Effects of radix polygoni multiflori components on tyrosinase activity and melanogenesis

Radix Polygoni multiflori is a herb used effectively to prevent graying and treat skin depigmentation diseases in traditional Chinese medicine but its active ingredients have not been discovered yet. In this investigation, we tested six compounds isolated from Radix Polygoni multiflori, to discover the active component on melanogenesis. Three experiments were performed in the present investigation: mushroom tyrosinase activity, melanin content B16 cell proliferation assay. Among all the six components tested, THSG showed the most potent effects on tyrosinase activation and melanogenesis; it was shown to be a potent tyrosinase activator and a melanogenesis stimulator in this study. On the other hand, we found that gallic acid significantly inhibited tyrosinase and, in addition, anthraquinones were cytotoxic to melanoma cells. They were both harmful to melanogenesis. Therefore, we propose that THSG acts as the active ingredient of Radix Polygoni multiflori on melanogenesis.     

Rapid molecular authentication of three medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum (Fallopia multiflorum), by the development of RAPD-derived SCAR markers and multiplex-PCR

Definitive identification of original plant species is important for standardizing herbal medicine. The herbal medicines Cynanchi Wilfordii Radix (Baekshuoh in Korean and Beishuwu in Chinese) and Polygoni Multiflori Radix (Hashuoh in Korean and Heshuwu in Chinese) are often misidentified in the Korean herbal market due to morphological similarities and similar names. Therefore, we developed a reliable molecular marker for the identification of Cynanchi Wilfordii Radix and Polygoni Multiflori Radix. We used random amplified polymorphic DNA (RAPD) analysis of three plant species, Polygoni multiflorum, Cynanchum wilfordii, and Cynanchum auriculatum, to obtain several species-specific RAPD amplicons. From nucleotide sequences of these RAPD amplicons, we developed six sequence characterized amplification region (SCAR) markers for distinguishing Polygoni Multiflori Radix and Cynanchi Wilfordii Radix. Furthermore, we established SCAR markers for the simultaneous discrimination of the three species within a single reaction by using multiplex-PCR. These SCAR markers can be used for efficient and rapid authentication of these closely related species, and will be useful for preventing the distribution of adulterants.     

In Vitro and in Vivo Melanogenesis Inhibition by Biochanin A from Trifolium pratense

Our previous study showed that a methanol extract from Trifolium pratense exerted potent inhibitory activity on melanogenesis in mouse B16 melanoma cells. In the present study, the active compound in this Chinese herb extract was isolated and identified as biochanin A by mass spectrum, ¹H-NMR, and ¹³C-NMR analysis. The inhibitory effects of biochanin A on melanogenesis were investigated in vitro in cultured melanoma cells and in vivo in zebrafish and mice. Biochanin A dose-dependently inhibited both melanogenesis and cellular tyrosinase activity in B16 cells and in zebrafish embryos. Application of a cream containing 2% biochanin A twice daily to the skin of mice also increased the skin-whitening index value after 1 week of treatment, and the increase continued for another 2 weeks. Biochanin A was confirmed as a good candidate for use as a skin-whitening agent in the treatment of skin hyperpigmentation disorders.     

Free‐Radical‐Scavenging,Antityrosinase, and Cellular Melanogenesis Inhibitory Activities of Synthetic Isoflavones

In this study, we examined the potential of synthetic isoflavones for application in cosmeceuticals. Twenty‐five isoflavones were synthesized and their capacities of free‐radical‐scavenging and mushroom tyrosinase inhibition, as well as their impact on cell viability of B16F10 murine melanoma cells and HaCaT human keratinocytes were evaluated. Isoflavones that showed significant mushroom tyrosinase inhibitory activities were further studied on reduction of cellular melanin formation and antityrosinase activities in B16F10 melanocytes in vitro. Among the isoflavones tested, 6‐hydroxydaidzein ( 2 ) was the strongest scavenger of both ABTS ^{. +} and DPPH ^. radicals with SC₅₀ values of 11.3±0.3 and 9.4±0.1 μM , respectively. Texasin ( 20 ) exhibited the most potent inhibition of mushroom tyrosinase (IC₅₀ 14.9±4.5 μM ), whereas retusin ( 17 ) showed the most efficient inhibition both of cellular melanin formation and antityrosinase activity in B16F10 melanocytes, respectively. In summary, both retusin ( 17 ) and texasin ( 20 ) exhibited potent free‐radical‐scavenging capacities as well as efficient inhibition of cellular melanogenesis, suggesting that they are valuable hit compounds with potential for advanced cosmeceutical development.     

