Chemical analysis of medical plant materials that contain alkaloids.
Alkaloids are complex organic compounds of basic nature containing nitrogen. They are vegetable origin (seldom animal origin) and have very strange, specific physiological effect on the body. The name “alkaloid” is derived from Arabian alkali – alkali and Greek eidos – similar to alkali.
A precise definitiono f the term ‘alkaloid’ (alkali-like) is somewhat difficult because there is no clear-cut boundary between alkaloids and naturally occurring complex amines. Typical alkaloids are derived from plant sources, they are basi, they contain one or more nitrogen atoms(usually in a heterocyclic ring) and they usually have a marked physiological action on man or other animals. The name ‘proto-alkaloid’ or ‘amino-alkaloid’ is sometimes applied to compounds such as hordenine, ephedrine and colchicine which lack one or more of the properties of typical alkaloids. Other alkaloids, not conforming with the general definition, are those synthetic compounds not found in plants but very closely related to the natural alkaloids (e.g. homatropine). In practice. Those substances present in plants and giving the standard qualitative tests outlined below are termed alkaloids, and frequently in plant surveys this evidence alone is used to classify a particular plant as ‘alkaloid-containing’.
HISTORY
The first isoltrtions of alkaloids in the nineteenth century followed the reintroduction into medicine of a number of alkaloid-containing drugs and were coincidental with the advent of the percolation process for the extraction of drugs. The French apothecary Derosne probably isolated the alkaloid afterwards known as narcotine in 1803 and the Hanoverian apothecary Serttilner further investigated opium and isolated morphine (1806, 816).Isolation of other alkaloids, particularly by Pelletier and Caventou. rapidly followed; stlychnine (1817). emetine (1817), brucine (1819), piperine (1819). caffeine (1819), quinine (1820), colchicine ( I 820) and coniine ( I 826). Coniine was the first alkaloid to have its structure established (Schiff. 1870) and to be synthesized (Ladenburg, 1889),but for othes, such as colchicine, it was well over a century before the structures were finally elucidated.
Modern methods and instrumentation have greatly facilitated these investigations, and it is interesting to note that the yields of ‘minor’ alkaloids. too small for further investigation, isolated by chemists during the first quarter of the lastc century would now be sufficient, several thousand times over, for a completes structure analysis. In the second half of the twentieth century alkaloids featured strongly in the search for plant drugs with anticancer activity. A notable success was the introduction of Catharanthus alkaloids and paclitaxel into medicine and there is much current interest in other alkaloids having anticancer properties as well as those exhibiting antiaging and antiviral possibilities.
DISIRIBUTION
Some I50 years of alkaloid chemistry had resulted by the mid-l940s in the isolation of about 800 alkaloids: the new technology of the next 50 vears increased this figure to the order of l0 000.
True alkaloids are of rare occurrence in lower plants. In the fungi the lysergic acid derivatives and the sulphur-containing alkaloids, e .g. the gliotoxins, are the best known. Among the pteridophytes and gymnosperms the lycopodium, ephedra and Taxus alkaloids have medicinal interest. Alkaloid distribution in the angiosperms is uneven. The dicotyledon orders Salicales. Fagales. Cucurbitales and Oleales at present appear to be alkaloid-free. Alkaloids are commonly found in the olders Centrospennae(Chenopodiaceae), Magnoliales( Lauraceae, Magnoliaceae). Ranunculales (Berberidaceac. Menispermaceae, Ranunculaceae), Papaverales( Paparreraceae, Fumariaceae), Rosales (Leguminosae sub family Papilionaceae), Rutales( Rutaceae), Gentialcs (Apocynaceae, Loganiaceae, Rurbiaceae) Tubiflorae (Boraginaceae, Convolvulaceae, Solanaceae) and Campanulales (Campanulaceae, sub-family Lobelioideae; Compositae, subfamily Senecioneae).
