ABSTRACT
Phytochemical are now a days used as powerful medicine to cure many diseases. A number of bioactive substances have been reported from plant tissue cultures. The present study pertains to the important naturally occuring metabolites such as alkaloids, antimicrobials, flavonoids, insecticides, sterols and steroidal sapogenins reported from in vitro tissue cultures of a number of plant species.
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How to cite this article
DOI: 10.3923/biotech.2005.79.93
URL: https://scialert.net/abstract/?doi=biotech.2005.79.93
INTRODUCTION
In recent years, phytotherapy is rapidly evolving throughout the world. The beneficial effects of medicinal plants to cure many dreadful diseases were discovered long before scientific advancement. With the advancement of technology new commercial organizations switch on to study hitherto illlknown medicinal plants. The study of modem herbalism has started selection of new seeds from cultures of plants in order to obtain qualitative and quantitative optimal yields and to elaborate new and scientifically tested drugs for the more effective treatment of specific illness. Phytochemical are naturally occuring biochemicals in plants that give plants their color, flavour, smell and texture. They may help to prevent diseases like cancer and heart diseases besides their role to inhibit the microorganisms causing many diseases in human beings. The active principles contained in the plants consist of a number of chemical compmmds, which have a specific action on the organs of our body.
Now a days, there is a great decrease in plant resources due to human disturbances of the natural envirornnent. Therefore, biotechnologists hope for a bypass to overcome this difficulty by introducing plant tissue culture technique and further multiplication of important plants by micropropagation technique. Now, the technique of tissue culture has been developed for large-scale cultivation of plant cell. The production of useful metabolites from plant tissue culture has created a new methodology for their commercialization. The useful metabolites from plant tissue cultures include alkaloids, antimicrobials, essential oils, flavonoids, pigments, proteins, phenols, pyrethrins, rotenoids, sterols and steroids. Several products are accumulated in cultured cells at a higher level than those in native plants e.g. shikonin by Lithospermum erythrorhizon and diosgenin by Dioscorea. For more than 30 years many researcher have been investigating plant cell cultures for the production of a variety of phytochemical. However, inspite of their many efforts only two products e.g. shikonin and ginseng cells are so far manufactured commercially. Mitsui Petrochemical Industries (Tokyo, Japan) has successfully produced shikonin, an antibacterial dye on commercial basis. A number of firms in US, Japan, Canada and Europe have been investigating intensively the production of a very promising anti-tumor compormd, Taxol using the cell cultures. Thus, the chances of exploitation of tissue culture technique for large-scale production of useful metabolites are bright.
The beginning of production of useful metabolites from tissue culture in India dates back to 1964 when Mitra and Kaul at National Botanical Research Institute, Lucknow for the first time reported the production of reserpine from Rauwolfia serpentina tissue culture. Later on other laboratories like Central Drug Research Institute, Lucknow, Central Institute of Medicinal and Aromatic Plants, Lucknow, Bhabha Atomic Research Centre, Trombay, National Chemical Laboratory, Prme and Regional Research Laboratory, Jammu=Tawi and Universities like M.S. University, Baroda and Jawaharlal Nehru Technology University, Delhi carried out study on various metabolites from plant tissue cultures.
A number of reviews on plant constituents have been written[1,22]. The prospects and problems in large-scale production of metabolites from plant tissue cultures has been discussed[9,21].
Natural plant metabolites: The naturally occurring plant metabolites have been divided into two groups.
Primary plant products: Which are of prime importance and essentially required for growth of plants e.g. ammo acids, ascorbic acid, carbohydrates, enzymes, lipids, nucleic acids and proteins, etc. They are of nniversal occurrence in plants.
Secondary plant products: Which are accessory, plant metabolites and seemingly not involved in the biosynthesis of primary metabolites. They are produced secondarily and derived biosynthetically from the metabolism of primary products such as carbohydrates, fats and amino acids. They are biosynthesized in selected few species of plants e.g. Alkaloids, cournanns, flavonoids, phenols, sterols and steroids. They are not of rmiversal occmence in plants.
Primary products from plant tissue cultures: Among pnmary products ammo acids, ascorbic acid, carbohydrates, lipids and protein have been reported from tissue culture of a number of plant species.
