2.8.6 Ergot Alkaloids
Ergot is a fungal disease very commonly and widely observed on a good number of wild as well as cultivated grasses, and is produced by different species of claviceps. This particular disease is usually characterized by the formation of hard and seedlike ‘ergots’ in place of the normal seeds. However, these specific structures are frequently termed as sclerotia, which represent the ‘resting stage’ of the fungus.
The generic name, ‘claviceps’, usually refers to the club-like nature of the sclerotium*, whereas purpurea signifies its purple colour. As these sclerotia are elongated and somewhat pointed in shape and appearance, hence the common name of spurred rye has been assigned to the drug.
Medicinal ergot is the dried sclerotium of the fungus Claviceps purpurea (Fries) belonging to the natural order Clavicipitaceae developed in the ovary of rye, Secale cereale (Germinae/Poaceae).
There are certain other species of Claviceps which have been found to produce ergots in the ovaries of other member of Graminae and Cyperaceae.
In fact, there exist four main categories of ergot alkaloids which may be distinguished, namely:
(a) clavine alkaloids, (b) lysergic acids, (c) lysergic acid amides, and (d) ergot peptide alkaloids.
There are, in fact, ten ergot peptide alkaloids which are: ergotamine, ergosine, ergocristine, ergocryptine, ergocornine, ergotaminine, ergosinine, ergocristinine, ergocryptinine, and ergocorninine; however, the last five alkaloids being isomers of the first five. The aforesaid alkaloids are beautifully typified by a structure comprising of four components, viz., lysergic acid, dimethylpyruvic acid, proline and phenylalanine strategically joined together in amide linkages as depicted below:
Interestingly, the poisonous properties of ergots in grain, specifically rye, for animal as well as human consumption, purposefully and unknowingly, have long been recognized. The dreadful causative agents are collectively termed as the ‘ergot alkaloids’, containing essentially an indole nucleus. These, group of alkaloids are also referred to as ‘ergolines’.
The three important and typical members of the ergot alkaloids (ergolines), namely: ergonovine, ergotamine and lysergamde (ergine) shall be discussed individually in the sections that follows:
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* Sclerotium A hardened mass formed by the growth of certain fungi. THe sclerotium formed by ergot on rye is of
medical importance due to its toxicity.
A. Ergonovine
Synonyms Ergometrine; Ergobasine; Ergotocine; Ergostetrine; Ergotrate; Ergoklinine; Syntometrine.
Biological Sources It is obtained from the seeds of Ipomea violaceae Linn. (Ipomea tricolor Cav.) belonging to family Convolvulaceae (Morning glory, Tlitliltzen, Ololiuqui); and also from the dried seeds of Rivea corymposa Hall. F. (Convolvulaceae) (Snakeplant).
The percentage of Ergometrine and Ergine present in the Rivea and Ipomea species are as given below:
Chemical Structure
[8β (s)]-9, 10-Diadehydro-N-(2-hydroxy-1-methylethyl)-6-methylergoline-8-carboxamide; (C19H23N3O2).
Isolation The following steps may be followed stepwise:
1. The seeds are dried, powdered, sieved and finally defatted with n-hexane in a Soxhlet apparatus.
2. The defatted mare is extracted with hot dilute sulphuric acid (6N) successively. The acid extract is then treated with on excess of barium sulphate, and the barium is removed with CO2 and subsequent filtration.
3. The resulting filtrate is then concentrated by evaporation under reduced pressure.
4. The concentrated solution is taken up in ethanol, made alkaline with NH4OH and subjected to extraction with chloroform successively.
5. The resulting chloroform extract is further extracted with dilute H2SO4 (6N). The acidic solution is made alkaline with ammonia and saturated with NaCl and then extracted with ether several times.
6. The solvent is removed from the ether extract under vacuo leaving the alkaloidal residue.
7. Ergonovine may be recrystallized from acetone.
It may also be prepared from D-lysergic acid and L (+)-2-amino-1-propanol by the method of Stoll and Hofmann.*
Characteristic Features
1. Ergonovine is obtained as tetrahedral crystals from ethyl acetate, and as fine needles from benzene. It tends to form solvated crystals having mp 162°C.
2. It has specific optical rotation [α]20D + 90° (in water).
3. Its dissociation constant is pKa 6.8.
4. It is found to be freely soluble in lower alcohols, acetone and ethyl acetate; more soluble in water than the other principal alkaloids of ergot; and slightly soluble in chloroform.
Identification Tests As per se the ergot alkaloids may be identified either by general precipitation and colour reactions or by preparing their derivatives as stated below:
(a) Precipitation Reactions
(i) The ergot alkaloids are readily precipitated by the alkaloidal reagents. However, Mayers reagent is regarded to be the most sensitive test whereby on opalescence in dilutions of 1 ppm can be obtained.
(ii) Iodine solution in KI also gives an instant precipitate with very dilute solutions of ergot alkaloids.
