This article throws light upon the two types of secondary metabolites.
The two types of secondary metabolites are: (1) Alkaloids and (2) Dioscorea. The biological functions and various types of alkaloids are discussed hereunder.
Secondary Metabolites # 1. Alkaloids:
Alkaloids have been known to man for several centuries. Morphine, an alkaloid of latex of the opium poppy was isolated by F.W. Serturner in 1806. Latter other alkali-like active principles were isolated and identified, e.g., narcotine in 1817 by Robiquet, emetine in 1817 by Pelletier and Magendie and so on.
The term ‘alkaloid’ was coined by W. Meibner, a German pharmacist, meaning ‘alkali like’. Latter it was demonstrated that the alkalinity was due to the presence of a basic nitrogen atom. The first alkaloid to be synthesized was coniine in 1886 by Ladenburg, which had already been isolated in 1827.
Definition:
The alkaloids are defined as ‘basic nitrogenous plant products, mostly optically active and possessing nitrogen hetero-cycles as their structural unit, with a pronounced physiological action’. Pelletier (1982) suggested following definition for alkaloids: ‘an alkaloid is a cyclic organic compound containing nitrogen in a negative oxidation state which is of limited distribution among living organisms’.
Examples of true alkaloids according to definition are: morphine (Papaver somniferum), the first alkaloid isolated, quinine (Cinchona species), and coniine (poison hemlock, the first alkaloid synthesized, from Conium maculatum; this plant alkaloid was given to Socrates in 400 B.C.) and reserpine (Rauwolfia serpentina).
Occurrence of Alkaloids:
Around 5000 alkaloids of all types have been known to occur in 15% of all land plants and in more than 150 families. The important families are: Apocynaceae, Papaveraceae, Papilionaceae, Ranunculaceae, Rubiaceae, Rutaceae and Solanaceae and less common lower plants and fungi (ergot alkaloids).
In plants, alkaloids generally exist as salts of organic acids such as acetic acid, oxalic, citric, malic, lactic, tartaric, tannic acid etc. Some weak basic alkaloids (nicotine etc.) occur free in nature. A few alkaloids also occur as glycosides of sugar (e.g., glucose, rhamnose and galactose) e.g., alkaloids of the solanum group (solanine) as amides (piperine) and as esters (atropine, cocaine) of organic acids.
Biological Functions of Alkaloids:
The biological functions of alkaloids within the plants are not clearly understood but it is clear that they are not produced in plants for a single function, but for many functions. The following functions have been observed in different plant species.
Alkaloids are considered as:
1. Reserve substances to supply nitrogen, but very little evidence is available about this function.
2. End products of the detoxification mechanism, otherwise their accumulation in plants might cause damage to the plants, e.g., in tobacco, 10% of carbon metabolism is directed to synthesize nicotine biosynthesis. Thus, it is an energy expensive process.
3. Poisonous substances to protect the plant itself from insects and animals. Nicotine has insecticidal properties. Sheep avoid grazing lupin plants with high alkaloid content. Some cacti repel fruit fly but Drosophila pachea is resistant and breeds on cactus.
4. Plant stimulants or regulators, e.g., alkaloids inhibit rye and oat seedling growth. Colchicine inhibits cell division.
5. Reservoirs for protein synthesis.
6. Excretory products of plant.
7. Inhibition of enzymatic activity by alkaloids is also known.
Classifications of Alkaloids:
There are several ways to classify the alkaloids, e.g., taxonomic (say solanaceous, papilionaceous alkaloids), pharmacological (say analgesic, cardio active alkaloids) and chemical classification. Since some families contain alkaloids of several types or the same alkaloids can produce various pharmacological effects in different systems, these classifications were confusing. Therefore classification based on chemical structure is universally acceptable. In the following pages text has been arranged on the basis of chemical structure.
i. Phenylalkylamines:
This class of alkaloids is synthesized from phenylalanine, an aromatic amino acid. These are ephedrine, pseudoephedrine, (Ephedra species), taxine (Taxus) and hordenine (Hordeum vulgare). Several species of Ephedra (Gnetaceae) contain the alkaloidal amine ‘ephedrine’. The ephedrines are L-ephedrine, d-pseudoephedrine, p-N-methyl ephedrine, p- nor-ephedrine, d-nor pseudoephedrine and d-N-methyl pseudoephedrine. Other plants known to contain ephedrine or its derivatives are Catha edulis and Taxus baccata.
