两种化妆品延缓衰老功效添加剂体外筛选方法的建立及应用研究
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摘要
随着生活水平的提高,抗皱祛皱,靓丽肤色等延缓衰老的新需求日益增长。基质金属蛋白酶衰老学说和非酶糖基化衰老学说的问世很好地解释了皱纹和肤色暗黄形成的深层次原因,随着相应化妆品研究开发的急速增长,省时、简便、准确、高效的体外功效评价筛选方法亟待建立。
本课题的研究主要包括两个方面:
(一)体外抑制基质金属蛋白酶-1(Matrix Metalloproteinase-1, MMP-1)实验方法的建立
1. MMP-1酶反应最佳实验条件研究采用单因素分析法确定MMP-1酶反应的最佳实验条件为:37℃下MMP-1酶原溶液(0.22μg/100μL)和酶原激活剂p-Aminophenylmercuric Acetate(APMA)溶液(30mmol/L)以10:1的体积比混合,恒温孵育45min,将MMP-1酶原激活,激活后的MMP-1溶液(0.20μg/100μL)与底物DQ-gelatin溶液(0.20μg/100μL)以体积比为1.0:1.3混合反应10min;采用荧光酶标仪法检测酶反应产物的荧光强度,确定最佳荧光检测条件为:激发波长460nm,发射波长520nm;
2.体外抑制MMP-1实验方法的建立取活化后的MMP-1溶液(0.20μg/100μL)40μL加入96孔酶标板中,再加入某种待测植物功效成分溶液8μL,荧光底物DQ-gelatin溶液(0.20μg/100μL)52μL,37℃恒温孵育600s,在激发波长460 nm,发射波长520 nm条件下检测反应体系的荧光强度,扣除待测植物功效成分自身荧光强度后,与使用缓冲液代替植物功效成分的空白组对比,可得该植物功效成分对MMP-1的抑制率;
3.运用体外抑制MMP-1实验方法对22种植物功效成分进行筛选,得到对MMP-1抑制率较高的四种植物功效成分(浓度均为50mg/mL)分别为:茶多酚(100.00%)、大黄提取物(100.00%)、马齿苋提取物(96.32%)和丁香酚(91.64%);
4.以增稠剂AVC 1%,甘油1%,1,3-丁二醇2%(均为质量分数)作为配方基质,分别添加质量分数均为5%的大黄提取物、马齿苋提取物和丁香酚(因茶多酚不稳定,将其排除),形成三款啫喱状化妆品样品，对三款化妆品样品及空白基质对照样品进行为期28天的人体皮肤纹理度功效评价实验,皮肤粗糙度的相对降低率分别为:大黄提取物(-15.0%),马齿苋提取物(-14.0%),丁香酚(-14.0%),空白对照(-4.0%);
5.人体皮肤纹理度功效评价实验结果与体外抑制MMP-1实验结果一致,证明体外抑制MMP-1实验方法可以用于抗皱祛皱化妆品功效添加剂的筛选与开发。
(二)体外抑制非酶糖基化(Non-Enzymatic Glycation,NEG)实验方法的建立
1. NEG最佳实验条件研究采用单因素分析法确定NEG最佳反应条件为:牛血清白蛋白与乙二醛质量比1.7:1.0,加叠氮化钠0.2%(质量分数),37℃恒温孵育72h;采用荧光分光光度法检测荧光产物的生成量,最佳荧光检测条件为:激发波长440nm,狭缝10nm,发射波长480nm,狭缝10nm,检测范围460-600nm;
2.体外抑制NEG实验方法的建立在100mL PBS中逐次加入1.7g的乙二醛,1.0g的牛血清白蛋白和0.2g的叠氮化钠,依次溶解混匀形成反应液,取1.0mL反应液与1.0mL一定浓度的植物功效成分溶液混合,于37℃恒温避光孵育72h后在激发波长440nm,狭缝10nm,发射波长480nm,狭缝10nm,检测范围460-600nm的检测条件下测定产物的荧光强度,扣除待测植物功效成分自身荧光强度后,与使用缓冲液代替植物功效成分的空白组对比,可得该植物功效成分对NEG的抑制率;
3.运用体外抑制NEG实验方法对13种植物功效成分进行筛选,得到对NEG抑制率较高的植物功效成分(浓度均为50mg/mL)为茶多酚(87%),马齿苋提取物(82%),厚朴提取物(76%);
4.以增稠剂AVC 1%,甘油1%,1,3-丁二醇2%(均为质量分数)作为配方基质,分别添加质量分数均为5%的厚朴提取物和马齿苋提取物(因茶多酚不稳定,将其排除),形成两款啫喱状化妆品样品，对两款化妆品样品及空白基质对照组样品进行为期28天的人体皮肤色度LAB值功效评价实验,皮肤色度L值的提高率分别为:厚朴提取物(1.4%),马齿苋提取物(1.5%),空白对照(0.1%);皮肤色度B值的降低率分别为:厚朴提取物(-4.9%),马齿苋提取物(-5.4%),空白对照(-0.1%);
5.人体皮肤色度LAB值测定实验结果与体外抑制NEG实验结果一致,证明体外抑制NEG实验方法可以用于去黄靓肤化妆品功效添加剂的筛选与开发。根据基质金属蛋白酶衰老学说和非酶糖基化衰老学说建立的两个体外功效检测实验为延缓衰老功效添加剂的筛选和开发提供了新方法,这两个方法可以用于筛选具有消除细纹、紧致肌肤、抗皱祛皱以及去黄靓肤、去除老年斑功效的添加剂,具有省时、简便、准确、高效等优点。
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.
