Study On Isolation And Identification Of Active Ingredients From Gynostemma Pentaphylium Makino And Their Bioactivities

Gynostemma Pentaphylium Makino,belonging to the family Cucurbitaceae,is a perennial creeping plant mainly distributed in the south of China,Korea,Japan,India and Southeast Asia.It has been widely used as vegetable,dietary supplement and herbal tea.Recent pharmacological studies have indicated that G.has various bioactivities such as antilipidermic,anti-inflammatory,hepatoprotective and sedative-hypnotic effects.Phytochemical studies on G.have revealed the presence of saponins,flavonoids,polysaccharides,proteins,fat and inorganic elements.Saponins and polysaccharides are the main components with healthy function.At present,researches of G.are focused on extraction process of active components and separation of gypenosides,there are few studies on other ingredients and pharmacological activities.This paper explored a systematical study on purification,structure characterization and biological assessment of the chemical constituents from G.Moreover,the anti-inflammatory activity and mechanism of total gypenosides were studied.The total crude extract was obtained by the extraction of heating reflux with 75% ethanol and then portioned with organic solvents of different polarity to yield four part extracts,namely petroleum ether fraction,ethyl acetate fraction,n-butanol fraction and water fraction.Different biological models(including anticancer and antioxidant experiments)were used to evaluated the activity of crude extract and four part extracts.Results revealed that active compounds with anticancer and antioxidant effects were concentrated in ethyl acetate fraction and n-butanol fraction.Considering the results comprehensively,the main active compounds were concentrated in ethyl acetate fraction and n-butanol fraction after solvent extractions.Therefore,ethyl acetate fraction and n-butanol fraction were selected for further research.The chemical compositions of ethyl acetate and n-butanol fraction were analyzed by silica gel column chromatography,Sephadex LH-20 gel column chromatography,polyamide column chromatography and ODS column chromatography.Eight compounds were obtained and their structures were characterized via physicochemical properties and modern spectroscopic techniques(including MS,1H-NMR,13C-NMR),namely Kaempferol 3-O-[2G-(E)-Coumaroyl-3G-O-?-D-glucosyl-3R-O-?-D-glucosylrutinoside],gypenoside XLVI,gypenoside LVI,3,4-dihydroxyphenyl-O-?-D-glucopyranoside,ginsenoside Rd,gypenoside LI,gypenoside L and 2?,3?,12?,20(S)-tetrahydroxy-25-hydroperoxy-dammar-23-ene-20-O-?-D-glucopyranosyl-(1?6)-?-D-glucopyranoside.Among them,Kaempferol 3-O-[2G-(E)-Coumaroyl-3G-O-?-D-glucosyl-3R-O-?-D-glucosylrutinoside](a flavonoids)and 3,4-dihydroxyphenyl-O-?-D-glucopyranoside(a polyphenolics),were isolated from G.for the first time.L02 cell was used to determine the toxicity of samples,anticancer and antioxidant activities of eight compounds isolated from ethyl acetate and n-butanol fraction in experimental concentration range.In the evaluation of cytotoxicity against A-549 and MCF cells,results showed that samples inhibited cell proliferation in a concentration dependent manner.The anticancer effects of compound 5,6 and 7 were better than 5-F,anticancer effect of compound 4 is equivalent to 5-F,whereas compound 3 and 8 were not sensitive to the two cancer cells.The inhibitory effect of compound 1 on A-549 was relatively stable similar to that of positive control,but it was not sensitive to MCF.The overall effect of compound 2 on MCF was similar to that of 5-F,but the inhibitory effect on A-549 was not so good.Results showed that compound 5,6 and 7 had good antitumor activities,compound 1 and 4 killed cancer cells with a certain target.Radical-scavenging and antioxidant activities were evaluated by DPPH,ABTS and FRAP assay,in all antioxidant studies,compound 1 and 4 showed strong antioxidant activity.Although other compounds had a certain ability to scavenge free radicals,the effect was not ideal.Total saponins from G.(GPMS)was obtained by organic solvent extraction and D101 macroporous resin concentration.Its content was 67.5%,determined by standard curve of ginsenoside Rb1.MTT experiments showed that GPMS had no toxic side effects on normal RAW 264.7 cells in the concentration range of 31.25~500 ?g/mL.GPMS could regulate the release of NO through decreasing the secretion of IL-1??IL-6,might suppress i NOS?IL-6 and IL-1? mRNA expression.It indicated that GPMS could play an anti-inflammatory role through inhibiting signaling pathways of MAPK and NF-?(33).