野木瓜总皂苷提取工艺优化及其美白活性组分筛选

目的:优化野木瓜中总皂苷的提取工艺,并为野木瓜总皂苷活性组分在美白化妆品中的后续开发应用奠定基础。方法:在单因素实验的基础上,以乙醇浓度、液料比、提取温度、提取时间为优化因素,总皂苷得率为评价指标,Box-Behnken法优化野木瓜总皂苷提取工艺;依次用不同极性溶剂对其提取物进行萃取,硅胶柱色谱层析进行分离,获得不同活性组分,MTT法检测B16细胞的增殖抑制能力,多巴氧化法检测酪氨酸酶活性。结果:各因素对野木瓜总皂苷得率影响顺序为:乙醇浓度>提取温度>液料比>提取时间;最佳提取工艺为乙醇浓度79%,液料比25:1 mL/g,提取温度81℃,提取时间89 min,总皂苷平均得率为2.16%;各萃取部位中正丁醇部位对细胞增殖和酪氨酸酶活性抑制效果最好,效果优于对照品熊果苷（P<0.05）,其IC₅₀分别为86.61和69.09 μg/mL;正丁醇部位分离得8个活性组分,其中组分Fr.6对B16细胞增殖和酪氨酸酶活性抑制效果最好,IC₅₀分别为80.93和58.99 μg/mL,且抑制酪氨酸酶活性的效果较分离前的正丁醇部位更好（P<0.05）。结论:本文探讨了野木瓜总皂苷的最佳提取工艺,获得的活性组分Fr.6具有良好的美白活性,在美白化妆品产品开发中具有较好的应用前景。     

酪氨酸酶活性的抑制研究及皮肤美白化妆品的研制

研究探讨了皮肤黑色的成因和皮肤美白机理。通过测定常用的6种生物美白剂对酪氨酸酶活性的抑制率，初步建立美白剂效果的评价体系，实验还表明自制中草药萃取物美白效果明显，抑制率可达95％。同时，开发了3种美白、祛斑、防晒等功能的化妆品，市场潜力很大。     

Flaniostatin, a new isoflavonoid glycoside isolated from the leaves of Cudrania tricuspidata as a tyrosinase inhibitor

In the course of screening program for skin whitening compounds, flaniostatin (FST) was isolated from the leaves of Cudrania tricuspidata as a novel inhibitor of tyrosinase. The structure of FST was determined by ESI-MS and NMR spectroscopic analyses as a new isoflavone glycoside. The FST was exhibited a tyrosinase inhibitory effect in a dose-dependent manner. This effect was higher than that of arbutin at the same concentrations. These results indicated that FST isolated from C. tricuspidata may be a positive tool for skin-whitening agent research.     

Schinus terebinthifolius Raddi extract and linoleic acid from Passiflora edulis synergistically decrease melanin synthesis in B16 cells and reconstituted epidermis

Several treatments for skin whitening are available today, but few of them are completely adequate, especially owing to the carcinogenic potential attributed to classical drugs like hydroquinone, arbutin and kojic acid. To provide an alternative and safer technology for whitening, we developed two botanical compounds originated from Brazilian biodiversity, an extract of Schinus terebinthifolius Raddi and a linoleic acid fraction isolated from Passiflora edulis oil. The whitening effect of these compounds was assessed using biochemical assays and in vitro models including cellular assays and equivalent skin. The results showed that S. terebinthifolius Raddi extract is able to reduce the tyrosinase activity in vitro, and the combination of this extract with linoleic acid is able to decrease the level of melanin produced by B16 cells cultured with melanocyte‐stimulating hormone. Furthermore, melanin was also reduced in human reconstituted epidermis (containing melanocytes) treated with the compounds. The combination of the compounds may provide a synergistic positive whitening effect rather than their isolated use. Finally, we demonstrated that the performance of these mixed compounds is comparable to classical molecules used for skin whitening, as kojic acid. This new natural mixture could be considered an alternative therapeutic agent for treating hyperpigmentation and an effective component in whitening cosmetics.     

5种天然植物提取物抗氧化性和酪氨酸酶抑制作用的比较

本研究检测了5种天然植物提取物体外抗氧化及酪氨酸酶活性抑制作用,并分析其黄酮和总酚含量,探讨提取物美白与抗氧化之间的关联。研究表明通过ABTS+、DPPH+、羟自由基、超氧阴离子自由基的清除能力测定得5种提取物的抗氧化能力强弱顺序虽有所差异,但四种抗氧化方法均表明提取物具有一定的抗氧化能力。同时酪氨酸酶活性抑制检测也表明它们均对酶活有一定抑制作用。5种植物提取物抗氧化及酪氨酸酶活性抑制能力均随样品浓度增加而增大,这些结果提示提取物抗氧化能力与其对酪氨酸酶活抑制作用相关联,可能是与提取物中黄酮和总酚物质有关。当采用ABTS+、DPPH+方法时,5种提取物的抗氧化性与酪氨酸酶抑制作用相关性更大,这可能与黄酮和酚类物质对酪氨酸酶活性抑制机理有关,研究结果可为天然美白植物原料的筛选和研发提供一定理论依据与借鉴。     