(b) Colour Tests: The most vital colour tests are given as under:
(i) Keller's Test: To a solution of the alkaloid in glacial acetic acid add a few mg of solid FeCl3 and then add 1-2 ml of concentrated sulphuric acid along the side of the tube. The appearance of an intense blue colouration is accomplished at the junction of the two layers.
(ii) Van Urk Test: When a solution containing an ergot alkaloid is mixed with Van Urk Reagent**, it gives rise to a characteristic deep blue colouration.
Note: (a) Van Urk Reagent may also be used in spraying developed paper chromatograms of the ergot alkaloids, and for this purpose 10% (v/v) HCl is used instead of H2SO4.
(b) The spectrophotometric assay for total ergot alkaloids is also based on the blue colour given with Van Urk Reagent.
(iii) Glyoxylic Acid Reagent Test: Ergot alkaloids gives a blue colouration with the addition of Glyoxylic acid reagent and a few drops of concentrated H2SO4.
(iv) Fluorescence Test: The aqueous solution of the salts of ergot alkaloids produce a distinct blue fluorescence.
(c) Derivatives of Ergonovine: The various derivatives of ergonovine are as follows:
(i) Ergonovine Maleate (Ergometrine Maleate) (C19H23N2O2.C4H4O4) [Synonyms Cornocentin; Ermetrine; Ergotrate Maleate]: It is obtained as crystals that decompose at 167°C. It has specific optical rotation [α]25D + 48° to + 57°. 1g dissolves in 36 ml water and 120 ml ethanol. It is almost insoluble in chloroform and ether.
(ii) Methylergonovine Maleate (C20H25N2O2.C4H4O4): It is a semisynthetic homologne of ergonovine and prepared from lysergic acid and 2-aminobutanol. It is obtained as a white to pinkish-tan microcrystalline powder.
(iii) Ergonovine Tartrate Hydrate (Ergometrine Tartrate Hydrate) [(C19H23N3O2)2. C4H6O6.H2O] (Basergin, Neofermergen): It is obtained as crystals that are slightly soluble in water.
Uses
1. Ergonovine is used as an oxytocic.
2. Ergonovine maleate also acts as an oxytocic and produces much faster stimulation of the uterine muscles as compared to other ergot alkaloids.
3. Methylergonovine meleate is observed to act as an oxytocic whose actions are slightly more active and longer acting than ergonovine.
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* Stoll, A., and Hofmann, A., Helv. Chim Acta, 26, 944 (1943).
** Van Urk Reagent Mix togetehr 0.125g of para-dimethylamino. benzaldehyde; 0.1 ml of FeCl3 soln. (5% w/v),
and 15% (v/v) H2SO4 to make 100 ml.
B. Ergotamine
Biological Source It is obtained from the seeds of Claviceps purpurea (Fr.) Tul. (Hypocreales) (Ergot).
Chemical Structure The chemical structure of ergotamine has been given in Section 7.2.8.6.
Isolation The method of Stoll* may be adopted as stated below:
1. The powdered dried ergot is first defatted with n-bexane or petroleum ether (40-60°)
2. The marc consisting of the defatted powdered ergot is thoroughly mixed with aluminium sulphate and water so as to fix the alkaloids by converting them into the double salts.
3. The resulting alkaloidal double salts are subjected to continuous extraction with hot benzene that removes the alkaloid exclusively on one hand; and the unwanted substances e.g., ergot oil, soluble acid, neutral substances like-phytosterol, colouring matter and organic acids on the other.
4. The benzene is removed under vacuo and the residue thus obtained is stirred for several hours with a large volume of benzene and subsequently made alkaline by passing NH3 gas.
5. The resulting solution is filtered and the benzene extract is concentrated under vacuo to approximately 1/50th of the original volume, whereupon ergotamine crystallizes out.
6. An additional quantity of ergotamine may also be crystallized from the mother liquour by treatment with petroleum ether.
7. Ergotamine may be further purified by crystallization from aqueous acetone.
Characteristic Features
1. It is obtained as elongated prisms from benzene that get decomposed at 212-214°C.
2. It usually becomes totally solvent-free only after prolonged heating in a high vacuum.
3. It is found to be highly hygroscopic in nature; and darkens and decomposes on exposure to air, heat and light.
4. It has specific optical rotation [α]20D - 160° (chloroform).
5. It is soluble in 70 parts methanol, 150 parts acetone, 300 parts ethanol; freely soluble in chloroform, pyridine, glacial acetic acid; moderately soluble in ethyl acetate; slightly soluble in benzene; and practically insoluble in petroleum ether and water.
Identification Tests The precipitation reactions and the colour tests are the same as described under ergonovine. However, the specific derivatives of ergotamine are as stated below:
1. Ergotamine Tartrate [(C33H35N5O5)2.C4H6O6] (Ergomar; Ergate; Ergotartrat; Ergostat; Exmigra; Fermergin; Lingraine; Gynergen; Lingran): It is normally obtained as solvated crystals e.g., the dimethanolate; also occurs as heavy rhombic plates from methanol having mp 203°C (decomposes). It has specific optical rotation [α]25D – 125° to – 155° (C = 0.4 in chloroform). One gram dissolves in either 500 ml of ethanol or water.
