Antimelanogenic and antioxidant properties of gallic acid

To find novel skin-whitening agents, the melanogenesis inhibitory action of gallic acid (GA) was investigated. In this current study, the effects of GA on mushroom tyrosinase, tyrosinase inhibitory activity, and melanin content were assessed in B16 melanoma cells (B16 cells). Results indicated that GA has a strong antityrosinase activity (IC50=3.59x10(-6) M). Furthermore, data on murine tyrosinase activity and melanin biosynthesis revealed that GA effectively suppressed murine tyrosinase action and the amount of melanin. To investigate the relation between GA's inhibition of melanogenesis and antioxidant activity, the effect of GA on reactive species (RS) generation and the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in were determined in B16 cells. Results indicated that GA effectively down-regulated the RS generation and enhanced the GSH/GSSG ratio. Based on these results, I propose that GA exerts antimelanogenic activity coupled with antioxidant properties by suppressing RS generation and maintaining a higher GSH/GSSG ratio.     

Inhibitory effects of macelignan isolated from Myristica fragrans HOUTT. on melanin biosynthesis

The aim of this study was to investigate the in vitro inhibitory effects of macelignan isolated from Myristica fragrans HOUTT. on melanogenesis and its related enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) in melan-a murine melanocytes. The IC50 values of macelignan for melanogenesis and tyrosinase were 13 microM and 30 microM, respectively, while those of arbutin as a positive control were 990 microM and 660 microM, respectively. In Western blot analysis, macelignan also significantly decreased tyrosinase, TRP-1, and TRP-2 protein expression. These results indicate that macelignan effectively inhibits melanin biosynthesis and thus could be employed as a new skin-whitening agent.     

Inhibitory effects of esculetin on melanin biosynthesis

To investigate the structure-activity relationship of coumarins for the inhibitory activity on mushroom tyrosinase, the 50% inhibitory concentration (IC50 values) of 18 coumarins and four cinnamic acid derivatives were measured. Among these compounds, esculetin had the strongest inhibitory activity (IC50=43 microM) on mushroom tyrosinase. Introduction of a hydroxy group to the C6 and C7 positions of the coumarin ring and no substitution on the lactone ring played an important role in the expression of the strong inhibitory activity of esculetin. We performed further studies to estimate the in vitro inhibitory effects of esculetin on melanogenesis. Esculetin 5 microM significantly suppressed melanin production in murine B16 melanoma cells without affecting cell growth. Furthermore, the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes in the split-epidermal sheets treated with 0.05% or 0.1% esculetin was significantly lower than that in the control. From these results, it is suggested that esculetin has inhibitory effects on tyrosinase activity in vitro. However, further detailed studies are necessary to understand the inhibitory mechanism of esculetin.     

Inhibitory effects of kurarinol, kuraridinol, and trifolirhizin from Sophora flavescens on tyrosinase and melanin synthesis

Previously, it was reported that some prenylated flavonoids contained in the dichloromethane fraction of the ethanolic extract of Sophora flavescens, such as kuraridin, sophoraflavanone G, kurarinone, and kushenol F, are tyrosinase inhibitors; however, based on the level of these inhibitors in the extract, its inhibitory effect on tyrosinase activity was higher than expected. This has led us to further investigate other possible constituents that may contribute to the extract's strong inhibitory activity. The results of this study indicate that kurarinol (1), kuraridinol (2), and trifolirhizin (3), from the ethyl acetate fraction of Sophora extract, can inhibit tyrosinase activity. Compared with kojic acid (16.22+/-1.71 microM), compounds 1-3 possessed potent tyrosinase inhibitory activity with IC(50) values of 8.60+/-0.51, 0.88+/-0.06, and 506.77+/-4.94 microM, respectively. These three compounds were further tested for their inhibitory effects on melanogenesis. In cultured B16 melanoma cells, 1-3 markedly inhibited (>50%) melanin synthesis at 50 microM. This is the first study indicating that 1-3 exert varying degrees of inhibition on tyrosinase-dependent melanin biosynthesis, and therefore, are candidates as skin-whitening agents.     

