A Micropropagation System for Tylophora Indica and Extraction of Tylophorine from Cultures and in Vitro Raised Plants

Abstract

Tylophora indica,(Burm.f.) Merill (Family Asclepiadaceae) is an important endangered medicinal plant which is used as a folk remedy for the treatment of a number of diseases and ailments. The roots and leaves of this plant contain pharmacologically active alkaloids tylophorine, tylophorinine and anticancerous tylophorinidine. Due to the lack of adequate propagation efforts and over exploitation of natural wild populations, Tylophora indica is threatened with extinction. We present an efficient and reproducible protocol for the mass propagation of this plant under in vitro conditions and extraction of tylophorinethe major secondary metabolite from callus, suspension cultures and in vitro regenerated plants. Leaf explants were excised from an elite field grown mature plant and thereafter planted on variously supplemented Murashige and Skoog’s medium for the de novo adventitious shoot formation and callus induction. Tylophora exhibited high degree of propensity of de novo shoot proliferation directly from leaf segments on MS medium supplemented with BAP (4.4μM - 22μM) either alone or in combination with adenine sulphate (1.35μM). Nodular meristemoids differentiated from the cut ends of leaf lamina after 10- 12 days of culturing and covered the whole surface of leaf explant within 3-4 weeks. Eventually, these meristemoids developed into green leafy shoots. Initially, fewer shoots were formed but number increased further to 50-60 shoots per flask on subsequent subculturing in about 85% of the cultures. Regenerated shoots when 4-5cm in length were separated and subjected to rooting on root inducing media. Microshoots cultured on basal MS medium resulted in the formation of long, healthy roots within 10-12 days and complete plantlets were formed. Plantlets were then transferred to moist cotton jars covered with polybags for initial acclimatization and were kept in growth room for 10-12 days. They were then transferred to the potting mixture of soil: vermicompost (1:1) and plants with newly formed leaves were shifted to green house for 2 weeks and eventually established in soil with 90% survival rate. Callus formation occurred from leaf explants when inoculated on different combinations of auxins and cytokinins. Murashige & Skoog’s medium supplimented with NAA (29.4μM) and Kn (4.65μM), hereby designated as NK medium turned out to be optimal for the initiation and sustained growth of calli. Leaf explants also showed callus induction on MS+ 2, 4-D (38.96μM) + Kn (4.65μM) but growth of callus was slow. Extraction of major secondary metabolite - tylophorine was carried out from the dried leaf powder of in vitro raised field established plants (2 years old), dried callus (3-4 months old) and dried suspension cultures raised on liquid NK medium. Extraction was carried out with chloroform using soxhlet apparatus followed by extraction with ethyl acetate. Thin layer chromatography was performed in the solvent system of different combinations of toluene: ethyl acetate and diethyl amine but best results were observed on 7:2:1 combination. For further fine purification of tylophorine, High Performance thin layer chromatography (HPTLC) was performed using the same solvent system as mobile phase. Three tracks 1, 2, 3 were obtained corresponding to samples of dried leaf powder, callus, and suspension powder respectively. Peak 7, peak 8 and peak 7 of track 1, 2 and 3 respectively showed Rf values comparable with the standard which confirmed the presence of tylophorine in the given samples.