Research On The Extraction Technology For Total Asponins And The Chemical Constituents Of Its Ethyl Acetate Extract From Trichosanthes Kirilowii Maxim

Objects:1. Trichosanthis Radix, derived from the roots of Trichosanthes kirilowii Maxim. or Trichosanthes rosthornii Harms, contains total saponins which can be used to treat ischemic cerebrovascular diseases. To optimize extraction process of total saponins from Trichosanthes kirilowii Maxim., this thesis uses response surface method to provide the theoretical basis for industrial preparation.2. Previous research has indicated that the ethyl acetate (EtOAc) fraction of Trichosanthes kirilowii Maxim, has neuroprotective effect at some degree. In that case, we intend to study the chemical compounds of EtOAc fraction and screen out active ingredients that can protect PC 12 Cells induced by glutamic.Methods:1. Response surface method, conbined with a central composite design, was applied for the optimization of extraction process of Trichosanthes kirilowii Maxim. with solid-liquid ratio, extraction time, ethanol concentration and extraction temperature as independent variables while the extraction rate of total saponins as the response value on the basis of single factor experiments.2. The root of Trichosanthes kirilowii Maxim. (38kg) was extracted with CH3OH by reflux for 8 hours. The resultant CH3OH extract was subjected to evaporation and suspended in water, followed by the successive solvent partition with PE and EtOAc. The EtOAc fractions were subjected to repeated column chromatography on silica gel, C18 gel and semi-preparative HPLC to afford 14 compounds. In addition, the chemical structures were deduced based on MS,’H-NMR,13C-NMR. Finally, MTT assay is used to study the cell viability in vitro of isolated compounds except (7) and explore the influence from isolated compounds to glutamic acid-induced injury in PC 12 Cells.Results:1. The optimum extraction technology conditions were as followings:solid-liquid ratio of 1:15, extracting time 120 min, ethanol concentration 48%, extraction temperature 88℃. Under these conditions, extraction ratio of total saponins from Trichosanthis Radix, was 0.93%.2. Fourteen compounds were obtained from the EtOAc fractions including soya-cerebroside Ⅰ (1), ligballinol (2), (22 E,24 R)-24-ethylcholesta-22-en-3-O-β-D-glucoside (3), stigmastanol glucoside, (4) hemslecin A (5), cucurbitacin R (6), cucurbitacin D (7), cucurbitacin B (8), hexadecanoic acid (9), pentandecane (10), behenic acid (11), n-octacos-9-enoic aid (12),9 (Z),12 (Z)-nonadecadienoic acid (13), (10 E,13 E)-octadeca-10,13-dienoic acid (14). Among these compounds,10 μmol/L compound 1 and 0.1 μmol/L compound 2 showed the most potent protective effects to glutamic acid-induced injury in PC 12 Cells.Conclusion:1. The optimum extraction process of total saponins from Trichosanthes kirilowii Maxim. is stable and practicable while the true value generally matches the predicted value from the established model.2. In this study, fourteen compounds were obtained from Trichosanthes kirilowii Maxim., eight of which (lignin 2, steroid saponin 3 & 4, triterpenes 5, fatty acids 10,11,12 & 13) were separated from Trichosanthes L. and two (fatty acids 9 & 14) were separated from Trichosanthis Radix at first time. Further more, lignin was first isolated from Trichosanthes L. The results of activity screening indicates that 10 μmol/L compound soya-cerebroside I and 0.1 μmol/L compound ligballinol has the most potent protective effects to glutamic acid-induced injury in PC 12 Cells.