Study On The Anti-Alcoholic Liver Injury Function Of Pine Pollen Water Extract And Its Enhancement

Alcoholic liver injury(ALD)is a critical social public health problem and is currently mostly treated with drugs.However,drugs mostly lead to some adverse effects,so there is a growing interest in developing functional foods of natural origin with the ability to intervene in ALD.Pine pollen is a traditional medicinal food raw material in China.It has been proven to have various effects such as anti-chemical liver injury.However,there are fewer reports on antiALD.Compared to alcoholic extracts,water extracts have the advantage of being used directly as common food ingredients.However,small molecule active ingredients such as flavonoids and phenols are mostly present in the bound state in pine pollen.Or their content in water extracts is low due to poor water solubility and limited functionality of water extracts.In this paper,the main functional components of pine pollen water extract(PPE)were analyzed.The mechanism of its action in protecting against ALD was explored by combining literature,network pharmacology and cellular models.PPE was graded using ultrafiltration.The liver protection effect of different molecular weight fractions was also compared using a cellular model.Thus,the enzymatic screening index was determined.The enzymatic method was then used for the assisted water extraction of pine pollen.The enhancement effect of enzymatic digestion on the hepatoprotective efficacy of PPE was evaluated by in vitro physicochemical indexes and cellular models.The main research contents and results are as follows:From the analysis of the basic components of the PPE combined with the previous research of the group,it was found that the total sugar content of PPE was 39.57% ± 1.67%,the crude polysaccharide content was 12.25% ± 0.33%,the total protein content was 12.11% ± 0.07%,the total phenol content was 5.36 ± 0.02 mg GAE/g PPE,the total flavonoid content was 1.95± 0.15 mg TE/g PPE.Eight small molecule active ingredients were identified by UPLC-QTOFMS,including 4-hydroxybenzoic 4-O-β-D-glucopyranoside,protocatechuic acid,coumaric acid,6-hydroxykynurenic acid,salicylic acid,2-coumaroylquinic acid,caffeic acid,and pcoumaric acid.The network pharmacological analysis inferred that the main pathways involved in the anti-ALD exerted by the small molecule active ingredients of pine pollen are PI3K-AKT signaling pathway,chemoattractant-receptor activation,lipid and atherosclerosis,chemoattractant-reactive oxygen species,Ras signaling pathway,MAPK signaling pathway,etc.Combined with the generalization of the studies on the liver protection mechanism of polysaccharides,proteins,and peptides in the literature.It is concluded that the most critical pathway of pine pollen anti-ALD is to improve the changes in expression of genes and proteins related to oxidative stress and apoptosis due to ROS overproduction,and may be highly correlated with the upstream pathways MAPK and PI3K/AKT.An ethanol-induced HepG2 cell model was established to investigate its hepatoprotective effect and verify its mode of action.The results showed that PPE could significantly reduce cellular LDH leakage and improve cell morphology thus showing a protective effect against cellular damage.PPE could significantly improve ethanol-induced cellular oxidative stress by reducing ROS and MDA while elevating GSH and SOD levels.RT-PCR and Western Blot results showed that PPE significantly elevated the expression levels of oxidative stress proteins and genes(Nrf2,HO-1,and Keap1),and significantly decreased the expression of apoptotic protein Bax and Cleaved Caspase3.PPE significantly improved ethanol-induced phosphorylation levels of AKT and MAPK(p38/JNK/ERK)in HepG2 cells.The improvement of cellular oxidative stress and apoptosis pathways by PPE was also verified to be MAPKrelated using inhibitors.Based on the determination of the anti-ALD efficacy of pine pollen,the PPE was graded using ultrafiltration to compare the content of total sugars and total protein in five fractions with relative molecular weights <1000,1000～50000,5000～10000,10000～100000,and >100000.The total sugars were found to be mainly concentrated in the fractions with relative molecular weights >10000.Proteins or peptides are mainly concentrated in fractions with relative molecular weights of 1000～10000,and flavonoids,polyphenols and other small molecules are mainly concentrated in the <1000 fraction.The differences in the antioxidant properties and the level of protection against HepG2 cell damage of the five fractions were compared.The results showed that the in vitro antioxidant activities of the five fractions were significantly different,with the smaller the molecular weight the stronger the antioxidant activity.However,the levels of ALT,AST,LDH,SOD and GSH showed that all five fractions had hepatoprotective effects,and the differences in the specific effects were not significant.It is suggested that several components of the PPE can exert hepatoprotective effects.Therefore,the simultaneous determination of the molecular weight distribution of the products and the content of polyphenols,flavonoids,and total sugar in the products will be considered in the enzymeassisted water extraction experiment.To enhance the solubilization capacity and hepatoprotective activity of the PPE,enzymatic digestion was used to assist in the water extraction of pine pollen.Proteases(acid protease,alkaline protease,and papain),cellulase,pectinase,and α-amylase were used for the enzymaticassisted PPE.The results showed that all six enzymes significantly enhanced the levels of yield,total phenolics,total sugars,and total antioxidant properties.Among them,protease and α-amylase contributed to a greater extent to the enhancement of total phenolics and total antioxidant properties.The total phenolic content was elevated by 37.36%,57.24%,31.11%,and 36.07% after acid protease,alkaline protease,papain and α-amylase assisted extraction under the enzyme high enzyme level condition,respectively.All six enzymes contributed to the enhancement of total sugar content.At high enzyme levels,pectinase,cellulase,α-amylase,papain,alkaline protease and acid protease assisted extraction enhanced the total sugar content by 95.11%,116.35%,46.91%,61.72%,94.60%,and 201.45%,respectively.Alkaline protease significantly changed the molecular weight distribution of the aqueous extract.At high enzyme levels,the content of fractions with relative molecular weights ≤1450 increased from 33.50%to 37.63%,and the content of fractions ≥5000 decreased from 19.90% to 8.27%.Combining the above results,the water extracts before and after enzymatic digestion by alkaline protease were used to compare the efficacy of liver protection.The expression levels of genes and proteins for anti-oxidative stress and apoptosis were significantly higher in the enzymatic digestion group compared with the non-enzymatic digestion group.The expression levels of pro-apoptotic genes and proteins were significantly lower.Therefore,enzymatic digestion could enhance the hepatoprotective efficacy of water extracts of pine pollen to some extent.