA, the Dietary Supplement and Health Education Act of 1994
recognised the class of phytotherapeutic agents derived from medicinal and
aromatic plants. Furthermore, under public pressure, the US Congress set up an
Office of Alternative Medicine and this office in 1994 assisted the filing of several
Investigational New Drug (IND) applications, required for clinical trials of some
Chinese herbal preparations. The significance of these applications was that each
Chinese preparation involved several plants and yet was handled as a single IND.
A demonstration of the contribution to efficacy, of each ingredient of each plant,
was not required. This was a major step forward towards more sensible
regulations in regard to phytomedicines.
My thanks are due to the staff of Harwood Academic Publishers who have
made this series possible and especially to the volume editors and their chapter
contributors for the authoritative information.

Roland Hardman

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 PREFACE

Ergot (Claviceps purpurea) is best known as a disease of rye and some other
grasses. However, it is probably the most widely cultivated fungus and it has
become an important field crop.
The main reason for its importance is ergot alkaloids, which are extensively
used in medicine. No other class of compounds exhibits such a wide spectrum of
structural diversity, biological activity and therapeutic uses as ergot derivatives.
Currently, ergot alkaloids cover a wide spectrum of therapeutic uses as the drugs
of high potency in the treatment of uterine atonia, postpartum bleeding, migraine,
orthostatic circulatory disturbances, senile cerebral insufficiency, hypertension,
hyperprolactinemia, acromegaly and parkinsonism.
Ergot—once dreaded pest and cause of epidemic intoxications has now
become a profitable crop for farmers. However, the danger of intoxication and
crop damage still persists. The fungus was already well known in the middle ages,
causing outbreaks of ergotism or “epidemic gangrene” called for example, St
Anthony’s fire.
Ergot alkaloids are traditionally obtained by extraction of ergot sclerotia
artificially cultivated on cereals. The parasitic cultures are not able to produce
some, e.g., clavine alkaloids necessary for most semisynthetic drugs. Crop
fluctuations and market demands lead to the development of submerged
cultivation in production plants. Present trends in ergot cultivation are the
development of saprophytic cultivation processes and improvement of field
production by, for example, introduction of new hosts and ergot strains. Even
though there is a constant effort to prepare ergot alkaloids synthetically their bio-
production is still much more competitive. In the contemporary economical crisis
of agriculture, especially in Europe, the ergot is a good and profitable alternative
crop for farmers. Thanks to the new advanced technologies it experiences a real
renaissance.
Various strains of Claviceps served as models for study of the fungal
metabolism, biogenesis, physiological and genetic aspects of ergot alkaloids
production. This interest continues because of good perspectives of submerged
and field production of ergot alkaloids.
The volume on the Claviceps genus should provide readers with both
biotechnological aspects of ergot alkaloid production, genetic and physiological
data but also with newly emerging dangers of toxicology and environmental
risks of ergot infection and contamination of food and forage. Chemistry and
pharmacology of ergot alkaloids will demonstrate both their use as classical drugs
and their newly discovered pharmacological applications.

Vladimír Køen

ix
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 NAMES OF ERGOT IN VARIOUS
COUNTRIES OF THE WORLD

Anyarozs Hungary
Bakkaku, Ergot Japan
Çacdar Mahmuzu Turkey
Centeio erspigado Brazil
Cornezuelo de centeno Argentina
Cornezuelo de centeno Chile
Cornezuelo de centeno Paraguay
Cornezuelo de centeno Spain
Cravagem de anteio Brazil
Cravagem de anteio Portugal
Cuernicillo de centeno Mexico
Ergot England
Ergot United States
Ergot de seigle Belgium
Ergot de seigle France
Erperao de anteio Brazil
Erüsi bôdês briza Greece
Grano speronato Italy
Meldröje Denmark
Meldröye Norway
Mjöldryga Finland
Moederkoorn The Netherlands
Mutterkorn Austria
Mutterkorn Germany
Námel Czech Republic
Razema glavnica Yugoslavia
Secara cornuta Romania
Segale cornuto Italy
Sporyn’ja Russia
Sporyzs Poland

