Study On Antioxidation And Mechanism Of Artificial Planting Of Ethanol Extract From Anoectochilus Formosanus In Vitro And In Vivo

Objective:With the development of the health industry of Anoectochilus Formosanus,its demand has been increasing year by year.Planting under forest can be an important means and approach to alleviate the severe shortage of wild resources of Anoectochilus Formosanus,and it is also beneficial to protect the wild resources of Anoectochilus Formos-anus.Therefore,this article discusses（1）the in vitro antioxidative activity of different solvent extracts of Anoectochilus Formosanus,and optimizes the extraction process of the drug;（2）Evaluating in vitro antioxidant activity of the ethanol extract of artificial Anoectochilus Formosanus using cell oxidative stress injury model.;（3）Evaluation of the antioxidant effects of AFEE on natural aging mice;（4）Evaluation of the antioxidant effects of AFEE on aging mice induced by D-galactose and possible mechanism has made a preliminary discussion.The above study will provide certain experimental basis for the development of the undergrowth plantation industry of Anoectochilus Formosanus.Method:1、Through the determination of total antioxidant capacity,hydroxyl radical（·OH）clearance rate,and superoxide anion（O₂^-·）clearance rate,the antioxidant activity in vitro of the different extraction processes of artificial planting Anoectochilus Formosanus was evaluated.2、Establish oxidative stress injury model by H₂O₂-induced LO2 cells,and explore the protective effect of AFEE on H₂O₂-induced LO2 liver cell injury.3、Effect of AFEE on natural aging mice:8 months old（old）KM mice were randomly divided into 5 groups according to body weight:natural aging group,positive control group（vitamin E group:0.1g/kg）,AFEE High-dose group（AFEE-H,4g crude drug/kg）,AFEE middle-dose group（AFEE-M,2g crude drug/kg）,and low-dose AFEE group（AFEE-L,1g crude drug/kg）were continuously administered for 30 days.The body weight of mice was determined before administration,14 days and 28 days after administration.At the end of the experiment,the MDA content and GSH-PX and SOD activity in serum and liver of the test animals were measured.4、Effect of AFEE on D-galactose-induced aging mice:8-week-old KM mice were randomly divided into 6 groups according to body weight:model group,blank control group,and positive control group（vitamin E group:0.1g;/kg）,AFEE high dose group（4g crude drug/kg）,AFEE medium dose group（2 g crude drug/kg）,AFEE low dose group（1 g crude drug/kg）.The mice were measured for body mass before administration,14 days and 28 days after administration.The day before the end of the experiment,the numbers of spontaneous activity of the mice were measured.After the end of the experiment,the brain,thymus,spleen,and other tissues of the mice were taken to measure the organ index.The liver and skin were examined by tissue section and HE staining.The levels of MDA,GSH-PX and SOD in the serum,liver and brain were measured,and the levels of ACH-E and MAO in the brain were measured.The qRT-PCR method was used to determine the GPx-1,GPx-4 and SOD-1 in the liver.gene expression.Result:1、AFEE and Anoectochilus Formosanus water extracts have certain scavenging ability for hydroxyl radical（·OH）,superoxide anion（O₂^-·）,and have a certain total antioxidant capacity,the IC₅₀0 of AFEE for hydroxyl radicals was 5.6 mg crude/mL,while the IC₅₀0 of Anoectochilus Formosanus water extracts against hydroxyl radicals was 19.3 mg crude/mL;Similarly,the IC₅₀0 of AFEE for superoxide anion is 13.2 mg crude/mL,while the IC₅₀0 of superoxide anion for Aqueous Aqueous extract is 77.9 mg crude/mL,indicating that the antioxidant activity of AFEE is stronger than that Anoectochilus Formosanus water extract in vitro.2、Protective effects of H₂O₂-induced oxidative damage on LO2 cells:Compared with the model group,AFEE high-and middle-dose groups can increase the survival rate of H₂O₂-injured LO2 cells（P<0.01 or P<0.05）;compared with the blank control group,H₂O₂ concentration of 400μmol/L stimulated the MDA content in the cell culture medium significantly（P<0.01）,SOD and GSH-PX content decreased significantly（P<0.01）,indicating that H₂O₂-induced L02 hepatocyte oxidative stress injury model has been set up.Compared with the model group,the activity of GSH-PX and SOD in LO2 cells was significantly increased in the high-dose AFEE group（P<0.01 or P<0.05）;the content of MDA in LO2 cells was significantly decreased in the high-and middle-dose groups of AFEE（P<0.01 or P<0.05）.3、Effects on the natural aging model:Compared with the natural aging group,there