Silymarin inhibits melanin synthesis in melanocyte cells

Objectives The aim was to search for inhibitors of melanogenesis from natural resources. Methods The inhibitory effect of silymarin on melanogenesis in a spontaneously immortalized mouse melanocyte cell line, Mel‐Ab, was studied. Key findings Silymarin significantly prevented melanin production in a dose‐dependent manner with an IC50 value (concentration producing 50% maximal inhibition) of 28.2 μg/ml, without effects on cell viability. Also, silymarin inhibited l ‐DOPA oxidation activity of tyrosinase, the rate‐limiting melanogenic enzyme, in cell based‐systems but it did not directly affect cell‐free tyrosinase activity. Furthermore, Western blot analysis indicated that silymarin decreased the expression of tyrosinase protein. Conclusions This study suggests that the depigmenting effect of silymarin might be attributable to inhibition of tyrosinase expression and that silymarin may be useful as a natural skin‐lightening agent.     

4-(6-Hydroxy-2-naphthyl)-1,3-bezendiol: a potent, new tyrosinase inhibitor

Tyrosinase is a key enzyme for melanin biosynthesis and known to be sensitive to ultraviolet light in the presence of oxygen. Therefore, finding effective tyrosinase inhibitors, either from synthetic or natural sources, can be beneficial in the treatment of melanin-related disorders. We synthesized 4-(6-hydroxy-2-naphthyl)-1,3-bezendiol (HNB), a new family of hydroxyl substituted phenyl naphthalenes, as the isosteres of oxyresveratrol. This study investigated inhibitory effects of HNB on tyrosinase activity. HNB inhibited mushroom tyrosinase with an IC(50) value of 0.07 microM, which is more potent than the anti-tyrosinase activity of kojic acid (IC(50)=38.24), a well-known tyrosinase inhibitor. The kinetic analysis of tyrosinase inhibition revealed that HNB is a competitive inhibitor (K(i) 4.78 x 10(-9) M at 0.125 microM and K(i) 6.21 x 10(-9) M at 0.25 microM). We further found that HNB also inhibited melanin production in B16F10 melanoma cells (B16 cells). In addition to tyrosinase inhibiting activity, melanin biosynthesis was inhibited by HNB in the B16F10 cells. These data strongly suggest that HNB can suppress the production of melanin via the modulation of tyrosinase activity.     

Piperlonguminine from Piper longum with inhibitory effects on alpha-melanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells

Skin hyperpigmentations such as melasma, freckles and senile lentigines can be subjectively treated by depigmenting agents. In our ongoing study to find melanogenesis inhibitors from natural sources, Piper longum L (fruits, Piperaceae) was discovered to have an inhibitory effect on alpha-melanocyte-stimulating hormone (alpha-MSH)-induced melanogenesis in melanoma B16 cells. Piperlonguminine has been identified as the melanogenesis inhibitor from P. longum by activity-guided extraction and isolation. The compound showed dose-dependent inhibitory effects with 85.1 +/- 4.9% inhibition at 25 microM, 62.1 +/- 6.1% at 12.5 microM, 36.4 +/- 4.6% at 6.3 microM and 18.4 +/- 5.1% at 3.1 microM on alpha-MSH-induced melanogenesis, showing an IC50 value of 9.6 microM. As a positive control, kojic acid exhibited an IC50 value of 44.6 microM on the melanogenesis. As to the mode of action, piperlonguminine showed an inhibitory effect on alpha-MSH-induced tyrosinase synthesis, documented by Western immunoblot analysis. However, piperlonguminine did not show an inhibitory effect on tyrosinase activity or a direct depigmenting effect of melanin.     