The alkaloid ephedrine and pseudoephedrine are largely used as antispasmodic and circulatory stimulants. They were used in China for at least 2000 years before being introduced into Western medicine in 1924. Ephedrine is largely used as substitute for epinephrine against bronchial asthma of allergic and reflexive types. It is also used orally and locally in patients suffering from hay fever, urticaria and other allergic reactions. E. foliata grows in the Thar desert contains traces of pseudoephedrine, E. geraradiana, a cold desert species growing in western Himalayas contains 2% alkaloids.
ii. Pyrrolidines, Piperidines and Pyridines:
Piperidine alkaloids such as coniceine, coniine and N-methyl coniine are present in Conium maculatum, but not detected in the cultures of this plant. Lobeline and other piperidine alkaloids are present in Lobelia inflata tissue cultures and intact plants. The alkaloids belonging to these groups are known to have divergent physiological activities. Apart from tobacco alkaloids, nicotinic acid and its derivatives are the major pyridine alkaloid present in plants. The most commonly occurring compound is trigonelline (N-methyl- nicotinic acid) present in Trigonella foenum-graecum. Plants and cultures of Nicotiana tabacum contain nicotine, anatabine, anabasine, myosmine and nicotelline.
Nicotine has been a major target of study among them. Nicotine and nicotinic acid are one of the most extensively studied secondary metabolites in plant tissue culture. Though nicotine production through plant tissue culture in not a viable programme, tobacco cultures were used as model system to develop technology for the production of secondary metabolites.
The practice of tobacco smoking was made known to Europeans about the year 1492 when they visited West Indies, after the discovery of New World (America). Since then tobacco was introduced in several countries of the world including India where it is extensively cultivated for its leaves used as tobacco in cigarette, bidis and other tobacco preparations. When plant is cultivated, plant tissue culture cannot compete for biomass production with a crop plant. Plant tissue culture is used to know biosynthetic pathway of nicotine and production of low nicotine containing plants.
Such plants will have aroma of tobacco but low tar and nicotine, which is injurious to health. In plants, nicotine is synthesized in roots and accumulated in leaves. This means nicotine is transported from roots to leaves and is energy dependent process. About a third of CO2 fixed in photosynthesis is used in nicotine synthesis. Tobacco cultures have been used for all types of studies ranging from cell cultures, cloning, bio-synthesis of nicotine, hairy roots cultures, and growth in bioreactors up to 2000 litres.
iii. Tropane Alkaloids:
The alkaloids hyoscyamine, atropine and hyoscine (scopolamine) are found principally in plants of the family Solanaceae and categorized as anticholinergics. More than 30 alkaloids are known to be present in Datura. Datura stramonium and D. innoxia are the main source of hyoscyamine and scopolamine, respectively.
Other species known to contain tropane alkaloids are Dubosia hybrids, Hyoscyamus niger (henbane), H. muticus, D. myoporoides, D. leichhardtii, and Atropa belladona (deadly nightshade). Cocaine in coca (Erythroxylon coca) was the first local anaesthetic to be discovered. Leaves of a few species of Erythroxylon indigenous to Peru and Bolivia, contain 0.6-1.8% cocaine.
The leaves of the plant have been used for centuries by the natives to increase endurance and to promote a sense of well-being. Cocaine was isolated in 1859 by A. Niemann for its Central Nervous System (CNS) stimulatory activity, which can lead to dependence liability, hence cocaine, has been used as drug of. These alkaloids are synthesized from tropic acid.
iv. Quinolizidine and Pyrrolizidine:
Quinolizidine alkaloids are common natural products of many Fabaceae and commonly called as lupin alkaloids because of their presence in all species of the genus Lupinus. This group of alkaloids is synthesized from lysine via cadaverine, e.g., lupanine, sparteine etc.
Isoquinoline type alkaloids show strong pharmacological activities like those of morphinane, protoberberine, and benzophenanthridine-type alkaloids; they are widely distributed in the plant kingdom, mainly in Papaveraceae, Berbidaceae, Ranunculaceae and Menispermaceae.
Papaver alkaloids require a special mention in isoquinoline alkaloids. The opium poppy, Papaver somniferum, is one of the man’s oldest cultivated plants. The therapeutic use of latex obtained from unripe capsules of poppy was recorded by Theophrastus in the third century B.C. Discorides (A.D.77) described the curative properties of the opium poppy and presented various uses for both latex and extracts of whole plants.
Purified alkaloids are used in modern medicine for treatment of pain, cough and diarrhea. Opium is the dried cytoplasm of a specialized internal secretoary system, the laticifer. Morphine content increases in the morning with decrease in codeine and thebaine content in the latex.
So capsules are incised in the morning to obtain morphine-rich latex. When the green unripe capsule (fruit) is cut, milky latex oozes out, which turns dark brown on drying and is collected as raw opium? This is a labour intensive manual process and requires strict vigilance and control to check illegal drug transport. Alkaloids are purified in laboratories (factories controlled by the Government).
