Constituents And Mechanism Of Elaeagnus Mollis Diels.on Acute Liver Failure

Elaeagnus mollis Diels.belongs to Elaeagnus in the family of Elaeagnaceae,which is an endemic species to China and originates from the Tertiary Period.E.mollis is mainly distributed in the hills and lower mountains of southern Shanxi and Shan’xi.E.mollis also possesses some pharmacological activity.For example,the seeds are rich in oleic acid and linoleic acid,which can be used medicinally for anti-hypertension,anti-hyperlipemia,and anti-atherosclerosis,and the leaves can be used for prevention and treatment of cerebrovascular ischemic damage,removal of oxygen free radicals and inhibition of pathogenic microorganism.The ethanol extract of leaves have been proved possess significant antibacterial and anti-inflammatory activities in our previous study.The chemical constituents and pharmacodynamic effects of E.mollis were further researched.Acute liver failure(ALF)is an inflammatory condition characterized by the development of severe liver injury.It is associated with high patient mortality,so there is no available therapy except for liver transplantation.At present,the natural plant extracts,effective frations and compounds,with few side effects,attracted our attention.Due to some chemical drugs are clinically used as hepatoprotective medications,which can be associated with producing certain hepatotoxicity.As a result,Chinese herbal medicine showed special advantage in hepatoprotective effect.As the leaves of Elaeagnus mollis have been used in treatment of anti-inflammatory and antiviral in folk medical uses.Meanwhile,ethanol extract have been showed protective effect on LPS induced injury to AML 12 cell.The above evidence implied that this plant probably possess anti-ALF activity.This dissertation is therefore conducted to investigate the anti-ALF activity of ethanol extract and flavonoids fraction from Elaeagnus mollis in vivo,followed by the isolation of the chemical constituents from its extract of branches and leaves.In order to determine the chemical responsible for the anti-ALF activity of this species,the experiments in vitro were used to test the anti-ALF activity of the isolated compounds.In addition,the Western blot assay was applied to detect the key protein expression in TLR4/NF-kappa B/Caspase signaling pathway of acute hepatic failure,so as to discuss the molecular mechanism of this action.1.Hepatoprotective effects of ethanol extract from Elaeagnus mollis in vivoThe anti-ALF effect of the ethanol extract of Elaeagnus mollis(400 mg/kg,200 mg/kg and 100 mg/kg)in D-galactosamine(GalN)and lipopolysaccharide(LPS)-induced hepatic failure model was investigated.The ALT/AST enzyme activity was detected in the serum of mice,H&E staining was used to observe the pathological changes of liver tissues;the main inflammatory factors TNF-alpha,IL-6 levels were detected in the tissues and serum to explore the effect of the drug through anti-inflammatory;the markers in oxidative stress including malondialdehyde(MDA)and Glutathione(GSH)were detected by liver tissue to examine the possible hepatoprotective mechanisms.The results showed that the ethanol extract of Elaeagnus mollis leaves had protective effects on acute liver injury,which inhibits the serum AST and ALT activity.The inflammatory cytokines level of TNF-alpha and IL-6 were inhibited by the alcohol extract,however,alcohol extract had a weak effect on MDA content and GSH enzyme activity,which suggested that the mechanism of the acute liver failure induced by D-GalN/LPS may be an anti-inflammatory mechanism.2.Chemical constituents of branches and leaves from E.mollisA total of sixty chemical compounds have been isolated and identified in the ethanol extracts of branches and leaves from E.mollis by using various chromatographic methods.Among these compounds,there were fourteen flavonoids