2. Ergotamine Hydrochloride [C33H35N5O5.HCl]: It is obtained as rectangular plates from 90% (v/v) ethanol which get decomposed at 212°C. It is found to be soluble in water-ethanol mixtures; and sparingly in water or ethanol alone.
3. Dihydroergotamine Mesylate (C33H37N5O5.CH3SO3H) (Agit;1 Dihydro-ergotamine methane sulphonate; Angionorm; DET MS; Dergotamine; D.H.E. 45; Diergo; Dihydergot; Dirgotarl; Endophleban; Ergomimet; Ikaran; Migranal; Morena; Ergont; Ergotonin; Orstanorm; Tonopres; Verladyn; Seglor): It is obtained as large prisms from 95% (v/v) ethanol having mp 230-235°C; and moderately soluble in water.
Note: (a) It is the salt of a semisynthetic alkaloid prepared from ergotamine by hydrogenation of the ∆9 double bond in the lysergic acid nucleus.
(b) It is mostly used in the treatment of migraine because it is found to be better in efficacy and more tolerated than the parent alkaloid.
Uses
1. It is employed as a potent antimigraine drug.
2. Ergotamine possesses oxytocic properties, but it is not employed for that effect.
3. Ergotamine tartrate is used invariably to prevent or abort vascular headaches, including migraine and cluster headaches. The mechanism of action is perhaps due to direct vasoconstriction of the dilated carotid artery bed with concomitant lowering in the amplitude of pulsations.
4. Ergotamine tartrate is also an antagonist of the serotonin activity.
5. Ergotamine tartrate is frequently used along with caffeine for the management and control of migraine headache. Both serve as cerebral vasoconstrictors; while the latter is considered to increase the action of the former.
6. Methylergonovine maleate is an oxytocic reported to be longer acting and more active than ergonovine.
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* Stoll, Helv. Chim. Acta 28, 1283, (1945)
C. Ergine
Synonyms Lysergamide; Lysergic acid amide;
Biological Sources It is obtained from the immature seeds of Argyreja nervosa (Burm.) Bojer (Convolvulaceae) (Wood Rose, Silver Morning Glory); Beeds of Ipomea Violaceae L. (Convolulaceae) (Tlitliltzen, Ololiuqui); seeds of Rivea corymbosa Hall. F. (Convolvulaceae) (Snakeplant); and also from the seeds of Ipomea tricolor Cav (Convolvulaceae).
Chemical Structure
9, 10-Didehydro-6-methylergoline-8β-carboxamide; (C16H17N3O).
Isolation It is isolated from the seeds of Rivea corymbosa (L.) and from Ipomea tricolor Cav. By the method of Hofmann and Tscherter.*
Characteristic Features
1. It is obtained as prisms from methanol which get decomposed at 242°C.
2. It has a specific optical rotation of [α]205461 + 15° (C = 0.5 in pyridine).
Identification Tests The precipitation reactions and the colour tests are the same as described under ergonovine (Section A).
Ergine may also be identified by forming its derivative as stated below:
Ergine Methane Sulphonate (C16H17N3O.CH3SO3H) It is obtained as prisms from a mixture of methanol and acetone that get decomposed at 232°C.
Uses It has a pronounced depressant action.
Note: It is a controlled substance listed in the U.S. Code of Federal Regulations. Title 21 Part 1308, 13 (1995).
Biosynthesis of Ergotamine The various steps involved in the biosynthesis of ergotamine are as enumerated below:
1. Three amino acids, viz., L-alanine, L-phenylalanine, and L-proline in the presence of ATP and enzyme SH; or D-(+)-lysergic acid in the presence of ATP and enzyme SH undergo two steps: first-activation via AMP esters, and secondly-attachment to the respective enzymes, thereby giving rise to an intermediate. It is worthwhile to observe that the enzyme is comprised of two subunits that essentially bind the substrates as indicated in the biosynthetic pathway given below.
2. The comparatively more complex structures comprising of the peptide fragments, such as: ergotamine are eventually formed by sequential addition of amino acid residues to the thioesterbound lysergic acid, yielding a linear lysergyl-tripeptide covalently attached to the enzyme complex.
3. The resulting complex undergoes lactam formation followed by release from enzyme. In other words, the cyclized tripetide residue is rationalized instantly by the formation of a lactam (amide) that releases ultimately the product from the enzyme.
4. This resulting product first affords hydroxylation then followed by generation of a hemeketallike linkage to give rise to the formation of ergotamine.
All these aforesaid steps (1) through (4) have been duly depicted in the following biosynthetic pathway.
Peptide Alkaloids in Ergot Interestingly, it has been observed critically that three amino acids, namely: alamine, phenylatine and proline, actually from the basis for the various structures which are encountered in the domain of the ‘ergot alkaloids’. Therefore, these known and established structures may be subdivided into three major groups which are: ergotamine group, ergoxine group, and ergotoxine group.
The various alkaloids having the peptide linkages found in ‘ergot’ are depicted as under.
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* Hofmann and Tscherter, Experientia, 16, 414 (1964).
