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Establishment Andapplication of Two in Vitro Screening Methods for Cosmetic Additives with Anti-aging Effect

Author: LaiJiXiang
Tutor: DongYinMao
School: Beijing Technology and Business University
Course: Biochemical Engineering
Keywords: Anti-aging In vitro screening methods The effectiveness of additives Matrix metalloproteinase-1 Non-enzymatic glycosylation
CLC: TQ658
Type: Master's thesis
Year: 2010
Downloads: 102
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With the improvement of living standards, anti-wrinkle wrinkle removal, beautiful complexion retard aging growing demand. The advent of matrix metalloproteinase theory of aging and non-enzymatic glycosylation theories of aging well explain the deep-seated reasons for wrinkles and skin color are yellowish formed, with the rapid growth of the corresponding cosmetic research and development, time-saving, simple, accurate, and efficient vitro efficacy evaluation of screening methods need to be established. The study of this project includes two main aspects: (a) Inhibition of matrix metalloproteinase the -1 (Matrix Metalloproteinase-1, MMP-1) the establishment of experimental methods 1. MMP-1 enzyme reaction best experimental conditions using univariate analysis determined the optimal experimental conditions for the MMP-1 enzyme reaction: 37 ° C zymogen solution of MMP-1 (0.22μg/100μL) and plasminogen activator p-Aminophenylmercuric Acetate (APMA) solution (30mmol / L) to 10: the volume ratio of a mixed thermostat incubated for 45min, the MMP-1 plasminogen activator, and after the activation of MMP-1 solution (0.20μg/100μL) of substrate DQ-gelatin solution (0.20μg/100μL) in a volume ratio of 1.0: 1.3 mixed reaction 10min; fluorescence intensity using a fluorescence microplate reader assay enzyme reaction product, to determine the optimal fluorescence detection conditions: excitation wavelength 460nm, emission wavelength 520nm; vitro inhibition taken after activation of the establishment of the MMP-1 Experimental Methods MMP-1 solution (0.20μg/100μL) 40μL added to 96-well microtiter plates, then add some sort of the tested plants effective ingredients solution 8 μL fluorogenic substrate DQ-gelatin solution (0.20μg/100μL) 52μL, 37 ℃ constant temperature incubation 600s, at the excitation wavelength of 460 nm, emitted fluorescence intensity of the detection of the reaction system under the conditions of a wavelength of 520 nm, after deducting the effect of tested plant component itself fluorescence intensity, with the use of buffer solution in place of the blank group comparison of the plant efficacy ingredient, the plant of efficacy component on the inhibition of MMP-1; 3. use of the in vitro inhibition of MMP-1 test method of 22 species effectiveness of the composition to be screened, to give higher MMP-1 inhibition rate of the four plants of effective ingredients (concentration are 50mg/mL ): tea polyphenols (100.00%), rhubarb extract (100.00%), purslane extract (96.32%) and eugenol (91.64%,); 4. thickener AVC 1%, glycerol 1% butanediol, 1,3 - 2% (both by mass) are 5% of the mass fraction of rhubarb extract, purslane extract were added and eugenol (due to the tea polyphenols unstable, as the formula matrix excluded), the formation of three gel-like cosmetic samples, human skin texture degree of efficacy of a 28-day evaluation of three cosmetic samples, control samples and blank matrix experiments, the relatively lower rate of skin roughness were: rhubarb extract (- 15.0%), purslane extract (-14.0%), the eugenol (-14.0%), blank control (-4.0%); human skin texture degree of efficacy evaluation of the experimental results with in vitro inhibition of MMP-1 experimental results proved in vitro inhibition of MMP-1 experimental methods can be used for screening and development of the efficacy of anti-wrinkle wrinkle removing cosmetics additives. (B) in vitro inhibition of non-enzymatic glycosylation (Non-Enzymatic Glycation NEG) the experimental method of establishing the NEG optimal experimental conditions of bovine serum albumin with ethylene using single factor analysis to determine the NEG optimal reaction conditions: aldehyde quality than 1.7:1.0, add sodium azide 0.2% (by mass) at 37 ℃ incubated for 72h; using the fluorescence spectrophotometric detection of fluorescent product generated amount, the best fluorescence detection: excitation wavelength of 440nm, slit 10nm, the emission wavelength of 480nm, slit 10nm, detection range 460-600nm; vitro inhibition of the establishment of the NEG experimental methods in 100mL PBS successively added 1.7g of glyoxal, 1.0 g of bovine serum albumin, and 0.2g of the stack sodium azide, followed by dissolving and mix to form a reaction solution, whichever 1.0mL reaction solution with 1.0mL the plants effectiveness of the composition a certain concentration of solution was mixed and incubated at 37 ° C thermostat dark after 72h at an excitation wavelength of 440nm, slit 10nm, emission wavelength 480nm slit 10nm, the detection under the conditions of the detection range of 460-600nm, the fluorescence intensity of the product was measured, after deducting the effect of tested plant component itself fluorescence intensity, with the use of a buffer instead of plant effectiveness of the composition of the blank set of contrast can be obtained the effective ingredients of the plant The inhibition rate of the NEG; 3. the use of in vitro inhibition NEG Experimental Method 13 species effective ingredients screened to obtain NEG inhibition rate higher plants the effectiveness of the composition (concentration of 50mg/mL) polyphenols (87%) purslane extract (82%), Magnolia extract (76%); 4 as thickeners the AVC 1%, 1% glycerol, 1,3 - butanediol, 2% (all mass fraction) formulation matrix, were added to the quality scores are 5% of the Magnolia extract and purslane extract (due to the instability of tea polyphenols excluded), two gel-like cosmetic samples, two cosmetics samples and blank matrix control group sample 28-day human skin chrominance LAB values ??Efficacy evaluation experiments to improve skin Color L values ??were: Magnolia extract (1.4%), purslane extract (1.5%), blank controls (0.1%); the skin chromaticity B value reduction rates were: Magnolia extract (-4.9%), purslane extract (-5.4%), the blank control (-0.1%); human skin chroma LAB value determination of the experimental results with in vitro inhibition of the NEG experimental results prove the in vitro inhibition the NEG experiments can be used to yellow beautiful skin cosmetics additive efficacy of screening and development. Provides a new method for delaying the aging effects of additives screening and development of matrix metalloproteinase theory of aging and non-enzymatic glycosylation two established theories of aging in vitro efficacy of detection experiments, the two methods can be used for screening with the elimination of fine lines, tighten cause skin, anti-wrinkle, wrinkle removal and Jane go yellow skin, remove the senile plaques efficacy of additives, with a provincial, simple, accurate and efficient advantages.

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