including(-)-epigallocatechin(22),Quercetin-3-O-α-L-arabinopyranoside(23),kaempferol-3-O-α-L-arabinopyranoside(24),Isorhamnetin-3-O-α-L-arabinopyranoside(25),Astragalin(26),trans-tiliroside(27),cis-tiliroside(28),kaempferol-7-O-α-L-rhamnopyranoside(29),kaempferol-3-O-α-L-arabinopyranosyl-7O-α-L-rhamnopyranoside(31),kaempferol-3-O-β-D-galactopyranosyl-7-O-α-L-rhamnopyranoside(32),kaempferol-3-O-β-D-glucopyranosyl-(1’"→2")-O-β-L-arabinopyranoside-7-O-αL-rhamnopyranoside(33),Kaempferitrin(34),rutin(35),kaempferol-3-O-rutinoside(36),eight lignans including(-)-fragransin A2(11),Nectandrin B(12),lariciresinol(13),erythroguaiacylglycerol-β-O-4-coniferyl ether(16),(7S,8R)-dehydrodiconiferyl alcohol-9’-β-D-glucopyranoside(17),syringaresinol(55),dehydrodiconiferyl alcohol(56),nitidanin(57),five monoterpenes and triterpenoid acids including(-)-loliolide(14),asiatic acid(30),Ursolic acid(37),arjunolic acid(44),Oleanic acid(45),eight nitrogen-containing compounds including neoechinulin A(15),adenosine(39),allantoin(40),2-(3-hydroxy-2-oxoindolin-3-yl)acetic acid(42),N,N’-diferuloyl-putrescine(46),N,N’-dicoumaroyl-putrescine(47),hippophamide(48),N-trans-feruloyl-3’-methoxytyramine(50),eleven phenolic acids including methyl 4-hydroxybenzoate(8),vanillic acid(9),p-hydroxybenzaldehyde(10),salicylic acid(18),Ferulic acid(19),3-Hydroxy-4-methoxycinnamic acid(20),p-Hydroxycinnamic acid(21),gallic acid(38),Benzoic acid(41),Protocatechuic acid(43),p-Hydroxybenzoic acid(49),one steroidal was β-sitosterol(5),six aliphatics including Octacosanol(1),1-Hexacosanol(2),Glycerin monostearate(3),Heptadecanoic Acid(4),(2S,3S,4R,8E)-2-[2’-Hydroxy-23 carbamyl amide]-octadecane-1,3,4-triol(6),9,12,15-Octadecatrienoicacid,methyl ester(7),three phenolic glycosides including chaenomeloidin(58),tremuloidine(59),salicyloyltremuloidin(60),one phenylpropanoid was Zhebeiresinol(54),and three other compounds including ethyl α-D-glucopyranoside(51),blumenol A(52),blumenol B(53).Compound 33 was new,compounds 11 and 42 were new natural product,compounds 6,7,12-17,23-25,29-32,34,40,44,46-48,50-54,56-60 were separated from this genus for the first time.A total of fourteen flavonoids were isolated from the ethyl acetate and n-butanol fraction and the content of flavonoids is higher than others,implied that flavonoids were the main constituents of E.mollis and probably the main chemical responsible for the hepatoprotective activity of the plant.3.In vitro anti-ALF evaluation of the compounds isolated from Elaeagnus mollisA total of nineteen compounds were screened for the proliferation and protective activity of hepatocytes.The stimulated effects on cell proliferation by using the mouse hepatocyte AML 12.The hepatocyte AML 12 was induced by LPS to sceen hepatoprotective effects of compounds.The results showed that fifteen compounds exhibited significant effects both on cell proliferation and hepatoprotective effects.In which,The hepatoprotective effect of flavonoids were significant.4.Extraction of flavonoids fraction from E.mollis leavesThe flavonoids fraction was collected by using solvent extraction combined with reversed-phase column chromatography.Then the HPLC method for qualitative quantitative detection of flavonoids fraction was established.The results showed that the main flavonoid constituents were kaempferol-3-O-β-D-glucopyranosyl-(1’"→2")-O-β-L-arabinopyranoside-7O-α-L-rhamnopyranoside,kaempferol-3-O-β-D-galactopyranosyl-7-O-α-L-rhamnopyranoside,kaempferol-3-O-α-L-arabinopyranosyl-7-O-α-L-rhamnopyranoside and trans-tiliroside,and the contents were 3.85±0.06%,5.59±0.17%,49.25±2.65%,11.57±0.867%,respectively.In view of the higher content of kaempferol-3-O-α-L-arabinopyranosyl-7-O-α-L-rhamnopyranoside,implied that this compound was the main constituent of E.mollis leaves,and probably the main chemical responsible for the biological activity of the plant.5.Hepatoprotective activity of flavonoids fraction from E.mollis leavesThis study examined the hepatoprotective effect of flavonoids fraction of E.mollis(FE)on D-galactosamine(GalN)and lipopolysaccharide(LPS)-induced hepatic