| Ethnic medicine is a treasure in Chinese medicine.It has unique curative effect in the treatment of various diseases,and has the advantages of multi-channel,multi-component and multi-target synergy.However,compared with traditional Chinese medicine,the study of national medicine is far lagging behind.Therefore,increasing basic research and development of ethnic medicines,to dig out the effective parts,monomer compounds,and lead compounds becomes inevitable.Swertia chirayita,the Tibetan medicine name“Jia Di”,a dry grass of the genus Swertia chirayita?Roxb.ex Fleming?H.Karst.It has a long history of clinical application and anti-inflammatory properties,liver protection,retreat heat and other effects.It is the dry grass as the main source of Tibetan medicine Tida and also the first choice for the treatment of icteric hepatitis,viral hepatitis and other liver.However,the Pharmacodynamic material basis for this medicinal plant is not yet clear.The previous research showed that the 85%ethyl acetate extract of S.chirayita had good anti-hepatic fibrosis activity in vivo and in vitro.In order to further clarify the pharmacodynamic material basis of this medicine,the chemical constituents of this fraction was first systematically isolated,purified and identified.Then,the main ingredient of monomer compounds were screened for HSC proliferation inhibition and study on anti-inflammatory activity;Finally,evaluated for equilibrium solubility and apparent oil-water partition coefficient of compound with anti-hepatic fibrosis activity?amarogentin?.It provides pharmaceutics basis for predict drug activity.The specific research content includes following four parts:1 Chemical constituents from ethyl acetate extract of S.chirayitaVarious chromatographic techniques were used to isolate from ethyl acetate extract of S.chirayita,and purify the compounds physicochemical properties and spectroscopic methods were used to elucidate their structures.32 compounds were isolated from it,18 compounds were identified,including 6 triterpenoids and glycosides,5 xanthones,3 secoiridoids,2 organic acids,1 flavonoids and 1 other compounds,identifiedasbellidifolin?1?,norbellidifolin?2?,1,7-dihydroxy-3,8-dimethoxyxanthone?3?,swertianolin?4?,swerpunilactone A?5?,nobiletin?6?,oleanolic acid?7?,4-epi-hederagenin?8?,2-epi-corosolic acid?9?,ursolic acid?10?,3?,23-dihydroxy-12-ene-28-ursolic acid?11?,?-daucosterol?12?.amarogentin?13?,swerimilegenin I?14?,erythrocentaurin?15?,pyrocatechol?16?,syringicacid?17?,and 4-hydroxy-3-methoxybenzoic acid?18?.Compounds 8,9,6,11and 17 are isolated from genus Swertia for the first time,compounds 3,5,14 and 15are found from S.chirayita for the first time.2 Screening of in vitro anti-hepatic fibrosis activity of ethyl acetate extract from S.chirayitaIn order to evaluate the anti-hepatic fibrosis effect of the compounds,MTT assay was used to observe the effects of main of monomer compounds on the proliferation of HSC-T6 cells stimulated by TGF-?in this experiment.The experimental results show that ursolic acid and oleanolic acid have significant anti-hepatic fibrosis cell activity in vitro,and with the increase of the concentration,the inhibition of HSC proliferation is enhanced.The rest compounds have no obvious effect in vitro.Therefore,ursolic acid and oleanolic acid are the main anti-hepatic fibrosis active substances in this fraction.3 A preliminary study on anti-inflammatory activity and mechanism of bellidifolinThe occurrence of inflammation is closely related to hepatic fibrosis.Therefore,it is necessary to study the anti-inflammatory activity and the mechanism of the compounds from the ethyl acetate fraction of S.chirayita.In this experiment,we studied the effects of main ingredient of bellidifolin on the RAW 264.7 macrophage cells induced by LPS,and the results showed that it can significantly increase cell viability when the concentration of bellidifolin was 216?M.It has a significant inhibitory effect on inflammatory cytokines TNF-?and IL-6,it can inhibit the expression of PGE2 and COX-2 proteins,and significant inhibition of phosphorylation level of protein Akt,IKK?,and NF-?B,p 65,p 38,ERK and JNK proteins.However,it had no significant effect on the protein expression of COX-1.The results suggested that bellidifolin has anti-inflammatory activity,and it is speculated that the mechanism of anti-inflammatory activity by regulating COX-2/NF-?B/MAPKs/Akt signaling pathways.4 Physicochemical properties of anti-hepatic fibrosis component of amarogentinThe dissolution,absorption,distribution,and excretion of drugs in the body are related to the water solubility and fat solubility of drugs.Therefore,it is necessary to study the equilibrium solubility and oil-water partition coefficient of compound in different pH buffers according to the characteristics of different body fluids in different parts of the body,it is helpful to predict its in vivo process and biotic properties.The equilibrium solubility and oil-water partition coefficient of ethyl acetate extract from S.chirayita,a main ingredient of secoiridoids compounds amarogentin were determined in this experiment.The chromatographic conditions were as follows:Waters C18?250 mm×4.6 mm,5?m?,mobile phase:methanol-water,detection wavelength:260 nm,flow rate:1.0 mL/min,column temperature:25oC,injection volume:20?L.The experimental results show that the equilibrium solubility of amarogentin in water is 150.74±0.78?g/mL.In the different pH PBS,the maximum is at pH 7.9,which indicates that the solubility of amarogentin in alkaline environment is large,and it is water insoluble compound.Apparent oil-water partition of amarogentin in water and PBS with different pH values is from large to small pH 2.5>pH 7.4>water>pH 2>pH 7.9>pH 6.8=pH5.8>pH 5.The results showed that amarogentin in pH 23 acidic environment and pH 77.4 neutral environment approximately had fat solubility,easy to enter the cell membrane transport,and conducive to absorption.In short,stematically isolated the chemical constituents of ethyl acetate from S.chirayita,screened the active ingredients and anti-inflammatory components anti-hepatic fibrosis,determined the active compounds,and conducted the preliminary physicochemical properties on main active compounds.Providing scientific basis for in-depth development and utilization of S.chirayita. |
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