Inhibitory activity of novel kojic acid derivative containing trolox moiety on melanogenesis

A novel kojic acid derivative containing a trolox moiety, (±)-5-hydroxy-4-oxo-4H-pyran-2-yl methyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (3a), was synthesized. The two biologically active compounds, namely, kojic acid and trolox, were conjugated via an ester bond as they are expected to behave synergistically. The antioxidant activity and the tyrosinase inhibitory activity of this novel kojic acid derivative on melanogenesis were evaluated. Compound 3a exhibited potent tyrosinase inhibitory activity and radical scavenging activity. Limited structure–activity relationship (SAR) investigations indicated that the tyrosinase inhibitory activities may originate from the kojic acid moiety, and the radical scavenging activity may be due to the phenolic hydroxyl group of trolox. Compound 3a also exhibited potent depigmenting activity in a cell-based assay. The limited SAR investigations revealed that the depigmenting activity of 3a may be due to the synergistic activities of kojic acid and its trolox moiety.     

Progressive Effects of Phloridzin on Melanogenesis in B16 Mouse Melanoma Cells

When we studied the effects of polyphenols from apple fruits on melanogenesis in B16 mouse melanoma cell lines, phloridzin had dose-dependent progressive effects on melanogenesis between 10 and 500 μg/ml without inhibiting cell growth. At a concentration of 500 μg/ml, phloridzin increased the melanin content in the cells to 181% of that in control cells. In contrast, phloretin, the aglycon of phloridzin, did not activate melanogenesis in the cells and was cytotoxic at a concentration of 5 μg/ml. Phloridzin increased the activity of tyrosinase to 223% of that in control cells. Furthermore, phloridzin inhibited the activity of protein kinase C (PKC), which is recognized to regulate tyrosinase activity. The inhibition of PKC activity continued for 120min from the addition of phloridzin. Therefore, we estimated that the activation of melanogenesis by phloridzin resulted from the increase of tyrosinase activity caused by the inhibition of PKC activity.     

Protective effect of polygoni cuspidati radix and emodin on <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> cytotoxicity and infection

Vibrio vulnificus, a good model organism of bacterial septicemia, causes fatal septicemia manifesting a fulminating course and a high mortality rate within days. In order to identify new natural substances preventing V. vulnificus infection, a plant library was screened for inhibiting cytotoxicity to host cells by using Trypan blue staining and LDH assay. We found that Polygoni Cuspidati Radix potently suppressed the acute death of HeLa and RAW264.7 cells in a dose dependent manner. Further studies revealed that Polygoni Cuspidati Radix inhibited V. vulnificus growth and survival in HI broth and seawater, respectively. We confirmed that Polygoni Cuspidati Radix contained high level of emodin by thin layer chromatography (TLC). Emodin showed direct antibacterial activity against V. vulnificus. In addition, emodin prevented the morphologic damages and acute death of HeLa cells caused from V. vulnificus. The safety of Polygoni Cuspidati Radix and emodin to host cells was confirmed by MTT assay. Polygoni Cuspidati Radix and emodin protected mice from V. vulnificus infection.     