was no statistically significant difference in the body weight before and 14 days and 28 days after dosing（P>0.05）;The activity of GSH-PX and SOD in the serum of vitamin E and AFEE high-and middle-dose groups was significantly increased（P<0.01 or P<0.05）.The content of serum MDA was significantly decreased in the vitamin E and AFEE high and middle dose groups（P<0.01 or P<0.05）.The activities of GSH-PX and SOD in the liver of vitamin E group and high dose of AFEE group were significantly increased（P<0.01）,and the content of MDA was significantly decreased（P<0.01）.The activity of SOD in liver of AFEE middle-dose group was increased（P<0.05）,MDA content was significantly reduced（P<0.01）.4、Effect on D-galactose induced aging model（1）Compared with the model group,there was no significant difference in body weight between the groups before and 14 days and 28 days after administration（P>0.05）;（2）Compared with the normal group,the number of locomotor activity in the model group was significantly reduced,and the number of autonomous activities in the drug group had a tendency to increase.However,there was no significant difference in the number of locomotor activities compared with the model group（P>0.05）.;（3）In the aging model group,the mouse epidermis became thinner,some of the cuticles fell off,and the structure was not complete.However,the skin structure of the mice in the drug group was relatively complete,and the cell layering was relatively clear.There were epidermal processes and dermal papilla;A large number of hepatocytes showed vacuolation and necrosis,and some hepatocytes had karyotypic nucleus dissolution in the model group.However,no significant abnormalities were found in the structure,size,and staining of hepatocytes and nuclei in the liver tissue of mice in the drug group.There was no expansion of hepatic sinusoids,and there was no abnormal hepatic cord structure.,indicating that the drug group has significantly improved the therapeutic effect;（4）Compared with the model group,there was no significant difference in the weight of the spleen,thymus,brain and liver organs and organ index（P>0.05）.（5）Effects on anti-oxidation related indicators in serum,liver,and brain:Compared with the model group,the activities of GSH-PX and SOD in serum of the drug groups increased significantly（P<0.01 or P<0.05）.)However,there was no significant difference in serum MDA levels between the drug groups（P>0.05）.Compared with the model group,GSH-PX activity in the liver of each drug group was significantly higher（P<0.01 or P<0.05）,but there was no significant change in the activity of SOD（P>0.05）.There was a significant difference in the content of MDA in the vitamin E group and each drug group（P<0.01 or P<0.05）.Compared with the model group,the activity of GSH-PX and SOD in the brain of the medium and high dose group was significantly increased（P<0.01 or P<0.05）.The content of MDA in the brains of the vitamin E and AFEE high and middle dose groups was significantly lower.The difference was statistically significant（P<0.01 or P<0.05）.Compared with the model group,the mRNA expression of GPx-1 and GPx-4 in the liver of the high-and middle-dose groups of AFEE and Vitamin E had statistical significance（P<0.01 or P<0.05）.No significant effect of SOD-1 mRNA expression in the liver of each drug was observed.（6）Effect on the activity of MAO and ACH-E in brain tissue:Compared with the model group,the activity of MAO in the brain of vitamin E group and each drug group were significantly lower（P<0.01 or P<0.05）;There was no significant difference in brain ACH-E activity between the drug groups and the model group（P>0.05）.Conclusion:1、There are some differences in the vitro antioxidative activities of different extraction processes of artificial Anoectochilus Formosanus,and the antioxidation activity of alcohol extracts outperforms the water extraction group.2、AFEE have the protective effect by antagonizing H₂O₂ oxidative stress on liver cell damage.3、AFEE has a significant antioxidant effect on natural aging model mice,and its mechanism may be related to the increase of SOD and GSH-PX activity and decrease of MDA content in natural aging mice.4、AFEE can improve the D-galactose induced aging skin and liver pathological changes,increase the activity of antioxidant enzymes,reduce MAO activity,AFEE have a certain degree of anti-oxidation in D-galactose induced aging model mice,the mechanism may be by increasing the expression levels of GPx-1 and GPx-4 mRNA,increase the activity of antioxidative enzymes such as GSH-PX in vivo,and thus play an antioxidative role in vivo.