More than 40 alkaloids have been identified from Papaver somniferum including 25 from latex. However, from the medicinal point of view, benzyl isoquinolines (papaverine and narcotine) and phenanthrines (morphanians: morphine and codeine) are important.
The demand for legal opium is estimated to be more than 1000 tons per annum. In India, cultivation and alkaloid isolation is a business completely controlled by the Government. World trade of opium is governed by the United Nations Opium Conference Protocol (1953).
Morphine was the first alkaloid isolated in pure form. Synthesis of morphine takes place from tyrosine via DOPA (Dihydroxy phenylalanine), nor-landanosoline to papaverine or reticuline. Reticuline is converted into thebaine, thebaine in to codeine and then morphine as the end product. Thus codeine is an intermediate product, not present in large quantities in the latex.
v. Quinoline Alkaloids of Cinchona:
Extracts obtained from the bark of Cinchona species have been of great therapeutic value. Alkaloids of the genus Cinchona, Rubiaceae, have been used to treat malaria for several hundred years in Western medicine. The use of Cinchona bark was first recorded in 1633. Quinine, isolated from this source, was the first efficacious treatment for malaria. At least 40 alkaloids have been isolated from this source.
Quinine is used as a standard for bitterness. Although these alkaloids have a quinoline structure, they are modified monoterpene-derived alkaloids. The biosynthetic pathway leading to quinine and related compounds is complex. Quinine intercalates with DNA, modulates ion channels, and inhibits glucose response in chemosensory cells. Quinidine, another alkaloid of this series, is used to treat heart conditions.
The alkaloid has been prepared by total synthesis but these procedures are too complex and not commercially viable. Quinidine, another major cinchona alkaloid, is used for the treatment of cardiac arrhythmia. It is also effective against malaria parasite.
Besides their pharmaceutical use, cinchona alkaloids are used frequently in the food and soft drink industry because of their bitter taste. The principal species are C. ledgeriana, C. pubescens (syn. C. succirubra) and C. officinalis.
Cinchona trees have been cultivated in plantations for more than 130 years for production of cinchona bark. Though the plant is native to certain parts of South America it is presently cultivated in India, Java and Indonesia. When trees are 7 to 12 years old, the bark of the tree is harvested; at this stage of maturity the alkaloid content is approximately 12-15%.
In all 35 alkaloids are found in bark. The important ones are quinine, quinidine, cinchonine, and cinchonidine, and their dehydro derivatives. Besides these, several indole alkaloids (aricine, cinchonamine, quinamine) are also present in the leaves and bark.
Plant tissue culture is used for the micro-propagation of high yielding clones. The first report on tissue culture and micro-propagation of high-yielding clones of Cinchona tree was published in 1975 by Chatterjee.
vi. Indole Alkaloids:
The monoterpene indole alkaloids represent a large and diverse group of plant products, mainly present in Loganiaceae, Apocynaceae and Rubiaceae. The monoterpene indole alkaloids are formally derived from a unit of tryptamine and a C9/C10 unit of terpenoid origin (secologanin). These alkaloids are classified in to three groups- Corynanthe-, Aspidosperma- and Iboga type on the basis of carbon skeleton.
Examples of Corynanthe type alkaloids are ellipticine, reserpilline and other alkaloids of this type in Ochrosia elliptica, reserpine and ajmaline in Rauwolfia serpentina and Catharanthus roseus, and cinchonamine, quinamine, aricine in Cinchona.
Conoflorine, tubotiwine are Iboga type alkaloids present in Tabernanthe iboga. Tabersonine, ichnericine and minovincine are Aspidosperma alkaloids present in Voacanga africana.
Large number of indole alkaloids produced by Catharanthus roseus (synonym Vinca roseus) has been identified. The plant is native to Madagascar but now cultivated in India. Several of these have been found to be valuable agents in the treatment of hypertension and others of cancerous growths.
In particular, vincristine and vinblastine, two dimeric indole alkaloids (0.0005% on dry weight basis obtained from roots) are used for the treatment of leukaemia and Hodgkin’s disease.
The antineoplastic properties of the alkaloidal constituents of Catharanthus were independently described by Canadian and American scientists. It contains more than 75 alkaloids; the other indole alkaloids present in large quantities are ajmalicine, serpentine, lochneridine, tabersonine and vindoline.
Medicinal properties of Catharanthus roseus have been described in traditional and folk medicine of several countries. Beneficial effect of its extract in diabetes mellitus was known, but later on active principles suppressing neoplasm were also isolated. The extracts yielded four active dimeric monoterpenoid indole alkaloids- vinblastine, vincristine, vinleurosine and vinrosidine.
