Bioassay guided fractionation of Cyclea peltata using in vitro RAW 264.7 cell culture, antioxidant assays and isolation of bioactive compound tetrandrine

BACKGROUND: Cyclea peltata is one of the herbs mentioned in ancient scriptures of Ayurveda and is used in different types of Ayurvedic gritham preparations. Moreover, in traditional/tribal medicine C. peltata is used as digestive, anti-inflammatory, diuretic and to treat jaundice, digestive disorders, etc. OBJECTIVE: Activity guided fractionation of C. peltata and in correlation with the levels of bioactive compound tetrandrine. MATERIALS AND METHODS: Preliminary phytochemical screening, estimation of total alkaloid content, preparation of different extracts of C. peltata (crude extract CP, hexane extract HCP, chloroform extract CCP, methanol extract MCP, alkaloid fraction ACP). In vitro anti-inflammatory studies using RAW 264.7 cells and in vitro antioxidant assays of the different extracts of C. peltata. HPTLC estimation of tetrandrine (TET) was carried out using solvent system toluene: ethyl acetate: diethylamine (7.2: 2: 0.8) and isolation of TET from ACP. RESULTS: Preliminary phytochemical studies of C. peltata showed the presence of alkaloid content in all extracts. Whereas, saponins, steroids and terpenoids were detected in CP and CCP. ACP and TET showed significant in vitro anti-inflammatory and antioxidant activity when compared to other extracts. ACP and TET (100 μg/ml) treatment significantly inhibited the mRNA expression of iNOS, COX-2, TNF-α in LPS treated RAW 264.7 cells. HPTLC estimation of bioactive compound tetrandrine was highest in ACP-228.4 μg/mg followed by CP-29.62 μg/mg, CCP-23.46 μg/mg, MCP-18.82 μg/mg and HCP-1.25 μg/mg. TET has been isolated from ACP. CONCLUSION: The results of the present in vitro assays revealed that the alkaloid fraction (ACP) is the most active fraction when compared to other extracts and has a positive correlation with the levels of bioactive compound tetrandrine.