Antioxidative Characteristics of Anisomeles indica Extract and Inhibitory Effect of Ovatodiolide on Melanogenesis

The purpose of the study was to investigate the antioxidant characteristics of Anisomeles indica methanol extract and the inhibitory effect of ovatodiolide on melanogenesis. In the study, the antioxidant capacities of A. indica methanol extract such as DPPH assay, ABTS radical scavenging assay, reducing capacity and metal ion chelating capacity as well as total phenolic content of the extract were investigated. In addition, the inhibitory effects of ovatodiolide on mushroom tyrosinase, B16F10 intracellular tyrosinase and melanin content were determined spectrophotometrically. Our results revealed that the antioxidant capacities of A. indica methanol extract increased in a dose-dependent pattern. The purified ovatodiolide inhibited mushroom tyrosinase activity (IC(50) = 0.253 mM), the compound also effectively suppressed intracellular tyrosinase activity (IC(50) = 0.469 mM) and decreased the amount of melanin (IC(50) = 0.435 mM) in a dose-dependent manner in B16F10 cells. Our results concluded that A. indica methanol extract displays antioxidant capacities and ovatodiolide purified from the extract inhibited melanogenesis in B16F10 cells. Hence, A. indica methanol extract and ovatodiolide could be applied as a type of dermatological whitening agent in skin care products.     

几种中药的美白作用研究

研究了黄芩、虎杖和地榆等中药提取物对B16黑色素细胞的增殖、细胞内酪氨酸酶活性及黑色素合成的影响。结果显示,各中药提取物能不同程度地抑制细胞增殖,对细胞内酪氨酸酶活性有明显抑制作用,能明显减少细胞内黑色素的含量。人体皮肤实验表明,用这几种中药制备的护肤品有较好的美白效果。本研究提示,这几种中药具有良好的美白功能,是较为理想的化妆品美白添加剂。     

Determination of the Thermodegradation of deoxyArbutin in Aqueous Solution by High Performance Liquid Chromatography

Tyrosinase is the key and rate-limiting enzyme responsible for the conversion of tyrosine into melanin. Competitive inhibition of tyrosinase enzymatic activity results in decreased or absent melanin synthesis by melanocytes in human skin. DeoxyArbutin (4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol), a novel skin whitening agent, was synthesized through the removal of hydroxyl groups from the glucose side-chain of arbutin. DeoxyArbutin not only shows greater inhibition of tyrosinase activity but is also safer than hydroquinone and arbutin. Hence, deoxyArbutin is a potential skin whitening agent for cosmetics and depigmenting drugs; however, stability of this compound under some conditions remains a problem. The lack of stability poses developmental and practical difficulties for the use of deoxyArbutin in cosmetics and medicines. Improving the thermostability of deoxyArbutin is an important issue for its development. In this research, we established an analytical procedure to verify the amount of deoxyArbutin in solutions using a high performance liquid chromatographic (HPLC) method. The results indicate that this novel skin whitening agent is a thermolabile compound in aqueous solutions. Additionally, the rate constant for thermodegradation (k) and the half-life (t(1/2)) of deoxyArbutin were determined and can be used to understand the thermodegradation kinetics of deoxyArbutin. This information can aid in the application of deoxyArbutin for many future uses.     

4种中草药乙醇提取物体外抗氧化及美白活性研究

采用乙醇浸提法从干燥的芦荟叶、白芷、怀山药和黄芩4种中草药中提取分离得到其乙醇提取物。应用自由基清除活性和总抗氧化活性检测方法评估这4种中草药乙醇提取物的抗氧化活性,应用酪氨酸酶活性抑制实验、黑色素B16细胞增殖抑制和黑色素生成抑制实验对其进行美白活性评估。结果表明:4种中草药的乙醇提取物虽然具有一定的抗氧化活性、清除自由基能力、抑制酪氨酸酶活性、抑制黑色素B16细胞增殖和黑色素形成作用,但与市售护肤品常用的抗氧化美白功效成分BHT(2,6-二叔丁基-4-甲基苯酚)相比,其活性水平偏低,需要进一步分离纯化,富集活性成分,提高抗氧化美白活性。     

Investigation of the Anti-Melanogenic and Antioxidant Characteristics of Eucalyptus camaldulensis Flower Essential Oil and Determination of Its Chemical Composition

