International Journal of Research and Engineering. Volume 2, Issue ISSN (Print) ISSN (Online)

Transcription

1 Estimation Of Phytochemicals In The Callus And Invivo Of (Artemisia Scorparia) Sandeep Kumar, Shriyansh Dixit NIMS Instituteof Engineering And Technology, Jaipur, Rajasthan,India Abstract: In the present work investigation was carried out in medicinal plants Artemisia scorpaia by culturing at in vivo conditions. The results obtained from the present study concluded that the plant parts of A.scorparia when cultured on the basal MS media supplemented with appropriate growth hormones can give rise to an unorganized mass of cells called callus. Callus induction from explant from both in vivo and in vitro raised plants, viz., seed, stems, from aerial shoots was initiated within 4 to 5 weeks of inoculation in basal medium supplemented with various concentration of auxin and cytokinins. Present study shows the importance of plant growth regulators in callus production and indirect regeneration. And in next section of study production of primary & secondary phytochemicals were extracted and observed from thin layer chromatography technique.a.scorparia plant is rich in primary metabolites. And in case of secondary metabolites quercetin&kaempferol were extracted as flavonoids whereas β-sitosterol&stigmasterol were extracted as carotenoids. Keywords: Artemisia scorparia, plant phytochemicals,quercetin, kaempferol, β-sitosterol, Stigmasterol, Asteracea INTRODUCTION Medicinal plants have been identified and used throughout human history. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against attack from predators such as insects, fungi and herbivorous mammals. At least 12,000 such compounds have been isolated so far; a number estimated to be less than 10% of the total. Chemical compounds in plants mediate their effect on the human body through processes identical to those already well understood for the chemical compounds in conventional drugs; thus herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines to be as effective as conventional medicines, but also gives them the same potential to cause harmful side effects [1]. Plant extracts are a valuable source of new therapeutically relevant natural products. Artemisia annua (Family- Asteraceae) has been used in traditional medicine for treating fever and malaria. There are several species of Artemisia. Known as aromatic fragrance plants that have a characteristic scent and taste. Some Artemisia species are used medically because they repel helminthes of intestines. For example, in folk medicine, for the treatment of worms, it is consumed as a type of tea for three consecutive days before going to sleep. In addition, other Artemisia species are recommended for neurological disorders. Some kinds of Artemisia are used in Iraqi folk medicine for the treatment of diabetes mellitus. Furthermore, a tea made from both leaves and flowers of Artemisia that grow in Egypt is used as repellent for intestinal gases and a gusher of menstruation. While a certain Artemisia species that grows in Saudi Arabia is used as a cure against rheumatism and to treat cold, another Artemisia species in Tunisia is used in traditional medicine as decoction for their antivenin, antiinflammatory, anti-rheumatic and antimicrobial properties. It has been reported that Artemisia species that grow wild in the uncultivated land in north India, and distributed from central Europe to Western Asia and Western Himalayas have medicinal properties like anticholesterolemic, antipyretic, antiseptic and used in the treatment of hepatitis, jaundice, and gall bladder inflammation [2]. Plants have been used in treating human diseases for thousands of years. Some 60,000 years ago, it appears that Neanderthal man valued herbs as medicinal agents, this conclusion is based on a grave in Iran in which pollen grains of eight medicinal plants were found [3]. One of these allegedly ancient medicinal herbs, yarrow, is discussed in this work as a modern medicinal plant.since prehistoric times, shamans or medicine men and women of Eurasia and the Americas acquired a tremendous knowledge of medicinal plants. All of the native plant species discussed in detail in this work was used by native people in traditional medicine. The fact that hundreds of additional species were also used by First Nations Canadians suggests that many of these also have important pharmacological constituents that could be valuable in modern medicine.there are a huge number of medicinal plants. In the US, almost 1800 medicinal plant species are commercially available. It has been estimated that about 13,000 species of plants have been employed for at least a century as traditional medicines by various cultures around the world [4]. Medicinal chemicals:- The medicinal qualities of plants are of course due to chemicals. Plants synthesize many compounds called primary metabolites that are critical to their existence. These include proteins, fats, and carbohydrates that serve a variety of purposes indispensable for sustenance and reproduction, not only for the plants themselves, but also for animals that feed on them. Plants also synthesize a dazzling array of additional components, called secondary metabolites, whose function has been debated. 6 ISSN (Print) ISSN (Online)