Hegnauer, who has made an intensive study of alkaloid distribution, while recognizing the undoubted potential chenotaxonomic significance of this group. is cautious about its use without due regard to all the other characters of the plant. Nevertheless it continues to be a popular area of research.
Nearly 300 alkaloids belonging to more than 24 classes are known to occur in the skins of amphibians along with other toxins. They include the potent neurotoxic alkaloids of frogs of the genus Phtyllobates. which are among some of the most poisonous substances known. Other reptilian alkaloids are strongly antimicrobial. Alkaloids derived from mammals include ones of indole and isoquinoline classes afew are found in both plants and animals.
PROPERTIES
Most alkaloids are well-defined crystalline substances which unite with acids to form salts. In the plant they may exist in the free state, as salts or as N-oxides (see below). ln addition to the elements carbon, hydrogen and nitrogen, most alkaloids contain oxvgen. A. f’ew, such as coniine from hemlock and nicotine from tobacco, are oxygen-free and are liquids.
Although coloured alkaloids are relatively rare, they are not unknown: berberine, for exarnple is yellow and the salts of sanguinarine are copper-red.
A knowledge of the solubility of alkaloids and their salts is of considerable pharmaceutical importance. Not only are alkaloidal substances often adrninistered in solution, but also the differences in solubility between alkaloids and their salts provide methods for the isolation of alkaloids from the plant and their separation from the nonalkaloidal substances also present. While the solubilities of diff’erent alkaloids and salts show considerable variation, as might be expected from their extremely varied structure, it is true to say that the free bases are frequently sparingly soluble in water but soluble in organic solvents; with salts the reverse is often the case, these being usually soluble in water but sparing soluble in organics olvents. For example, strychnine hydrochloride is much more soluble in water than is strychnine base. It will soon be realized that there are many exceptions to the above generalizations, caffeine (base) being readily extracted from tea with water and colchicines being soluble in either acid. neutral or alkaline water. Again, some alkaioidal salts are spalingly soluble-for example. quinine sulphate is only soluble to the extent of I part in 1000 parts of water, although 1 part quinine hydrochloride is soluble in less than 1 part of water.
Types of classification
Biogenic classification
Veritable alkaloids are formed of aminoacids and have heterocycle with nitrogen atom in molecule.
Proalkaloids are formed of aminoacids and don’t have heterocycle (biogenic amines, aminoalkaloids)
Pseudoalkaloids: the group of alkaloids, genetically bind with terpenoids (isoprenoids). They are divided into monoterpens (actinidine), sesquiterpens, diterpenoids, steroid pseudoalkaloids.
Biosynthetic classification– after aminoacids name that alkaloids are formed of.
Pharmacological classification – after pharmacological properties: alkaloids – narcotic analgetics; alkaloids that stimulate Central Nervous System (CNS)
Phylogenetic classification – according to the close principles of botanical and chemical relation (alkaloids ipecacuanha).
Chemical classification (suggested by Oryekhov) – alkaloids are divided into groups accoring to nitrogen bearing heterocycle.
There are:
Pyrolydine derivatives
Pyperidine derivatives
Pyridine derivatives
Pyrolysidine derivatives
Quinolysidine derivatives
Quinoline derivatives
Isoquinoline derivatives
Indole derivatives
Imidazole derivatives
Acridine derivatives
Purine derivatives
Tropine derivatives
Aminoacids are biogenic precursors of alkaloids:
Ornithine group – pyrolydine, pyrolysidine alkaloids.
Lysine – quinolysidine alkaloids of Fabaceal Famile and some pyperidine alkaloids.
Tyrosine – isoquinoline alkaloids.
Tryptophan – indole, quoinoline cinchonine, some pyridine and piperidine alkaloids.
Histidine – imidazole alkaloids like pilocarpine
Glycine and asparagin acid – purine alkaloids.
Nicotine acid participates in some alkaloids synthesis.
