Studies On The Chemical Constituents From The Leaves Of Ilex Centrochinensis And Screening Of The Substances With Anti-inflammatory Activities In Vitro

Ilex centrochinensis is one of species of the genus Ilex,which belongs to the family of Aquifoliaceae.It has the function of detoxification,detumescence and analgesia,etc.It is mainly used to treat hepatitis and injury,especially on rheumatoid arthritis?RA?.Some experts had found that the alcohol extract of Ilex centrochinensis had good anti-inflammatory activities.In order to develop and utilize the Tujia folk medicine,the chemical constituents and anti-inflammatory activity in the leaves were studied systematically in this paper.To screen the chemical constituents from the leaves of Ilex centrochinensis,various chromatography techniques such as column chromatography on silica gel,MPLC,HPLC,LH-20,ODS,TLC etc.were used to isolate and purify the compounds from ethyl acetate?EtOAc?extracted parts and their structures were identified by the spectral data?such as 1H-NMR,13C-NMR,IR,1H-1H COSY,HMBC,HSQC,HR-ESI-MS,UV,CD,etc.?and physicochemical properties.Twelve compounds were identified as?2S?-7-methoxy-3',4'-dihydroxy-5,8-quinoflavan?1?,?2S,7''S?-7-methoxy-3',4'-dihydroxy-flavan-[5,6-e]-?3",4"-dihydroxyph-enyl?-dihydro-7"?8"H?-pyranone?2?,?2S,7"R?-7-methoxy-3',4'-dihydroxy-flavan-[5,6-e]_{3",4"-dihydroxyphenyl}-dihydro-7"?8"H?-pyranone?3?,ursol-icacid?4?,?2S?-5-hydroxy-7,3',4'-trimethoxyflavane?5?,kaempferol?6?,?2S?-5,3',4'-trihydroxyl-7-methoxyflavane?7?,3',4',5-trihydroxyl-7-methoxy-flavonone?8?,quercetin?9?,3 '-O-methylcatechin?10?,3-O-a-L-arabinopyranosyl-19a-hydroxy lursolic acid?11?,Sitosterol-3-O-?-D-glucopyranoside?12?.8,10 and 11 were isolated from the Ilex centrochinensis for the first time.1,2 and 3 were new compounds.In order to evaluate and screen the basis of anti-inflammatory substances in the leaves of Ilex centrochinensis,the cytotoxicity was determined by MTT method.RAW264.7 cells induced by LPS and MH7A cells induced by TNF-a was used as inflammatory model,respectively.The release of IL-1? and The production of NO in culture supernatant were measured by Elisa kit.Different extraction parts,total flavonoids and the partial compounds were screened and evaluated on anti-inflammatory activities.The results showed that the PE parts,EtOAc parts and total flavonoids were less than 100 ?g/ml,50 ?g/ml,50 ?g/ml,respectively.There were no obvious toxicity to RAW264.7 cells.Within the safe range of administration concentration,the PE parts,EtOAc parts,and total flavonoids could obviously inhibit the release of inflammatory factors.Among them,When the concentration of the PE parts,EtOAc parts,total flavonoids was 50 ?g/mL,the inhibition rate of NO production was 30.7%?**p<0.01?,66.1%?**p<0.01?and 68.8%?**p<0.01?respectively.N-BuOH parts and water parts had no obvious toxicity to RAW264.7 cells at 10 ?g/ml.N-BuOH parts could inhibit the production of NO in RAW264.7 inflammatory cells induced by LPS to some extent,but the effect of water fraction was not obvious.When the concentration of compound 1 was not more than 8 ?M,there was no obvious toxicity to RAW264.7 cells.Compound 1 decreased the production of NO and the release of IL-1? on LPS induced RAW264.7 cells in a dose-dependent manner within the safe range of administration concentration.Compound 1,7 and 8 was not toxic to MH7A cells at the concentration of 80,100,100 ?M,respectively.Compound 1,7 decreased the production of NO on TNF-? induced MH7A cells in a dose-dependent manner.Therefore,the PE parts,EtOAc parts and the total flavonoids are the main active sites of Ilex centrochinensis,and compound 1,7 is an important active component of anti-inflammatory activity in the leaves of Ilex centrochinensis.