Piceatannol inhibits melanogenesis by its antioxidative actions

In our efforts to find new skin lightening agents, piceatannol (PICE) was investigated for its antioxidative property and ability to inhibit melanogenesis. In this study, PICE's effect on inhibition of mushroom tyrosinase, and tyrosinase inhibiting activity and melanin content were assessed utilizing the B16F10 melanoma cell (B16 cell) culture system. Results indicated that PICE has a strong antityrosinase activity (IC(50)=1.53 microM). To evaluate the relative efficacy of PICE compared to other tyrosinase inhibitors, its inhibitory effect was compared and showed that PICE was significantly stronger than kojic acid (IC(50)=50.1 microM) and resveratrol (IC(50)=63.2 microM). Furthermore, PICE was shown to down-regulate melanin content. To document PICE's antioxidative property, which is known to influence melanogenic activity, we assessed reactive species (RS) generation, reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in these B16 cells. The results showed that PICE suppressed RS generation and enhanced the GSH/GSSG ratio. In conclusion, our results indicated that the antimelanogenic action of PICE is likely exhibited by the combined effect of PICE's antioxidative property and its ability to suppress RS generation while increasing the GSH/GSSG ratio.     

Inhibitory effects of 1,3‐thiazine derivatives on melanogenesis

Objectives The aim of this study was to identify a novel skin‐depigmenting agent from synthetic 1,3‐thiazine derivatives. Methods We investigated the inhibitory effects of six kinds of 1,3‐thiazine derivative on melanogenesis by examining their effects on tyrosinase activity and melanin biosynthesis in melan‐a cells and the zebrafish model. Key findings Of the six compounds, 4‐hydroxy‐2,6‐dimethyl‐5,6‐dihydro‐4H‐1,3‐thiazine (TZ‐6) had the strongest anti‐melanogenic effects in cultured melan‐a cells (30.4% inhibition at 100 μM). In addition, TZ‐6 exhibited an inhibitory effect on mushroom and cellular tyrosinase. Based on the results of Western blotting, TZ‐6 reduced the expression of tyrosinase at 100 mM. Additionally, TZ‐6 reduced body pigmentation and inhibited tyrosinase activity in the zebrafish model. Conclusions The results have provided useful information for the development of a skin whitening agent.     

Cytotoxic and antimutagenic stilbenes from seeds of Paeonia lactiflora

Cytotoxic and antimutagenic effects of a novel cis-epsilon-viniferin and five known stilbenes, transresveratrol, trans-epsilon-viniferin, gnetin H, suffruticosols A and B, isolated from the seeds of Paeonia lactiflora Pall. (Paeoniaceae) were determined against five different cancer cell lines, and mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium TA100, respectively. Six stilbenes showed cytotoxic activity in a dose-dependent manner, and especially did potent cytotoxic activity against C6 (mouse glioma) cancer cell with IC50 values ranging from 8.2 to 20.5 microg/ml. trans-Resveratrol showed significant cytotoxic activity against HepG2 (liver hepatoma) and HT-29 (colon) human cancer cell lines with IC50 values of 11.8 and 25.2 g/ml, respectively. In contrast, trans-epsilon-viniferin and cis--viniferin, and gnetin H exhibited marked cytotoxic activity against Hela (cervicse) and MCF-7 (breast) human cancer cell lines with IC50 values of 20.4, 21.5, and 12.9 microg/ml, respectively. However, suffruticosol A and B had less cytotoxic effect against all cancer cells except C6. Meanwhile, six stilbenes exerted antimutagenic activity in a dose-dependent fashion. Of them, trans-resveratrol exhibited the strongest antimutagenic effect against MNNG with IC50 value of 27.0 microg/plate, while other five resveratrol oligomers also did moderate antimutagenic activity with IC50 values ranging from 31.7 to 35.2 microg/plate.     