Alkaloids spreading in vegetable life and their biological functions
Today alkaloids comprise 10% of world flora. They are spread irregulary in vegetable life.
Alkaloids are rarely in lower plants (they are in Lycopodium – Lycopodium selago Family) and seadless. There are no alkaloids in Salicales, Fagales, Cucurbitales, Oleales. They are spread in Poppy, Parsley, Lily, Celery, Legume, Madder Families. Higher plants worked out the so-called metabolic extraction during the evolution. They are able to accumulate secondary compounds out of metabolic centres – in vacuoles and cell wall. Alkaloids are accumulated mainly in tissues of four types:
1. tissues of active growth;
2. epidermal and hypodemal;
3. the coats of vascular fascicles;
4. latex vessels.
Alkaloids are in vacuoles and they are not determined in young cells till vacuolization. They are rarely found in mortified tissues. Their content ranges from thousands particles of per cents to some per cents. The bark of cinchona tree contains from 15 to 20% of alkaloids. Quantitative content of alkaloids is species characteristic. Each plant contains as a rule the mixture of several alkaloids. There are 15-20 alkaloids sometimes. They are similar for their structure. There are 26 alkaloids in opium. There are some plants that have only one alkaloid (ricinine in ricin). Each plant sometimes in different organs has different number of alkaloids. But there are some organs that do not contain alkaloids. For example Opium Poppy contains alkaloids in all organs besides seeds. Different parts of the plants may contain identical alkaloids or alkaloids different structure.
Biological Effect and Application of Alkaloids
Alkaloid-bearing plants are used in Pharmacy and Medicine with different purposes. They are used directly at the Chemist’s shops for manufacturing of extracts and decoctions (thermopsis – Thermopsis Lanceolata). The others are used for manufacturing galenicals tinctures, extracts, neogalenicals. Alkaloids are produced from raw stuff in pure form and then manufactured as: tablets, ampoules, drage.
Medical significance of alkaloids:
– analgetics (Poppy remedies)
– hemostatics (Claviceps purpurea medicines )
– cardiovascular remedies (ephedra)
– anticancer (autumn crocus remedies )
– spasmolytic (beladonna).
It is not possible to describe all types of phamacological effects. Effects of some alkaloids on the human body are studied well. These substances effect on specific receptors or influence enzyme activity.
Alkaloids have strong influence on enzyme activity. Some of them are connected with induction or reduction of enzyme activity. For example physostigmine, neostigmine and other anticholinesterase remedies decrease acetylcholine.
Alkaloid-analeptics directly or by means of refluxes stimulate vitally active centres of medulla. They are used in inhibition of Central Nervous System, asphyxia, collapse, cardiac insufficiency.
Ten years experience gained in medical application of alkaloids is monitored experimentally and contributes to the development of new remedies.
Alkaloids biosynthesis
Alkaloids are the products of nitrogenous metabolism in the plants. They are grouped according to the formal chemical principles – existence of nitrogen in the molecule. Aminoacids are precursors of rational alkaloids and protoalkaloids. Anthranilic and nicotinic acids, multicarbonic units (for example acetates) play the role of precursors.
Biosynthesis means of proteinogenic aminoacids from pyruvate (lysin, alanine), oxoloactase (aspargine acid), 2-oxoglutaminase (ornithine) are studied. Aminoacids are formed in Calvin cycle or from shikimate acid. Exchange links exist between these groups.
The presence of heterocycles (pyrolydine, pyperidine, pyridine) or combining these simple heterocycles with carbo or others heterocycles (forming more complex polycyclic compounds) is common for the majority of Alkaloids. Small amount of structure elements that are synthesized from common primary precursors comprises the base of alkaloid structure.
The origin of alkaloids has some general (universal) features. It was established experimentally by means of specific precursors, introduced into the plant.
Decarboxylation, oxidation-deamination or aminoacid transamination are primary reactions of biosynthesis. Then the direct transmethylation of obtained intermediate compounds comes and precursors aliphatic chains cycle into various hetero and carbocyclic structures takes place.
