Study On Eucommia Active Ingredient Screening Of Anti-Aging Skin And Its Mechanism

Eucommia ulmoides?Eucommia ulmoides Oliv.? is a unique precious economic tree species in China. It has a variety of functions, such as anti-aging, lowering blood pressure, anti-oxidation,anti-fatigue, anti-virus, enhancing immunity, anti-osteoporosis, anti-tumor, nursing liver, lowering blood sugar. In this paper, we mainly study the cell protective effect and the possible mechanism of the active components in eucommia ulmoides on the aging skin fibroblasts cell?ESF-1?. The thesis consists of four parts.First, the condition of H₂O₂ and UV?UVA+UVB? damage model were optimization by MTT assay. We found the dose of 4×10-4mol/L H₂O₂ in the role of 3 hours on cell injury was moderate. UV phototherapy apparatus and cell distance fixed for 15 centimeters in irradiation time was 2 minutes damage degree of cell and time is the most appropriate.Second, 18 compounds of eucommia ulmoides were preliminary screened by MTT method.The results showed that: at the dose of 2×10-6 mol·L-1, 7 compounds has significant protective effect on ESF-1. They were EUB-4? aucubin?, ET-11? quercetin-3- O-?-L-arabinosyl-?1 ?2?-?-D-glucoside?, EUB-8?quercetin-3-O-?-D-glucosyltransfera- se-?1?2?-?-D-glucoside?,EUB-11?quercetin?and EUB-15?isoquercitrin?, EUB-2?asperuloside?, EUB-17?eucommia A?.The protection of EUB-4 was more obvious on H₂O₂ injury than UV damage. And ET-11,EUB-8, EUB-11, EUB-15, EUB-2 and EUB-17 were more significant on UV damaged.Third, in order to further study of its mechanism, the H₂O₂ damage aging model wasadopted.Different concentrations of ET-11, EUB-8, EUB-11, EUB-15, EUB-2, EUB-17 was used to pre-protect ESF-1 cells.Then 4×10-4 mol·L-1 H₂O₂ and UV irradiation damage aging model were established respectively. Cell senescence was detected by cell senescence beta-gal staining?SA-?-Gal?. Cell activity was detected by MTT assay. The kit was used to detect cell superoxide dismutase?SOD? activity and lipid peroxidation products?MDA? content. Elisa method was used to detect the content of type?collagen?COL??, type?matrix metalloproteinase?MMP-1? and type ? matrix metallo- proteinase inhibitor?TIMP- ?? in supernatant of cell supernatant.Compared with the control group, SA-?-Gal blue staining rate significantly increased after the model group cells from oxidative injury, indicating damage model to build successful. Compared with the model group, the treatment group could reduce the stained blue rate of damaged cells.The order of anti aging activity level was EUB-17 > EUB-2 > EUB-11 > EUB-15 >EUB-8, ET-11.Cell proliferation showed, compared with the model group, the treatment group could significantly improve the activity of damaged cells, and 24 h proliferation effect was better than 48 h. EUB-17 greater than EUB-8 and EUB-2 better than ET-11, EUB-15 and EUB-11. The results of antioxidant activity showed that drug preconditioning can enhance cell SOD activity and decrease the MDA content, and active in descending order was EUB-2>EUB-11 and EUB-15> EUB-17 >ET-11 and EUB-8. Elisa test results showed that COL? content in culture supernatants of each treatment group were higher than model group,and curative effect of EUB-17 was best, EUB-2times, after successively for EUB-8, ET-11, and EUB-15, EUB-11. They could inhibit the cell injury of MMP-? secretion and improve the TIMP-? secretion, and action in descending order was EUB-2 > EUB-11 > EUB-15, EUB-8 > EUB-17, ET-11.Fourth, using UV irradiation?UVA and UVB? damage aging model studied the mechanismof above 6 kinds compounds against photo-aging.Compared with the control group, SA-?-Gal blue staining rate of model group increased significantly than the cells from UV damage, showing damage model to build successful. Compared with the model group, the treatment group could reduce the stained blue rate of damaged cells. The order of anti aging activity level was EUB-17,EUB-2 > EUB-11 and EUB-15 > EUB-8, ET-11. Cell proliferation activity Showing: compared with the model group, the treatment group could significantly improve the activity of damaged cells, but 24 h was no more than 48 h, which EUB-17, EUB-2 effect quite, higher than ET-11,superior to EUB-11, EUB-15,EUB-8. The results of antioxidant activity showed that drugs can enhance the protection of pre-injury cell SOD enzyme activity and decrease the content of MDA,and active in descending order was EUB-2>EUB-17, ET-11>EUB-8, EUB- 11 and EUB-15. Elisa test results showed that COL ? content in culture supernatants of each treatment group were higher than model group, and curative effect of EUB-17 was best, EUB-2 times, after successively for EUB-8, the EUB-15, ET-11, EUB-11. They can inhibit the cell injury of MMP-?secretion and improve the TIMP-?secretion. The action in descending order was EUB-2 > EUB-15 > ET-11 >EUB-8, EUB-11 > EUB-17.ET-11, EUB-8, EUB-11,EUB-15,EUB-2,EUB-17 pre-protection on ESF-1 cell senescence damaged by H₂O₂ and UV has a good protective effect. The mechanism may be antagonistic damage caused by superoxide dismutase?SOD? activity changes, reduce lipid oxidation product malondialdehyde?MDA?, then protecting human skin fibroblast?ESF-1? against oxidative damage.They could inhibit the secretion of MMP-? and increase the secretion of TIMP-?, which can reduce the degradation of COL?, thus protect the skin and delay aging.