三种木质素降解酶对皮肤和毛发黑素的脱色活性比较

Decolorization of skin and hair-derived melanin by three ligninolyfic enzymes

目的比较木质素过氧化物酶（LiP）、锰过氧化物酶（MnP）和漆酶对优黑素和褐黑素的脱色活性并观察外用LiP对308nm准分子光诱导豚鼠皮肤色沉斑的影响。方法碱溶法从人毛发及皮肤黑素瘤组织中提取褐黑素并商业购买优黑素，将等量（0．02％）黑素与相同活性单位（200U／L）的3种木质素降解酶孵育3h，使用酶标仪测定反应液A475，值变化；用308nm准分子光诱导豚鼠皮肤色沉斑模型，将LiP与加热灭活LiP反应液外涂于色沉斑处，同时用3％氢醌乳膏与乳膏基质作对照。用皮肤色度计测定L值变化；胶带法采集脱落角质层细胞，Fontana—Masson嗜银染色观察脱落角质层细胞与皮肤组织中黑素颗粒的分布与数量。结果3种木质素降解酶对优黑素及褐黑素均有脱色活性，其中LiP脱色效率为40％～70％，MnP约为22％～42％，漆酶约为9％-21％；体内实验显示，LiP反应液与3％氢醌乳膏均可使色沉斑颜色变浅。脱落角质层细胞中黑素颗粒的平均数量，LiP组为199±11，低于空白组（923±12）和酶热灭活假处理组（989±13）。组织学检查发现氢醌组表皮增厚，全层黑素颗粒减少，而LiP组表皮正常，仅表皮浅层黑素颗粒减少，深层仍有黑素颗粒存在。结论LiP对皮肤及毛发黑素脱色作用最强，优于MnP和漆酶，能否替代氢醌制剂成为新一代高效安全的皮肤增白剂用于临床有待进一步证实。

Objective To compare the decolorization efficiency of lignin peroxidase （LiP）, manganese peroxidase （MnP） and laccase on eumelanin and pheomelanin, and to investigate the effect of topical administration of LiP solution on hyperpigmented guinea pigs skin induced by 308 nm excimer light. Methods Pheomelanin-enriched specimens were prepared from human hair and cutaneous melanoma tissue using alkaline lysis method. Synthetic eumelanin was purchased from a commercial supplier. The same amount （0. 02% ） of melanin was incubated with the equal enzyme activity （0. 2 U/ml ） of ligninolytic enzymes for 3 h respectively. The absorbance at 475 nm （A475 ） in the enzyme-catalyzed solution was measured using ELISA microplate reader. The experimental hyperpigmentation model was established in the dorsal skin of brownish guinea pigs using 308 nm excimer light radiation. LiP and heat-inactivated LiP solution were topically applied at each site. Meanwhile, 3% hydroquinone and vehicle cream were used as control. The skin color （L value ） was recorded using a CR-10 Minoha chromameter. Corneocytes were collected using adhesive taping method. The amount and distribution of melanin in the comeocytes and skin tissues was visualized by Fontana-Masson staining. Results All three ligninolytic enzymes showed various degree of eumelanin and pheomelanin decolorization activity. The decolorization activity of LiP, MnP and laecase was 40% - 70% , 22% - 42% and 9% - 21% , respectively. The similar lightening was shown in the skin treated with LiP solution and 3% hydroquinone. The amount of melanin granules in the corneocytes was 199 ± 11 by LiP, which was less than that in untreated control （923 ± 12） and heat-inactive control （989 ± 13 ）. The amount of melanin was decreased in the whole epidermis treated with hydroquinone, the epidermis thickness was increased as well. In contrast, melanin of LiP group was decreased only in thesuperficial epidermis, the epidermis thickness seemed to be normal. Conclusion LiP exerts a potent decolorization activity for hair- or skin-derived pheomelanin as well as eumelanin. It remains to be further investigated whether LiP serves as a substitute for hydroquinone in skin lightening products.