Amino acids are the building blocks of proteins and many other secondary products. The concentration and amino acid composition of proteins from plant cells grown in suspension cultures has been reported[23]. The amino acids have been reported from tissue culture of a number of plant species such as Datura mete!, D. tatula, Momordicacharantia and Trigonellafoenum� graecum[24], Tephrosia urpurea, T. vogelii, Emblica officinalis and Sesamum indicum[25], Scopolia japonicd [26] Ephedra foliatd 21J, Tagetes erectd [28].The effect of various amino acids on the production of tropane alkaloids in Scopolia species has also been investigated and it has been demonstrated that tryptophan is most effective amino acid in producing scopolamine and hyoscyamine[29].
Chemically ascorbic acid is more related to the monosaccharides as it is a hexose derivative. It controls the cholesterol metabolism and helps in the absorption and utilization of iron. It is widely distributed in plants mainly in citrus fruits. Production of endogenous ascorbic acid and effect of exogenous ascorbic acid on grovvth and metabolism has been reported in tissue cultures of Emlica officina/is and Momordica charantid [30], Datura mete! and D. tatuld [31] Trigonella foenum graecum[32], Crotolaria burhid [33], Tagetes erectd [34], Ephedra foliata, Helianthus annuus, Tephrosia purpurea and T. vogelli [35], Papaver somniferum [36] and Solanum nigrum[37].
Carbohydrates have been reported from tissue cultures[38,39]. All culture media require carbohydrates as a source of carbon. Utilization and metabolism of carbohydrates in cell and callus cultures has also been reported[40].
Fatty acid composition of lipids in various cell cultures have been reported[41-43]. Lipids in plant tissue cultures have been reported from a number of plant species[4l-47] and the fatty acid composition oftriglycerides in tissue cultures of rape and turnip rape[47] Corchorous, Yucca, Dioscorea, Withania and Rived[48] have been investigated. Major lipid components in tissue cultures of Euphoria species has also been studied[49]. Biosynthesis of neutral lipids from malonate alongwith cardenolides in Digitalis lanata have been reported[50l].
A hypoglycemic polypeptide-p, has been isolated from fruits, seeds and tissue culture of Momordica charantid 51·52l. Polypeptide-p, when administered subcutaneously (1.8 mg mL-1) is very effective hypoglycemic agent[53].
The enzymes have been reported from tissue cultures of a number of plant species[54-55]. Physiology of enzyme production by plant cell cultures has also been investigated. Medora et al.[55] has reported proteolytic enzymes in papaya tissue cultures.
Secondary products from plant tissue cultures: The biosynthesis of secondary products from plant tissue cultures is of considerable interest and has become very important these days. A number of important secondary products such as alkaloids, flavonoids, pyrethrins, rotenoids, sterols and steroids have been isolated from different plant species grown in vitro.
Alkaloids: Alkaloids, a heterogenous group of basic nitrogen containing heterocyclic compormds are produced by plants as secondary metabolites. Some exceptions are colchicine and ricinine which are not basic and ephedrine and muscarine which are not nitrogen containing heterocycles. Their inclusion among the alkaloids is based mainly on their biosynthetic similarity with the true alkaloids. Alkaloids are very important in the medical world and are used as powerful drugs mainly due to their sedative properties and powerful effect on the nervous system. A variety of alkaloids have been used as pharmaceuticals. Alkaloids are produced in actively growing tissue and rarely occur in dead tissue. Research on production of useful alkaloids by plant tissue culture has also been carried out for more than 25 years. However, industrial production has not yet succeeded because of low producing ability of cultured cells. Vinblastine, an anti-tumor alkaloid is most likely to be produced commercially by a Japanese company using a combination process of plant cell culture and that of chemical synthesis, which was initially investigated by a Canadian company, Allelix Inc. At present time more than 3000 alkaloids are known which are distributed among ahnost 4000 plant species. A particular alkaloid is usually of limited distribution and confined to a specific genus or closely related group of plants. A number of alkaloids have been reported from tissue cultures of different plants.
