INTRODUCTION A large number of chemical compounds including fragrances, flavours, pigments, natural sweeteners, antimicrobials and pharmaceuticals are obtained from plants. In most cases, these compounds belong to a broad metabolic group, collectively referred to as secondary products. Plant cell cultures can be established from an array of plant species, including most that produce secondary products of commercial value (Berlin, 1984). Phyllanthus amarus (Euphorbiaceae) finds a reputed place, especially, in the Indian Pharmacopoeia (Kamboj, 2000). It has been traditionally used in the treatment of a variety of ailments, including hepatic disorders (Nadkarni, 1976). It is a potential diuretic, hypotensive and hypoglycaemic drug (Raphael, 2002). It has immense medicinal properties by virtue of containing several phytochemicals, viz., securinine, norsecurinine, epibubbialine and isobbubialine (Foo and Wang, 1992), lignans like phyllanthin and hypophyllanthin (Row et al, 1966), phenolics like gallic acid; polyphenolics like ellagic acid, phenazine and phenazine derivatives (Foo, 1995). About 300 million people worldwide are estimated to be carriers of the Hepatitis B virus. The plant has therapeutic potential for treating Hepatitis B virus by inhibiting polymerase activity and decreased episomal DNA content. It has also been shown to exhibit antihepatotoxic activity against carbon tetrachloride and galactosamine in Estimation of anti-hepatic viral compounds in Phyllanthus amarus in vitro cultures R. Chitra, E.Vadivel1 and K. Rajamani Horticulture College and Research Institute Tamil Nadu Agricultural University, Coimbatore 641 003, India E-mail: chitra.varadharaj@gmail.com ABSTRACT Phyllanthus amarus Schum. and Thonn (Euphorbiaceae) is recognized commonly as ‘Bhumyamlaki’ in the Indian system of medicine and has been traditionally used for treating a variety of ailments, including hepatic disorders. Anti-hepatic viral compounds such as phyllanthin and hypophyllanthin were evaluated in different types of in vitro cultures of Phyllanthus amarus by High Performance Liquid Chromatography (HPLC). Among the cultures, in vitro plantlets regenerating from the nodal segment recorded higher amounts of phyllanthin and hypophyllanthin. Key Words: Phyllanthus amarus, HPLC, phyllanthin and hypophyllanthin 1Directorate of Extension Education, TNAU, Coimbatore 641 003 primary cultured rat hepatocytes (Syamasunder et al, 1985). Knowledge about Phyllanthus amarus especially on its anti- viral property, has elicited a great interest in this plant, and has triggered its large-scale collection from natural flora. Availability of this plant is subject to seasonal variations, which leads to uncertainty in supply of the plant material when required (Rajasubramanian and Pardha saradhi, 1997). In vitro secondary metabolite extraction has been a precision tool for studying organic compounds in plants even when present in trace quantities. The study of cell suspension culture, hairy root culture and other in vitro cultures is an ideal method to investigate the rare compounds and, especially, many active, unknown compounds within a short period. In this background, the study was taken up to explore a lignans from in vitro culture of Phyllanthus amarus. In field grown crops, it takes about six months for extraction of these lignans whereas this time lag is just three months in in vitro cultured plantlets. MATERIAL AND METHODS Indirect organogenesis Murashige and Skoog (1962) medium was used for induction of callus from leaf bits, stem pieces, shoot tips and nodal segments of Phyllanthus amarus. Sucrose (3.0%), agar (0.8%), cytokinins, viz., kinetin (3.0 mgl-1) J. Hortl. Sci. Vol. 3 (1): 62-65, 2008 page 62 63 and BAP (3.0 mgl-1) and auxins, viz., 2, 4-D (4.0 mgl-1) and NAA (0.4 mgl-1) were added to the medium. Cultures containing 2, 4-D was inoculated under darkness by covering culture racks with a black cloth and the remaining cultures were incubated at 25±2oC in light: dark cycle of 16:8 h, respectively. Direct organogenesis For direct organogenesis by axillary shoot proliferation or by adventitious shoot formation, the explants, viz., shoot tip and nodal segments, were inoculated onto MS basal medium supplemented with BAP (2.0 mgl-1) along with GA 3 (1.0 mgl-1). After separating the multiple shoots, each individual shoot was sub-cultured onto half strength MS medium containing two auxins, IAA (0.5 mgl-1) and IBA (0.5 mgl-1). The Cultures were maintained in a growth room at 24±2oC under 16 h light and 8 h dark photoperiodic regime. Estimation of anti-hepatic viral compounds For estimation of lignans, different types of cultures were used as follows: Cultures used for estimation of anti-hepatic viral compounds Treatment Source of culture Culture medium on which (Nature of culture) the culture was initiated T 1 (Multiple shoot Shoot tip MS + BAP (2.0 mgl-1) + clumps with GA 3 (1.0 mgl-1) basal callus) T 2 (Multiple shoot Nodal segment MS + BAP (3.0 mgl-1) + clumps with GA 3 (1.0 mgl-1) basal callus) T 3 (Micro shoots Shoot tip ½ MS + IBA (0.5 mgl-1) + with roots) IAA (0.5 mgl-1) T 4 (Micro shoots Nodal segment ½ MS + IBA (0.5 mgl-1) + with roots) IAA (0.5 mgl-1) T 5 (Green callus) Shoot tip MS + BAP (3.0 mgl-1) + Kin (3.0 mgl-1) T 6 (Green callus) Nodal segment MS + BAP (3.0 mgl-1) + Kin (3.0 mgl-1) T 7 (White callus) Stem pieces MS + 2,4-D (4.0 mgl-1) + NAA (0.4 mgl-1) T 8 (White callus) Leaf bits MS + 2,4-D (4.0 mgl-1) + NAA (0.4 mgl-1) Analysis of anti-hepatic viral compounds The above in vitro