Anti-melanogenesis potential of a new series of Morita-Baylis-Hillman adducts in B16F10 melanoma cell line

Melanin is a natural polymer pigment which provides skin photoprotection against ultraviolet radiation. An excessive synthesis of melanin leads to hyperpigmentation disorders. Tyrosinase catalyzes the rate limiting steps on melanogenesis. Therefore, tyrosinase inhibitors have potential applications in medicine and cosmetic fields. We carried out herein the screening of a family of cyclic Morita-Baylis-Hillman adducts (MBH) to find out their effects on tyrosinase activity and on melanogenesis in murine melanoma B16F10 cell line. Kinetic analysis of tyrosinase inhibition showed that compounds 1a (2-hydroxymethyl) cyclohex-2-enone) and 3f (diethyl (1-(6-oxocyclohex-1-en-1-yl) ethyl-phosphonate) were competitive inhibitors, whereas the compound 2b (6-oxocyclohex-1-en-1-yl) ethyl acetate) was a non-competitive one. Additionally we have found that (1a, 2b and 3f) compounds had a strong melanogenesis inhibition effect in isobutylmethylxanthine (IBMX)-treated murine melanoma B16F10 cells when tested at low and non cytotoxic dose (10–50 µM), by attenuating the melanin production, intracellular tyrosinase activity and tyrosinase expression. Thus, we suggest that these compounds could be used as effective skin-whitening agents.     

2-Aminothiazole-Oxadiazole Bearing N-Arylated Butanamides: Convergent Synthesis,Tyrosinase Inhibition,Kinetics, Structure-Activity Relationship,and Binding Conformations

A multi-step synthesis of novel bi-heterocyclic N-arylated butanamides was consummated through a convergent strategy and the structures of these medicinal scaffolds, 7a–h , were corroborated using spectral techniques. The in vitro analysis of these hybrid molecules revealed their potent tyrosinase inhibition as compared to the standard used. The kinetics mechanism was investigated through Lineweaver-Burk plots which exposed that, 7f , inhibited tyrosinase enzyme non-competitively by forming the enzyme-inhibitor complex. The inhibition constants K_i calculated from Dixon plots for this compound was 0.025 μM. Their binding conformations were ascertained by in silico computational studies whereby these molecules disclosed good binding energy values (kcal/mol). So, it was anticipated from the current research that these bi-heterocyclic butanamides might be probed as imperative therapeutic agents for melanogenesis.     

Up‐regulation of Tyrosinase Gene by Nitric Oxide in Human Melanocytes

Ultraviolet light (UV) radiation causes skin‐tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO‐induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S‐nitroso‐N‐acetyl‐ l ‐arginine. An increase of tyrosinase activity was also detected time‐dependently within the 24‐hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3′,5′‐monophosphate (cGMP)‐dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO‐induced melanogenesis.     

HPTLC Autography Based Screening and Isolation of Mushroom Tyrosinase Inhibitors of European Plant Species

In the course of this project, 133 plants were evaluated on their ability to inhibit tyrosinase, a key enzyme in melanogenesis. The screening was performed by means of a HPTLC autographic assay, resulting in the selection of three plants, Asplenium trichomanes, Pinus uncinata, and Scutellaria altissima, with promising tyrosinase inhibiting activities. With the aid of the HPTLC assay, it was not only possible to select the most interesting plant extracts, but also to monitor the activity‐guided fractionation which, in a relatively short time period, led to the isolation of active principles. Benzoic acid, roseoside, and dihydrovomifoliol‐O‐β‐d ‐glucopyranoside could be identified as tyrosinase inhibitors present in P. uncinata. Globularin turned out to be the active principle of S. altissima, and 4‐ethenylphenyl 6‐O‐(6‐deoxy‐α‐l ‐mannopyranosyl)‐β‐d ‐glucopyranoside was detected as tyrosinase inhibitor of A. trichomanes. The pure compounds were tested also in a 96 well‐plate assay in order to determine their IC₅₀ values. The lowest IC₅₀ value (42 μm ) could be obtained for globularin, whereas the other compounds, e. g., benzoic acid exhibited a rather high IC₅₀ value (IC₅₀=552 μm ). This stood in clear contrast to the autographic assay, but is has to be taken into account that the outcome of the autography assay is not only depending on the IC50 value of a compound, but also on the content of the respective constituent in the extract.     

Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO)

Background

Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase.

Methods

The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo.

Results

MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling.

Conclusions

Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders.

General significance

MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.     