Based on their chemical nature alkaloids can be classified into following 7 groups:
1. | Pyrrolidine Alkaloids-This is a small group of alkaloids which can be further classified into 2 groups: (1) Simple Pyrrolidines e.g. Hygrine, hygroline, cuscohygrine, stachydrine, betanicine and turicine and (ii) Tropane alkaloids e.g. Atropine, hyocyamine, scopolamine, cocaine, etc. |
These two types are related biogenetically and sometimes occur together in the same plant.
2. | Pyrrolizidine alkaloids e.g. Senecio alkaloids |
3. | Pyridine and Piperidine alkaloids e.g. Nicotine and Anabasine |
4. | Polyacetyl Alkaloids e.g. Coinine |
5. | Isoquinoline Alkaloids e.g. Morphine and codeine |
6. | Indole Alkaloids e.g. reserpme, strychnine, vinblastine and quinine |
7. | Steroidal Alkaloids e.g. solasodine and solanine. They are usually classified with terpenoids as their C-skeleton is furnished by 5 C nnit. |
Alkaloids biosynthesis is a subject of very much greater inherent diversity due to their complex structure. They are derivatives of amino acids which supply nitrogen and C-skeleton. Amino acids-ornithine, lysine, phenylalanine, tyrosine and tryptophan are some of the primary metabolites from which various groups of alkaloids are formed.
Among pyrrolidine alkaloids tropane alkaloids are very important pharmaceutically particularly atropine and hyoscyamine which dilates the pupil of the eyes and therefore finds extensive use in ophthalmology. Tropane alkaloids act as sedatives and have been fonnd in solanaceae, convolvulaceae and erythroxylaceae. The name comes from the tropane skeleton in their structure and each is the ester of an organic acid with a bicyclic base. Their biosynthesis starts from an amino acid, ornithine which is converted in pyrrolidine system. Biosynthesis of tropane alkaloids has been studied in root culture of Atropa belladonnd[56-58], Brugmansia candidd[59], Datura innoxid [60], Datura stramonium[61-64], effect of amino acids on the production of atropine in suspension cultures of Atropa belladonna. Dmitruk[76] has also studied the effect of various amino acids on the production of tropane alkaloids in Scopolia sp.[76] and demonstrated that tryptophan was most effective in producing scopolamine and hyoscyamine. Cordan[77] has written a review on the production of tropane alkaloids in tissue cultures. Production of tropane alkaloids in genetically engineered root cultures was also investigated[78]. Kitamura et al.[79] reported that root differentiated from cultured cells accumulate scopolamine, hyoscyamine and nicotine but not accumulated in leaves of the regenerated plantlets[79]. The effect of various growth hormones on growth and production of tropane alkaloids in tissue cultures of Datura mete! has been reported[80]. Sarker et al.[81] has studied the elicitation of tropane alkaloids in suspension culture of Hyoscyamus, Datura and Atropa by osmotic stress. Elicitation of tropane alkaloid biosynthesis in transformed root cultures of Datura stramonium has been studied[82]. Effect of jasmonic acid and aluminium on the production of tropane alkaloids in hairy root cultures of Brugmansia candida has also been investigated[83]. Inspite of many efforts to increase the yield using various approaches the concentration of tropane alkaloids in cultured cells are generally very low. Therefore, plant cell culture has not yet been employed to manufacture these alkaloids.
Pyrrolizidine alkaloids show a wide spectrum of biological activities against tumor and are hypotensive and muscle relaxant. They are known for their anti-tumor, carcinogenic, hepatotoxin and mutagenic properties. Pharmacodynamic importance of semisynthetic derivative of these alkaloids has been reported[84]. These alkaloids constitute a large family based on the pyrrolizidine nucleus and distributed in a number of plants belonging to asteraceae, boraginaceae and fabaceae families.