Inhibitory effects of 6-(3-hydroxyphenyl)-2-naphthol on tyrosinase activity and melanin synthesis

As a part of an ongoing project searching for new skin-lightening agents, the inhibitory property of 6-(3-Hydroxyphenyl)-2-naphthol (HPN) on melanogenesis was investigated. The inhibitory action of HPN (IC₅₀=15.2 μM) on mushroom tyrosinase was revealed. To further explore the action of HPN on melanogenesis, the inhibition of tyrosinase and melanin levels were measured in B16 melanoma cells (B16 cells). Results show that HPN inhibited tyrosinase activity and reduced melanin in B16 cells. Therefore, our data indicate HPN as a new candidate for depigmentation reagents. Contributed equally to this work.     

Inhibitory effects of active compounds isolated from safflower (Carthamus tinctorius L.) seeds for melanogenesis

In order to develop a new skin whitening agent, safflower (Carthamus tinctorius L.) seeds were evaluated for melanogenesis inhibitory activity and its active principles were identified following activity-guided isolation. The 80% aqueous methanol extract and ethyl acetate fraction from safflower seeds showed a significant inhibition for mushroom tyrosinase. Three active compounds, N-feruloylserotonin, N-(p-coumaroyl)serotonin, and acacetin, were isolated from the ethyl acetate fraction as the active principles. Compared with arbutin (IC50=0.223 mM), the IC50 values of these compounds were 0.023, 0.074, and 0.779 mM for N-feruloylserotonin, N-(p-coumaroyl)serotonin, and acacetin, respectively. It was also found that N-feruloylserotonin and N-(p-coumaroyl)serotonin strongly inhibited the melanin production of Streptomyces bikiniensis and B16 melanoma cells in comparison with a known melanogenesis inhibitor, arbutin.     

Barbarin as a new tyrosinase inhibitor from Barbarea orthocerus

A tyrosinase inhibitor was isolated from the whole plant of Barbarea orthocerus Led. (Brassicaceae) by activity-guided fractionation, and identified as (R)-5-phenyl-2-oxazolidinethione (barbarin) by structural analysis followed by comparison with reported spectral data. The compound exhibited significant inhibitory effects on mushroom and murine tyrosinases at more than 1.6 x 10(-5) M. Barbarin exhibited IC50 values of 4.2 x 10(-5) M on mushroom tyrosinase and of 4.8 x 10(-5) M on murine tyrosinase. Kojic acid as a positive control exhibited IC50 values of 3.4 x 10(-5) M and 6.0 x 10(-5) M on mushroom and murine tyrosinases, respectively. Therefore, barbarin exhibited a similar level of inhibitory potency with kojic acid used as a positive control. In a kinetic study with various concentrations of L-dopa as the substrate, barbarin was identified as an uncompetitive inhibitor and kojic acid as a mixed inhibitor of both mushroom and murine tyrosinases. Barbarin exhibited KEIS values of 3.3 x 10(-5) M and 3.6 x 10(-5) M on mushroom and murine tyrosinases, respectively. Kojic acid exhibited KEIS and KEI values of 2.4 x 10(-5) M and 2.2 x 10(-5) M on mushroom tyrosinase and those of 8.9 x 10(-5) M and 7.2 x 10(-5) M on murine tyrosinase, respectively.     

Depigmentation of melanocytes by isopanduratin A and 4-hydroxypanduratin A isolated from Kaempferia pandurata ROXB

