Study On The Chemical Constitutions Of The Ethyl Acetate Extract From Alpinia Oxyphylla And Avtivity Of The Chrysin

Alpinia Oxyphylla is the dry and ripe fruit of Alpinia oxyphylla Miq.Alpinia Oxyphylla,Morinda officinalis How,Areca catechu L.and Fructus Amomi are also known as the"Four Southern Drugs".It is mainly distributed in Guangxi,Hainan and Guangdong of China.Mild efficacy,it can act on the spleen and kidney and has the functions of warming the kidney and the spleen,reducing urine,and stopping diarrhea.It is mostly used for the symptoms of kidney deficiency and enuresis,frequent urination,spermatorrhea,diarrhea,abdominal cold,salivation and other symptoms^[1].Because of the chemical constituents of Alpinia oxyphylla are complex and difficult to separate,and the reports of its chemical constituents in literature are limited,this study will hopefully enrich the types of natural products,and will provide the basis and reference for the future research on its activity and pharmacological effects.Chrysin（5,7-dihydroxyflavone）is a flavanone compound with pharmacological activities such as anti-cancer,anti-anxiety,anti-inflammatory and anti-oxidation^[2].It can play a neuroprotective role by up-regulating Nrf2-ARE signaling pathway^[3],up-regulating p-ERK and down-regulating the expression of NF-κB and p-P38[4].However,there is no report on how chrysin can repair damaged neurons in cerebral ischemia-reperfusion injury.In order to enrich the kinds and quantities of chemical constituents in Alpinia oxyphylla and find other active ingredients,the chemical constituents of ethyl acetate fraction of Alpinia oxyphylla were studied,and the in vivo and in vitro activity of the chrysin was tested in this paper,which provided a new idea for the treatment of cerebral ischemia and had potential clinical application and research value.The dried fruits of Alpinia Oxyphylla were crushed and extracted with 95% ethanol.The extract was concentrated under vacuum.After the concentrated liquid is dispersed with water,petroleum ether,ethyl acetate and n-butanol are added.After extration and concentration,the corresponding parts are obtained.A total of ethyl acetate extract 784.16g was obtained,and then separated by repeated use various separation methods,such as gel Sephadex LH-20,semi prepared HPLC,normal silica gel column chromatography,reverse phase silica gel column chromatography,recrystallization and so on,until the monomeric compounds were obtained.Finally,the structures of 20 monomer compounds were identified by combining physical and chemical properties,TLC,LC-MS,¹H NMR and ¹³C NMR.And the structures of 14 monomeric compounds were confirmed,including 7 flavonoids（compounds 1-7）,2 diphenylheptane（compounds 8-9）,1 xanthone（compounds 10）,2 phenols（compounds 11-12）,1eucalyptus alkane sesquiterpene（compound 13）,1 triterpenoid（compound 14）and 1steroid（compounds 15）.they were named as:3,5-dihydroxy-7,4’-dimethoxyflavone（1）;chrysin（2）;tectochrysin（3）;kaempferol（4）;baicalein（5）;wogonin（6）;myricetin（7）;yakuchinone A（8）;1-（3’’,5’’-dihydroxy-4’’-methoxy-phenyl）-7-phenyl-3-heptanone（9）;mangiferin（10）;protocatechuic acid（11）;vanillic acid（12）;teuhetenone A（13）;oleanolic acid（14）;β-sitosterol（15）.Among the compounds,compound 5、6、7 was isolated from the Alpinia Oxyphylla for the first time.In addition,the neuroprotective effect of chrysin on cerebral ischemia-reperfusion injury and its possible mechanism were studied.Firstly,MTT assay was used to detect the protective effect of chrysin on glutamate-induced nerve injury cells in vitro.Furthermore,immunofluorescence staining was used to detect the expression of neurotrophic factors related to bone marrow mesenchymal stem cells（MSC）in rats induced by chrysin.In vivo pharmacodynamic evaluation,cerebral ischemia-reperfusion model of rats was established by middle cerebral artery occlusion（MCAO）.In vivo evaluation indexes were used to evaluate the effect of chrysin on cerebral ischemia-reperfusion injury,including neurological function score,cerebral infarction volume,water content,HE staining to observe pathological tissues,immunofluorescence staining to detect the expression levels of brain-derived nutrient factor（BDNF）and nerve growth factor（NGF）in rat brain tissue.The results showed that chrysin had no cytotoxicity to normal PC12 cells at the experimental concentration,and chrysin（drug concentration was 10μM）had protective effect on injured cells in glutamate（10mM）injury model.After chrysin induced MSC,surface nerve marker proteins on the cell surface such as galactose ceramide（GalC）,nerve growth factor（NGF）,neuron-specific enolase（NSE）,Nestin and Vimentin were expressed in varying degrees in different time.In vivo experiments,compared with the model group,the experimental group treated with chrysin improved the neurological deficit symptom score,reduced the volume of cerebral infarction,improved the tissue damage of ischemic penumbra,and increased the expression of NGF and BDNF protein in rat brain.It is concluded that chrysin can play a neuroprotective role in cerebral ischemia-reperfusion injury by up-regulating the expression of NGF and BDNF.