Establishment Andapplication Of Two In Vitro Screening Methods For Cosmetic Additives With Anti-aging Effect

With the improvement of living standards, people aging put forward a number of new demands, such as the elimination of dull, beautiful color of skin. However, there is no in vitro testing means related to inhibition on matrix metalloproteinase activity and inhibition of non-enzymatic glycation reactions, only through the human body method for screening of raw materials, both time-consuming and laborious, but also need a large number of volunteers, inefficient. Therefore, in vitro screening methods that can evaluate the effectiveness of raw materials need to establish urgently. The research projects mainly include two parts:(A) Establishment of experimental methods by the in vitro inhibition of matrix metalloproteinase -1;1. By single factor analysis to establish in vitro inhibitory activity of MMP-1 detection methods, the optimum experimental conditions as follows: combining MMP-1 enzyme solution (0.22μg/100μL) with p-Aminophenylmercuric Acetate(APMA)with a volumn ratio of 10:1 under 37℃, the reaction time is 45min to react MMP-1. Combine the reacted MMP-1 solution (0.20μg/100μL) with DQ-gelatin solution (0.20μg/100μL) with a volumn ratio of 1.0:1.3 and react for 10 min.Fluorescence detection conditions for excitation wavelength 460nm and emission wavelength of 520nm.2. The establishment of in vitro anti-MMP-1 experiment method: add 40μL active MMP-1 solution (0.20μg/100μL), 8μL tested sample and 52μL DQ-gelatin (0.20μg/100μL) to 96 well plates in order, react for 600s under 37℃, and test the Fluorescence strength in 460nm excitation wavelength and 520nm emission wavelength. Compared with the blank of PBS, and debate the fluorescence strength of the sample, the MMP-1 inhibition ratio of the sample can be got.3. 22 kinds of natural plant ingredients have been screened with the method and the result showed that tea polyphenols (100.00%), rhubarb (100.00%), portulaca oleracea extract (96.32%) and eugenol (91.64%) have a significant inhibitory effect to MMP-1.4. Based on the formulation of 1% AVC, 1% glucery, 2% and 1,3-butandiol, three gels are formulated with 5% rhubarb, portulaca oleracea extract and eugenol separately. The results of 28d human body test on skin showed that the derease ratios of skin rough were as bellow: rhubarb (-15.0%), portulaca oleracea extract (-14.0%) and eugenol (-14.0%), blank (-4.0%).5. In vivo method had a good correlation with the in vitro method, so the in vitro anti-MMP-1 experiment can be used to screen and develop the anti-aging ingredients.(B) Establishment of experimental methods by the in vitro inhibition of non-enzymatic glycation;1. Research on the best conditions for NEG experiment: By single factor analysis to establish in vitro inhibitory of NEG reaction methods, the best experimental conditions as follows: bovine serum albumin and glyoxal mass ratio of 1.7:1.0, add sodium azide 0.2%, reaction under 37℃and detected after 72h incubation, fluorescence detection conditions for the excitation wavelength 440nm, slit 10nm, emission wavelength 480nm, slit 10nm, the detection range 460-600nm.2. The establishment of in vitro anti-NEG experiment method: add 1.7g glyoxal, 10g bovine serum albumin and 0.2g sodium azide to 100ml PBS in order to form the reaction solution. Combine 1.0mL reaction solution with the sample in certain concentration, reaction under 37℃and detected after 72h incubation, fluorescence detection conditions for the excitation wavelength 440nm, slit 10nm, emission wavelength 480nm, slit 10nm, the detection range 460-600nm. Compared with the blank of PBS, and debate the fluorescence strength of the sample, the NEG inhibition ratio of the sample can be got.3. By screening the effectiveness of 13 raw materials obtained citrus aurantium extract, Portulaca oleracea extract (82%), Magnolia officinalis extract (76%), and tea polyphenols (87%), Chamomile extract on non-enzymatic glycation inhibitory effect is remarkable.4. Based on the formulation of 1% AVC, 1% glucery, 2% and 1, 3-butandiol, two gels are formulated with 5% Portulaca oleracea extract and Magnolia officinalis extract separately. The results of 28d human body test of LAB value showed that the increase ratios of L value were as bellow: Portulaca oleracea extract (0.83), Magnolia officinalis extract (0.80) and blank (0.06), and the derease ratios of B value were as bellow: Portulaca oleracea extract (0.65), Magnolia officinalis extract (0.60) and blank (0.02).5. In vivo method had a good correlation with the in vitro method so the in vitro anti-NEG experiment can be used to screen and develop the whitening ingredients.These two in vitro methods provides a new approach for the development of additives for anti-aging cosmetics and can be used to screen additives with the effect to eliminate of fine lines, compact skin, anti-wrinkle, removal senile plaque, with the province no need human test volunteers, convenient, economic and other advantages.