The effects of areca catechu L extract on anti-inflammation and anti-melanogenesis

Ethanolic extract (CC-516) from Areca catechu L was prepared and its various biological activities were evaluated, CC-516 showed potent anti-oxidative, free radical scavenging, and anti-hyaluronidase activity. Anti-oxidative effect of CC-516 (IC _ 50: 45.4 mug/ml) was lower than butylated hydroxytoluene (IC _ 50: 5mug ml), but similar to tocopherol and higher than ascorbic acid. Especially, CC-516 exhibited relatively high free radical scavenging activity (IC _ 50: 10.2 mug/ml) compared to control. CC-516 inhibited effectively hyaluronidase activity (IC _ 50: 416 mug/ml), showed inhibition in vivo on delayed hypersensitivity as well as croton-oil induced ear edema in mice when it was topically applied. These results strongly suggest that CC-516 may reduce immunoregulatory/inflammatory skin trouble. Also, from the results, we have elucidated that CC-516 showed anti-allergic and anti-cytotoxicity activity. The whitening effect of CC-516 shown by the inhibition of mushroom tyrosinase activity with IC _ 50 of 0.48 mg/ml and of melanin synthesis in B16 melanoma cells. This study indicates that CC-516 is effective on anti-inflammatory/anti-melanogenesis, and can be used as a new agent for cosmetics.     

玫瑰发酵液的抗氧化及美白功效探究

通过对玫瑰发酵液的抗氧化和美白功效及皮肤安全性进行评价,探究美白机理。利用酿酒酵母发酵玫瑰获得玫瑰发酵液,检测其对自由基的清除作用以及对酪氨酸酶活性与黑色素合成的影响,利用基因芯片检测其美白机理,采用人体斑贴试验评估其皮肤安全性。结果表明,玫瑰发酵液具有较强的清除DPPH自由基能力,对细胞外酪氨酸酶活性的抑制作用与浓度呈正比,对B16细胞内酪氨酸酶活性以及黑色素合成都有一定的抑制作用;玫瑰发酵液能够抑制黑色素瘤通路上游的NGF和FGF2基因表达;人体斑贴试验显示玫瑰发酵液未引起阳性刺激反应。玫瑰发酵液具有较高的皮肤安全性,同时具有抗氧化、抑制酪氨酸酶活性与黑色素生成的功效,其通过抑制NGF和FGF2的表达发挥美白作用。     

A new mechanism of action for skin whitening agents: binding to the peroxisome proliferator-activated receptor

Synopsis Octadecenedioic acid is known as a skin whitening agent but its activity is not mediated via a direct inhibition of tyrosinase. Based on the secondary properties of this molecule, such as its anti-inflammatory and anti-ageing effects, we postulated that octadecenedioic acid interacted with the peroxisome proliferator-activated receptor (PPAR) as this nuclear receptor also mediates these effects. Using reporter gene technology, we were indeed able to demonstrate binding of octadecenedioic acid to all three PPAR subtypes, in particular PPARgamma with an EC(50)-value of approx. 1 x 10(-6) m. Binding to PPARgamma of octadecenedioic acid or rosiglitazone, a known pharmaceutical PPARgamma agonist, led to reduced melanogenesis. Subsequently also tyrosinase mRNA (as measured by real-time polymerase chain reaction) and tyrosinase levels (as measured by Western blot) were reduced, suggesting the existence of a complete novel mechanism of skin whitening agents: binding to PPARgamma results in reduced tyrosinase mRNA expression which in turn results in less tyrosinase being formed. This in turn leads to reduced melanogenesis both in vitro and in vivo Because octadecenedioic acid binds not only to PPARgamma but also to PPARalpha and PPARdelta, other efficacies mediated via these receptors may also be expected.     

