Analysis On Chemical Constituents In Ethyl Acetate Extract And Quality Standard Research Of Weigela Japonica Thunb.Var.Sinica (Rehd.) Bailey

Objective:We systematicaly studied on the chemical compositions in ethyl acetate extract from Weigela japonica Thunb.var.sinica（Rehd.）Bailey（W.J.）that is the plant’s of Weigela Thunb.in caprifoliaceae.The antitumor activity of the alcohol extract and the polarity parts,including the two major monomer compounds from ethyl acetate fraction,were evaluated in vitro,which lays theoretical foundation of screening for its valid and active ingredients.The quality standard of W.J.was drew up,which provides effective basis for the quality control and the development and utilization.Methods:（1）First of all,the chemical constituents of the roots of W.J.was extracted with 95%Et OH,then the alcohol extract partitioned sequentially against PE,Et OAc and n-Bu OH,which obtained the extracts of different polar parts by concentrating apart.The ethyl acetate fraction was separated with the column chromatography of normal phase silica gel,Sephadex LH-20,and preparative HPLC,which tracting with TLC and HPLC method at the same time,to separate and purify monomer compounds.The structures of the compounds were identified by using modern spectroscopy technology(¹H-NMR,¹³C-NMR)and combining with related literatures.（2）the effect of the alcohol extract and different extraction parts of W.J.on different tumor cells was evaluated by MTT method in vitro,then determined which was the active part,and tracked it,so that it is capable of searching for its effective anti-cancer active ingredients.（3）Depending on the basic methods of the medicinal materials quality control,the quality standard of W.J.was studied,and the item of research contains identification of properties,microscopy and TLC,checking about moisture,ash and extract contents,as well as content determination:scopoletin as reference substance,the content of that and total coumarins were determined by HPLC and UV method.Results:（1）Five coumarin compounds wree isolated from the ethyl acetate parts,which is from 95%alcohol extract of the roots of W.J.,and identified as:scopoletin,cleomiscosin B,fraxein,fraxin and scopolin.（2）There was a selectivity on different tumor cells for different extracts of W.J.,as for as human endometrial cancer cell line RL95-2 and cervical cancer U14 cell line in mice,the extraction of parts all showed cytotoxic effect,in which the extract of petroleum ether and ethyl acetate had better activity for the cells of RL95-2,and the IC₅₀ value was 65.55and 77.11μg·m L^-1,respectivly.In terms of the human gastric cancer cell line HGC-27,only the ethyl acetate part had possesed more activity in dose-dependent and the IC₅₀ value was 84.03μg·m L^-1.The anti-tumor activity of the monomer compounds of scopoletin and fraxetin were not relying on cytotoxic effect.While fraxetin was poisonous to the cell of U14,and the inhibition of that enhanced along with the increase of drug concentration and action time.（3）the characteristics of W.J.was typical.Powder microscopic identification indicated that the main features reflecting fibers,stone cells,oil chamber,common bordered pit catheter and the crystal fiber made by calcium oxalate,and so on.TLC identificaton of 10 batches of W.J.showed that the spots were clear and not interfered with other impurities spots,while it had good separating degree and the same R_fvalue with the reference substance spot.Drew up the content of moisture,total ash and acid insoluble ash of W.J.less than 12.0%,2.0%and 2.0%,respectively,while the contains amount of solubility in water and alcohol soluble extract of which no less than 5.0%and 4.5%,apart.The concentrations of scopoletin in the range of 1.25～40.0μg/m L（r=0.9997）and the content of total coumarin in 2.0～64.0μg/m L（r=0.9999）all had good line relatios,and the average sample recovery rate was 98.50%（RSD=0.6%）,0.6%（RSD=1.06%）,respectively.Conclusions:（1）There are 5 compounds that were separated from ethyl acetate of the roots of W.J..They were all coumarins and were identified as scopoletin（1）,cleomiscosin B（2）,fraxein（3）,fraxin（4）and scopolin（5）.Compounds above are obtained from the plant for the first time.（2）The ethyl acetate parts of W.J.had better inhibitory effect on cells of RL95-2 and HGC-27,and the value of IC₅₀ was 77.11and 84.03μg·m L^-1,separately.Meanwhile,U14cells proliferation were inhibited by fraxetin,which was obtained from the ethyl acetate extract of W.J..（3）The quality standard of W.J.was formulated,which will be record in the 2018edition of the hubei province traditional Chinese medicine quality standard.So that provides a legal basis for the quality control and reasonable use of the plant.