The reactions that lead to the formation of N-heterocycle structures have general significance. They are connected with the formation of C-N-bind as a result of different reactions. The reactions of azomethanes formation (shif bases)? And the reaction of Mannih condensation type are the most significant. Azomethanes can be formed spontaneously or by fermentation from amino- and carbon groups.
Reactions of C-N-bind formation
Amines that form Shiff’s bases (A) are synthesised in aminoacids decarboxylation. Carbonic compounds are synthesised due to transamination and oxidation-deamination. In Mannih condensation C-N- bind formation of similar functional groups occurs in interstitial creation of N-hydroxymethyl derivative or acid amine depending on the application of carbonil compound: aldehyde (B) or acetyl KoA (C).
The processes of aliphatic chains cycle in heterocycles are supplemented by condensation processes at the next stages: separate rings are combined and more complex structures are formed, sometimes polyciclic. The formation of new A are combined with splitting of previously formed cyclic structures due to C-C, C-N or C-O binds breaking. Skeletal complication is achieved by internal molecular regrouping with old binds breaking and formation of new C-C; C-N binds.
Limited cyclic variants and regrouping in biosynthesis of A. are combined mainly with including of functional groups and substitutes during different stages of metabolism, it leads to the development of different structural types of alkaloids iature.
THE EXTRACTION OF ALKALOIDS
The extraction of alkaloids is based, as a general rule, on the fact that they normally occur in the plant as salts and on their basicity, in other words on the differential solubility of the bases and salts in water and organic solvents. Petroleum ether and hexane are well suited for defatting of the crushed drug: alkaloids are soluble in these solvents only in exceptional cases, when the medium is neutral.
The powdered defatted drug is mixed with an alkaline aqueous solution that displaces the alkaloids from their combinations as salts; the free bases are then extracted with an organic solvent. Alkalinization is very often achieved with aqueous ammonia. If the structure of the alkaloids to be extracted contains a fragile element, for example, an ester or lactone function, aqueous ammonia must be replaced by an alkaline carbonate solution. The organic solvent can be a chlorinated solvent (dichloromethane, chloroform), ethyl acetate, or diethyl ether.
The organic solvent containing the alkaloids as bases is separated from the residue and if necessary, partially concentrated by distillation under reduced pressure. The solvent is then stirred with an acidic aqueous solution, the alkaloids go into solution in the aqueous phase as salts, whereas the neutral impurities remain in the organic phase. The operation is repeated as many times as necessary until the organic phase no longer contains any alkaloids. Many acids are used (e.g., hydrochloric, sulphuric, sulphamic, citric, tartaric), but always in very dilute solutions (1-5%).
The aqueous solutions of the alkaloid salts, combined, and if necessary, “washed” with an apolar solvent (hexane, diethyl ether) are aUcalinized with a base in the presence of an organic solvent immiscible with water. The alkaloids as bases precipitate and dissolve in the organic phase. The extraction of the aqueous phase continues until the totality of the alkaloids has gone into the organic phase. Finally, the organic solvent containing the alkaloids as bases is decanted, freed from possible traces of water by drying over an anhydrous salt (for example, sodium sulphate), and evaporated under reduced pressure. A dry residue is left: the total basic alkaloids.
Isolation of the individual alkaloid can be obtained by direct crystallization: one example is quinine which is crystallized as a basic sulphate by simply neutralizing the acidic extraction medium with sodium carbonate to pH 6. In other cases, the various alkaloids in the mixture have different basicities and this allows the design of back-extractions by non – miscible solvents at various pHs. In many cases, it is necessary to resort to the classic methods of resolution of complex mixtures, particularly to chromatographic techniques (on silica gel, alumina, ion- exchange resins).