xi
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 CONTRIBUTORS

Martin Buchta Alexandr Jegorov

Galena Pharmaceutical Company Galena Pharmaceutical Company
74770 Opava 9 Research Unit
Czech Republic Branišovská 31
e-mail: [email protected] 37005 Èeské Budìjovice
Czech Republic
Petr Bulej e-mail: [email protected]
Galena Pharmaceutical Company
74770 Opava 9
Czech Republic Ullrich Keller
e-mail: [email protected] Max-Volmer-Institut für
Biophysikalische Chemie
Nicoletta Crespi-Perellíno und Biochemie
Department of Pharmaceutical Fachgebiet Biochemie und
Sciences Molekulare Biologie
University of Bologna Technische Universität Berlin
Via Belmeloro 6 Franklinstrasse 29
40126 Bologna 10587 Berlin-Charlottenburg
Italy Germany
e-mail:
Ladislav Cvak [email protected]
Head of R&D
Galena Pharmaceutical Company
747 70 Opava 9 Anatoly G.Kozlovsky
Czech Republic Laboratory of Biosynthesis of
e-mail: [email protected] Biologically Active Compounds
Institute of Biochemistry and
Eckart Eich Physiology of Microorganisms
Institut für Pharmazie II Russian Academy of Sciences
Freie Universität Berlin 142292 Pushchino
Koenigin-Luise-Strasse 2 Moscow Region
14195 Berlin (Dahlem) Russia
Germany e-mail: [email protected]
Anna Fišerová
Department of Immunology and Vladimír Køen
Gnotobiology Laboratory of Biotransformation
Institute of Microbiology Institute of Microbiology
Academy of Sciences of the Academy of Sciences of the
Czech Republic Czech Republic
Vídeòská 1083 Vídeòská 1083
142 20 Prague 4 142 20 Prague 4
Czech Republic Czech Republic
e-mail: [email protected] e-mail: [email protected]

xiii
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xiv CONTRIBUTORS

Zdìnek Malinka Miloslav Pospíšil

Galena Pharmaceutical Company Department of Immunology and
74770 Opava 9 Gnotobiology
Czech Republic Institute of Microbiology
e-mail: [email protected] Academy of Sciences of the
Czech Republic
Anacleto Minghetti Vídeòská 1083
Department of Pharmaceutical 142 20 Prague 4
Sciences Czech Republic
University of Bologna e-mail: [email protected]
Via Belmeloro 6
40126 Bologna Richard A.Shelby
Italy Department of Plant Pathology
e-mail: [email protected] 209 Life Sciences
Auburn University
Éva Németh AL 36849
University of Horticulture and USA
Food Industry e-mail: [email protected]
Villányi str. 29–43
1114 Budapest Klaus B.Tenberge
Hungary Institut für Botanik
e-mail: [email protected] Westfälische Wilhelms-Universität
Schlossgarten 3
Douglas P.Parbery 48149 Münster
Faculty of Agriculture, Germany
Forestry and Horticulture e-mail: tenberg@uni-muenster
University of Melbourne
Parkville 3052 Paul Tudzynski
Australia Institut für Botanik
Westfälische Wilhelms-Universität
Sylvie Pazoutová Schlossgarten 3
Institute of Microbiology 48149 Münster
Academy of Sciences of the Germany
Czech Republic e-mail: [email protected]
Vídeòská 1083
14220 Prague 4
Czech Republic
e-mail: [email protected]

Heinz Pertz
Institut für Pharmazie II
Freie Universität Berlin
Koenigin-Luise-Strasse 2
14195 Berlin (Dahlem)
Germany

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 1. THE HISTORY OF ERGOT

ANACLETO MINGHETTI and NICOLETTA CRESPI-PERELLINO

Department of Pharmaceutical Sciences,
University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy

