Study On The Chemical Constituents And Related Activities Of Alpinia Oxyphylla Miq.

Background:Alpinia oxyphylla Fructus is the dry ripe fruit of Alpinia oxyphylla Miq.,which belongs to Zingiberaceae and is grown in Guangdong,Guangxi,Hainan and Yunnan provinces of China.It is considered as one of four major Chinese traditional herbs in South China and also a highly safe medicinal and edible plant resource.At present,the compounds isolated from Alpinia oxyphylla Miq.are diarylheptanoids,terpenoids,flavonoids,steroids and other kinds of components,among which diarylheptanoids and sesquiterpenes are the major bioactive ingredients.Modern pharmacological studies have shown that Alpinia oxyphylla Miq.has various biological activities including anti-cancer,neuroprotection,anti-oxidation,improving immunity,anti-inflammation,protecting gastrointestinal tract and so on.The chemical composition of ethyl acetate fraction of Alpinia oxyphylla Miq.has been preliminarily studied.The crude fraction was divided into fifteen fractions from A to O and the A to D fractions were separated and purified.And seventeen compounds have been isolated and identified,including diarylheptanoids 7,flavonoids 2,terpenoids 4 and others 4.To find the active components of Alpinia oxyphylla Miq.and develop the medicinal value,we further study the chemical composition and biological activity of ethyl acetate fraction.Objective:The main aim of this study is to find active compounds which can be used for anti-tumor,anti-inflammation,anti-oxidation and other functions on the research of chemical constituents of ethyl acetate fraction of Alpinia oxyphylla Miq.Methods:（1）Compounds were isolated and purified using silica gel column,MCI column,polyamide column,Sephadex LH-20 gel column and semi-prepared liquid chromatography.And the structures were all identified according to the physicochemical constants,¹H-NMR and ¹³C-NMR.（2）The CCK-8 method was used to study the cytotoxicity of crude extracts and isolated compounds from different polar parts of Alpinia oxyphylla Miq.（3）The antioxidation capacity of the isolated compounds was evaluated by DPPH·scavenging method,Fe³⁺reduction ability assay and H₂O₂induced oxidative stress model.（4）The Griess method was used to investigate the anti-inflammatory activity of two terpenoids of Alpinia oxyphylla Miq.Results and Conclusions:（1）Twenty-one compounds were isolated from Alpinia oxyphylla Miq.Compound 14 was first isolated from the plant and compounds 6,10,15-21 were first isolated from Zingiberaceae.They were respectively dibutyl phthalate（1）,yakuchinone A（2）,chrysin（3）,kaempferol（4）,7-epi-teucrenone（5）,armerillone（6）,oxyphyllenodiol B（7）,（4a S,7S）-7-hydroxy-1,4a-dimethyl-7-（prop-1-en-2-yl）-4,4a,5,6,7,8-hexahydronaphthalen-2（3H）-one（8）,11-hydroxy-valenc-1（10）-en-2-one（9）,veadeiroic acid（10）,daucosterol（11）,β-sitosterol（12）,protocatechuic acid（13）,ferulic acid（14）,4-（2-hydroxyethyl）-1,2-benzenediol（15）,4-hydroxyphenethyl alcohol（16）,4-methoxy-1,3-benzenediol（17）,（E）-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid（18）,（S）-5-hydroxy-3,4-dimethyl-5-pentylfuran-2（5H）-one（19）,4-hydroxy-3-methoxy-benzeneacetic acid methyl ester（20）,methyl-5-[4-methoxy-3-[2-（methoxycarbonyl）ethyl]-phenyl]pentanoate（21）.（2）All 5 tumor cells（4T1,B16,Hep G2,Hgc-27,Pc-3）were inhibited by different polar regions of Alpinia oxyphylla Miq.Among them,the inhibition rate was above80%,when the petroleum ether and ethyl acetate extract were at a concentration of200μg/m L.From the result,we can find petroleum ether and ethyl acetate fraction had the strongest inhibitory effect on Pc-3 cell,with IC₅₀values were 12.90±2.03μg/m L and 30.97±1.01μg/m L.At the same time,the effects of monomeric compounds on three tumor cells（B16,Hgc-27,Pc-3）were also measured.The results showed that compound 2-4 had strong inhibitory effects on 3 tumor cells;compounds5-8 and 10 had moderate inhibitory effects on Pc-3 cell and these five compounds were terpenoids.So it can be inferred that terpenoids may be the main active substances in the anti-prostate cancer effect of Alpinia oxyphylla Miq.extract.（3）The results showed that compounds 2,13-16 had strong DPPH·clearance ability and Fe³⁺reduction ability.The protective effect of compound 15 intervention H₂O₂induced oxidative stress in H9c2 cell was determined.The results showed that the sample was significantly different from that of the model group（P<0.05）when it was at a concentration of 100μg/m L.In terms of compound type,phenolic compounds may be the main active substances in the antioxidant effect of Alpinia oxyphylla Miq.extract.（4）Compounds 6 and 10 inhibited LPS induced RAW 264.7 mouse macrophages.The results showed that the inhibitory effect of compound 6 on NO release was relatively weak and compound 10 on NO release was relatively strong.Compared with the model group,when the sample concentration was 25μg/m L,there was significant difference（P<0.05）.Compound 10 was the first diterpenoid isolated from this genus.The above results suggested that it may have some anti-inflammatory activity in vitro.