本课题的研究主要包括两个方面:
(一)体外抑制基质金属蛋白酶-1(Matrix Metalloproteinase-1, MMP-1)实验方法的建立
1. MMP-1酶反应最佳实验条件研究采用单因素分析法确定MMP-1酶反应的最佳实验条件为:37℃下MMP-1酶原溶液(0.22μg/100μL)和酶原激活剂p-Aminophenylmercuric Acetate(APMA)溶液(30mmol/L)以10:1的体积比混合,恒温孵育45min,将MMP-1酶原激活,激活后的MMP-1溶液(0.20μg/100μL)与底物DQ-gelatin溶液(0.20μg/100μL)以体积比为1.0:1.3混合反应10min;采用荧光酶标仪法检测酶反应产物的荧光强度,确定最佳荧光检测条件为:激发波长460nm,发射波长520nm;
2.体外抑制MMP-1实验方法的建立取活化后的MMP-1溶液(0.20μg/100μL)40μL加入96孔酶标板中,再加入某种待测植物功效成分溶液8μL,荧光底物DQ-gelatin溶液(0.20μg/100μL)52μL,37℃恒温孵育600s,在激发波长460 nm,发射波长520 nm条件下检测反应体系的荧光强度,扣除待测植物功效成分自身荧光强度后,与使用缓冲液代替植物功效成分的空白组对比,可得该植物功效成分对MMP-1的抑制率;
3.运用体外抑制MMP-1实验方法对22种植物功效成分进行筛选,得到对MMP-1抑制率较高的四种植物功效成分(浓度均为50mg/mL)分别为:茶多酚(100.00%)、大黄提取物(100.00%)、马齿苋提取物(96.32%)和丁香酚(91.64%);
4.以增稠剂AVC 1%,甘油1%,1,3-丁二醇2%(均为质量分数)作为配方基质,分别添加质量分数均为5%的大黄提取物、马齿苋提取物和丁香酚(因茶多酚不稳定,将其排除),形成三款啫喱状化妆品样品，对三款化妆品样品及空白基质对照样品进行为期28天的人体皮肤纹理度功效评价实验,皮肤粗糙度的相对降低率分别为:大黄提取物(-15.0%),马齿苋提取物(-14.0%),丁香酚(-14.0%),空白对照(-4.0%);
5.人体皮肤纹理度功效评价实验结果与体外抑制MMP-1实验结果一致,证明体外抑制MMP-1实验方法可以用于抗皱祛皱化妆品功效添加剂的筛选与开发。
(二)体外抑制非酶糖基化(Non-Enzymatic Glycation,NEG)实验方法的建立
1. NEG最佳实验条件研究采用单因素分析法确定NEG最佳反应条件为:牛血清白蛋白与乙二醛质量比1.7:1.0,加叠氮化钠0.2%(质量分数),37℃恒温孵育72h;采用荧光分光光度法检测荧光产物的生成量,最佳荧光检测条件为:激发波长440nm,狭缝10nm,发射波长480nm,狭缝10nm,检测范围460-600nm;
2.体外抑制NEG实验方法的建立在100mL PBS中逐次加入1.7g的乙二醛,1.0g的牛血清白蛋白和0.2g的叠氮化钠,依次溶解混匀形成反应液,取1.0mL反应液与1.0mL一定浓度的植物功效成分溶液混合,于37℃恒温避光孵育72h后在激发波长440nm,狭缝10nm,发射波长480nm,狭缝10nm,检测范围460-600nm的检测条件下测定产物的荧光强度,扣除待测植物功效成分自身荧光强度后,与使用缓冲液代替植物功效成分的空白组对比,可得该植物功效成分对NEG的抑制率;
3.运用体外抑制NEG实验方法对13种植物功效成分进行筛选,得到对NEG抑制率较高的植物功效成分(浓度均为50mg/mL)为茶多酚(87%),马齿苋提取物(82%),厚朴提取物(76%);
4.以增稠剂AVC 1%,甘油1%,1,3-丁二醇2%(均为质量分数)作为配方基质,分别添加质量分数均为5%的厚朴提取物和马齿苋提取物(因茶多酚不稳定,将其排除),形成两款啫喱状化妆品样品，对两款化妆品样品及空白基质对照组样品进行为期28天的人体皮肤色度LAB值功效评价实验,皮肤色度L值的提高率分别为:厚朴提取物(1.4%),马齿苋提取物(1.5%),空白对照(0.1%);皮肤色度B值的降低率分别为:厚朴提取物(-4.9%),马齿苋提取物(-5.4%),空白对照(-0.1%);
5.人体皮肤色度LAB值测定实验结果与体外抑制NEG实验结果一致,证明体外抑制NEG实验方法可以用于去黄靓肤化妆品功效添加剂的筛选与开发。根据基质金属蛋白酶衰老学说和非酶糖基化衰老学说建立的两个体外功效检测实验为延缓衰老功效添加剂的筛选和开发提供了新方法,这两个方法可以用于筛选具有消除细纹、紧致肌肤、抗皱祛皱以及去黄靓肤、去除老年斑功效的添加剂,具有省时、简便、准确、高效等优点。
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.