1.1. THE EARLIER ALKALOIDS: ISOLATION AND STRUCTURES

Years ago a review on ergot alkaloids (EA) appeared with the title: “The
biosynthesis of ergot alkaloids; the story of the unexpected” (Floss, 1980). I
don’t believe that any other title could be more appropriate since the entire
history of EA research, from the discovery of ergotamine almost a century ago
until the present, has truly been the history of the unexpected.
The beginning of modern ergot alkaloid research dates back to 1918 when
A.Stoll isolated in crystalline form ergotamine (Stoll, 1945), an alkaloid present
in the sclerotia of the Claviceps purpurea fungus and patented it. In 1917 the
Sandoz pharmaceutical company of Basel granted Stoll, then a young Swiss
chemist and student of R.Willstaetter, already distinguished in the field of natural
products, the responsibility of setting up a laboratory and developing new drug
research (Stoll, 1965). Stoll proposed the goal of isolating the oxytocic active
principle present in Claviceps sclerotia, universally used in post partum
hemorrhages and now known as ergometrine. He hoped to do exactly as
Sertumer had done a century earlier in isolating the active principle morphine
from opium. Unfortunately, unlike morphine, ergometrine was not easily
extractable with solvents due to its tendency to remain in the aqueous phase
and, above all, because it was present in scarce quantities in the mixture of
alkaloids produced by the Claviceps sclerotia: often one tenth in comparison
with the production of ergotamine (Hofmann, 1964). This explains how Stoll
ended up finding ergotamine, the major and the most lipophilic alkaloid in the
extracted mixture, while looking for ergometrine. Nevertheless, ergotamine was
used for some time as an oxytocic drug but with poor results. In fact, the crude
drug (ground sclerotia) was used for many years in spite of serious dosage
problems. Stoll was credited with being able to isolate in the pure state the first
alkaloid of a series of almost one hundred products the majority of which were
present in traces in the sclerotia collected in the Black Forest region. Stoll isolated
from the mother liquor of ergotamine, also ergotaminine, a more liposoluble
alkaloid of the same elementary composition as ergotamine, but which was
dextrorotatory.
At the beginning of research on EA scientists had the following means for
characterizing a product: elemental analysis, melting point, characteristic
chromatic reactions and measurement of optical rotation. Elemental analyses

1
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2 ANACLETO MINGHETTI AND NICOLETTA CRESPI-PERELLINO

were the most reliable data while the melting point could vary by a few units
due to impurities or traces of the solvent present in the crystals used. The
characteristic chromatic reactions included entire classes of compounds, and
two reagents, Keller’s (1896) and Van Urk’s (1929), specific for the indoles
substituted in position 4, were used for EA. Optical rotation, largely used in the
characterization of isolated compounds, in the case of EA initially provided
more confusion than help. It is now known that all EA are levorotatory in
nature and that during extraction and isolation procedures these compounds
can turn to be dextrorotatory as a result of temperature, light or pH. It was for
this reason that Stoll, after having isolated ergotamine, found in the mother
liquor a notable quantity of ergotaminine which was for a long time believed to
be a natural product. In addition, dextrorotatory EA, when treated with acids
as in the case of the transformation of bases to salts, can retroisomerize to the
levorotatory state. Hence, the discrepancy which exists in literature between
the previously reported values of optical rotation for various isolated products
can be explained.
Other researchers had been trying to isolate the alkaloids present in Claviceps
for 50 years, and upon Stoll’s success/failure with ergotamine, this goal was
reached step by step. In 1875 C.Tanret, in the hopes of isolating ergometrine,
had obtained the so-called “ergotinine cristallisée” which was, contrary to its
name, a mixture of almost all the solvent extractable alkaloids and composed
mainly of peptide alkaloids (Barger, 1931). Tanret also dedicated himself to the
systematic research of the compounds present in C. purpurea sclerotia which
contain a significant quantity of lipids. He isolated several fatty acids and
identified two “sterines” in the non-saponifiable fraction, one with a higher
melting point which he called ergosterol and the other with the lower melting
point, phytosterol. These products were later found to be ubiquitous in the
plant kingdom. Regarding nitrogenous compounds, Tanret, isolated
ergothioneine (the betaine of thiolhistidine) histidine, choline, and betaine.
Trisaccharides such as clavicepsine, made of glucose and mannitol, as well as
disaccharides such as trehalose and free mannitol were also identified from the
sclerotia (Barger, 1931). Another researcher who was very active in the study of
the components of C. purpurea was F.Kraft, a Swiss pharmacist, who was
attracted by the purple color of the mature sclerotia and identified several
pigments including ergochrisine and ergoflavine as derivatives of secalonic acid
(Frank et al., 1973). In addition, Kraft extracted a fraction composed mainly of
ergotoxine group alkaloids which he called hydroergotinine (Hofmann, 1964).
Subsequent progress in isolating alkaloids produced by C. purpurea was made
by G.Barger and F.Carr (1907). These researchers isolated ergotoxine, which
was at first considered a pure product, but later was recognized as a mixture of
three alkaloids (ergocornine, ergocristine and ergocryptine). After the isolation
of ergotoxine, a mixture which was rarely constant, the interesting
pharmacological activities of other EA different from ergometrine, began to be
discovered.

