The Extraction And Activities Of Alkaloids In Tylophora Ovata (Lindl.) Hook.ex Steud

Tylophora ovata(Lindl.)Hook.ex Steud belongs to Asclepiadaceae.It was a Chinese traditional toxic medicine.The alkaloid components of Tylophora ovata (Lindl.)Hook.ex Steud possessed very strong antitumor activitiesaccording to literature.It has played an important role as an ancient medicine for centuries. Particularly today the medicinal use of Tylophora ovata(Lindl.)Hook.ex Steud was widespread and growing.A wide array of therapeutic effects of Tylophora ovata (Lindl.)Hook.ex Steud such as antimicrobial,anticancer,have been reported.It was well known that Tylophora ovata(Lindl.)Hook.ex Steud contains alkaloids such as tylophoridicine A,tylophorinine,O-methyl tylophorinidine and tylophorinidine, etc.All of them were said to have most of the medical effects of Tylophora ovata (Lindl.)Hook.ex Steud.The main studies and results in the paper were mentioned as the following;1.The data of nutrition and contents of nutrients in Tylophora ovata(Lindl.) Hook.ex Steud.The results were;(g/100 g,dry mass)water,9.44,flash,6.30,total sugar,2.67;fibre,17.03,protein,14.02,fat,11.32.GC-MS was used to study many components in the extracts with SFE-CO₂ for the first time.The results showed that Hexadecanoic acid,ethyl ester,n-Hexadecanoic acid,Hexadecanoic acidmethyl ester, 2-Nitro-4-(trifluoromethyl)phenol,8-Methoxy-2-methylquinoline,1-(12-methyl-1-oxo tetradecyl)-1-Pyrrolidine and 1-Phenyl-1-azaspiro[4,5]decane were the main components.2.The total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.were determined by Bromophenol Blue acid dye colorimetry method.The suitable conditions for colorimetry were obtained;1.5 mL of Bromophenol Blue acid dye,2 mL of CHCL₃ and 4.0 mL of Na₂HPO₃-citric acid at pH 4.2.The color of the treated samples was stable within 60 min.The results showed that the developed method with high precision could be simple,correct and reproducible for the determination of the contents of total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud. 3.The solvent extraction technology,microwave assisted extraction technology and the CO₂ supercritical fluid extraction technology of total alkaloids in Tylophora ovata(Lindl.)Hook.ex Steud.were optimized by orthogonal experiments respectively.The experiment results showed that the optimal experimental parameters of the solvent extraction technology are 90% ethanol,90℃of the extraction temperature, 1;25 of the ratio of solid/liquid,extracted for twice,2 h each time.The yield of the total alkaloids were extracted from Tylophora ovata(Lindl.)Hook.ex Steud exceeds 0.386% on the optimal condition.SFE-CO₂ was used to extract alkaloid from Tylophora ovata(Lindl.)Hook.ex Steud for the first time.In this paper,the effect of working parameters on the extraction process was studied respectively,including extracting pressure,extracting temperature,extracting period and modifiers(alcohol)concentration for the ratio of total alkaloids.The optimal experimental parameters of the CO₂ supercritical fluid extraction technology were 50℃of the extraction temperature,25 MPa of the extraction pressure,80% of the ethanol as modifier,extraction for 90 min.The results showed that the yield of the total alkaloids extracted from Tylophora ovata(Lindl.)Hook.ex Steud was 0.249%,which was lower than the yield by the solvent extraction technology but the content of the impurities was also lower.The experiment results showed that the optimal experimental parameters of microwave assisted extraction technology were 90% ethanol,90℃of the extraction temperature,1;20 of the ratio of solid/liquid,extracted twice,15 min each time.The yield of the total alkaloids were extracted from Tylophora ovata(Lindl.)Hook.ex Steud exceeds 0.436% on the optimal condition.,which was higher than the yield by the solvent extraction technology and the CO₂ supercritical fluid extraction technology.4.A LC-MS method was established for qualitative analysis of the total alkaloids that extracted from Tylophora ovata(Lindl.)Hook.ex Steud It indicated that in the future the similar qualitative analysis and research can be carried out by the same method without standard sample.5.Silica gel column chromatography was used to isolate and purify the components from Tylophora ovata(Lindl.)Hook.ex Steud.UV,IR,ESI-MS,EI-MS, ¹H-NMR and ¹³C-NMR were used to identify these components.The results showed four sterols had been isolated and purified and their structures were identified as component(1)and component(2).All the Compounds were isolated from Tylophora ovata(Lindl.)Hook.ex Steud for the first time.6.The growth of cells was inhibited when Hela cells cultured in vitro were treated with the component(1)and component(2)from Tylophora ovata (Lindl.)Hook.ex Steud.for 48 h.The dose and effect appeared positive correlation. 50% inhibiting concentration(IC₅₀)were 98μg/mL and 218μg/mL by the MTT assay.Dosedependently component(1)and component(2)exhibited good antiproliferation activity to Hela.The bacteriostasis activity of the total alkaloids from Tylophora ovata(Lindl.)Hook.ex Steud.at different concentrations was evaluated by through the filter paper method.The results showed that the total alkaloids from Tylophora ovata(Lindl.)Hook.ex Steud exhibited good bacteriostasis activity to Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Aerobacter aerogenus, Proteus vulgaris,Saccharomy cescerevisiae.