Pyridine (e.g. Nicotine and Nomicotine) and Piperidine (e.g. Anabasine) alkaloids occurs ahnost nniversally in tobacco plants. Nicotine and nornicotine consist of a pyridine ring to which a pyrrolidine ring is attached and they are the chief alkaloids of Nicotiana tabacum. In Anabasine, piperidine ring rather than a pyrrolidine ring is joined to the pyridine nucleus and it is the chief alkaloid of Nicotiana glauca. Nicotine is the principal constitute of tobacco leaves and occurs to the extent of 0.5-8%. The smoke of a cigarette can yield 6-8 mg of nicotine. It is a natural liquid alkaloid and is colorless, volatile and strongly alkaline. On exposure to air it turnsbrown and acquires the odour of tobacco. It has no Tropane alkaloids from tissue culture of Atropa belladonna 1s shown by a number of therapeutic application and increases the heartbeat rate and raises blood pressure. The production of nicotine and eanabasinem tobacco callus tissue has been investigated[85] and the regulation of nicotine biosynthesis by auxins has also been studied[86]. Regulation of nicotine biogenesis by urea in tobacco tissue cultures has also been investigated[87-88]. Pyridine alkaloids from cell cultures of Nicotiana tabacum has also been reported[89]. Trigonelline, another pyridine alkaloid has been reported from Trigonella foenum-graecum root callus culture[90] and the yield of this alkaloid was further increased by feeding the tissue with different concentrations of nicotinic acid[91].
Polyacetyl alkaloids includes coniine, Lycopodium and muscopyridine and carpaine alkaloids. Coniine, an alkaloid present in Conium maculatum (hemlock) is very poisonous and the extracts were used in ancient times for the execution of criminals. Lycopodium alkaloids are fmmd in club mosses and muscopyridine is one of the few alkaloids from animals rather than plant source.
Isoquinoline alkaloids include most important benzylisoquinoline alkaloids. This group of alkaloids include major alkaloids of the genus Papaver e.g. morphine, codeine, thebaine, papaverine and narcotine which are pharmaceutically very important. Among these alkaloids morphine is purely narcotic and is normally reserved for severe pain when other analgesics fail to give relief. Codeine is widely used by general public as a mild analgesic because it is less toxic than morphine. The baine has almost the same analgesic effect as morphine but it is the most poisonous of the opium alkaloids and is scarcely used as such in therapy but is used in the form of its derivatives. Papaverine is used in the treatment of astlnna and angina pectoric and narcotine is widely used in the preparation of cough linctus. Opium, the inspitated milky juice from mipe capsules of Papaver somniferum contains nearly 25 alkaloids. These alkaloids are absent in the seeds but are synthesized as soon as the plant is grown. Maximum amormt is present in the capsules. Production of six major opium alkaloids has been reported from callus tissue cultures of Papaver somniferum[92-97], P. bracteatum[98], P. rhoeas[94, 99, 100]and four Papaver species[97]. Furuya et al.[101] have reported nine alkaloids from the callus tissue of P. somniferum and Ikuta et al. have reported the presence of same alkaloids and the absence of morphine alkaloids in the callus tissues[102] and redifferentiated plantlets of eleven species of papaveraceae. All these alkaloids are benzophenanthridine, types of alkaloids. protopine and aporphinetypes
Formation of thebaine in the suspension culture of Papaver bracteatrum has also been reported[ [103] Effect of ascorbic acid, tyrosine and auxins on the production of these alkaloids is also studied in callus culture of P. somniferum[36-93]. Tetraploid tracheid containing callus of P. somniferum reportedly produced codeine, morphine and thebaine[104]. The high yielding tissues having large cells containing amorphous alkaloid contents were observed inP. somniferum and P. rhoeas [94] Effect of tyrosine (a known precursor of opium alkaloids) on the production of alkaloids in high yielding cell strains of P. somniferum and P. rhoeas was also studied[95-105] Effect of temperature stress on the production of alkaloids in cell suspensions from four Papaver sp. has also been investigated[97].
Berberine, a benzyl-isoquinoline (non-tryprophan indole alkaloid), used as a tonic and in stomachache. It is highly toxic to bacteria and is an intestinal antiseptic. Therefore, it is used for intestinal disorders. Berberine is obtained mainly from roots of Coptis japonica (Ranrmculaceae) and it takes 5 to 6 years to produce Coptis roots as raw material. Furuya et al.[100]have investigated the production of berberine by Coptis japonica cell cultures. High berberine producing cultures of Coptis japonica were also reported[106-109]. Mitsui petrochemical has improved the productivity by addition of gibberellic acid into medium which stimulated berberine productivity upto 1.66 g L-1 of medium. Khanna et al.[110]have reported berberine from Argemone maxicana tissue and cell cultures.
