Study On The Chemical Constituents Of Uighur Medicine Gulina And Anti-inflammatory Activities

In uyghur,GuLiNa is a kind of commonly used medicine,also known as pomegranate flower,dried petals of Punicaceae pomegranate of the genus granatum.In uyghur medicine,it is used to treat neurasthenia,ophthalmic diseases and nausea;In traditional Chinese medicine,pomegranate flower was used to treat bleeding,tongue sores,prolapsed hemorrhoids,bad breath,toothache,itching skin;Pomegranate flower has the effect of anti-inflammatory hemostasis,can be used to treat suppurative otitis media.In China,it is mainly produced in Xinjiang Yecheng,Henan Xinxiang,Shandong Zaozhuang,Anhui Huaiyuan,Yunnan and other places.Pomegranate flowers bloom three times in May and June,and most of the second and third flowers will naturally fall off,so its resources are quite rich.Modern studies show that the active components of pomegranate flower are mainly polyphenols and flavonoids,and the main parts are ethyl acetate and n-butanol.At present,there are many researches on the chemical constituents of the leaves and fruits of pomegranate,but the reports on the chemical constituents of the flowers are few.In order to further study the effective components of pomegranate flowers,this study isolated,purified and identified the extracts of n-butanol and petroleum ether from pomegranate flower.And the anti-inflammatory activities of seven polyphenols and flavonoids in pomegranate flower were determined.Object: This study mainly includes the following two aspects: 1.To study thechemical constituents of pomegranate flowers,in order to discover new compounds and to find out the physical basis of pomegranate flowers.2.In vitro inflammatory model of RAW264.7 cells induced by LPS was established,after successfuL cell modeling,seven polyphenols and flavonoid monomer compounds in pomegranate flower were given,NO inhibition rate was used as anti-inflammatory activity index to screen anti-inflammatory activity components.Methods: In this experiment,mineral ether and n-butyl fractions from pomegranate flower were isolated and purified by silica gel,Sephadex LH-20,ODS column chromatography,semi-preparative HPlC and recrystallization,structures of the pure compounds were determined by NMR and other modern spectral techniques combined with references;In addition,anti-inflammatory activity and cytotoxicities for seven compounds were evaluate.The cytotoxicity was investigated by MTT and CCK-8 assay and anti-inflammatory activity was investigated based on the inhibition of NO generation.These seven compounds are: ?-1,4,6-tri-O-galloyl-D-glucose,butylbrevifolin carboxylate,methy brevifolincarboxylate,corilagin,tricetin,ellagic acid,gallic acid and 8-O-methylretusin.In addition,seven monomer compounds were taken from pomegranate flower: ?-1,4,6-tri-O-galloyl-D-glucose,butylbrevifolin carboxylate,methy brevifolincarboxylate,corilagin,tricetin,ellagic acid,gallic acid and 8-O-methylretusin,the cytotoxicity of 7 monomers was detected by MTT method and cck-8 method,and inhibition rate of NO was detected by microplate method.Result: Petroleum ether and n-butanol from ethanol extract from pomegranate ?flower,twenty compounds were isolated,and identified respectively as ?-1,4,6-tri-O-galloyl-D-glucose(1),butylbrevifolin carboxylate(2),methy brevifolincarboxylate(3),brevifolin(4),1,6-di-O-galloyl-?-D-glucopyranose(5),ellagic acid(6),biochanin A(7),retusin-8-methylether(8),taraxerol(9),sitosterol(10),?-sitosterol palmitate(11),crispin A(12),formononetin(13),tricetin(14),D-dulcitol(15),taraxerone(16),gallic acid(17),daucosterol(18),?-sitosterol(19),ursolic acid(20);In terms of anti-inflammatory experiments,data showed that only the corilagin inhibited the growth of RAW264.7 cells and had cytotoxicity.These compounds,butylbrevifolin carboxylate,methy brevifolincarboxylate,tricetin,ellagic acid and 8-O-methylretusin,could inhibit the synthesis of NO in RAW264.7 cells induced by LPS,and have varying degrees of anti-inflammatory activity.Conclusion: In this experiment,the chemical composition of n-butanol and petroleum ether parts of pomegranate flower was preliminarily explored,and a total of 20 compounds were separated and identified,compounds 1-3 as well as 7-12 were firstly isolated from this genus,and compounds 4 were isolated from pomegranate flowers for the first time.Polyphenolic compounds of butylbrevifolin carboxylate,methy brevifolincarboxylate,ellagic acid,flavonoids compounds of tricetin and 8-O-methylretusin have anti-inflammatory effects,they are the anti-inflammatory activity of pomegranate flowers.In this study,seven compounds from pomegranate flowers were screened for their anti-inflammatory activity,other known chemical components may also be used to study the anti-inflammatory activity.In addition,in vitro anti-inflammatory experiments,only one indicator,NO,was determined,Subsequently,more indicators,such as IL-6?TNF-??INF-??IL-1? can be detected by ElISA reagent according to the existing data.In addition,real-time quantitative PCR can also be used to detect the mRNA level to clarify the anti-inflammatory mechanism of pomegranate flower.In this study,ten compounds were isolated from pomegranate flowers for t he first time,and in vitro anti-inflammatory activities of compounds ?-1,4,6-triO-galloyl-D-glucose,butylbrevifolin carboxylate and methy brevifolincarboxylate were studied.Studies showed that compounds butylbrevifolin carboxylate and b revifolincarboxylate can inhibit the synthesis of NO in cells,so they have antiinflammatory effects.Therefore,this experiment laid a theoretical foundation for the material basis and pharmacological action of pomegranate flower,and provi ded a reference for its further development and application.