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 THE HISTORY OF ERGOT 3

However, the problem concerning the oxytocic activity of Claviceps remained

unsolved because in vitro pharmacological tests to follow the activity in the
various steps of the extraction were not available. Stoll, after having realized
that ergotamine was not the active principle of Claviceps, ceased to work on
this topic for 16 years. But he suddenly woke up when he knew that an English
physician, C.Moir, had found the oxytocic activity to be present in the exhausted
aqueous phase after extraction of the alkaloids with organic solvents. Since
that moment Stoll devoted himself to the search of new Claviceps alkaloids. He
carried out this task so thoroughly that, after having isolated in less than one
decade four alkaloid couples (ergometrine/ inine, ergocristine/inine, ergokryptine/
inine and ergocornine/inine), he is now considered by everybody as “the father”
of the EA.
As reported above, C.Moir (1932) made the unexpected discovery that the
Claviceps oxytocic activity was present in the aqueous phase which remained
after the extraction with solvents of the main alkaloids. It should not be surprising
that in all that time many researchers had continued to look for ergometrine in
the organic extracts after having thrown it down the drain with the aqueous
phase. This is a rather frequent occurrence in research. As soon as Moir realized
his discovery, he published it and, with the help of the chemist H.W.Dudley,
devoted himself to the compound’s extraction. He achieved this goal three years
later (Dudley et al., 1935), and along with defining the chemical-physical
characteristics gave this new product the name ergometrine (Dudley et al., 1935)
with obvious reference to its activity on the endometrium. However, three other
laboratories were also working independently on the isolation of this compound
and as a result four papers describing a product isolated from Claviceps with
oxytoxic activity appeared in the literature in 1935. The authors include
M.Kharash and W.Legault from Chicago who named the product ergotocine
(Kharasch et al., 1935), M.Thompson from the Johns Hopkins University in
Baltimore who called his product ergostetrine (Thompson, 1935) and, finally,
A.Stoll from Sandoz in Basel who, being a chemist and not a physician, baptized
his long-sought-after compound ergobasine (Stoll et al., 1935) in reference to
its basic characteristics. The chemical-physical data for the isolated compounds
reported by the various authors were similar enough to infer that they referred
to the same molecule. In any case, after a great deal of polemics documented in
several articles (Thompson, 1935; Stoll, 1935) in order to settle the controversy,
the four researchers exchanged their respective products for chemical and
pharmacological comparison. The four groups came to the unanimous
conclusion that the slight differences in melting point and optical rotation were
due to the differing degrees of purity and that they had all found the same
compound. In short, ergometrine, ergotocine, ergostetrine and ergobasine were
synonyms (Kharasch et al., 1936). However, which name to adopt for this
molecule so as to avoid confusion in the literature remained to be decided. The
American authors opted for ergonovine as a substitution for ergotocine and
ergostetrine but the English and Swiss researchers didn’t agree and so today,

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4 ANACLETO MINGHETTI AND NICOLETTA CRESPI-PERELLINO

even after sixty years, the three synonyms ergonovine, ergobasine and
ergometrine (now the prevailing name) can be found in the literature. This
international race to identify such a compound demonstrates how great the
need was at the time for a drug which could save many mothers from dying for
post partum hemorrhage. After the isolation of ergometrine the classification
of the EA into “liposoluble alkaloids”, including those products with a peptide
chain such as ergotamine, and “water soluble alkaloids” such as ergometrine
was adopted. Given, however, that in reality ergometrine is not much more
water soluble than ergotamine, it is now preferred to define the first group as
ergopeptines and the second as “simple amides of lysergic acid”. The discovery
of ergometrine gave a significant boost to ergot alkaloid research, especially in
the Anglo-Saxon scientific world which was more interested in natural products
than its Germanic counterpart in which synthetic chemistry research was at
that time at its zenith.
The second half of the thirties was one of the most productive periods
regarding EA structural elucidation. From Tanret’s ergotinine English researchers
S.Smith and G.M.Timmis isolated a new peptide alkaloid, ergosine (Smith et
al., 1937). It is noteworthy that Smith in his first report, assigned no name to
his new alkaloid, (the third after ergotamine and ergometrine) “in order to
prevent possibly later unnecessary complications” (Smith et al., 1936). In fact,
the debate on ergometrine had just ceased. Almost contemporary Stoll isolated
from ergotoxine ergocristine (Stoll et al., 1937; Stoll et al., 1951). In the same
years also a great development in EA chemistry occurred. Smith and Timmis
while trying to understand the structure of EA refluxed ergotinine, ergotoxine
(Smith et al., 1932), ergotamine and ergotaminine (Smith et al., 1932) with
metanol in IN KOH and obtained, from all four products, a crystallized basic
compound which they named ergine. Although ergine had a much lower
molecular weight than the starting materials and no biological activity, it showed
all the reactions characteristic of the Claviceps alkaloids (Keller’s and Van Urk’s)
and therefore it was guessed that it had to be the fundamental nucleus of all
these compounds. The same researchers found ergine to be also the degradation
product of ergometrine which had a slightly higher molecular weight (Smith et
al., 1935). From this discovery it was clearly understood that ergometrine was
composed of ergine and a short side chain which lacked nitrogen.
Contemporaneously W.A.Jacobs and L.C.Craig, researchers at the Rockefeller
Institute in New York, carried out the degradation of ergometrine and found 2-
amino-propanol (Jacobs et al., 1935).
Since the structure of EA was still unknown, the same authors performed
nitric oxidation, acidic and basic hydrolysis, and pyrolysis of ergotinine in order
to elucidate its structure. It is not clear as to why these authors chose to use
ergotinine, known to be a mixture, rather than ergotamine, the only pure alkaloid
known at the time, perhaps because ergotamine was patented while ergotinine
was readily available. Nitric oxidation brought about the possible existence of
a methylindole ring as well as of a nucleus of methylhydroquinoline in the

