| Alcoholic liver disease(ALD)is a progressive liver injury disease caused by long-term excessive drinking.ALD has become a worldwide disease,which is serious harm to human health and even life.Studies have shown that the rich phenolic compounds of bee pollen can intervene and treat liver injury diseases to some extent.Schisandra chinensis bee pollen,Rape bee pollen and Silibinin bee pollen were used as materials in this study,Firstly,the phenolic compounds of each bee pollen were obtained by ultrasonic assisted extraction of ethanol and enrichment and separation with macroporous resin.After that,the antioxidant activities of crude extracts and components were evaluated through antioxidant models in vitro,and then the phenofic components from bee pollen with strong antioxidant activity were screened out,which were pretreated with alcohol injury cells after the model of alcohol induced oxidative damage in Hep G2 cells was established to evaluate the protective effects of phenolic compounds from bee pollen on liver cell injury induced by alcohol.The main research contents and experimental results are as follows:1.Extraction,enrichment and component analysis of phenolic compounds from three bee pollens.Phenolic compounds in Schisandra chinensis bee pollen,Rape bee pollen and Silibinin bee pollen were extracted by 80%ethanol with ultrasonic assisted extraction,and separated into five fractions by AB-8 macroporous adsorption resin with different ratio of ethanol/water elution.The effect of separation and enrichment was evaluated by HPLC,and the total phenols and total flavonoids in crude extracts and their fractions were determined by colorimetry.The results showed that the total phenols content of crude extracts of Schisandra chinensis bee pollen,Rape bee pollen and Silibinin bee pollen were 2.54 g/100g,6.93 g/100g and 0.41 g/100g respectively,and the total flavonoids content was 3.35 g/100g,2.74 g/100g and 1.95 g/100g respectively.After the crude extracts of bee pollen separated by AB-8 macroporous resin,the elution fraction of Schisandra chinensis bee pollen reached better enrichment of phenolic compounds,the total phenolic content of fractions from Schisandra chinensis bee pollen ranged from1.23 g/100g to 46.04 g/100g,and the total phenols in 40%and 60%ethanol/water elution fraction were 46.04 g/100g and 43.69 g/100g respectively,which was about 13 times higher than that of crude extracts.And the total flavonoids content of fractions from Schisandra chinensis bee pollen ranged from 0.44 g/100g to 6.39 g/100g,and the total flavonoids content of 40%and 60%fractions were 6.39 g/100g and 5.55 g/100g respectively,which was about 2.5 times higher than that of crude extracts.The total phenolic content of elution fractions from Rape bee pollen ranged from 2.93 g/100g to38.07 g/100g,and total flavonoids content ranged from 0.53 g/100g to 7.08 g/100g,and the total phenols content of 40%and 60%fractions were 9 times and 14 times higher than that of crude extracts respectively.The total phenolic content of elution fractions from Silibinin bee pollen ranged from 1.84 g/100g to 11.19 g/100g,and total flavonoids content ranged from 0.35 g/100g to 1.67 g/100g,and the total phenols content of the 40%ethanol/water elution fraction was 11 times higher than that of crude extracts.2.Comparison of antioxidant activity of phenolic compounds from three bee pollens.The antioxidant activities of crude extracts and their elution fractions from Schisandra chinensis bee pollen,Rape bee pollen and Silibinin bee pollen were studied by the antioxidant models in vitro including the 1,1-diphenyl-2-picrylhydrazyl radical(DPPH·)scavenging activities,diammonium2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS~+)scavenging activities,reducing power and anti-lipid peroxidation.It was found that the antioxidant activities of Schisandra chinensis bee pollen were better than those of Rape bee pollen and Silibinin bee pollen.Among them,the crude extracts,40%and 60%ethanol/water elution fraction of Schisandra chinensis bee pollen had strong antioxidant activity in vitro.The semi inhibitory concentration(IC50)indicated that the radical scavenging capacity of the crude extracts was the strongest,and the IC50 values of DPPH·and ABTS~+scavenging activities were 4.32?g/m L and 10.71?g/m L respectively.The reducing power results showed that the activities of crude extract and 40%ethanol/water elution fraction were higher than other components,and theirreducing power under 200?g/m L concentration was 2 to3 times heigher than that of other components.The 40%and 60%fraction had stronger lipid peroxidation resistance,and the IC50 values were2.09?g/m L and 6.05?g/m L respectively.The crude extracts and fractions of Rape bee pollen and Silibinin bee pollen also had different antioxidant activities,but they were weaker than that of Schisandra chinensis bee pollen on the whole.According to the comparative evaluation of the antioxidant activity in vitro,Schisandra chinensis bee pollen had strong antioxidant activity,so the choice of 40%,60%,80%elution fractions and crude extracts of Schisandra chinensis bee pollen were chosen to further study the protective effect of phenolic compounds from Schisandra chinensis bee pollen on liver injury induced by oxidative stress.3.The protective effect of phenolic compounds from Schisandra chinensis bee pollen on hepatocytes injury induced by alcohol.The model of alcohol-induced Hep G2 cells injury was established,and then protective effects of 40%,60%,80%ethanol/water elution fraction and crude extracts of Schisandra chinensis bee pollen in the suitable concentration on alcohol-induced hepatocytes injury were evaluated by the determination of glutamic-oxaloacetic transaminase(AST),alanine transaminase(ALT),glutathione(GSH),superoxide dismutase(SOD)and reactive oxygen species(ROS).The experimental results showed that the antioxidant system of cells in the model group was affected after Hep G2 cells were treated with 500 m M ethanol for 24 h,resulting in GSH depletion,SOD enzyme activity decreased,free radical scavenging capacity decreased,and intracellular ROS content increased,causing cell damage.After 40%and 80%elution fractions(0.5 to 20?g/m L),and crude extracts and 60%elution fractions(25?100?g/m L)were pretreated with the cells,damage of Hep G2 cells induced by alcohol was alleviated,which show a dose effect.Meanwhile,viability of cells was improved,the levels of AST,ALT,and ROS were obviously decreased,and the levels of GSH and SOD were obviously increased,which reduced the lipid peroxidation of liver cells and enhanced the vitality of the anti-oxidative system in cells,reduced the liver injury caused by oxidative stress.Experimental results illustrates that phenolic compounds from Schisandra chinensis bee pollen have protective effect on alcoholic liver injury. |
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