引文
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[15] Esrefoglu M, Gul M. Ultrastructural findings and tumor necrosis factor-alpha and intercellular adhesion molecule-1 expression in psoriasis patients before and after oral cyclosporin A therapy[J].Ultrastruct Pathol, 2006; Jan-Feb, 30(1):95-102.
[16]赵斌,黄云超. MMP-9与膀胱癌的研究进展[J].昆明医学院学报,2008;(2B):112-116.
[17] Brinckerhoff CE, Matrisian LM. Matrix metalloproteinases: a tail of a frog that became a prince[J]. Nat Rev Mol Cell Biol,2002;3(3): 207-214.
[18] Sudbeck BD, Pilcher BK, Pentland AP, et al. Modulation of intracellular calcium levels inhibits secretion of collagenase-1 by migrating keratin ocytes[J]. Molec Biol Cell,1997;8:811-824.
[19] Soo C, Shaw WW, Zhang X, et al. Differential expression of matrix metalloproteinases and their tissue-derived inhibitors in cutaneous wound repair[J]. Plast Reconstr Surg, 2000;105:638-647.
[20] Saarialho-Kere UK. Patterns of matrix metalloproteinase and TIMP expression in chronic ulcers[J]. Arch Dermatol Res,1998;290:47-54.
[21]陈华江,王军杰.基质金属蛋白酶的结构及其调节机制[J].国外医学肿瘤学分册,2001;28:20-23.
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[25] McCawley LJ, Matrisian LM. Matrix metalloproteinnases: they’re not just for matrix anymore [J]. Curr Opin Cell Biol, 2001; 13(5):534-540.
[26] Hatamochii A.Analysis of collagen gene expression by cultured fibrob-lasts in morphea [J]. Br J Dermatol,1992;126: 216-221.
[27] Lim H, Kim HP. Inhibition of mammalian collagenase, matrix metalloproteinase-1, by naturally-occurring flavonoids [J]. Planta Med, 2007, 73: 1267-1274.
[28]张红梅.基质金属蛋白酶抑制剂筛选方法的研究与应用[D].山东:山东大学,2005.
[29]顾振华,汪俊军.基质金属蛋白酶-1的研究进展[J].医学研究生学报,2006,19(11):1030.
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[31]边芳,王惠芳.晚期糖基化终产物与糖尿病周围神经病变[J].实用医技杂志.2006,13(20):3648-3651.
[32]鲍恒,侯筱魁.糖尿病大鼠椎间盘组织中戊糖甙素与细胞凋亡的关系[J].中国临床康复.2004,8(23):4875-4877.
[33]贺桦,许顶立,谭家余,等.青年人急性心肌梗死血清晚期糖基化终末产物N^ε-羧甲基赖氨酸含量的变化[J].中国动脉硬化杂志.2004,12(2):221-222.
[34]印大中.衰老研究的新纪元[J].生命科学研究,2000,4(2):95~101.
[35] Niwa T.Dialysis-related amyloidosis:pathogenesis focusing on AGE modification[J].Semin Dial,2001,4(2):123~6.
[36] Dyer D G , Dunn JA ,Thorpe SR , etal. Accumulations of mail lard reaction products in skin collagen in diabetes and aging [J]. Clin Invest, 1993, 91(6)2463-2467.
[37] Niwa T.Dialysis-related amyloidosis:pathogenesis focusing on AGE modification[J].Semin Dial,2001,4(2):123~6.
[38] YIN D.Biochemical basis of lipofuscin, ceroid, and agepigment-like fluorophores[J].Free Rad Biol Med, 1996,21(6):871-888.
[39]龚萍,李国林,印大中.老年色素类荧光物质形成的生化机理[J].生命科学研究,2001,5(1):19~24.
[40] Bucala R ,Vlassara H ,Cerami A.Advanced glycosylation endproducts :role in diabetic and non-diabetic vascular disease[J].Drug DevRes,1994,32 (1):77 ~89.
[41]蔡志忠,陈皓君*.生物体内美拉德反应的化学与其抑制[J].Chemistry,2002,60(3):335~349.