2. Aim and Objectives

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1 AIM AND OBJECTIVES

2 2. Plants represent unlimited sources of natural products. Biologically active compounds are accumulated in plants especially as secondary metabolites that have been used as a source of major phytopharmaceuticals ranging from anticancer activity, phosphodiesterase inhibition to cytotoxicity against HIVinfected cells. There has been an explosive demand for herbal plants and extracts which can be used to improve human health and well being. In recent times focus on plant research has increased all over the world and a large body of evidence has collected to show immense potential of medicinal plants used in various traditional systems. Biotechnological approaches, specifically plant tissue culture plays a vital role in search for alternatives to production of desirable medicinal compounds from plants. There has been considerable interest in plant cell cultures as a potential alternative to traditional agriculture for the industrial production of secondary metabolites. The capacity for plant cell, tissue and organ cultures to produce and accumulate many of the same valuable chemical compounds as the parent plant in nature has been recognized almost since the inception of in vitro technology. The strong and growing demand in today's marketplace for natural, renewable products has refocused attention on in vitro plant materials as potential factories for secondary phytochemical products, and has paved the way for new research exploring secondary product expression in vitro and also provides an excellent forum for in-depth investigation of biochemical and metabolic pathways, under highly controlled micro environmental regimes. Different strategies using cell culture systems have been extensively studied with the objective of improving the production of bioactive secondary metabolites. There are many approaches described in the literature to increase Page 24

3 or enhance the secondary metabolites production in the cell and tissue cultures. Of the different approaches, precursor feeding, elicitor-mediated enhancement (both by biotic and abiotic means), hairy root cultures and metabolic pathway engineering occupy the prominence. Compared to metabolic pathway engineering the three former approaches are feasible. The accumulation of secondary metabolites in plants is part of the defense response against pathogenic attack which is triggered and activated by elicitors, the signal compounds of plant defense responses. Therefore, the treatment of plant cells with biotic and/or abiotic elicitors has been a useful strategy to enhance secondary metabolite production in cell cultures. Gymnema sylvestre R. Br. is a valuable and multipurpose medicinal plant belonging to family Asclepiadaceae. It is a slow growing perennial woody climber distributed over most parts of India and Africa. Gymnema Sylvestre has been used in folk, ayurvedic and homeopathic systems of medicine since ancient times. It is regarded as a plant with potent anti-diabetic properties. It is also used in the treatment of asthma, eye complaints, inflammations, family planning and snakebite. In addition, it possesses anti-microbial, antihypercholesteromic, hepatoprotective activities. In spite of its wide distribution it is considered as an endangered plant due to its overexploitation to meet the requirement of pharmaceuticals for its active principles. Thus various reports on its multiple uses, particularly as potent remedy for diabetes attracted attention for utilization of this plant for its active principles, gymnemic acids. All these functions of Gymnema sylvestre are due to the presence of oleanane type triterpenoid saponins collectively known as gymnemic acid. Indeed, saponins synthesized in Gymnema sylvestre have found use in management of diabetes and obesity. The leaves and roots of Gymnema sylvestre R.Br, possess a mixture of active principles named as "gymnemic acids(i-vii)" (triterpenoid saponins), oleanone type triterpenoid saponins, with Page 25

4 different sapogenin structure such as gymnemagenins. Gymnemic acids (GA) have various physiological effects. They suppress taste sensitivity to sweetness, inhibit intestinal glucose absorption and lower the plasma glucose and insulin levels. There are however, few reports about molecular interaction of gymnemic acids with proteins. Crude saponins factors from Gymnema sylvestre have been shown to possess potent S-GLUT-1 (Na*-GLUT-1) mediated inhibition of glucose transport, anti-hyperglycemic activity. Since the gymnemic acid fraction is allegedly beneficial for treating diabetes and obesity, preparations containing Gymnema sylvestre plants of Indian origin are now commercially available in Japan, Germany and USA as health foods. In Indian domestic market the leaf powder of Gymnema sylvestre is available as Gymnema tea for the benefit of diabetic patients. In the recent past there is a very good boom for the herbal drug industry worldwide. Commercial preparations containing Gymnema sylvestre leaf powder are widely traded through the internet as a nutritional supplement, mainly from Indian and American sources. The United States Food and Drug Administration has not established a recommended dosage for Gymnema sylvestre. The quality varies between and within brands. Quality Gymnema sylvestre should be standardized to a minimum of 25% gymnemic acid. A single 500 mg capsule standardized to 25% yields 125 mg of active gymnemic acid per capsule. In India export of leaf powder varied from 2002 to 2006 between 10,600 kg to 278,960 kg. In domestic and international market Gymnema extracts are available in different forms or as ingredients in various Ayurvedic and medicinal formulations. Some of the products are, Gymnema sylvestre extract in the form of capsules 450 mg (60 capsules) per container price $ 8.95, Gymnema herbal tea Net Wt. 125 gms price $15.50, Dia-Botica Capsules 500 mg (60 Capsules) in which Gymnema sylvestre extracts are present 100 mg/400 Page 26

5 mg wt. along with other plant extracts, price $ In domestic market one of the purified product of the Gymnema triterpenoids gymnemagenin is priced at Rs.9,600/- per 10 mg quantity (as provided by Natural Remedies, Bangalore; w w w.naturalremedy. com). But, unfortunately the less availability of the compounds in the natural sources and purification costs made these compounds costly (gymnemagenin is priced at Rs.9,600/- per 10 mg) in their pure form, the price is not affordable by the common man. The total yield of gymnemic acid fraction crude extracts of Gymnema leaves (collected from different parts of India) vary from 0.96% to 1.56% (0.96 gm/100 gm to 1.56 gm/100 gm of dried leaf powder, and the gymnemagenin (major sapogenin) content is 3.9 to 4.6% of total gymnemic acid fraction. Currently, in domestic market one of the purified product of the Gymnema triterpenoids gymnemagenin is priced at Rs.9,600/- per 10 mg quantity. The yield gymnemagenin is 40 mg per 100 gms of dried leaf material according to published reports. 40 mg of gymnemagenin would cost nearly Rs.38,400/-. In spite of the numerous studies concerning the occurrence, chemical structure and biological activities of Gymnema saponins, all the gymnemic acids synthesized by G. sylvestre were also not isolated and purified till-date. There are limited published evidences so far pertaining to the micropropagation of Gymnema sylvestre. Though there are reports on production of gymnemic acid in cell and tissue cultures of Gymnema sylvestre, but, very few attempts have been made towards the increase or enhancement of gymnemic acid production in cell and tissue cultures, particularly with regard to elicitor enhanced production of gymnemic acid. Till-date no attempts in the direction of genetic transformation have been made in the literature about Gymnema sylvestre. _

6 2.1. Aim of the present study: The main aim of the present study is to fill the lacuna or bridge the gap found in the pertinent literature cited above by developing protocols for the enhancement of gymnemic acid production in cell and tissue cultures Gymnema sylvestre R.Br, and also develop protocols for its genetic transformation. In view of all these facts and findings and lacuna, the following objectives have been drawn to design and carry out the present study on Gymnema sylvestre for the active principle production and to confirm the accessibility of these plant species for genetic transformation by Agrobacterium Objectives: < To develop and standardize protocols for the in vitro propagation from ex vitro explants > To optimize the culture conditions for rapid propagation to increase the biomass production Biochemical monitoring of explant proliferation and regeneration Optimization of medium and culture conditions for the enhancement of active principle production by different approaches Biochemical characterization of regeneration and genetic transformation using Agrobacterium. Page 28

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