The effects of essential oil from Eucalyptus camaldulensis flowers oil on melanogenesis and the oil’s antioxidant characteristics were investigated. Assays of mushroom and cellular tyrosinase activities and melanin content of mouse melanoma cells were performed spectrophotometrically, and the expression of melanogenesis-related proteins was determined by Western blotting. The possible signaling pathways involved in essential oil-mediated depigmentation were also investigated using specific protein kinase inhibitors. The results revealed that E. camaldulensis flower essential oil effectively suppresses intracellular tyrosinase activity and decreases melanin amount in B16F10 mouse melanoma cells. The essential oil also exhibits antioxidant properties and effectively decreases intracellular reactive oxygen species (ROS) levels. The volatile chemical composition of the essential oil was analyzed with gas chromatography–mass spectrometry (GC/MS). The chemical constituents in the essential oil are predominately oxygenated monoterpenes (34.9%), followed by oxygenated sesquiterpenes (31.8%), monoterpene hydrocarbons (29.0%) and sesquiterpene hydrocarbons (4.3%). Our results indicated that E. camaldulensis flower essential oil inhibits melanogenesis through its antioxidant properties and by down-regulating both mitogen-activated protein kinases (MAPK) and protein kinase A (PKA) signaling pathways. The present study indicates that the essential oil has the potential to be developed into a skin care product.     

Protocatechuic Aldehyde Inhibits α-MSH-Induced Melanogenesis in B16F10 Melanoma Cells via PKA/CREB-Associated MITF Downregulation

Protocatechuic aldehyde (PA) is a naturally occurring phenolic compound that is a potent inhibitor of mushroom tyrosinase. However, the molecular mechanisms of the anti-melanogenesis activity of PA have not yet been reported. The aim of the current study was to clarify the melanogenesis inhibitory effects of PA and its molecular mechanisms in murine melanoma cells (B16F10). We first predicted the 3D structure of tyrosinase and used a molecular docking algorithm to simulate binding between tyrosinase and PA. These molecular modeling studies calculated a binding energy of −527.42 kcal/mol and indicated that PA interacts with Cu400 and 401, Val283, and His263. Furthermore, PA significantly decreased α-MSH-induced intracellular tyrosinase activity and melanin content in a dose-dependent manner. PA also inhibited key melanogenic proteins such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2 in α-MSH-stimulated B16F10 cells. In addition, PA decreased MITF expression levels by inhibiting phosphorylation of cAMP response element-binding protein (CREB) and cAMP-dependent protein kinase A (PKA). These results demonstrate that PA can effectively suppress melanin synthesis in melanoma cells. Taken together, our results show that PA could serve as a potential inhibitor of melanogenesis, and hence could be explored as a possible skin-lightening agent.     

Depigmenting effect of <Emphasis Type="Italic">Sterculia lynchnophera</Emphasis> on B16F10 melanoma and C57BL/6 melan-a cells

To develop a novel skin depigmenting agent from natural sources, the inhibition of melanogenesis by the Chinese herb, Sterculia lynchnophera (SL), was evaluated. Treatment of B16F10 melanoma cells and melan-a cells with SL exhibited a 32.9% and 68.2% inhibition of melanin synthesis without cytotoxicity at a concentration of 200 μg/ml, respectively. This herb possessed a high free radical scavenging activity with IC₅₀=11.02 μM. The methanol extract of SL slightly inhibited in vitro mushroom tyrosinase activity (23.4% at a concentration of 200 μg/ml) and had a significant inhibitory effect on cellular tyrosinase activivity (48.65% and 88.56% inhibition at the concentration 200 μg/ml in B16F10 cells and C57BL/6 melan-a cells, respectively). From the western blotting results, SL inhibited the expression of tyrosinase and tyrosinase related protein 1 (TRP-1). Taken together, we suggest that SL may be a safe and effective depigmentation agent.     

Melanogenesis inhibitory effect of dehydroevodiamine isolated from fruits of <Emphasis Type="Italic">Evodia rutaecarpa</Emphasis>

Dehydroevodiamine, an alkaloid, was isolated from the fruit of Evodia rutaecarpa and melanin production, and tyrosinase inhibition in B16F10 melanoma cells treated with the isolated dehydroevodiamine was investigated. The compound decreased melanin synthesis significantly without promoting cytotoxicity. The IC₅₀ value of dehydroevodiamine for melanogenesis and cell viability were 59.8 μM and 90.0 μM, respectively. The L-dopa oxidase activity of mushroom tyrosinase was reduced after dehydroevodiamine treatment by about 22.4% at a concentration of 33.2 μM. However, there was no effect on cellular tyrosinase activity. These results indicate that the observed decrease in melanin content after treatment with dehydroevodiamine was attributed to the direct inhibition of tyrosinase activity, rather than the suppression of tyrosinase gene expression. Dehydroevodiamine may be a promising new agent for use in cosmeceutical application.     

