Study Of Chemical Constituents And Hepatoprotective Activity Of Coryrlus Heterophylla Flowers

Coryrlus heterophylla flowers is the flowers of Coryrlus heterophylla Fish.C.heterophylla flowers widely distributed in the northeast,north China and shanxi,gansu and other places.Fruits and flowers can be used as medicine.C.heterophylla flowers has anti-inflammatory,swelling,analgesic effects.It can be used for skin inflammation,frostbite and topical treatment of various purulent infections.It mainly contain chemical components such as flavonoids,diphenylheptanes and terpenes.It has been reported that these compounds have various biological activities such as anti-cancer,anti-viral,anti-oxidation and anti-bacterial.It's also used in treatment of liver cirrhosis ascites.In this experiment,we studied the chemical composition and hepatoprotective activity in order to provide basis for the further development and utilization of C.heterophylla flowers.In this experiment,C.heterophylla flowers were soaked in methanol solution overnight and extracted three times,the extract was separated with petroleum ether,ethyl acetate and n-butanol were extracted three times,and each layer of the extract was concentrated and subjected to various physical and chemical methods such as recrystallization,silica gel column chromatography,reversed phase silica gel column chromatography,medium pressure preparative chromatography,and so on.30 compounds were isolated from the petroleum ether extact,ethyl acetate extact and n-butanol extract.On the basis of physicochemical characters and spectroscopic analysis such as NMR and MS,30 compounds were identified as ?-sitosterol(1),daucosterol(2),7,22-diene-3-?-ergosterol(3),7,22-diene-3,5-di-?-ergosterol(4),7,22-diene-3-ergosterol-?-D-glucopyranoside(5),p-hydroxy benzoic acid(6),3,4-dihydroxy benzoic acid(7),1,2,4-benzenetriol(8),2-hydroxy-4-undecyl-benzaldehyde(9),methyl gallate-3-O-?-D-glucopyranoside(10),2-(3,4-dihydroxyphenyl)-ethyl-?-D-glucopyranoside(11),alusenone 1a(12),alusenone lb(13),hirsutanone(14),hirsutanonol(15),oregonin(16),5(S)-hirsutanonol-5-O-?-D-glucopyranoside(17),1,7-bis(4-hydroxyphenyl)-5-(?-D-xylopyranosyloxy)-(5S)-3-heptanone(18),alnuside A(19),alnuside B(20),carpinontriol B(21),coryluside A(22),kaempferol(23),kaempferol-3-O-a-L-rhamnoside(24),kaempferol-3-O-?-L-(4"-E-p-coumaroyl)-rhamnoside(25),kaempferol-3-O-?-D-glucopyranoside(26),quercetin(27),quercetin-3-O-?-D-glucopyranoside(28),quercetin-3-O-?-D-galactopyranoside(29),quercetin-3-O-?-D-glucopyranosyl-(1-6)-?-D-galactopyranoside(30).Compound 22 is a new compound,and Compounds 4,5,8,9,10,11,16,17,18,19,20 and 30 are isolated from C.heterophylla for the first time.Because this experiment did not isolate the low polar volatile components of C.heterophylla flowers,we used gas-purged micro-injector extraction(GP-MSE)to obtain the volatile components of C.heterophylla flowers.Gas chromatography-mass spectrometry(GC-MS)was used to analyze the volatile components of C.heterophylla flowers and the relative content of chemical components was determined by normalization method.Supplementary experiments were performed on this part.130 peaks were detected in the volatile components of C.heterophylla flowers and were identified as linoleic acid methyl ester(11.49%),hexadecanoic acid(10.92%),9,12-octadecadienoic acid(9.75%),9,12,15-octadecatrienoic acid ethyl ester(8.80%),methyl hexadecanoate(7.37%),n-tetradecane(6.02%),and so on.Next,in order to determine the pharmacological activity of C.heterophylla flowers extract,we used the DPPH method to determine the antioxidant activity of the extract.It can be seen that the total extract of C.heterophylla flowers,as well as petroleum ether,ethyl acetate and n-butanol layer extracts with good antioxidant activity,the antioxidant activity of the ethyl acetate extract was better than positive group.Therefore,we continued to investigate the activities of extract and compounds of C.heterophylla flowers in vitro and in vivo.In vitro activity experiment,we used a human hepatoma cell line as a model tu evaluate hepatoprotective activity of 30 compounds which isolated from C.heterophylla flowers.The compounds were prepared into five concentrations(80 pM,40 ?M,20 ?M,and 10 ?M).Curcumin was used as a positive control drug and tacrine was used to induce hepatotoxicity.The cell proliferation rate was measured by MTT assay.The results indicated that compounds 16,17,19,20,21,23,24 and 27 had a certain hepatoprotective activity.The EC50 values of compounds 19 and 20 were lower than curcumin.In vivo activity experiment,the extracts of C.heterophylla flowers were given to mice for intragastric administration.After 7 days,we used CCl4 to induce acute liver injury model in mice.and measured AST,ALT in serum and SOD,GSH and MDA in liver homogenate,liver sections of mice were stained by H-E staining.It can be seen that C.heterophylla flowers have protective effect on CC14-induced liver injury model.