grown materials were dried and ground to a fine powder using a mortar and pestle. Each powdered sample (1 g) was macerated with lime (300 mg) and HPLC grade water (2.5 ml) at room temperature and kept in a shaker for 18 hours. Thirty ml of methanol containing 3% Potassium hydroxide was added to the macerated material and kept in boiling water-bath for 30 min. The material was filtered the residue washed 3 times with 5 ml methanol and the volume of the combined filtrate and washings was made up to 50 ml. A sample (10 µl) of this solution was injected in to HPLC column and the lignans were estimated. A Varian Chromatographic system comprising of L.C.8A Model dual pump and UV detector was employed. A µ Bondapak C 18 column (30 cm X 3.9 mm) with isocratic run of solvent system of methanol: water (66:4), v/v at the rate of 1.8 ml/min flow rate and UV detection at 230 nm was used for resolving and analysing phyllanthin and hypophyllanthin. The quantification was carried out using external standards of phyllanthin and hypophyllanthin (Sigma Aldrich chemicals) and values were expressed as percentage. RESULTS AND DISCUSSION The lignans were detected and quantified in the in vitro cultures of Phyllanthus amarus. Among the various cultures, in vitro grown plants recorded highest phyllanthin (0.921%) and hypophyllanthin (0.396 %) on ½ MS medium containing IBA (0.5 mgl-1) and IAA (0.5 mgl-1) as compared to the field grown plants. Phyllanthin (0.709 w/w dry basis) and hypophyllanthin (0.271 w/w dry basis) content was estimated by the method of Anupum Sharma et al (1993) in fieldgrown Phyllanthus niruri. Similar finding was also reported by Mahalakshmi et al (2006) in Phyllanthus amarus genotypes. This was supported by the findings of Ara Kirakosyan (2003) in Hypericum perforatum and Sharma Tripti, (2006) in Artemisia annua. The contents of phyllanthin (0.714 %) and hypophyllanthin (0.261%) was low in leaf-bit derived white callus on MS medium supplemented with 2,4-D (4.0 mg l-1) and NAA (0.4 mgl-1). The level of hypericin in Hypericum perforatum callus was very low, representing only 0.11% of that found in field- grown plants (Kirakosayan, 2003). Callus initiated from stamens of H. perforatum showed only traces of hypericin or pseudohypericin (Kirakosyan et al, 2000). In general, an increase in the level, of auxins such as 2, 4-D in the medium stimulates dedifferentiation of cells and consequently diminishes the level of secondary metabolites. This is the reason that auxins are commonly added to the medium for callus induction, but used at low concentrations or omitted altogether for production of metabolites. Zenk et al (1977) reported that cytokinins stimulated alkaloid synthesis which was induced by removing auxin from the medium of a cell line of Catharanthus roseus. This was J. Hortl. Sci. Vol. 3 (1): 62-65, 2008 Estimation of anti-hepatic viral compounds 64 supported by the findings of Shiio and Ohta (1973) along with Takahashi and Yamada (1973). They reported that lower concentrations of auxins viz., IAA, NAA and 2,4-D promoted nicotine synthesis in tobacco cell cultures while higher concentrations inhibited nicotine synthesis. In the present study, the quantity of phyllanthin and hypophyllanthin was found to be higher in in-vitro grown plants than in the callus. Thus, it seems that in many cases morphological differentiation may be necessary to obtain higher yields of secondary metabolites. Hiraoka and Tabata (1974) investigated the correlation between stage of morphological differentiation and tropane alkaloid producing ability in Datura meteloides, and found that roots forming shoots produced higher amounts of tropane alkaloids. Dhar and Pal (1988) demonstrated that more pyrethrin was being synthesized in Chrysanthemum cinerariaefolium in vitro shoots than the roots and its content was even lower in undifferentiated callus culture. Chromatographic analysis of the regenerated plants of Phyllanthus amarus showed antiviral content higher than that found in field-grown plants, suggesting that an in vitro culture system could be used for this plant which may significantly reduce the cost, time and resources required for field-production of antiviral compounds, as well as enable growers to produce high quality, standard antiviral products. REFERENCES Anupum Sharma, R., Singh, S., Sukhdev, S. Handa.,1993. Estimation of phyllanthin and hypophyllanthin by HPLC in Phyllanthus amarus. Phytochem. Anal., 4: 226-229 Berlin, J.1984. Plant cell cultures – a future source of natural products. Endeavour 8: 5-8 Table 1. Estimation of anti-hepatic viral compounds from different types of cultures Sl.No Nature of culture & Phyllanthin Hypophyllanthin Source of culture (%) (%) 1. Multiple shoot clumps with 0.745 0.298 basalcallus (Shoot tip) 2. Multiple shoot clumps with 0.723 0.289 basal callus (Nodal segment) 3. Microshoots with roots 0.892 0.325 (Shoot tip) 4. Microshoots with roots 0.921 0.396 (Nodal segment) 5. Green callus (Shoot tip) 0.716 0.294 6. Green callus 0.731 0.309 (Nodal segment) 7. White callus (Stem bit) 0.728 0.314 8. White callus (Leaf bit) 0.714 0.261 Mean 0.771 0.311 SEd 0.027 0.016 CD (0.05) 0.054 0.034 3rd Peak - Hypophyllanthin (Retention time 54.33 minutes) 4th Peak - Phyllanthin (Retention time 56.87 minutes) Fig 1. HPLC chromatogram of Phyllanthus amarus Dhar, K, Pal A. 1988. Pyrethrin content in unorganized and organized callus cultures of Chrysanthemum cineraraefolium Vis. Fitoterapia LXIV., 336-340 Foo, L. Y. 1995. 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