Studies On Separation And Purification Of The Antioxidant Component From Phellinus Pini S.l.,and Its Mechanism

Cardiovascular disease is one of the diseases with the highest mortality rate in the world.At present,there still exist some problems in the clinical use of combination therapy due to its drug resistance and easy recurrence,so the exploration and development of high-efficiency and low toxicity natural drugs have become a research hotspot for the scholars at home and abroad.According to reports,most heart and vascular diseases are closely related to the imbalance between oxidation and antioxidation in biological system,or the production and elimination of reactive oxygen species.The excessive generation of reactive oxygen species(ROS)will disrupt the balance between oxidation system and anti-oxidation system in vivo,resulting in cell oxidative stress.Oxidative stress can lead to over oxidation of protein or enzyme,resulting in the loss of function of protein or enzyme.Therefore,oxidative stress can lead to oxidative damage associated with chronic diseases such as cardiovascular diseases,neurodegenerative diseases and inflammation.Mushrooms have been used as functional food and medicine because they can produce a variety of bio-active primary and secondary metabolites,such as polysaccharides,proteins,polyphenols,flavonoids,triterpenoids and steroids.In recent years,scholars at home and abroad have conducted extensive research on the chemical composition,pharmacological activity and clinical application of mushrooms,and found that most of the polyphenols,flavonoids,triterpenes and polysaccharides contained in mushrooms have good antioxidant activity,which has important medicinal and health value.In this study,the inhibition activities of erythrocyte hemolysis and lipid peroxidation and total polyphenol contents of 12 strains of mushroom fruit bodies were screened,and Phellinus pini was selected as the research object.Then,the components with antioxidant activity were isolated and purified from the fruiting body of P.pini,and the cardioprotective effect and mechanism of action of the components were evaluated,that is,the relationship between the cardioprotective effect and the activation of Nrf2/Keap1 signaling pathway and the subsequent induction of HO-1.In this paper,a small molecule active component PP-S4-1 was isolated and purified from P.pini by 70%methanol shaking extraction,AB-8 macroporous adsorption resin,Sephadex LH-20 gel chromatography and high performance liquid chromatography(HPLC).After gradient operation on AB-8 macroporous adsorption resin and Sephadex LH-20 gel chromatography,the total phenolic content of active component PP-S-4increased 4.8 folds from 128.6mg(GE)/g _extract,the original value.621.24 mg(GE)/g _extract increased the inhibition rate of red blood cell lysis and lipid peroxidation from59.11%and 56.45%to 87.7%and 83.3%respectively,at the dose of 150?g/m L.In addition,the inhibition rate of erythrocyte hemolysis and lipid peroxidation was proportional to the total phenolic contents of eluting components.PP-S4-1 and PP-S4-2 were obtained from PP-S-4 by semi-preparative high performance liquid chromatography.At 100?g/m L,each component had different degrees of antioxidant effect,but only at 50?g/m L,PP-4-1 had the strongest inhibitory activity on erythrocyte hemolysis and lipid peroxidation,with inhibition rates of 91.9%and 87.6%,respectively(p<0.01).In addition,PP-S4-1 had strong scavenging activity for superoxide anion,and the scavenging rate of superoxide anion reached 90.61%at the concentration of 200?g/m L.PP-S4-1 was identified as catechin(molecular weight 290.015,C₁₅H₁₄O₆)by ESI-MS,¹H-NMR,¹³CNMR and HPLC retention time comparison with the corresponding standard.It was the first time to isolate and purify catechin from P.pini.More and more researchers pay attention to the role of cell death caused by oxidative stress in the occurrence and development of diseases.Therefore,it is very important to study the mechanism of oxidative damage caused by reactive oxygen species and the signaling pathway of antioxidant active components.In this study,the oxidative stress model of H9c2 cells was established by H₂O₂induced oxidative damage.PP-S4-1,an active component isolated from P.pini,was used as the experimental object to evaluate the protective effect of natural antioxidants on oxidative damage cells.During the study,the concentration of H₂O₂ and PP-S4-1 were determined.H9c2cells were exposed to 200,250,300,350 and 400?M H₂O₂ for 6 h,and the cell viability decreased to 75.63%,69.81%,59.60%,50.10%and 43.11%respectively.Therefore,350?M and 6 h were selected as the conditions of oxidative stress model.In the concentration range of 12.5?g/m L-100?g/m L,no cytotoxicity was found in H9c2 cells treated with PP-S4-1.In order to evaluate the protective effect of PP-S4-1 on oxidative damage of H9c2 cells,the cells were pretreated with 12.5,25,50 and 100?g/m L PP-S4-1 