白藜芦醇对酪氨酸酶体外抑制活性的动力学研究

在30℃,pH 6.8的Na2HPO4-NaH2PO4的缓冲体系中,采用酶促动力学方法,研究了白藜芦醇对酪氨酸酶单酚酶和二酚酶的抑制作用。结果表明,白藜芦醇对酪氨酸酶、单酚酶和二酚酶均有抑制作用,对单酚酶抑制活性的IC50值(抑制率达到50%时的白藜芦醇质量浓度)约为5.1mg/mL,对二酚酶抑制活性的IC50值约为5.6 mg/mL。此外,白藜芦醇可延长单酚酶的迟滞效应,8 mg/mL的白藜芦醇能使迟滞时间从22 s延长至62 s,而对二酚酶则无此迟滞作用。Lineweav-er-Burk图分析表明,白藜芦醇对酪氨酸酶的抑制作用为混合型抑制,对游离酶的抑制常数(KI)和对酶-底物络合物的抑制常数(KIS)分别为3.4 mg/mL和35.98 mg/mL。     

Artocarpus lakoocha heartwood extract as a novel cosmetic ingredient: evaluation of the in vitro anti-tyrosinase and in vivo skin whitening activities

The heartwood extract of Artocarpus lakoocha Roxb. was evaluated for the in vitro tyrosinase inhibitory activity and the in vivo melanin-reducing efficacy in human volunteers. The IC(50) of the extract and oxyresveratrol, its major active ingredient, against mushroom tyrosinase was determined to be 0.76 and 0.83 mug mL(-1), respectively. The extract dissolved in propylene glycol was subsequently tested in female volunteers using a parallel clinical trial with self-control (n = 20 per group). The first group received the 0.25% w/v A. lakoocha solution as the test solution, whereas the second and the third group, respectively, received 0.25% licorice extract and 3% kojic acid as the reference solutions in the same solvent. The subjects in each group twice daily applied the test (or reference) solution in one of her upper arm, whereas the remaining arm was treated with only propylene glycol (self-control) for 12 weeks. The melanin content of each application site was measured using Mexameter every week and calculated as % reduction in melanin content relative to the initial melanin value (% whitening). The value of % whitening was then compared between the product-treated and the propylene glycol-treated arms within the same subject using paired t-test (alpha = 0.05). The A. lakoocha extract was the most effective agent, giving the shortest onset of significant whitening effect after only 4 weeks of application (P < 0.05), followed by 3% kojic acid (6 weeks) and 0.25% licorice extract (10 weeks). The effect also increased with time with maximum whitening observed at week 12 for A. lakoocha extract. When the extract was formulated as an oil-in-water emulsion, its whitening efficacy was further enhanced. Daily application of 0.1% w/w A. lakoocha lotion to the upper arms (n = 25) and cheeks (n = 15) of volunteers produced significant whitening over the lotion base after 2 and 3 weeks, respectively (P < 0.05). Thus, the preliminary study suggested that the heartwood extract of A. lakoocha may have a promising potential for use as an effective and economical skin-whitening agent.     

Whitening effects of 4-hydroxyphenethyl alcohol isolated from water boiled with Hizikia fusiformis

Isolation of phenolic compounds from water boiled with Hizikia fusiformis and purificartrion of the extracts from solvent fractions was achieved using column chromatography. The simple phenolic compound, 4-hydroxyphenethyl alcohol (4-HPEA) with an elemental composition C₈H₁₀O₂ was identified on the basis of spectroscopic data. The whitening effects of 4-HPEA, inhibitory activity against mushroom tyrosinase, and inhibitory activity against melanin production in B16 melanoma cells were evaluated using an in vitro assay system. The whitening effect of 4-HPEA was also measured using a colorimeter and visual assessment of UVB induced hyperpigmentation of brown guinea pig skin. Topical application of 4-HPEA promoted depigmentation of UVB-induced hyperpigmented spots. 4-HPEA has a potential for use as a safe and effective ingredient for whitening in cosmetic and medical applications.     

Inhibitory effects of chestnut inner skin extracts on melanogenesis

Antioxidant capacities (ABTS radical scavenging assay and ferric reducing/antioxidant power assay), mushroom tyrosinase inhibitory effect, and α-melanocyte stimulating hormone (α-MSH) induced melanogenesis inhibitory effect of 60% methanol extracts and ethylacetate fractions from chestnut inner skin in B16F10 cells were investigated to inspect whitening effect. Above research showed that 60% methanol extracts and ethylacetate fractions from chestnut inner skin resulted in a dose-dependent manner on in vitro antioxidant effects. Especially, the ethylacetate fractions inhibited enzyme activity of mushroom tyrosinases with an IC₅₀ value of 160 μg/mL. Ethylacetate fractions from chestnut inner skin also decreased cellular melanin synthesis in B16F10 melanoma cells. Expression of tyrosinase showed that ethylacetate fractions from chestnut inner skin significantly decreased cellular melanogenesis. Consequently, these results suggest that chestnut inner skin extracts can be considered for a whitening agent of human skin.     