Indole alkaloids posses an indole nucleus often in reduced form and in case of some Cinchona alkaloids, altered almost beyond recognition. These alkaloids has received a great deal of attention from the pharmacologists, physiologists and physicians. The interest in indole alkaloids evolved from the discovery of the remarkable physiological properties of lysergic acid diethylamide on one hand and of reserpine, the sedative principle used for the treatment of hypertension, headache, astharna and dermatological disorders of Indian plant Rauwolfia, on the other hand. The extensive investigations of indole alkaloids as well as other constituents of the large family of flowering plants, Apocynaceae, were highlighted a few years ago by the introduction of two Vinca alkaloids, vinkaleukoblastine and leurocristine into the treatment of Hodgkin's disease and acute lukemia respectively. Indole alkaloids are fmmd in plants of families apocynaceae, loganiaceae, rubiaceae and euphorbiacea. Production of indole alkaloids from callus cultures of Catharanthus roseus has been reported[111-115]. Influence of various chemical factors on the production of indole alkaloids in tissue cultures of Ipomea, Rives andArogrela has been reported [116].
Another class of alkaloids is pseudoalkaloids resulting from the oxidation followed by the alkylation or acylation of certain amino acids. Such alkaloids are called protoalkaloids. Ephedrine, a phenylalkylamine alkaloid which dilates the bronchiolar muscle is very helpful in astlnna. Pseudoephedrine, an isomer of ephedrine and an effective anti-aesthematic drug has been reported from tissue culture of Ephedra foliatd [117]. An alkaloid, momordicine has also been reported in traces from tissue culture of Momordica charantid [118]
Antimicrobials: Important characteristics of chemical antimicrobial substances are their capability of inhibiting bacterial colonization, adhesion and their capacity to affect plaque growth metabolic activity. Plant products are very powerful antimicrobial agents[119]. Antimicrobial activity has also been shown in tissue cultures[120-129]. Khanna et al.[125]have screened ten plant species- Atropa belladonna Linn., Brassica nigra Koch., Datura metal, D. tatula, Emblica officina/is, Hyoscymus niger, Momordica charantia, Sesamum indicum, Tagetes erecta and Trigonella foenum-graecum grown in vitro as static cultures against a Gram positive and two Gram negative Bacteria and a frmgus-Candida albicans. Later on, Khanna et al.[122] have screened tissues of ten other plant species-Agave wightii, Aregemone mexicana, Calendula o.fficinalis, Cheiranthus cheiri, Crotolaria burhia, Dahlia pinnata, Lycopersicon esculentum, Papaver rhoeas, Solanum luteum, S. tuberosum and Trigonella corniculata for their antimicrobial activity against Escherichia coli, Staphylococcus albus, Streptococcus faecalis and a frmgus Candida albicans. They have isolated and identified the possible antibacterial substances produced by them such as Apigenin, isorharnnetin, kaemferol, negretein, quercetine and scopoletin.
Flavonoids: Flavonoids are water soluble pigments which occur ahnost nniversally in higher plants and contribute to the flower and fruit colour. They impart mostly red yellow, blue and violet colour to plant organs. Chemically they are phenolic compormds and most of them have flavone nucleus with two side aromatic rings. Flavones occur as glycosides in plants. Flavonoids are classified on the basis of the oxidation state of the central heterocycle of the flavone nucleus. Each subgroup is further grouped according to the pattern of substitution in side aromatic ring. Flavones such as luteolin and apigenin have the central heterocycle with two double bonds whereas flavonones such as naringenin and eriodictyol have one double bond. Flavonol result from the addition of a hydroxyl group at Carbon-3 of the central heterocycle. Kaempferol, quercetin and myericetin are common examples of flavonol. The precursors of flavonoid biosynthesis include shikimic acid, phenylalanine, cinnamic acid and p-coumaric acid.
The distribution of flavonoids in plant kingdom is more or less of taxonomic significance. Algae, ftmgi and bacteria lack any kind of flavonoid, whereas mosses have a few types of them. Fems and gymnosperms have many types of simple flavonoids whereas angiosperms have a whole range of flavonoids. Highly complex forms of flavonoids e.g. quercetagetin occur in the highly evolved families like compositae. Production of flavonoids has been reported from tissue cultures of a number of plant species such as Cicer arietinum[130], Citrus[131-132], Crotolaria junced [133], Datura sp.[134], Emblica officinalis[135], Haplopappus gracilis[136]Trigonella foenum-grecum[137], Tephrosia purpured [138], Tylophora indicd [139].