failure.Mice were given an administration of FE(50,100 and 200 mg/kg)for 7 days before GalN(700 mg/kg)/LPS(40 μg/kg)injection and were killed 6 h after GalN/LPS treatment.D-GalN/LPS markedly increased mortality and serum aminotransferase activity,both of which were attenuated by FE.FE also attenuated increases in serum and liver tumor necrosis TNF-α,IL-6,MDA levels by D-GalN/LPS.On the contrary,FE significantly increased liver and serum GSH level induced by LPS/GalN.The protective effect of FE on acute liver failure caused by D-GalN/LPS was more significant than that of alcohol extract,suggesting that FE was the effective component of the E.mollis leaves on hepatoprotective effect.6.Hepatoprotective effect and molecular mechanism of kaempferol-3-O-α-L-arabinopyranosyl-7-O-αL-rhamnopyranoside(EM31)on D-galactosamine and lipopolysaccharide caused hepatic failureTo elucidate the effective constituent of the anti-hepatic injury of E.mollis leaves,the anti-ALF activity of kaempferol-3-O-α-L-arabinopyranosyl-7-O-α-L-rhamnopyranoside(EM31)(25,50 or 100 mg/kg)in model of D-galactosamine(GalN)and lipopolysaccharide(LPS)-induced hepatic failure was investigated.The results showed that pretreatment with EM31 could increase the survival rate in a dose-dependent manner.The pathological changes including extensive hemorrhage,neutrophil infiltration could be markedly alleviated by pretreatment with EM31.Serum AST and ALT levels increased significantly after D-GalN/LPS treatment and this could be alleviated by pretreatment with EM31.The production of TNF-α,IL-1β and IL-6 in the serum and liver tissues were significantly increased after D-GalN/LPS treatment,and this could be attenuated when pretreatment with EM31 in a dose dependent manner,which suggest that EM31 plays an important role in protecting against D-GalN/LPS-induced increasing of inflammatory cytokines.Furthermore,the expression level of oxidase was effectively interfered with EM31 that the level of GSH significantly decreased in the serum and liver tissues and this could be reversed when pretreatment with EM31,and EM31 could attenuate the levels of MDA and ROS in the serum and liver tissues,which suggested that EM31 is the effective compound of the E.mollis leaves on hepatoprotective effect.The study further investigate the protective mechanism of EM31 in D-GalN/LPS-induced acute liver injury.Studies have shown that the NF-kappa B signaling pathway plays a crucial role in the inflammatory response induced by D-GalN/LPS.Recently,it is reported that TLR4 plays important roles in D-GalN/LPS induced liver injury and inhibiting the activity of TLR4 signaling is a potential therapy.Previously,we observed that D-GalN/LPS treatment could induce the apoptosis of hepatocytes,and it is demonstrated that dysregulation of apoptosis is associated with acute hepatic failure.Caspases protease activation is the crucial process of inducing apoptosis and it is closely related to the pathogenesis of acute liver damage.In this study,the expression of TLR4,NF-κB signaling pathway and apoptosis-related proteins,including cytochrome c,Bax,Bcl-2,Caspase-3,Caspase-8 and Caspase-9,p-IκBα,p65,TLR4,TRAF6,MyD88,TRIF in liver tissues were detected using western blotting assay.The results indicate that EM31 inhibits the apoptosis of hepatocytes and reduces the expression of TLR4 and NF-κB signaling pathway related proteins induced by D-GalN/LPS treatment.These findings suggest that the anti-oxidative,anti-inflammatory and anti-apoptotic effects of EM31 on D-GalN/LPS-induced acute liver injury were performed through down-regulating the activity of TLR4 and NF-κB signaling pathway.In conclusion,the leaves of Elaeagnus mollis exhibited a definite anti-ALF activity,the chemical responsible for this effect are flavonoids compounds,and kaempferol-3-O-α-Larabinopyranosyl-7-O-α-L-rhamnopyranoside(EM31)is the main active constituent,and the anti-ALF activivity is related to the TLR4/NF-κB/Caspase signaling pathways.