The catharanthus alkaloids are cell-cycle specific agents, similar to colchicine and podophyllotoxin, block mitosis and cause metaphase arrest. Though vincristine and vinblastine have anti-proliferative properties, but both have different patterns of cytotoxic effect and are used in combination for the last 40 years (received FDA approval in 1963 and 1965, respectively).
The plant is extensively investigated for the production of indole alkaloids but, ajmalicine and serpentine are major alkaloids present in cell cultures. Shoot cultures of Catharanthus are reported to contain vincristine and vinblastine.
However, the cell culture have been associated with various technological developments for the production of secondary metabolites as whole, such as cloning, two stage culture system, gene cloning and transfer to other indole alkaloid containing plants, enzymes of biosynthetic pathway and elicitors use.
The Catharanthus alkaloids, navelbine (trade name Vinorelbine), vinblastine (trade name Velban), and vincristine (trade name Oncovin) are currently used clinically. Vinblastine introduces a wedge at the interface of two tubulin molecules, thus interfering with tubulin assembly.
vii. Purine Alkaloids:
Purine alkaloids are widely distributed within the plant kingdom (Fig. 2.12) and have been detected in at least 90 species belonging to 30 genera. Their occurrence, however, is limited to dicots. Caffeine and threobromine, methylated derivatives of xanthine, are generally the main purine alkaloids and are regularly accompanied in low concentrations by the two methylxanthines- theophylline and paraxanthine as well as by methylated uric acids such as theacrine, methylliberine and liberine.
Since purine alkaloids are present in tea and coffee are widely consumed in human diet across the continents. Plant species from different families are made in to a pleasant stimulant, e.g., coffee (Coffea arabica, C. robusta), tea (Camellia sinenesis), Cocoa (Theobroma cacao), mate (Ilex paraguayiensis) and cola (Cola nitida).
viii. Tropolone Alkaloids:
The neutral alkaloid colchicine present in the bulbs of Colchicum autumnale and tubers of Superb Glory (Gloriosa superba), Liliaceae is an example of a tropolone- type alkaloid. Colchicine is synthesized from phenylalanine via cinnamic acid and sinapic acid. Other important secondary metabolites categorized as alkaloids are acridone alkaloids present in plants of the family Rutaceae and steroidal alkaloids present in plants of the family Solanaceae.
Secondary Metabolites # 2. Dioscorea:
Dioscorea deltoidea (family Dioscoreaceae) related to D. batata commonly known as Ratalu. This has attained importance due to population explosion in the Asian countries. The plant contains diosgenin used in the biosynthesis of hormones for contraceptive pills for females. Micro-propagation technology has been developed by National botanical research institute, Lucknow in the leadership of Dr H.C. Chaturvedi.
Diosgenin, a steroid sapogenin, is the product of hydrolysis by acids, strong bases, or enzymes of saponins, extracted from the tubers of Dioscorea wild yam. The sugar-free (aglycone), diosgenin is used for the commercial synthesis of cortisone, pregnenolone, progesterone, and other steroid products. Diosgenin is converted into natural progesterone scientifically.
There are no enzymes in the human body that will convert diosgenin, which is the active component of wild yams into progesterone. Diosgenin is still very useful in the body and has been used by phytotherapists for centuries as an adaptagen. Whatever the effects of wild yam, it does not have the same benefits as Natural progesterone.
Diosgenin, which is a sapogenin chemical very similar to cholesterol, progesterone and DHEA – the precursor to testosterone. Diosgenin provides about 50% of the raw material for the manufacture of cortisone, progesterone, and many other steroid hormones and is a multi-billion dollar industry.
The major breakthrough in the development of progesterone for birth control was the synthesis (or semi-synthesis) of progesterone from diosgenin, the major spirostan found in yams. In 1934, Schering Laboratories, a major player in the pharmaceutical applications of steroids then and now, was able to isolate 20 mg of progesterone from 625 kg of ovaries obtained from 50,000 sows.
This was hardly an economically feasible process. However, in 1940 Russell E. Marker developed the process, initially for Parke Davis and Co., for degrading sapogenins to C21 steroids. This process was applied by Marker to convert diosgenin obtained from a Japanese Dioscorea species into progesterone.
Marker’s investigation of the Mexican yam, Dioscorea macrostachy known locally as cabeza de negro, showed that it contained large amounts of diosgenin, and that this diosgenin could be easily converted into progesterone by his method, known as the Marker degradation. Marker was unable to convince Parke Davis to apply for foreign patents for this process and establish a hormone industry in Mexico.