2 Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including: The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits. To quickly produce mature plants. The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds. The regeneration of whole plants from plant cells that have been genetically modified. The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens. The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchid. Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationaryphase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobilephase) is drawn up the plate via capillaryaction. Because different analytes ascend the TLC plate at different rates, separation is achieved.thinlayer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. 1. MATERIALS AND METHODS Plant Artemisia Scorpariawere collected from university of Rajasthan,Jaipur,afterward seed & stem were crushed to powered form for extraction process, in soxhlet apparatusmethanol was used as extraction solvent. The present investigation was carried out on the biochemical studies of medicinally important plant viz. Artemisia scorparia in vivo and in vitro. Establishment of tissue culture of the selected plants Artemisia scorparia plants were collected from nursery of Rajasthan University.Healthy mature leaves, nodel segment and young stem were used for tissue culture. It was washed with tap water and cut into small pieces of about 1 cm length from various regions along with the margin center and tip. These pieces washed with distilled water and used as explants. The various explants as described above were separately surface sterilized by treating with 0.1% mercuric chloride for various time periods i.e., leaves and mature stem for 2 to 5 min and nodal segment for 3 to 4 min. The sterile explants were then aseptically inoculated in 100 ml flasks containing 35 ml of [5] MS medium and B 5 medium supplemented with different concentration of auxin and cytokinin and 0.8% Agar. The resulting calli in successful cases were maintained by frequent subculture after every 6 to 8 weeks. In first few subsequent transfer the peripheral tissue mass was preferred for sub-culturing. Production of primary metabolites. Dried plant crushed in mortar and pastle (leaves,seed,stem,fruit,) of plant A.scorparia. Extraction was done with 80% methanol in water bath at C,Left overnight for separation,after that filetation was done using Whatmannfilterpaper. Production of secondary metabolites. Extraction of Alkaloid: Artemisinin was extracted from different plant parts of A. scorporia(stem, root, seed)(2, 4, 6 and 8 weeks old as well as tissue samples) TLC for alkaloid Thin glass plates (20x20cm) were coated with Silica gel-g (250 m thick). The freshly prepared plates were dried at room temperature, There after these were kept at 100 C in an oven for 30 min to inactivate the enzymes and then cooled at room temperature.each of the extract was cochromatographed with authentic alkaloids, artemisinin as a marker. These plates were developed in an airtight chromatographic chamber saturated with solvent mixture (NH 4 OH: methanol, 98.5: 1.5). The developed plates were air dried and visualized under UV light and by exposure to ammonia fumes. Extraction of Flavonoids: The methanol soluble fractions were filtered, concentrated in vacuo and the aqueous fractions were fractioned by sequential extraction with petroleum ether (Fr-1), ethyl ether (Fr-II) and ethyl acetate (Fr-III) separately.each step was repeated thrice for complete extraction, fraction I was discarded because it contained fatty substances, whereas fraction II and III were concentrated and used for determining free and bound flavonoids respectively.fraction III was further hydrolyzed by refluxing with 7% sulphuric acid (10 ml/g plant material for 2 hr), filtered and filtrate was extracted thrice with ethyl acetate.all ethyl acetate layers were pooled together separately, neutralized by distilled water with repeated washings, and concentrated in vacuo. Both fraction II and fraction III were taken up in small volume of ethanol (2-5 ml) before chromatographic examination. TLC for Flavanoids Each of the extract was co-chromatographed with authentic samples of flavonoids (Kaempherol and Quercitin) as markers.these plates were developed in an air-tight chromatographic chamber saturated with solvent mixture (Benzene : Acetic Acid : Water :: 125 : 72 : 3). [6].The developed plates were air dried and visualized under UV light and by exposure to ammonia fumes.the mouth of a 100 ml bottle containing concentrated NH 4 OH was held in contact with each spot for about 5-10 seconds and fluorescent spots corresponding to that of standard markers were marked. The developed plates were also sprayed with 5% FeCl 3, 0.1% alcoholic AlCl 3 and kept in I 2 chamber separately. The coloured spots thus developed were noted and the Rf value of each spot was calculated. 7 ISSN (Print) ISSN (Online)