Effect of p-aminophenols on tyrosinase activity

Tyrosinase is involved in the synthesis of melanin in the skin and hair as well as neuromelanin in the brain. This rate limiting enzyme catalyzes two critical steps (reactions) in melanogenesis; the hydroxylation of tyrosine to form DOPA and the subsequent oxidation of DOPA into dopaquinone. Several new aminophenol derivatives have been synthesized based on structure–activity relationship studies of N-(4-hydroxyphenyl)retinamide (1), a derivative of retinoic acid. In order to find new tyrosinase inhibitors, we investigated the effects of these p-aminophenols, including p-decylaminophenol (3), on the activity of mushroom tyrosinase. Compound 3 was the most potent agent, showing significant inhibition as compared with control. The inhibitory effects of 3 on tyrosinase activities were greater than seen with kojic acid, a well-known potent inhibitor of tyrosinase activity, which also causes adverse effects, including rash and dermatitis. A Lineweaver–Burk kinetic analysis of inhibition showed that 3 suppresses tyrosinase activity in a non-competitive fashion for both substrates, tyrosine and DOPA. These results suggest that 3 might be a useful alternative to kojic acid as a tyrosinase inhibitor.     

Isolation of tyrosinase and melanogenesis inhibitory flavonoids from Juniperus chinensis fruits

ABSTRACT

A new biflavonoid, amentoflavone-7-O-β-D-glucoside, and thirteen known flavonoids were isolated from the fruits of Juniperus chinensis using a bioactivity-guided method and their tyrosinase inhibitory effects were tested using a mushroom tyrosinase bioassay. Two isolates, hypolaetin-7-O-β-D-glucoside and quercetin-7-O-α-L-rhamnoside, were found to reduce tyrosinase activity at a concentration of 50 μM. Quercetin-7-O-α-L-rhamnoside attenuated cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells. Molecular docking simulation revealed that quercetin-7-O-α-L-rhamnoside inhibits tyrosinase activity by hydrogen bonding with residues His85, His244, Thr261, and Gly281 of tyrosinase.

Abbreviations: EtOH, ethanol; CH2Cl2, dichloromethane; EtOAc, ethylacetate; n-BuOH, n-butanol; MeOH, metanol; CHCl3,chloroform; DMSO, dimethylsulfoxide; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; α-MSH, α-melanocyte stimulating hormone; L-DOPA, L-3, 4-dihydroxyphenylalanine     

Melanogenesis inhibition by tetrahydropterines

There is controversy in the literature concerning the action of tetrahydropterines on the enzyme tyrosinase and on melanogenesis in general. In this study, we demonstrate that tetrahydropterines can inhibit melanogenesis in several ways: i) by non-enzymatic inhibition involving purely chemical reactions reducing o-dopaquinone to L-dopa, ii) by acting as substrates which compete with L-tyr and L-dopa, since they are substrates of tyrosinase; and iii) by irreversibly inhibiting the enzymatic forms met-tyrosinase and deoxy-tyrosinase in anaerobic conditions. Three tetrahydropterines have been kinetically characterised as tyrosinase substrates: 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydropterine and 6,7-(R,S)-dimethyl-5,6,7,8-tetrahydropterine. A kinetic reaction mechanism is proposed to explain the oxidation of these compounds by tyrosinase.     

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKCα had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKCα in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1. © 1996 Wiley-Liss, Inc.     

Chemical Constituents of the Seed Cake of Camellia oleifera and Their Antioxidant and Antimelanogenic Activities

There is a growing interest in the exploitation of agricultural byproducts. This study explored the potential beneficial health effects from the main biowaste, tea seed pomace of Camellia oleifera Abel (Theaceae), produced when tea seed is processed. Eighteen compounds were isolated from the 70% EtOH extract of the seed cake of C. oleifera. Their structures were determined by ESI‐MS, ¹H‐ and ¹³C‐NMR together with literature data. All fractions and compounds were evaluated for the antioxidant and melanogenesis inhibitory activities. As the result, AcOEt fraction has the best in vitro antioxidant and antimelanogenesis activities, compounds 7 – 12 and 15 showed remarkable antioxidant activity, compounds 4 , 6 , 8 , and 15 – 17 exhibited superior inhibitory activities against melanogenesis. Furthermore, tyrosinase inhibitory activity assay suggested that compound 8 could suppress melanogenesis by inhibiting the expression of tyrosinase.     

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.