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 THE HISTORY OF ERGOT 5

structure of ergotinine (Jacobs et al., 1932). Alkaline degradation was more

fruitful and, instead of ergine, gave an acid which crystallized easily and which
was named lysergic acid (Jacobs et al., 1934). Elemental analysis of this acid
revealed that its structure corresponded to that of ergine, minus a nitrogen
atom. When subjected to identical alkaline hydrolysis conditions ergine gave
lysergic acid and ammonia, thus demonstrating that ergine was the amide of
lysergic acid and that it contained three of the five nitrogens of ergotinine (Jacobs
et al., 1934). Lysergic acid was shown to contain only one carboxylic group
and a-N-CH3 group and gave all the chromatic reactions characteristic of
ergotinine (Jacobs et al., 1935). At the same time Smith and Timmis also isolated
lysergic acid from ergine (Smith et al., 1934) and two years later isolysergic
acid (Smith et al., 1936; Smith et al., 1936), thus indicating where in the EA
structure the isomerization point was located. Stoll later admitted that the
American researchers were more bold than him since he, knowing the sensitivity
of the EA, would have never thought of treating them with an aqueous solution
of boiling 7% potash (Stoll, 1965).
The structure of lysergic acid, which relation with tryptophan was already
evident from the Ehrlich and Van Urk reactions, remained to be established.
Jacobs and Craig from the fragments obtained by ergine degradation inferred
the tetracyclic ergoline structure (Jacobs et al., 1936) which they confirmed
with an elegant chemical synthesis (Jacobs et al., 1937; Jacobs et al., 1939). In
order to have the definitive structure of lysergic acid, the positions of the carboxyl
group (Jacobs et al., 1938) and of the double bond needed to be defined. Position
4 was assigned to the carboxyl group, as reported in Figure 1. Later position 7
was considered, but experimental data finally confirmed that this group was
located in position 8 (Uhle et al., 1945). The double bond had to be assigned to
the position conjugated with the indole aromatic ring, therefore between carbons
10–5 or 9–10 where rings C and D join. Although the former position was
initially believed to correct (Craig et al., 1938), the latter was finally confirmed
(Uhle et al., 1945). These structure-defining experiments led to the logical
attempts to chemically synthesize lysergic acid, a goal which was achieved 20
years later, with extremely discouraging yields (Stoll et al., 1954; Kornfeld et

Figure 1 The first structure of lysergic acid as reported by Jacobs et al. (1936)

Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the
Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.
6 ANACLETO MINGHETTI AND NICOLETTA CRESPI-PERELLINO