This study was carried out to investigate the in vitro effects of isopanduratin A and 4-hydroxypanduratin A isolated from Kaempferia pandurata ROXB. on melanin biosynthesis and tyrosinase activity. Two chalcone compounds, isopanduratin A and 4-hydroxypanduratin A, were isolated from the ethyl acetate fraction of ethanol extract as the active principles. Compared with phenylthiourea (IC(50)=34.3 microM) as a positive control, the depigmentation IC(50) values for isopanduratin A and 4-hydroxypanduratin A were 10.6 microM and 23.2 microM, respectively. The compounds also significantly inhibited the activity of tyrosinase, the enzyme that converts DOPA (3,4-dihydroxyphenylalanine) to dopachrome in the biosynthetic process of melanin. The IC(50) values of isopanduratin A and 4-hydroxypanduratin A for tyrosinase were 10.5 microM and >30 microM, respectively, while that of phenylthiourea was 47.6 microM. The tyrosinase protein level was also significantly decreased by isopanduratin A and 4-hydroxypanduratin A. The results indicate that isopanduratin A and 4-hydroxypanduratin A isolated from K. pandurata ROXB. are promising compounds that could be useful for treating hyperpigmentation as skin-whitening agents.     

Inhibitory effect of p-hydroxybenzyl alcohol on tyrosinase activity and melanogenesis

Tyrosinase is a key enzyme catalyzing the rate-limiting step for the biosynthesis pathway of melanin pigment, which is the most important determinant of the color of skin. Inhibiting tyrosinase and repressing melanocyte metabolism can reduce melanin production. Among the possible melanin reducing compounds, tyrosinase inhibitors are most promising for treating pigmentation and are used as skin-whitening agents in the cosmetic industry. In our investigation, some new tyrosinase inhibitors from plants have been identified to have high tyrosinase inhibitory activity. Specifically, p-hydroxybenzyl alcohol (4HBA) was found to inhibit the monophenolase activity of mushroom tyrosinase. When 4HBA binds with the enzyme, conformation of the enzyme is altered and its activity decreases. The inhibitory effect of 4HBA on melanogenesis has been studied using cultured mouse melanoma cells. Melanin synthesis in cell culture with 4HBA at 1.0 mM was decreased to 45% of control and below 1.0 mM there was no effect on cell growth. The inhibitory effects of 4HBA on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity, rather than to the suppression of tyrosinase gene. These results showed that 4HBA is a promising and safe agent for skin whitening.     

Identification of tyrosinase inhibitors from Glycyrrhiza uralensis

Tyrosinase is a key enzyme in the production of melanins. Phytochemical studies of a Glycyrrhiza uralensis extract were performed by measuring the tyrosinase and melanin synthesis inhibitory activity. Glycyrrhisoflavone and glyasperin C were identified as tyrosinase inhibitors for the first time. Glyasperin C showed a stronger tyrosinase inhibitory activity (IC (50) = 0.13 +/- 0.01 microg/mL) than glabridin (IC (50) = 0.25 +/- 0.01 microg/mL) and a moderate inhibition of melanin production (17.65 +/- 8.8 % at 5 microg/mL). Glycyrrhisoflavone showed a strong melanin synthesis inhibitory activity (63.73 +/- 6.8 % inhibition at 5 microg/mL). These results suggest that glyasperin C and glycyrrhisoflavone could be promising candidates in the design of skin-whitening agents.     

4,4'-Dihydroxybiphenyl as a new potent tyrosinase inhibitor

The color of mammalian skin is determined by many factors, for which visible ones are the degree and distribution of melanin pigmentation. Because tyrosinase, (polyphenol oxidase) is the key enzyme for melanin biosynthesis, the use of various tyrosinase inhibitors is a common practice for whitening purpose in cosmetics. In the present study, the inhibition of tyrosinase by 4,4'-dihydroxybiphenyl (44'-BP) was investigated. In addition to tyrosinase inhibiting activity, melanin biosynthesis was assessed in B16F10 melanoma cells (B16 cells). The results showed that 44'-BP exhibits a strong anti-tyrosinase activity with IC50=1.91 microM. The kinetic analysis of tyrosinase inhibition revealed that 44'-BP acts a competitive inhibitor (Ki=4.0 x 10(-4) M at 2.5 microM and Ki =21 x 10(-5) M at 5 microM). Furthermore, data on melanin biosynthesis indicated that the amount of melanin was clearly suppressed by 44'-BP.