Insecticides: Naturally occuring insecticides fall nnder two major groups-Pyrethrins and rotenoids. Both are very toxic to insects and harmless to mammals. Thus, can be used safely as domestic insecticides.
Pyrethrins: Pyrethrin are economically most important of natural insecticides and are currently derived from Pyrethrum plant. Some biotechnology companies studied the possibility of industrial production of pyrethrins. Whether they are able to overcome the biological and technical constraints is still rmclear.
Pyrethrins are the esters used in domestic insecticidal sprays as they are considered to be very toxic to flying insects and harmless to mammals and plants. They have an rmusual paralytic effect, 'knock downl', on flying insects and inhibit the mitochondrial electron transfer system of insects at characteristic site. Pyrethrins are present m floral heads of Chrysanthemum cinerariaefolium Vis. commonly called Pyrethrurn[140] and C. coccineum Willd. Four closely related esters (Pyrethrin I and II and Cinerin I and II). Later on, Godin et al.[141-143]to the flower and fruit colour. They impart mostly red, yellow, blue and violet colour to plant organs. Chemically they are phenolic compormds and most of them have flavone nucleus with two side aromatic rings. Flavones occur as glycosides in plants. Flavonoids are classified on the basis of the oxidation state of the central heterocycle of the flavone nucleus. Each subgroup is further grouped according to the pattern of substitution in side aromatic ring. Flavones such as luteolin and apigenin have the isolated and identified two additional pyrethrins (Jasmolin I and II) from the extracts of Chrysanthemum cinerariaefolium. Now, it is clear that there are at least six structurally related compormds collectively called 'Pyrethrins' responsible for the insecticidal activity of Pyrethrum flowers. All the six pyrethrins (Pyrethrin I and II, Cinerin I and II, Jasmolin I and II) have been reported from seeds, floral heads and tissue cultures of central heterocycle with two double bonds whereas flavonones such as naringenin and eriodictyol have one double bond. Flavonol result from the addition of a hydroxyl group at Carbon-3 of the central heterocycle. Kaempferol, quercetin and myericetin are common examples of flavonol. The precursors of flavonoid biosynthesis include shikimic acid, phenylalanine, cinnamic acid and p-coumaric acid. The distribution of flavonoids in plant kingdom is more or less of taxonomic significance. Algae, ftmgi and investigated the effect of ascorbic acid on the production of pyrethrins from in vitro tissue cultures of Tagetes erecta and reported that ascorbic acid plays an important role in grovvth and production of pyrethrins in T. erecta tissue culture. Later on, investigations into possible new sources of pyrethrins from a number of plant species such as Calendula o.fficinalis, Dimorphotheca sinuata, Zinnia elegans, Z. linearis[147]and Vernonia species[148]belonging to family asteraceae were done in vivo and tissues of Chrysanthemum cinerariaefolium has also been carried out [149-150]. Studies of pyrethrum plant derived in vitro cultures revealed that rmorganized tissue culture do not have secondary metabolism characteristics of the corresponding intact plant and only organized shoot cultures could be considered for pyrethrins production. Biological and technological obstacles have prevented the development of a large scale industrial process based on shoot cultures so far. Natural pyrethrins and biotechnological alternatives have been described[151]. Hitrni et al.[153] have written a critical review [152]on the production of pyrethrins by plant cell and tissue cultures of Chrysanthemum cinerariaefolium and Tagetes species. According to them although technology for plant cell culture exists, industrial applications have to date been limited due to both low economical viability and technological feasibility at large scale. Bioconservation of readily available precursors looks more attractive but more research is needed before this technique is used for industrial production of pyrethrins.
