Qualitative and quantitative determinations of anticonvulsants in centella asiatica (L.) urban

Abstract

Ethyl acetate extract of the aerial part of Centella asiatica (L.) Urban (CA) was found to possess the anticonvulsant activity in the PTZ test model. Using the fractionation of the extract by conventional chromatographic technique together with the standard PTZ antiepileptic screening model, it was found that saturated very long-chain fatty acids (VLFAs) were the active principle in CA. Spectroscopic techniques were used to elucidate the chemical structure, IR and NMR data confirmed that active principle are VLFAs with the GC-MS technique to analyze the composition of the VLFAs, revealed that the sample contain C12-C28 chain length acids, which contain C16 and C28 chain length acids as main component. A thin-layer chromatographic (TLC) method was developed to determine VLFAs by using silica gel plate GF254 as stationary phase and hexane: ethyl acetate: glacial acetic acid (14:6:1) as developing solvent, oleanolic acid as internal standard. The detection of the TLC spot was developed by spraying with 3% phosphomolybdic acid in methanol and scanned with densitometer at wavelength 550 nm. This method was validated according to ICH guideline. The percentage recovery was in the range of 98.68-102.99 and percentage of RSD was not more than 2. The developed method was applied to analyze the CA that collected from 12 locations of Thailand, such as Prachin Buri, Nakhon Si Thammarat (Ban Bo Lo local growth and Ban Bo Lo trade), Ubon Ratchathani, Trat, Rayong, Chon Buri, Nakhon Ratchasima, Sukhothai, Chiang Mai (green petioles), Chiang Mai and Phitsanulok. The maximum content of VLFAs was founded 0.27% w/w (Rayong), 0.24% w/w (Sukhothai and Nakhon Ratchasima). Furthermore, the content of those compounds in CA that collected from the commercial crops of Nakhon Pathom, Ubon Ratchathani and Nakhon Si Thammarat provinces. The maximum VLFAs of CA in sample collected from Nakhon Pathom province was observed also annually determine in November, from Ubon Ratchathani in September and from Nakhon Si Thammarat province was observed in May. Information obtained may be utilized as criteria for plant varieties selection and breeding programs for commercial cultivars of optimal VLFAs content for further isolation of pure VLFAs.