| 摘要: | 纖維母細胞受陽光之紫外線曝曬會引起基質金屬蛋白酶大量表現，其為調節光老化重要因子，進而降解細胞外基質。多酚類具有抗氧化活性，能抑制膠原蛋白酶與彈力蛋白酶活性，若能抑制該等酵素活性即可用於預防光老化。本研究探討使君科植物大葉欖仁水萃物 (TCLW) 與咖啡酸 (CaA)、綠原酸 (ChlA)、香豆酸 (p-CA)、肉桂酸 (CA)、鞣花酸 (EA)、阿魏酸 (FA)、沒食子酸 (GA)、沒食子酸丙酯 (PG)、白藜蘆醇 (Res)、香草酸 (VA) 與香草醛 (Van) 11種酚類化合物是否具有抑制UVB引起之光老化活性。首先進行明膠分解試驗、膠原蛋白酶與彈力蛋白酶抑制試驗，具活性者再以紫外線B照射纖維母細胞誘導基質金屬蛋白酶 (MMPs) 產生之模式，探討對MMPs與第I型膠原蛋白作用，進而評估對絲裂原活化蛋白激酶 (MAP kinases) 磷酸化影響。
大葉欖仁水萃物萃取率為22.5%。明膠分解試驗初篩結果顯示：大葉欖仁甲醇萃取物 (TCLM)、大葉欖仁水萃物及其水解物於濃度1 mg/mL時抑制率皆大於100%；11種酚類化合物於濃度100 μM時抑制率皆小於60%。
膠原蛋白酶活性抑制試驗結果顯示：TCLW序列稀釋水萃物(10~500 μg/mL) 之抑制率為82.3~101.0%，抑制效果具濃度依存性；酚類化合物CaA、ChlA、GA、PG 與FA於濃度100 μM時抑制率皆大於70%，其餘酚類化合物p-CA、CA、EA、Res、VA和Van對膠原蛋白酶抑制率皆小於50%。然而，TCLW與11種酚類化合物對彈力蛋白酶皆無抑制效果。
抑制UVB誘導基質金屬蛋白酶1、3、9活性試驗結果顯示：TCLW分別於濃度25、50及25 μg/mL；PG分別於濃度10、100及100 μM；FA分別於濃度25、5及25 μM能抑制MMP-1、-3與-9表現，而GA之抑制濃度均為50 μM。此外，TCLW、GA與PG均能促進第I型膠原蛋白原增生。
TCLW係透過抑制ERK、JNK與p38之磷酸化而抑制MMP-1、-3、-9之表現；GA與PG係透過抑制p38之磷酸化而抑制MMP-1、-3、-9之表現；FA係經由抑制ERK與p38磷酸化路徑降低而抑制MMP-1、-3、-9之表現。TCLW對纖維母細胞無毒性，對皮膚及眼球亦無刺激性。
由上述研究顯示TCLW、GA、PG與FA具有抑制MMP-1、-3、-9之活性，具有開發成抗老化化妝品之潛力。
Matrix metalloproteinases (MMPs) are key factors of the skin photoaging process that is overexpressed by exposure to ultraviolet (UV) irradiation in human fibroblasts. Polyphenols have antioxidant characteristics to inhibit the activities of MMPs and elastase. Thus, it is plausible that MMPs and elastase inhibitors could prevent skin photoaging. This study investigated the capacity of Terminalia catappa L. water extract (TCLW) and phenolic compounds, CaA, ChlA, p-CA, CA, EA, FA, GA, PG, Res, VA and Van to prevent photoaging in UVB-irradiated human dermal fibroblasts. Gelatin digestion assay, MMP activity assay by fluorescence and elastase assay were applied for screening. Those which showed good activity through the screening test will be applied on western blot to confer variations of MMPs and type I procollagen by exposure to UVB irradiation in human fibroblasts. And evaluate the effect of MAP kinase phosphorylation further.
The extraction yield of TCLW was 22.5% (w/w). In gelatin digestion assay, the inhibition rate of TCLM, TCLW and it’s hydrolysates were greater than 100% at the concentration of 1 mg/mL. However, the inhibition rate of all the phenolic compounds at the concentration of 100 μM were all less than 60%.
The results indicated that serial dilution of TCLW (10~500 μg/mL ) would inhibit collagenase activity and the inhibition rates were 82.3 ~ 101.0% in a dose-dependent manner. The results of CaA, ChlA, GA, PG and FA were greater than 70% when the concentration was 100 μM; p-CA, CA, EA, Res, VA and Van showed not good effect on collagenase activity, the inhibition rate were lower than 50%. However, TCLW and phenolic compounds had no significantly inhibition on elastase activity.
The results showed that TCLW at the concentrations of 25, 50 and 25 μg/mL, GA at the concentrations of 50 μM, PG at 10, 100 and 100 μM and FA at 25, 5 and 25 μM could inhibit the expression of MMP-1, -3 and -9. In addition, TCLW, GA and PG could promote the expression of type I procollagen.
In MAP kinases phosphorylation inhibition, the results showed that TCLW through the inhibition of ERK, JNK and p38 phosphorylation to reduce the expression of MMP-1, -3, -9; GA and PG, respectively, through the inhibition of p38 phosphorylation to reduce the expression of MMP-1, -3, -9; FA through the inhibition of ERK and p38 phosphorylation to reduce the expression of MMP-1, -3, -9. TCLW was non-toxic in human fibroblasts, which proceeded safety evaluation, no toxicity at high concentration on the skin and eye irritation test.
These findings suggest that TCLW, GA, PG and FA could inhibit the activity of MMP-1, -3 and -9, to developed a potential of anti-aging cosmetics. |