Rotenoids: Rotenoids, a group of ketonic compmmds have become of agricultural and horticultural importance due to their insecticidal and pesticidal activity as well. The rotenoids are of special value for control of leaf chewing beetles, caterpillars and specially where toxic residues are not desired. With low toxicity and relatively long residual action to warm blooded animals rotenoids are also used as a fish poison. In South America rotenoids were used to control leaf-eating caterpillars one and a half century ago and three centuries prior to that to paralyse fish. Rotenoids are respiratory enzyme inhibitors acting between NAD+ (a coenzyme involved in oxidation and reduction in metabolic pathways) and coenzyme Q (a respiratory enzyme responsible for carrying electrons in some electron transport chains) resulting in failure of respiratory frmctions. Chemically they contain cis-fused tetrahydrochromeno [3,4-b] chromenenucleus. Many rotenoids contain an additional ring e.g. rotenone.
Rotenoids are mainly produced in roots of two genras of fabaceae (Leguminosae) family-Derris and Londrocarpus grown in South America. Now, rotenoids have been reported in vivo and in vitro from a number of plant species such as Crotolaria burhid 153l, Cicer aeritinum[154], Derris sp.[155], Indigofera tinctorid [156], Abrus precatorius[157], Tephrosia purpured [158-159], T. vogeliz[158-160], T. falaformis[161], T. strigosd [162] and Trigonella foenum-graecum[163].
Steroids: Steroids are the compormds known as terpenoids or isoprenoids. Terpenes are formed by the triterpenes or triterpenoids. The term triterpene refers to a group of natural products containing 30 carbon atoms which are derived from six isoprene (5 C) rmits. Most terpenes posses carbon content in multiples of 5 C. Before the common biosynthesis of this class of products was recognized the terpenes was introduced for those compormds containing 10 C atoms and this base is still used for the modern classification of such natural products. The classification divides terpenes into six groups:
I. | Hemiterpenes-(1 x C5) |
2. | Monoterpenes-(Cl 0 2 x C5) |
3. | Sesquiterpenes-(Cl5 3 x C5) |
4. | Diterpenes-(C20 4 x C5) |
5. | Sesterpenes-(C25 5 x C5) |
6. | Triterpenes-(C30 6 x C5) |
Hrmdreds of isoprenoids have been formd and the actual number existing in the plant kingdom is probably in the thousands. Many of these are of interest because of their commercial uses and because they illustrate the ability of plants to synthesize a vast complex of compormds not formed by animals. Some steroids important to living world are:
1. | Sterols |
2. | Bile acids |
3. | Steroid hormones (e.g., sex hormones and hormones of the adrenal cortex) |
4. | The Vitamins ofD group |
5. | Steroid saponins (Saponin = Sapogenin + Sugar; Sapogenins are used in the commercial preparation of steroidal hormones) |
6. | Heart glycosides |
7. | Steroid alkaloid |
All of these substances have skeletal structure of sterane or cyclopentoperhydrophenanthrene, which is then subjected to modifications which vary from group to group.
The sterols are most often discussed steroids in the plant literature. They are crystalline steroids which contain an alcoholic group and may be either saturated or rmsaturated. Sterols have at least 2 functions.
As precursor in the formation of other steroids e.g., cholesterol and sitosterols are precursors in the formation of saponins As components of cell membranes e.g., cholesterol and phospholipids m animals and phytosterols, phospholipids, glycolipids and sulpholipids in plants.
Depending on their origin, they are called Zoosterol (from animals), phytosterols (from plants), mycosterols (from fungi) and marine sterols (from marine organisms e.g. sponges). Phytosterol have been isolated from a large number of plant species. They are also reported from tissue cultures of a number of plant species such as Datura metef! 64l, Digitalis lanatd [165], Dioscorea tokord [166], Helipterum roseum[167], Lindera strychnifolid [168], Momordica charantid [169], Sesamum indicum[170], Stephania cepharanthd [171], Tephrosia purpured [172], Tobacco[173], Yucca glaucd [174], Uncaria tomentosd[175].
Steroidal sapogenins are of economic importance as main precursors of many medicinally useful steroidal hormones such as sex hormones, corticosteroids and oral contraceptives. Economically steroidal sapogenins are isolated mainly from species of Agave, Dioscorea and Yucca. They have been reported from several other plant genra such as Aspargus, Balanites, Costus, Lycopersicon, Solanum, Tribulus, Trigonella and Veleriana. Tissue cultures of a number of plant species also produce them. Furuya et al.[176] have isolated saponins and sapogenins from callus tissue of Panax ginseng.
