Screening Of Chinese Herbal Medicine And The Study On Anti-Oxidative Stress Properties Of Maydis Stigma's Extract

Diseases such as aging,angiocardiopathy,inflammation,tumor,etc related to free radical injury have attracted more and more public concern.At the same time,further studies on roles of natural antioxidants and free radical scavengers are being conducted.Theoretically,searching efficient natural antioxidants can further confirm the relation of their antioxidative abilities and preventive abilities of diseases related to free radical injury.Practically,searching efficient natural antioxidants will contribute to further studies on their effective constituents,developing health food and medicine to prevent or cure diseases.Our study was divided into 2 parts and 4 levels(including gene and protein, deuto-cell,cell and animal) to research the reaction characteristics and action ways of many traditional Chinese herbal medicine extracts on their scavenging free radicals and anti-oxidative stress injury activities.Maydis stigma extract (MSE) was found to have multi-level anti-oxidative stress function and exhibited a concentration-dependent relation,so it deserves a furher development and utilization. In the part of in vitro test,we effectively screened 11 ethanol extracts from traditional Chinese herbal medicines employing chemiluminescence and microsomal lipid peroxidation models.We have investigated their direct scavenging effects on hydroxyl radicals(^·OH) and superoxide anions(O2^·-),as well as their anti-lipoperoxidative capacity on rat liver microsomal membrane peroxidation induced in vitro by three different sources of stimulators: Fe²⁺/ascorbate,cumene hydroperoxide(CHP),and CCl₄/reduced form of nicotinamide-adenine dinucleotide phosphate(NADPH).The result indicated that all the tested extracts had antioxidant effect at some extent,but Maydis stigma extract was firstly found to have much stronger and broader,more efficient and systematic antioxidant ability compared with the others.It could scavenge free radicals obviously at concentrations as low as 3.55μg/ml.And between the final concentration of 6.25 and 2000μg/ml,it could effectively inhibit lipid peroxidation with concentration-dependent relations(r≥0.79, P＜0.05).In the part of ex vivo and in vivo test,we took L929 cells and BABL/c mices as research subjects respectively.The cells and animals are divided into 5 groups: control group,radiation group,radiation+low dose MSE group, radiation+middle dose MSE group,and radiation+high dose MSE group.We made use of ⁶⁰Co-γray irradiation which produces a large mount of free radicals and reactive oxygen species to cause oxidative stress injury on the experimental subjects.(1) Ex vivo studies:L929 cell was irradiated by ⁶⁰Co-γray with a single dose of 20Gy.MSE was added to the cells immediately after irradiation with a final concentration of 10,50,250μg/ml respectively.4 hours after irradiation,cell samples were collected and oxidative stress related indicators were tested.We found that a single dose of 20Gy irradiation could induce free radical-mediated oxidative stress injury reaction noticeably,which was marked by the increase of TBARS,decrease of GSH and GSH/GSSH ratio and reduction of CAT,SOD.Giving different dose of MSE could prevent L929 cell from oxidative injury by lowering oxidative stress level,which was showed as decrease of TBARS,increase of GSH/GSSH ratio and CAT,SOD activities.Especially,the middle dose of MSE showed the most dictinct effect on improving various indicators. Meanwhile,the high dose of MSE was found to play some GSH consumption roles.(2) Gene and protein level studes:the total RNA was extracted from the oxidative injury L929 cells.The mRNA level changes of Nrf2,TGFβand antioxidant enzymes including CAT,SOD and GCS were observed by RT-PCR.The protein level change of TGFβwas observed by cell immunofluorescence.The results are as follows.â‘ Down-regulation of Nrf2 and up-regulation of TGFβwere observed after irradiation.However, CAT,SOD,GGS mRNA level didn't change obviously.â‘¡Middle dose of MSE could down-regulate SOD,CAT and TGFβwhile up-regulate Nrf2 and GCS.â‘¢Low and high dose of MSE had no obvious effect on Nrf2 and TGFβmRNA level,but they could up-regulate SOD while down-regulate CAT.In addition,the effect of low-dose MSE on GCS mRNA level was up-regulation,while the effect of high-dose MSE on it was down-regulation.(3) In vivo studies:BABL/c mice were irradiated with 20Gy single dose of ⁶⁰Co-γray.The mice were oral administrated with MSE(75,150 and 300mg/kg/d) 1 day before irradiation and once a day for 10 days after irradiation.All mice were sacrificed at a predetermined time of ten days postradiation.The livers and kidneys homogenates and blood serum were prepared for testing MDA,GSH,GSSH and CAT,SOD activity.The livers protein level changes of TNFαand antioxidant enzymes including CAT,SOD were observed by Western Blot.Pathological changes of livers and kidneys were observed by H&E staining.WBC,RBC,HGB and PLT were also tested in whole blood.The results showed that low dose irradiation could increase oxidative level in the mice's whole body.Liver seemed to be the most sensitive tissue to oxidative stress while kidneys to be the less sensitive.After MSE oral administration,decrease of TBARS and increase of antioxidant enzymes activity were observed in livers and serum.However,no obvious changes of GSH and GSSG were observed in livers and serum.Meanwhile,MSE had no obvious protective effect on kidneys,low and high dose of MSE even induced the generation of TBARS.The result of Western Blot in livers showed that expressions of SOD and TNFαwere altered but that of CAT was not altered.In addition, the decrease of RBC,HGB and PLT caused by irradiation could be significantly reversed by MSE in a dose-react relationship,but that of WBC could not be reversed by MSE.These results indicated that chemiluminesence systems and microsomal lipid peroxidation models are efficient and reliable technique platform for screening antioxidants.On this platform,Maydis stigma's extract was demonstrated to possess multi-level anti-oxidative stress property and have dose-effect relation in both in vitro and in vivo experiments.However,as a byproduct of agriculture, Maydis stigma's health care and medical value has not been fully exploited,the research progress on its pharmacodynamics is limited.So Maydis stigma enjoys a great potential for research and development.Our results will provide important experimental evidence for its therapeutic action on free radical-related diseases as well as useful bio-information for its further development and utilization...