3 Several other solvent systems such as n-butanol, acetic acid, water (4:1:5, upper layer), n-butanol, acetic acid, water (3:1:1) were also tested, but the solvent system containing benzene, acetic acid and water (125 : 72 : 3) gave better results. Extraction of Sterols Dried and powdered plant test materials were defatted in petroleum ether (60-80 o C) for 24 hr on a water bath.defatted material was air-dried and hydrolyzed in 30% HCl (v/v) for 4hrs.Each hydrolyzed sample was washed with water till ph 7 was obtained and dried.the dried preparation was again extracted with benzene for 24 hrs.the extract was filtered and dried in vacuo. The crude extract was dissolved inchloroform before chromatographic examination [7]. TLC for sterols Each of the extract was co-chromatographed separately with authentic sterols ( -sitosterol and stigmasterol) standard.these plates were developed in an air-tight chromatographic chamber, saturated with solvent mixture (hexane: acetone: 8: 2) [8]. Other solvent systems such as benzene and ethyl acetate (85 : 15) [9] benzene : ethyl acetate (3:1,)[42] were also used but hexane acetone (8 : 2) gave better separation. These plates were air-dried and visualized under UV light and fluorescent spots corresponding to that of standard markers were marked.these developed plates were sprayed with 50% H 2 SO 4.[10] and anisaldehyde reagent, separately and heated at 110 o C for 10 min. 3.RESULTS AND DISCUSSIONS 3.1. Tissue culture study The aim of present study was to establish the protocol for the callus culture of medicinally important plants of Artemisia Scorparia in vitro for their biochemical estimations. The Results obtained from the present study concluded that the plant parts of A.scorparia when cultured on the basal MS media supplemented with appropriate growth hormones can give rise to an unorganized mass of cells called callus. This proliferation of cells can be maintained more or less indefinitely by sub-culturing the callus on to fresh medium periodically. Growth indices: - The maximum growth index was observed in 6 weeks old tissues grown as static cultures in both the plants, which decreased subsequently in 8 weeks old tissues. (Tables 3.1.1). The maximum growth index (1.27) in six weeks old and minimum (0.26) in 2-weeks old cultures were observed in A.scorparia the maximum growth index (1.12) was found in 6 weeks old and minimum (0.25) in 2-week old cultures. In the present study stem, taken from field grown plants of A.scorparia Callusing and regeneration was observed from all type of explants and best response was from Nodal explants A.scorparia All the cultured explants showed callus formation. About 60% of the nodal segment explants, 54% of the stem explants, 63% of the shoot bud explants, 76% of the seed have successfully produced calluses after 5 weeks. Present study shows the importance of plant growth regulators in callus production and indirect regeneration. It is likely that different explants require specific growth regulators at various concentrations in various combinations for exhibiting such morphogenetic responses as callusing, shooting, rooting, shoot and root elongation. Not only the source of explants but their inheritance and age are likely to influence in vitro morphogenesis. One more factor is the levels of endogenous growth regulators in each explant just before culture that often is not taken into consideration while discussing the effects caused by growth regulators. The synergistic and antagonistic effects of the growth regulators used in combination must also be taken into account. Subculture of callus on the same medium after 4-5 weeks showed induction of large number of somatic embryos, which was confirmed with histological studies. Development of embryoids in plantlet took place when the embryogenic callus was transferred to basal MS (Murashige and Skoog, 1962)[5] medium supplemented with BAP (5 mg/l), CH (Charcoal) (50 mg/l) + CW (Coconut water) (20%). Roots were developed by sub culturing them on the medium containing kinetin or BAP (5 mg /l ) and IBA (4 mg /l). Plantlets were successfully transferred to pots containing mixture of soil, sand and farmyard manure (2:1:1). Callus induction from explant from both in vivo and in vitro raised plants, viz., seed, stems, from aerial shoots was initiated within 4 to 5 weeks of inoculation in basal medium supplemented with various concentration of auxin and cytokinins. The best results obtained were in the medium supplemented with a combination of NAA 2mg/l + KIN 0.5mg/l All the cultured explants showed callus formation. About, 65% of the stem explants, 66% of the shoot bud explants, 53% of the seed have successfully produced calluses after 5 weeks. The callus was maintained on MS + NAA 1.0 mg/l mg/l KIN and growth indices calculated. FIG. Showing induction of shoot bud after 4 days 8 ISSN (Print) ISSN (Online)

4 Table Growth indices (GI) of tissue culture of A.scorparia S. No. Age of Tissue (in Weeks) Growth Indices (GI) Multiple shoot induction in A.scorparia through nodal segments Auxillary bud provides a rapid multiplication system in which the number of potential plants is increased exponentially by repetitive reculturing. Nodal segments with axillary buds from mature plants were used as primary explants. MS medium lacking growth regulators failed to induce shoots formation from nodal explants. Both cytokinins (BAP, Kinetin) facilitated axillary bud initiation. Number of newly formed buds depends on the concentration and type of cytokinin used to analyse the impact of BAP on shoot proliferation, it was supplemented to the MS medium at mg/l. However, at concentration of BAP (2.0 mg/l), 7-8 shoots were produced by the nodal segments. Further increase in the BAP concentrations was not found to elicit favorable responses.however, the number gradually increased up to with the formation of adventitious shoots on MS medium supplemented with BAP (2.0 mg/l), Kn (3.5 mg/l). Increased concentration of BAP and Kn in combination minimized the number of shoots. These in vitro obtained multiple shoots were healthy and dwarf in nature. These shoots were elongated on same medium supplemented with gibberelic acid (0.5 mg/l). The caulogenic effect of BAP in combination with Kn observed in the present study is in consonance with other workers[11][12] Further in vitro raised multiple shoots after separation from the bunch of shoots were elongated on MS medium incorporated with BAP and Kn along with gibberelic acid, elongation of shoots on gibberelic acid favoured The optimal role of IBA for rooting in the regenerated plantlets has also been reported by several workers [12]. In conclusion, the present results of micropropagation of A.scorparia.through tissue culture techniques were encouraging as these eliminates labour intensive, shorten time period required for the development of plantlets and overcome the problems arising due to indiscriminate harvest from wild population and rapid loss of germinability of the seeds. The finding described here will provide help in efforts on large scale production of identical plants of A.scorparia. Medium : MS + Sucrose (3.0%) + BAP / Kn ( mg/l) 1) Inoculum : Nodal segments Incubation : At 26±2 C under 16 hours photoperiod for three-four weeks Cytokinin levels (mg/l) Kn No. of shoot buds per explant (Average 10 explants) Control Nil Nil BAP 0.5 Nil Table Effect of BAP in combination with Kn on axillary bud proliferation Table Effect of cytokinins on axillary bud proliferation 9 ISSN (Print) ISSN (Online)

5 Medium : MS + Sucrose (3.0%) + BAP (2.0 mg/l) + Kn ( mg/l) Inoculum : Nodal segments Incubation : At 26±2 C under 16 hours photoperiod for three-four weeks Con. of Kn (mg/l) No. of shoot buds per explant (Average 10 Explants) Nil 3.2. Estimation of Primary metabolites of A.scorparia:- Carbohydrates Quantitative estimation of glucose showed that content of glucose was more in stem6.65 mg/gdw, and minimum amount i.e., 3.80 mg/gdw in seed. Starch The maximum amount of starch was found in stem (7.12 mg/gdw) and minimum amount was observed in seed (2.3 mg/gdw) Protein The maximum amount of protein was observed in seed (44.40 mg/gdw) and minimum amount was observed in stem (22.62 mg/gdw) Lipid Results obtained from quantitative estimation of lipid showed that seeds of A.scorpariacontains more lipids i.e., (72.20 mg/gdw) and stem part contain lowest amount of lipid i.e., (21.13 mg/gdw). Phenol The darkening of cut or dying plant parts is caused by the reaction of phenols. Stem contains highest amount of phenol (72.46 mg/gdw) and seed contain lowest amount (19 mg/gdw). The chemical analysis of primary metabolites in A.scorpariahas not been reported. The present investigation deals with the evolution of primary metabolites.a.scorpariaplant is rich in primary metabolites. Most of natural products are compounds biosynthetically derived from primary metabolites such as amino acids, carbohydrates, fatty acids and are generally categorized as secondary metabolites. Table Primary metabolites from various plant parts of A.scorparia S. N o. Pla n t p a Carbohydr ates mg/gdw Starc h mg/ gd w Prote in mg/ gd w Lipid mg/ gd w Phen ol mg/ gd w rt s 1 Ste m 2 See d Fig. Micropropagation of A.scorparia showing multiplication of shoots 10 ISSN (Print) ISSN (Online)