Among the steroidal sapogenins, diosgenin is most important and highly investigated. Diosgenin, is a major raw material in the commercial production of steroidal contraceptives and corticosteroids and is principally obtained from the rmdergrmmd portions of various Dioscorea species in 4-5% concentration on dry weight basis. Plant tissue cultures are the ideal system for studying the frmdamental aspects of biosynthesis of diosgenin. Diosgenin has been reported from tissue culture of a number of plant species such as Agave wightizi[177], Costus speciosu}[178], Dioscorea sp.[179-186], Daucus carotd [187], Holarrhena antidysenfericd [188], Solanum sp.[189-198] Trigonella foenum-graecum[199], Yucca aloefolid [200], Y. glaucd [201], Y. shidigerd [202].
Increase in the yield of steroids, mainly steroidal sapogenins by incorporation of precursors had been a field of interest to tissue culturists. Effect of cholesterol, a precursor of sapogenins has been extensively investigated which showed an increase in diosgenin content in suspension cultures of Costus speciosus, Dioscorea floribundaSolanum aviculare, S. xanthocarpus[203], S. elaeagnifolium[204]. Effect of hormones on diosgenin biosynthesis in Dioscorea floribunda Solanum aviculare, S. xanthocarpu:f.[203], S. elaeagnifolium[204]. Effect of hormones on diosgenin biosynthesis in Dioscorea deltoided[184] and Trigonella foenum-grecum[205-207] has also been formd to increase the diosgenin and other steroid levels in tissue cultures.
In 1785, the English physician Withering used the red foxglove (Digitalis purpurea) as a remedy for heart diseases for the first time in Europe. Since then the genus Digitalis has become an indispensable accessory to the physicians. The efficacious substances of the plants are called glycosides after their field of application. They consist of a glycone with 23 carbon atoms which are linked with a varying number of sugars. The aglycone of the heart glycosides are known as cardinolides or cardiac glycosides as they have been employed in treatment of heart disease. A steroid with 21 carbon atoms, pregnenolone, derived from tmknown intermediates is converted to digitoxigenin and other cardenolides. There are a number of papers describing production and biotransformation of cardiac glycosides in Digitalis purpurea and D. lantana tissue cultures[208-212]. Biosynthesis of cardenolide drom malonate in Digitalis lanata was studied by Groenveveld[213] and the effect of precursors and inhibitors on cardenolide metabolism in D. lanata was studied by Milek et al.[214]. Effects of digitoxigenin, digoxigenin and varwus cardiac glycoside[215] and effects of various pregnanes and two 23-nor-5-cholenic acids[216] on cardenolide accumulation in cell and organ cultures of D. lanata has also been reported. Moldenhaur et al.[217] have reported cardenolides in Digitalis lanata cells transformed with Ti plasmid. Although there are a number of papers describing production of cardiac glycosides in Digitalis purpurea and D. lanata tissue cultures, generally the yield was very low and moreover, during successive transfers of the cultured cells the amormt of cardenolides often decreased and disappeared completely.
Steroidal alkaloids are the alkaloids whose carbon skeleton is finished exclusively by 5 C rmit. They have a fairly complex nitrogen containing nucleus and usually classified structurally with alkaloids but biosynthetically with terpenes. Two important classes of steroidal
alkaloids are:
1. | Solanum type-are formd in the form of glycosides which are ethers that join a non-carbohydrate moiety the aglycone, by an ester bond to a carbohydrate. Solanidine is the nucleus (i.e. aglycone) for two important glycoalkaloids, solanine and chaconine occuring in plants of solanaceae family. |
2. | Veratrum type-There are more than 50 veratrum alkaloids including veratrrnincyclopamine, cycloposine, jervine and muldarnine occuring in plants of Veratrum sp. |
Isolation and characterization of steroidal alkaloids from tissue cultures of some solanaceous plants such as Solanum dulcamara, S. jasminoides, S. khasianum,
S. nigrnm, S. xanthocarpum, has been reported[[198,218,224] Vagujfalvi et al.[225] have reported absence of solasodine but presence of diosgenin in tissue cultures of Solanum laciniatum[225].
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