al., 1954; Kornfeld et al., 1956). In fact, in spite of its high cost and demand as
an intermediate for new drugs, the only source of lysergic acid is up to now the
natural one.
The elucidation of the structure of the side chain of the alkaloids present in
ergotinine or ergotoxine was performed mainly by Jacobs and Craig who in a
few years produced a lot of work on this topic. After alkaline hydrolysis of
ergotinine, by which lysergic acid was removed, the building blocks of the
aminoacidic moiety were found to be: proline, phenylalanine, pyruvic and α-
hydroxyisovaleric acids (Jacobs et al., 1935). The residue from alkaline
hydrolysis was treated with hydrochloric acid and was found to contain a
dipeptide which after acidic hydrolysis gave an equimolecular mixture of proline
and phenylalanine (Jacobs et al., 1935). This dipeptide originated from
ergocristine which was probably the major component of the starting material.
After hydrogenation of ergotinine the same authors found isobutyric, formic,
and α-hydroxyvaleric acid (Jacobs et al., 1938). The isolation of all these
compounds meant a solution to the ergotinine, or rather ergotamine,
composition puzzle, but not in a correct sequence, as reported in Figure 2.
Research on new alkaloids present in C. purpurea was being carried out in
parallel with EA structure research. Stoll, besides ergotamine, isolated two other
peptide alkaloids from ergotinine: ergocornine and ergokryptine (Stoll et al.,
1943), the latter as its name indicates being the most difficult to isolate since it
was a minor component in the mixture. In 1943 Stoll published the isolation
and the structures of these two new alkaloids (Stoll et al., 1943), perhaps with
the thought that he had almost exhausted the series of alkaloids present in
Claviceps and also being thankful for having worked in the peaceful island
known as Switzerland. Surely he never imagined that at that time a new and
unexpected chapter in EA history was being opened in war-torn England. Here
indeed, the era of microbial fermentation had begun.
Nineteen forty-three was a historic year in EA research thanks to the unexpected
discovery of the hallucinogenic properties of lysergic acid diethylamide (LSD) a
synthetic derivative of lysergic acid. LSD had already been synthesized in 1938 in
a screening of compounds with oxytocic activity, and tested in comparison with
ergometrine, but it was impossible to note its effects on the central nervous system

Figure 2 The first structure of ergotamine as reported by Jacobs et al. (1938)

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Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.
 THE HISTORY OF ERGOT 7

since trials were carried out on laboratory animals. It was found only to have less
oxytocic activity than ergometrine. In the spring of 1943 A. Hofmann, in the
Sandoz labs in Basel, resynthesized LSD in order to further investigate its analeptic
activity owing its structural analogy to nikethamide (Stoll et al., 1943). Without
realizing it, Hofmann contaminated himself with the product. Returning home
from work he noticed that he was having reactions and sensations completely
new and unusual for an orderly and methodical person like himself. He understood
immediately that what he was experiencing was the result of something he had
come into contact with at work and since he hadn’t voluntarily inflicted anything
on himself he concluded that traces of a product from the laboratory were causing
the strange effects. It is not unusual for a chemist, even one as precise as Hofmann,
to become contaminated by working at the lab bench. In fact, the cases of self-
contamination are much more frequent than one would expect but are rarely
evident because of the low biological activity of the product as well as the dose,
usually less than a milligram, taken. The effect Hofmann experienced was so
strong with respect to the dose ingested that he, like any curious researcher, couldn’t
keep himself from discovering which product had caused the sensations. He
therefore separately ingested every product in small doses he had handled that
day. He began by carefully taking 0.25 mg of each substance, and after ingesting
the product labeled LSD 25 the hallucinations and feelings that he had experienced
before burst inside him again. In his personal trial he took a quantity of LSD
which corresponded to 5 times the active dose. It would be funny to think about
how things would have been if Hofmann, had stopped at a bar with his friends,
the habit of many researchers, instead of going directly home. He would have
attributed the observed anomalies to something else and we would have never
known about LSD, whose effects on the central nervous system would have been
felt only by the few laboratory animals treated in 1938 and 1943. LSD remained
a molecule used only in psychiatry and under strict medical control for 20 years.
This was due mainly to the limited availability of lysergic acid which was obtainable
only from C. purpurea sclerotia. However, its numerous and peculiar biological
activities increased the reputation of lysergic acid year by year for its use by the
pharmaceutical industries interested in new drug research.

1.2. THE FERMENTATION ERA

The discovery of penicillin and its production in large scale caused the explosion
of microorganism fermentation technology in the major pharmaceutical
companies which, in the second half of the 1940’s, began research on new
antibiotics. The earliest attempts to obtain EA from C. purpurea in saprophytic
conditions were performed by several authors, all in academic institutions. Their
aim was to investigate the best growth conditions in connection with the
production of alkaloids (Bonns, 1922; Kirchhoff, 1929; McCrea, 1931; Baldacci,
1946). Despite the luxuriant growth of Claviceps in some cases, only traces of

Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the
Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.