6 Quercetin(0.13 mg/gdw),so net amount of bound flavonoids in stem was (0.36 mg/gdw). TLC results of flavonoids under U.V illuminator:- Fig Primary metabolites from various plant parts of A.scorparia Fig. Under U.V illuminator showing sample of seed 3.3. Estimation of secondary metabolites Flavonoids The qualitative and quantitative estimation of identified flavonoids (kaempferol, quercetin) from A,scorparia in vivo and in vitro. The presence of two flavonoids, kaempferol and quercetin was confirmed by thin layer chromatography. Two spots conciding with of kaempferol and quercetin in Rf values (kaempferol 0.86 and Quercetin 0.78) and the colour reaction tests, when sprayed with 5% ethnolic FeCl 3 (kaempferol, bright yellow and quercetin yellow). Further Identification of isolated flavonoids was done` by mp (kaempferol, C, quercetin C) Free flavonoids content in seed & stem of A.scorparia The concentration of free flavonoids in seed Kaempferol(0.52% mg/gdw), and concentration of free flavonoids Quercetin (0.33% mg/gdw). So total amount of free flavonoids in seed of A.scorpariacalculated was (0.85% mg/gdw ). The concentration of free flavonoids in stem part was observed (0.2 mg/gdw)kaempferol, and concentration of Quercetin was (0.16 mg/gdw). So total amount of free flavonoids in stem part of A.scorpariawas calculated (0.36 mg/gdw). Bound flavonoids content in seed & stem of A.scorparia The concentration of bound flavonoids in seed Kaempferol(0.45 mg/gdw), Quercetin(0.15 mg/gdw),so total amount of bound flavonoids present in seed of A.scorpariawas (0.6 mg/gdw). The concentration of bound flavonoids in stem Kaempferol(0.23 mg/gdw) &stem Fig. Comparing between standard (Quercetin) & sample Sterols In A.scorparia extracts, when the plates were visualized under UV lamp two of the spots gave characteristic fluorescence and their Rf values were comparable to their respective standard compounds. (β-sitosterol - pinkish grey, Rf 0.90; Stigmasterol - greyish violet, Rf 0.83). The characteristic colours were also developed when TLC plates were sprayed with anisaldehyde reagent (β-sitosterol pink; Stigmasterol - greyish violet) and with 50% sulphuric acid (β-sitosterol - pink; Stigmasterol - greyish violet) corresponding to their authentic standard compounds. Melting points (β-sitosterol CStigmasterol C) were also measured and compared with authentic standard compounds. IR spectra and authentic sample standard. Quantitative data revealed that in A.scorpariathe maximum amount of total sterols (β-sitosterol and Stigmasterol) in seed (22.12 mg/gdw) and minimum in stem (15.82 mg/gdw). However, till date there was no report on the presence of sterols from A.scorparia In the present study β-sitosterol and Stigmasterol have been confirmed in plant species, namely A.scorparia. 11 ISSN (Print) ISSN (Online)

7 Total sterol content ( β-sitosterol + Stigmasterol) (mg/gdw) in various plant part of A.scorparia S. N o. Plant part s β-sitosterol (mg/gdw) Stigmasterol (mg/gdw) 1 Seed Stem Total Sterol ( β-sitosterol + Stigmaster ol) content (mg/gdw) of sterols Fig. Standard and sample 4. CONCLUSIONS & RECOMMENDATIONS Fig. Total sterol content ( β-sitosterol + Stigmasterol) (mg/gdw) in plant part of A.scorparia TLC results of sterols under U.V illuminator :- Fig. Sterols contents in A.scorpariaunder uv illuminator The main motive of study with this desired planta.scorpariadue to it s culturing properties and medicinal properties. Callus induction from explant from both in vivo and in vitro raised plants, viz., seed, stems, from aerial shoots was initiated within 4 to 5 weeks of inoculation in basal medium supplemented with various concentration of auxin and cytokinins. All the cultured explants showed callus formation. About, 65% of the stem explants, 66% of the shoot bud explants, 53% of the seed have successfully produced calluses after 5 weeks. Primary metabolites Primary metabolites participate in growth, development, and reproduction of the plants. The primary metabolite is typically a key component in maintaining normal physiological processes; thus, it is often referred to as a central metabolite. Primary metabolites are typically formed during the growth phase as a result of energy metabolism, primary metabolites which are present in plant(a.scorparia) was carbohydrates,proteins, lipids, phenols. A.scorpariaplant