Tyrosol and Its Analogues Inhibit Alpha-Melanocyte-Stimulating Hormone Induced Melanogenesis

Melanin is responsible for skin color and plays a major role in defending against harmful external factors such as ultraviolet (UV) irradiation. Tyrosinase is responsible for the critical steps of melanogenesis, including the rate-limiting step of tyrosine hydroxylation. The mechanisms of action of skin hypopigmenting agents are thought to be based on the ability of a given agent to inhibit the activity of tyrosinase and, hence, down regulate melanin synthesis. Tyrosol and its glycoside, salidroside, are active components of Rhodiola rosea, and in our preliminary study we found that Rhodiola rosea extract inhibited melanogenesis. In this study, we examined the effects of tyrosol and its analogues on melanin synthesis. We found that treatment of B16F0 cells to tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), 2-hydroxyphenylacetic acid (7), or salidroside (11) resulted in a reduction in melanin content and inhibition of tyrosinase activity as well as its expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5) and 2-hydroxyphenylacetic acid (7) suppressed MC1R expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) inhibited α-MSH induced TRP-1 expression, but salidroside (11) did not. All the compounds did not affect MITF and TRP-2 expression. Furthermore, we found that the cell viability of tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) at concentrations below 4 mM and salidroside (11) at concentrations below 0.5 mM were higher than 90%. The compounds exhibited metal-coordinating interactions with copper ion in molecular docking with tyrosinase. Our results suggest that tyrosol, 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, and salidroside are potential hypopigmenting agents.     

Dual Bioactivities of Essential Oil Extracted from the Leaves of Artemisia argyi as an Antimelanogenic versus Antioxidant Agent and Chemical Composition Analysis by GC/MS

The study was aimed at investigating the antimelanogenic and antioxidant properties of essential oil when extracted from the leaves of Artemisia argyi, then analyzing the chemical composition of the essential oil. The inhibitory effect of the essential oil on melanogenesis was evaluated by a mushroom tyrosinase activity assay and B16F10 melanoma cell model. The antioxidant capacity of the essential oil was assayed by spectrophotometric analysis, and the volatile chemical composition of the essential oil was analyzed with gas chromatography-mass spectrometry (GC/MS). The results revealed that the essential oil significantly inhibits mushroom tyrosinase activity (IC₅₀ = 19.16 mg/mL), down-regulates B16F10 intracellular tyrosinase activity and decreases the amount of melanin content in a dose-dependent pattern. Furthermore, the essential oil significantly scavenged 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzthiazoline- 6-sulphonic acid) ABTS radicals, showed an apparent reduction power as compared with metal-ion chelating activities. The chemicals constituents in the essential oil are ether (23.66%), alcohols (16.72%), sesquiterpenes (15.21%), esters (11.78%), monoterpenes (11.63%), ketones (6.09%), aromatic compounds (5.01%), and account for a 90.10% analysis of its chemical composition. It is predicted that eucalyptol and the other constituents, except for alcohols, in the essential oil may contribute to its antioxidant activities. The results indicated that essential oil extracted from A. argyi leaves decreased melanin production in B16F10 cells and showed potent antioxidant activity. The essential oil can thereby be applied as an inhibitor of melanogenesis and could also act as a natural antioxidant in skin care products.     

Inhibition of melanogenesis by Erigeron canadensis via down-regulating melanogenic enzymes in B16F10 melanoma cells

The effects of Erigeron canadensis extract on melanogenesis and cell toxicity in cultured B16F10 mouse melanoma cells were investigated. E. canadensis extract down regulated melanin synthesis effectively at a non-toxic concentration. Its extract was fractionated by using a recycling HPLC with GS310 column (21.5×500 mm, 10–15 μM) into five fractions. The fraction 1 showed melanin inhibition by 48.0% at 100 mg/ml which was 2.5 times more efficient than the depigmenting effect of commercial arbutin (17.5%) and also did not show cell toxicity. To elucidate the depigmenting mechanism of fraction 1, in vitro and cellular tyrosinase activity, antioxidant activity, and protein level of the main melanogenic enzymes, such as tyrosinase, TRP-1 and TRP-2 were evaluated. Fraction 1 inhibited melanin synthesis in B16F10 melanoma cells by decreasing protein levels of melanogenic enzymes, especially tyrosinase. In conclusion, we suggest that this fraction may be a safe and effective depigmentation agent.     

Novel Chemically Modified Curcumin (CMC) Analogs Exhibit Anti-Melanogenic Activity in Primary Human Melanocytes

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.     

棕榈酰三肽-5皮肤美白功效及机制研究

采用MTT法检测细胞存活率,利用α-促黑素(α-MSH)诱导的B16小鼠黑色素瘤细胞模型和UVB照射诱导豚鼠皮肤色素沉着模型对棕榈酰三肽-5的美白功效进行评价。研究了其对细胞内酪氨酸酶活性、小眼畸形相关转录因子(MITF)和酪氨酸酶m RNA水平的影响。结果表明,棕榈酰三肽-5能够显著降低上清及细胞内的黑色素水平且无细胞毒性。进一步研究发现,棕榈酰三肽-5通过抑制酪氨酸酶活性,下调MITF和酪氨酸酶的mRNA水平,从而抑制黑色素的生成。在动物模型中,棕榈酰三肽-5能够显著降低UVB照射诱导的豚鼠皮肤色素沉着和黑色素分布区域灰度值(P0.05)。