桑白皮中白藜芦醇、氧化白藜芦醇和桑皮苷的抗氧化活性

二苯乙烯类化合物具有很好的抗氧化和抑制酪氨酸酶的活性,以熊果苷、VC、VE为参照,对桑白皮中的3种二苯乙烯类化合物(氧化白藜芦醇、白藜芦醇、桑皮苷)的抗氧化和清除自由基的生物活性进行比较研究。结果表明：还原力强弱：VC＞白藜芦醇＞氧化白藜芦醇＞VE＞熊果苷＞桑皮苷;清除DPPH自由基能力：VC＞VE＞氧化白藜芦醇＞白藜芦醇＞桑皮苷＞熊果苷;清除ABTS＋ ·能力：白藜芦醇＞氧化白藜芦醇＞熊果苷＞桑皮苷＞VC ＞VE。     

Inhibitory Mechanism and Kinetics Study of Apple Polyphenols on the Activity of Tyrosinase

Tyrosinase is the key enzyme in the biosynthesis of melanin. In order to investigate the effect of apple polyphenols on tyrosinase activity, the enzyme kinetic method was used. The concentration of apple polyphenols, which caused the tyrosinase activity to be lost by 50%, was 1.0 mg ml^?1 for monophenolase and 0.9 mg ml^?1 for disphenolase. When the concentration of apple polyphenols was 2.0 mg ml^?1, the lag time before reaction of monophenolase was extended from 1.2 to 4.5 min. Inhibition kinetics analysis suggested that apple polyphenols were reversible and mixed inhibitors for tyrosinase with an inhibition equilibrium constant 2.3 mmol l^?1. The results indicated that apple polyphenols were ideal tyrosinase inhibitors and could be used in food and cosmetics.     

柚皮苷二氢查尔酮的抗氧化活性研究

利用4种常见的抗氧化方法和分子对接,研究柚皮苷二氢查尔酮的抗氧化活性.通过测定酪氨酸酶活性,研究其美白活性,并用MTT试验测定其细胞毒性.试验结果表明:柚皮苷二氢查尔酮有较好的抗氧化性和美白活性,对小鼠黑色素瘤B16细胞的安全浓度为小于等于500μmol/L.     

酪氨酸酶特异性卵黄抗体Fab’片段的制备及其对酶活力的抑制性能

针对导致黑色素沉淀的酪氨酸酶,制备了特异性卵黄抗体Fab’片段,研究其对酶活力的抑制效果并从动力学角度进行分析。结果表明,胃蛋白酶法制备Fab’片段的最适条件为：pH?4.0、反应比（卵黄抗体-胃蛋白酶）1∶150（mg/U）、反应时间8?h。经免疫亲和柱纯化后可得理化性质稳定、纯度大于95%的Fab’片段;该片段通过非竞争性结合抑制酪氨酸酶活力,IC50为11.16?μmol/L,结合常数为5.79×105,抑制效果相比完整的卵黄抗体分子大大提高。本研究可为卵黄抗体Fab’片段作为潜在的酶活力抑制剂在食品、化妆品和医药等行业中应用提供理论参考。     

Biological screening of 100 plant extracts for cosmetic use (I): inhibitory activities of tyrosinase and DOPA auto-oxidation

The aim of this study was to evaluate several plant extracts with a view to developing melanogenesis inhibitors. In this study, 100 plant extracts were screened to elucidate their whitening effects using in vitro inhibition of tyrosinase and DOPA auto-oxidation activity. Several plant extracts such as Chaenomeles speciosa, Dryopteris crassirhizoma, Gastrodia ellata, Glycyrrhiza glabra, Morus alba, Myristica fragrans, Rheum palmatum and Sophora japonica showed inhibition of mushroom tyrosinase activity. Plant extracts including Bupleurum falcatum, Caragana sinica, Morus alba and Tussilago farfara showed inhibition of DOPA auto-oxidation activity.     

辐照红景天乙醇提取物的抗氧化作用及美白作用研究

目的:探讨60Co-γ射线辐照处理对红景天乙醇提取物抗氧化及美白作用的影响。方法:本试验所用红景天经不同剂量(5、10、20、30 kGy)的60Co-γ射线辐照处理后,采用1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率、2,2'-联氨-(3-乙基苯并噻唑啉-6-磺酸)二氨盐(ABTS)自由基清除率及铁离子还原能力(FRAP)、酪氨酸酶抑制实验、小鼠B16黑色素瘤细胞实验,评价辐照红景天提取物的抗氧化活性及美白活性。结果:与未辐照组相比,辐照组红景天提取物的抗氧化活性及美白活性均增加,其中20 kGy辐照组红景天提取物效果最佳,清除DPPH自由基的IC₅₀值为3.75 μg/mL、清除ABTS自由基的IC₅₀值为44.88 μg/mL、铁离子还原能力FRAP值为(1.90±0.05) mmol/mg、抑制酪氨酸酶的IC₅₀值为244.10 μg/mL、能够有效降低小鼠黑色素瘤细胞中黑色素的合成,并在浓度为0.50%以下时无细胞毒性。结论:20 kGy的60Co-γ射线辐照处理红景天乙醇提取物的抗氧化活性最强,并具有良好的美白功效。