is rich in primary metabolites. Most of natural products are compounds biosynthetically derived from primary metabolites such as amino acids, carbohydrates, fatty acids and are generally categorized as secondary metabolites. Secondary metabolites Secondary metabolites are typically organic compounds produced through the modification of primary metabolite syntheses. Secondary metabolites do not play a role in growth, development, and reproduction like primary metabolites do, and are typically formed during the end or near the stationary phase of growth.alkaloids present in A.acorparia is atremisinin which shows outstanding antimalarial properties Artemisinin is considered as the natural, active and potent antimalarial drug Artemisia annualinn. is the only known source for the production of artemisinin. A. scoparia Waldstet Kit. to find out an alternative of A. annuafor the production of artemisinin 12 ISSN (Print) ISSN (Online)

8 Flavonoids Different plant parts as well as tissue samples ( 2, 4, 6 and 8 weeks old) of A.scorpariawere air dried and powdered, separately. Each of these were extracted separately with 80% methanol on a water bath [40] for 24 hr. The methanol soluble fractions were filtered, concentrated in vacuo and the aqueous fractions were fractioned by sequential extraction with petroleum ether (Fr-1), ethyl ether (Fr-II) and ethyl acetate (Fr-III) separately. Sterols Glass plates coated with silica gel-g as described above were used. Each of the extract was co-chromatographed separately with authentic sterols ( -sitosterol and stigmasterol) standard. These plates were air-dried and visualized under UV light and fluorescent spots corresponding to that of standard markers were marked. These developed plates were sprayed with 50% H 2 SO 4 andanisaldehyde reagent, separately and heated at 110 o C for 10 min.[45] 5. ACKNOWLEDGEMENTS I am very grateful to all the people who in one way or the other have helped me to finally put this thesis together. I specifically want to acknowledge: I would like to express my sincere gratitude to my dissertation supervisor Dr. Sandeep Kumar (Professor) for giving me the opportunity to work on this topic. His ability to probe beneath the text is a true gift, and his insights have strengthened this study significantly. I benefited a lot from his wisdom, knowledge, and deep concern, not only for me, but for all those who are in touch with him. Without his encouragement, friendly attitude, vision, meticulous planning and efforts for making things run smoothly, it would not have been possible for me to complete this work. I am very thankful to Dr. Sandeep Kumar for accepting me for this project. I also thank him for teaching me many concepts and also helping me while working in the lab. I want to thank National Institute of Engineering & Technology, Jaipur for providing necessary chemicals and equipment during my work. Last but not the least I want to thank my parents, brother and sister for their moral supports. (5) Murashige T, Skoog F, A Revised Medium For Rapid Growth And Bioassays With Tobacco Tissue Cultures. Physiol. Plant. 15, 1962, (6)Wong E, Francis CM. Flavonoids In Genotypes Of Trifoliumsubterraneum-I-The Normal Flavonoid Pattern Of The GeraldtonVariety. Phytochemistry. 7, 1968, (7). Kaul B, Staba EJ. Dioscoreatissuecultures. I. Biosynthesis And Isolation Of DiosgeninFromDioscoreadeltoideaCallus And Suspension Cells. Lloydia., 31, 1968, (8). Fazli FRY, HardmanR,TheSpice, Fenugreek (TrigonellaFoenumgraecom): Its Commerciall Var. Of Seed As A Source Of Diosgenin.Trop.Sci.10, (9)Heble MR, Narayanaswamy S, Chadha MS. DiosgeninAnd Β-Sitosterol: Isolation FromSolanumxanthocarpumTissue Cultures. Sci. 161, 1968, (10)Heftmann E. Thin Layer Chromatography OfSteroids.Chromatogr.7,1965, (11)Murashige T, Skoog F, A Revised Medium For Rapid Growth And Bioassays With Tobacco Tissue Cultures. Physiol. Plant. 15,1962, (12) Siddique F M, Anis, M. In vitro rapid regeneration of plantlet from nodal explants of Mucunapruriens- a valuable medicinal plant. Annals of Applied Biology ,pages: 1-6. (13)Sanatombi, K,Sharma, GJ. MicropropagationOfCapsicum FrutescensL. Using Axillary Shoot Explants. ScientiaHorticulturae113,2007, REFERENCES (1) Petrovska BB. Historical Review Of Medicinal Plants, PharmacognRev. 6(11),2012, 1-5. (2) Azza AN, Tawashi A, Mohammad AH, Al- TallaZAH, Rifai NAL, ComparisionOf Artemisia Annua Bioactivities Between Traditional Medicine And Chemical Extrat, CurrBioac Compd,9(4), (3) Solecki, R.S. Shanidar IV, A Neanderthal Flower Burial In Northern Iraq, Science. 190(28)1975, (4) ArnasonTR,J Hebda, T Johns.Use Of Plant For Food And Medicine By Native Peoples Of Eastern Canada. Canadian Journal Of Botony 59(11), 1981, ISSN (Print) ISSN (Online)