Detection. The detection methods currently in use are preceded by an extraction and consist, most generally, in precipitating the alkaloids by using fairly specific reagents: the “general reagents for alkaloids”. The preliminary extraction can be a “classic” alkaloid extraction or an alcoholic maceration, which takes less time: the alcoholic solution is evaporated and the residue redissolved in acidic water; after filtering, the alkaloids are characterized in the filtrate.
The general reactions of precipitation are based on the fact that alkaloids form combinations with metals and metalloids: bismuth, mercury and iodine. In practice, what is used is a solution containing iodine and iodide, or a solution containing potassium iodide and mercuric chloride – known as Mayer’s reagent – or a reagent containing bismuth nitrate and potassium iodide, better known as Dragendorff s reagent. The BP uses as a general test for alkaloids a modified Dragen- dorif s reagent.
Other reagents are available to characterize alkaloids, particularly those that give colour reactions characteristic of subgroups of alkaloids: the Vitali-Morin reaction for the esters of tropic acid, я-dimethylaminobenzaldehyde for the ergot alkaloids and pyrrolizidine alkaloids.
General precipitate reactoins on alkaloids
1. With Major’s reagents (bichloride mercury solution on potassium iodide solution) – white or yellow precipitate.
2. With Vagner’s and Bushard’s reagents (iodine solution in potassium iodide) – brown precipitates that are the compounds of alkaloids hydroiodides with iodine.
3. With Drehendorf’s reagent (bismuth subnitrate solution potassium iodide and acetic acid) – orange-red or brick-red sediment.
4. With Marmer’s reagent (cadmium iodide solution in potassium iodide) – white or yellowish sediment, often soluble in surplus reagent.
5. With tannin solution – whitish or yellowish amorphic precipitate.
6. With picric acid solution – yellow precipitate.
7. With Zonnenshtein’s reagent (phosphoromolybdenic acid) – yellowish amorphic precipitate, getting dark-blue or green colour.
8. With silicon tugsten acid – whitish precipitate.
Chromatographical analysis. The reactions listed above show the presence of alkaloids, but are not sufficient to verify the identity of a drug; they also do not provide information on the composition of mixtures. To this end, and as in the case of many other secondary metabolites from plants, the methods currently used are TLC and HPLC, oormal or reverse phase (with solvents of the water-methanol or water-acetonitrile type).
Dragendorffs reagent, the iodine-iodide solution (or iodine vapours), potassium iodoplatinate, or cerium and ammonium sulphate are commonly used to visualize TLC plates. Often used scrming systems for silica gel plates comprise toluol – ethyl acetate – diethylamine (70: 20: 10) or ethyl acetate – methanol – water (100: 13,5: 10)
Quantitative determination. Individual methods for quantification of alkaloids in each MPM are developed. They may be classified as follows: acidic – basic titration ion – water solvents for all forms of alkaloids; neutralization; gravimetry; methods, based on individual chemical characters of analized alkaloids; physicochemical methods (photometry, polarography, polarometry, spectrophotometry). The SP method for quantitative determination of tropane alkaloids is reverse titration. The EP method for quantification of tropane alkaloids in Belladonna leaf include titration of the excess of acid with sodium hydroxide using methyl red mixed solution as indicator.
TLC connected with densitometry is often used for quantitative determination of isoquinoline alkaloids. GLC is often coupled with MS for the simultaneous quantification and identification of alkaloids.
REFERENCES
1. Antonyuk V. O. A Laboratory manual on Pharmacognosy /V. O. Anthonyuk, Lysyuk R. M., Anthonyuk L. Ya. ― Lviv, 2012. ― 220 p.
2. William Charles Evans Pharmacognosy / William Charles Evans. ― [16th edition] ― China : Elsevier, 2009. ― 603 p.
3. Pharmacognosy / [V. S. Kyslychenko, L. V. Upyr, Ya. V. Dyakonova etc.]; edited by V. S. Kyslychenko. ― Kharkiv : NUPh «Golden Pages», 2011. ― 551 p.
Prepared by: assistant Kozachok S.S.
