Preparation Of Active Components From Hippophae Rhamnoides Linn.and Study On Doxorubicin-induced H9c2 Myocardial Injury

Hippophae rhamnoides Linn.subsp.Sinensis Rousi（Seabuckthorn）,a plant of Hippophae L.in Elaeagnus family,is a kind of medicine food plant,mainly distributed in Asia and Europe,which has been widely used in China.Seabuckthorn not only contains six essential nutrients for human body,water,inorganic salt,vitamin,oil,protein and carbohydrate,but also contains a variety of bioactive components,mainly including flavonoids,alkaloids,terpenes,etc.Seabuckthorn has many functions and clinical application value,such as anti-oxidation,anti fatigue,anti radiation,hypoglycemia and Hypolipidemia.In this study,the chemical components of seabuckthorn seed residue were separated systematically.Multiple chromatographic techniques（Silica gel,MCI gel,Thin layer preparation chromatography,Semi preparative-HPLC,HPLC and other methods）had been used to separate and preparation of 75%ethanol aqueous extract from seabuckthorn seed residue.Using UV,¹H-NMR,¹³C-NMR,HMBC,HSQC,HHCOSY,TOCSY and other organic spectral analysis techniques and comparative literature data,18 monomers were identified,including 2 phenolic acids（2E,4E）-8-Hydroxy-2,7-dimethyldeca-2,4-dienedioic acid,isovanillin),2 sesquiterpenes（（6R,9R）-9-Hydrox-4,7-megastigmadien-3-one-9-O-β-D-apiofuranosyl-（1→6）-β-D-glucopyranoside,（6S,9R）-9-Hydrox-4,7-megastigmadien-3-one-9-O-β-D-apiofuranosyl-（1→6）-β–D-glucopyranoside）,2 flavones with isorhamnetin asthe mother nucleus（isorhamnetin3-O-α-L-rhamnopyranosyl-（1→6）-β-D-galactopyranoside7-O-α-L-rhamnopyanoside,isorhamnetin 3-O-β-D-glucoside-7-O-α-L-rhamnoside）,1 flavone with quercetin as the mother nucleus（Quercetin-3-O-（4′′′′-O-E-sinapoyl）-α-rhamnopyranosyl-（1′′′→2′′）[α-rhamnopyranosyl-（1′′′′→6′′）]-β-glucopyranoside）and 11 flavones with kaempferol as the mother nucleus（kaempferol3-O-β-glucopyranoside-7-O-（6-trans-feruloyl）-β-glucopyranosyl-（1→2）rhamnopyranoside,acacetin,kaempferol 3-（2^G-rhamnosylrutinoside）,kaempferol-3-O-β-D-glucoside（1→2）-β-D-glucoside-7-O-（3-O-trans-sinapoyl）-α-L-rhamnoside,kaempferol 3-O-β-D-glucosyl（1→6）-O-β-D-glucoside,kaempferol-3-rutinoside,kaempferol-3-O-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside,kaempferol-3-O-（2-O-β-D-glucoside）-β-D-glucoside-7-O-α-Lrhamnoside,kaempferol-1,kaempferol-2,kaempferol-3）.Four of the 18 compounds are new compounds,which are compound 4,compound 17,compound 18 and compound 19.In addition,10 compounds,such as compound 1,compound 3,compound 5,compound 7,compound 8,compound 9,compound 10,compound 11,compound 13 and compound14,are isolated for the first time from Hippophae rhamnoides.Flavonoids are the main active components in Hippophae rhamnoides.A method for the simultaneous determination of 8 phenolic acids（gallic acid,protocatechuic acid,catechin,caffeic acid,p-coumaric acid,ferulic acid,kaempferol-3-O-rutoside and isorhamnetin-3-O-glucoside-7-O-rhamnoside）in seabuckthorn leaves was established.The results showed that the linear ranges of the eight compounds were 0.012-0.870μg,0.696-3.913μg,0.203-4.174μg,0.203-3.043μg,0.035-2.609μg 0.023-3.478μg,0.565-8.478μg,0.522-7.826μg（R²≥0.9992）,respectively.The average recoveries were 93.58%-108.11%,RSDS≤1.81%.The leaves of Hippophae rhamnoides from different habitats all contain the above 8 phenolic acids,but there are differences in content.A model of doxorubicin（DOX）induced injury of H9c2 cardiomyocytes in vitro was established.The optimal concentration of DOX in the experiment was determined to be 3μm by observing cell morphology,MTT assay,Griess assay,microplate assay and Western blot assay.On this basis,the model of DOX induced injury of H9c2cardiomyocytes was established,and the activity of different kinds of monomer compounds in Seabuckthorn was screened and evaluated.MTT method was used to detect cell viability,Griess method to detect no content in cell supernatant,microplate method to detect LDH content in supernatant and Western blot to detect apoptosis factors.The results show that triterpenoids have a significant effect on the In DOX induced injury of H9c2 cardiomyocytes,it has a certain protective effect,but the mechanism is not clear;in phenolic acids compounds,Syringate and rosmarinic acid have a protective effect on DOX induced injury of H9c2 cardiomyocytes,and the effect is rosmarinic acid>Syringate;in flavonoids,rutin,quercetin,kaempferol,isorhamnetin,kaempferol,kaempferol,kaempferol,kaempferol and epicatechin Apigenin,naringin,Narcissus,quercetin,isoquercetin,catechin,k-1,K-2 and K-5 all have protective effects on DOX induced injury of H9c2 cardiomyocytes.