In Vitro Culture, Chemical Composition Profile and Biological Activities of Paris polyphylla var. chinensis (Franch.) Hara

Abstract

Bioactive compounds from rhizome of Paris polyphylla var. chinensis (PPC) have been reported to have many pharmaceutical activities such as antitumor, anticancer and hemostatic. However, PPC had become a severe declined in population due to deforestation and overharvesting. The present research aimed to determine suitable conditions for in vitro culture of PPC, examine its chemical constituents, and test its bioactive properties. In vitro culture of PPC in the present research was conducted on various plant parts including seed, pseudostem, and rhizome. In the investigation on PPC seed, after treated with 30 % Clorox disinfectant solution, coat was removed from the seeds which subsequently were cultured on E-20, MS, Gamborg B5, and water media, at 15 °C in dark condition. The decoated PPC seeds cultured on water had 100 % germination rate within 15 months. Culturing on the other three media could not induce the germination of the decoated seeds at all. Other regulating factors like temperature and GA3 were thus examined to determine their effect in breaking seed dormancy of PPC. For the study on the effect of temperature, PPC seeds after disinfectant treatment and coat removal were cultured on water at the temperatures of 5, 10, 12, 14, 16 and 18 °C. It was found that under 14 and 16°C conditions, 100 % germination could be attained in 15 months. The experiment on treatments by alternate cold and warm temperature regimes, 4°C and 22 °C with 4 replicas, provided the result that PPC seeds failed to germinate. The study on the effect of GA3 at 0, 5, 10, 15, 20 mg/l concentrations in breaking PPC seed dormancy also found no success in seed germination. Thus, it could be concluded that PPC seed was indeed characterized as having very deep physiological dormancy and GA3 could not break the dormancy. In vitro culture of PPC rhizome shoot tip was limited by bacterial contamination in normal tissue culture laboratory. Therefore, environmental factors such as temperature were studied on the propagation of shoot tip explants of PPC in MS free hormone medium. They were cultured in a gradient incubator at 5 to 18 °C for 45 days. It was found that temperature is a crucial factor controlling in vitro culture of PPC, and the temperatures between 14 to 16 °C were better than other temperature treatments in controlling endophytic bacteria and PPC growth. The shoot tip explants cultured at this range of temperature grow up to a stem of 7.5 cm length. At a temperature lower than 14 °C, the shoot tip explants could not grow. At the temperature higher than 18 °C, the shoot tip explants did not grow because the explants were contaminated by endophytic bacteria. It could be concluded that the temperature between 14 to 16 °C was the suitable culturing condition for shoot tip culture of PPC. The effects of BA concentrations were studied and it was found that PPC shoot tip cultured at temperature 15 °C on MS medium supplemented with BA 5 mg/l and PPM 2 ml/l had multiplication rate at 1 : 1.2 and highest height at 7.0 cm over 90 days. And adenine sulfate at all concentrations had no effect in PPC shoot tip multiplication. PPC in vitro microrhizome induction was studied by increasing sucrose concentrations from 0 – 120 g/l and no effect was found to induce microrhizome no significant difference in diameter of shoot. Also in in vitro microrhizome induction various NAA concentrations appeared to have no effect and there existed no significant difference in diameter of PPC shoots. It has been concluded that PPC seed embryo rescue is a appropriate method for propagation and could break the dormancy from 24 – 36 months to 15 month with 100% germination rate. Chemical constituent study by thin-layer chromatography (TLC) revealed presences of steroids and alkaloids from the dichloromethane crude extract (DiCE) of PPC, while methanol crude extract (MCE) afforded saponins, flavonoids and alkaloids. The evaluation of antioxidant activity by DPPH scavenging assay exhibited the highest EC50 value at 3.46+1.23 µmol TE/g from the DiCE of rhizomes collected from Mae-Rim district. MCE of rhizomes from Mae-rim district exhibited the best IC50 of cytotoxic MTT assay against HL-60 cell line at 0.77+0.1 µg/ml. These values suggested potential pharmaceutical application of PPC extracts.