before being exposed to H₂O₂.The results showed that the cell viability increased from 50.10%to 82.95%after 50?g/m L(p<0.05).Next,we studied the effects of PP-S4-1 pretreatment on different oxidative indexes,such as ROS level,lactate dehydrogenase(LDH)release,malondialdehyde(MDA)content,antioxidant enzyme activities,glutathione(GSH)level,mitochondrial membrane potential(MMP),cytoplasmic calcium ion,cell morphological changes and DNA damage.The results showed that pretreatment with PP-S4-1 could significantly reduce the release of lactate dehydrogenase(LDH)(p<0.01)and intracellular calcium overload(p<0.01),significantly reduce the production of malondialdehyde(MDA)(p<0.01),and increase the level of superoxide dismutase(SOD)(p<0.01).In addition,PP-S4-1could significantly inhibit the decrease of catalase(CAT)(p<0.05),glutathione peroxidase(GSH-Px)activity and glutathione(GSH)level in H9c2 cells induced by H₂O₂(p<0.01).After exposure to H₂O₂,the level of ROS in H9c2 cells was significantly increased(p<0.01),and the mitochondrial membrane potential(MMP)was significantly decreased(p<0.01).Pretreatment with PP-S4-1 could significantly reduce the level of ROS in H9c2 cells(p<0.01),and enhance the decrease of MMP induced by H₂O₂.The morphological changes were observed by Hoechst/PI double staining dye and DAPI staining dye.The results showed that compared with the normal control group,the nucleus of cells in the oxidative stress model group agglutinated,the cell membrane ruptured,and a large number of cells were in the late stage of apoptosis,showing red fluorescence.PP-S4-1 pretreatment could significantly change the chromatin agglutination caused by oxidative damage,reduce the number of red stained cells,and improve the cell morphology.Comet assay was used to evaluate the degree of DNA damage in H9c2 cells.The results showed that H₂O₂caused serious DNA damage,but PP-S4-1 pretreatment could protect H9c2 cells from H₂O₂ induced damage.In order to further study the protective effect of PP-S4-1 on oxidative damage of H9c2 cells,this study used Keap1/Nrf2/HO-1 signaling pathway as the breakthrough point to explore the mechanism of its antioxidant activity.It was found that H₂O₂destroyed the balance of oxidation and antioxidation in H9c2 cells,inhibited the normal function of mitochondria and led to DNA damage.After H9c2 cells were exposed to H₂O₂,Western blotting and q RT-PCR results showed that the protein expression levels of antioxidant enzymes(SOD1,CAT and GPx-1)and phase II enzymes(HO-1,NQO1,GCLM and GCLC)decreased,but PP-S4-1 pretreatment increased the protein expression levels,and the gene expression levels of antioxidant proteins Hmox-1,Nqo1,Gclc and Gclm.Nrf2 can regulate the expression of enzyme genes,including antioxidant genes,phase II enzyme genes(Hmox-1,Nqo1,Gclc and Gclm),transporters and so on.On the basis of proving that PP-S4-1 can enhance the protein expression of antioxidant enzymes and phase II enzymes,the changes of Nrf2 protein expression in H9c2 cells were discussed.The nuclear translocation of Nrf2 was evaluated by Western blotting analysis of cytoplasm and nucleus and immunofluorescence staining of Nrf2 and Keap1.It was found that PP-S4-1 could promote the translocation of Nrf2 into the nucleus.Western blotting results showed that Nrf2 content increased in the presence of PP-S4-1,indicating that Nrf2 expression level in nucleus induced by PP-S4-1 was higher than that in cytoplasm.These results indicate that PP-S4-1 activates the Keap1/Nrf2 pathway in H9c2cells.In order to verify the role of Nrf2 in the regulation of HO-1 expression by PP-S4-1,cells were treated with Brusatol,a specific inhibitor of Nrf2,and then treated with PP-S4-1.The results showed that Brustol significantly inhibited the expression of Nrf2protein,and effectively terminated the expression of HO-1 protein by making Nrf2,which means that PP-S4-1 may destroy the interact between Keap1 and Nrf2(PPI),thus translocating the free Nrf2 into the nucleus,thus up-regulating the expression of HO-1 protein.In conclusion,a small molecule PP-S4-1 with obvious antioxidant activity in vitro was isolated and purified from P.pini,which was identified as catechin with a molecular weight of 290.015.Furthermore,the antioxidant mechanism of PP-S4-1 was further studied,which revealed that PP-S4-1 could specifically inhibit H₂O₂ induced oxidative damage of H9c2 cells by inhibiting excessive accumulation of reactive oxygen species,improving permeability of mitochondrial membrane,inhibiting intracellular calcium overload,and inhibiting DNA damage;the mechanism of PP-S4-1 was through activating Nrf2 translocation to the nucleus,HO-1 was induced to mediate the expression of antioxidant enzymes and phase II enzyme related genes,and Keap1/Nrf2 and Nrf2/HO-1 signaling pathways were regulated to protect H9c2 cells from oxidative stress.This study laid a solid theoretical foundation for the application of active components of P.pini.