Protective effects of ginkgo biloba leaves extract on peroxide-induced oxidative stress damage in PC12 cells

BACKGROUND： Extracts of ginkgo biloba leaves （EGB） and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress. OBJECTIVE： To investigate protective effects of EGB on peroxide （H2O2）-induced oxidative stress damage in PC12 cells. DESIGN： Observational contrast study. SETTING： Department ofPathophysiology, Guangdong Pharmacological College. MATERIALS： EGB was provided by Xi＇an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium （MTT）, trypsin and dimathyl sulfoxide （DMSO） by Sigma Company; PC12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase （LDH） kit by Nanjing Jiancheng Bioengineering Research Institute. METHODS： The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005. ①Cell culture： PC12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1 × 10^7 L^-1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall. ② Grouping and intervention： PC12 cells （1 × 10^8L^-1） were plated in 96-well plates with the density of 200 μ L/hole and divided into three groups： normal control group （routinely adding media）, H2O2 group （treating with media and H2O2 for 20 hours） and EGB group （adding media, 100μmol/L EGB and 100 μmol/L H2O2）. ③ MTT assay： PC12 cells （1 × 10^8L^-1） were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT （100μL） and cultured for 4 hours. And then, 200 μ L DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly ④ Experimental evaluation： Absorbance （A） at 630 nm was measured and LDH activity was measured at the same time. MAIN OUTCOME MEASURES： Results of MTT assay and LDH activity. RESULTS： ① Results of MTT assay： A value was lower in the H2O2 group than that in the normal control group （P 〈 0.01）, while A value was higher in the EGB group than that in the H2O2 group （P 〈 0.01）. ② LDH activity： LDH activity was higher in the H2O2 group than that in the normal control group （P 〈 0.01 ）, while LDH activity was lower in the EGB group than that in the H2O2 group （P 〈 0.01）. CONCLUSION： EGB can inhibit H2O2-induced oxidative stress damage in PC12 cells possibly by preventing damage to the cell membrane.

A Simple High-Performance Liquid Chromatography Method for the Assay of Flavonoids in Ginkgo biloba Leaves

Objective:Ginkgo biloba leaves,as an herbal medicine or dietary supplement,have been widely used worldwide.In this study,an integrated analytical method was established for the comprehensive analysis of flavonoids in G.biloba leaves.Materials and Methods:A practical chromatographic method combining high-performance liquid chromatography fingerprint analysis and quantitation was used to simultaneously determine 11 flavonoids(6 flavonol glycosides and 5 biflavones)in G.biloba leaves from different regions.Results:A total of 11 characteristic peaks were identified accurately,and the similarity of fingerprints ranged from 0.944 to 0.996.Methodology validation revealed appropriate linearity(R^(2)≥0.9997),precision,repeatability,stability,and recovery.The total contents of the six flavonol glycosides and five biflavones were within the range of 2.142-8.378 mg/g and 3.759-5.675 mg/g in 19 batches of samples,respectively.Among them,two coumaroyl flavonol glycosides were the predominant components.Conclusions:In this study,a convenient and reliable approach was successfully employed for the comprehensive evaluation of flavonoids in G.biloba leaves,which also provided a reference for its quality standard.

Shuxuening Injection Inhibits Apoptosis and Reduces Myocardial Ischemia-Reperfusion Injury in Rats through PI3K/AKT Pathway

Objective: To investigate the main components and potential mechanism of Shuxuening Injection(SXNI) in the treatment of myocardial ischemia-reperfusion injury(MIRI) through network pharmacology and in vivo research. Methods: The Traditional Chinese Medicine Systems Pharmacology(TCMSP) and Pharm Mapper databases were used to extract and evaluate the effective components of Ginkgo biloba leaves, the main component of SXNI. The Online Mendelian Inheritance in Man(OMIM) and Gene Cards databases were searched for disease targets and obtain the drug target and disease target intersections. The active ingredient-target network was built using Cytoscape 3.9.1 software. The STRING database, Metascape online platform, and R language were used to obtain the key targets and signaling pathways of the anti-MIRI effects of SXNI. In order to verify the therapeutic effect of different concentrations of SXNI on MIRI in rats, 60 rats were first divided into 5 groups according to random number table method: the sham operation group, the model group, SXNI low-dose(3.68 mg/kg), medium-dose(7.35 mg/kg), and high-dose(14.7 mg/kg) groups, with 12 rats in each group. Then, another 60 rats were randomly divided into 5 groups: the sham operation group, the model group, SXNI group(14.7 mg/kg), SXNI+LY294002 group,and LY294002 group, with 12 rats in each group. The drug was then administered intraperitoneally at body weight for 14 days. The main biological processes were validated using in vivo testing. Evans blue/triphenyltetrazolium chloride(TTC) double staining, hematoxylin-eosin(HE) staining, terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) assay, enzyme-linked immunosorbent assay(ELISA), and Western blot analysis were used to investigate the efficacy and mechanism of SXNI in MIRI rats. Results: Eleven core targets and 30 Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways were selected. Among these, the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) pathway was closely related to SXNI treatment of MIRI. In vivo experiments showed that SXNI reduced the myocardial infarction area in the model group, improved rat heart pathological damage, and reduced the cardiomyocyte apoptosis rate(all P<0.01). After SXNI treatment, the p-PI3K/PI3K and p-AKT/AKT ratios as well as B-cell lymphoma-2(Bcl-2) protein expression in cardiomyocytes were increased, while the Bax and cleaved caspase 3 protein expression levels were decreased(all P<0.05). LY294002 partially reversed the protective effect of SXNI on MIRI. Conclusion: SXNI protects against MIRI by activating the PI3K/AKT signaling pathway.