Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica

The tyrosinase inhibitory activity of methanol extracts of the leaves of 39 plant species growing on the seashore of Iriomote island (Okinawa, Japan) was investigated. The extracts of Hibiscus tiliaceus, Carex pumila, and Garcinia subelliptica showed potent activity among them. The inhibitors in the extract of Garcinia subelliptica were purified by assay-guided fractionation to give two biflavonoids. These were known compounds (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone and 5,7,4',5',7',3',4'-heptahydroxy[3-8'] biflavanone), although their strong inhibitory activity toward tyrosinase is revealed for the first time in this work. One of these biflavonoids (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone) showed much stronger activity (IC50 2.5 microM) than that of kojic acid (IC50 9.1 microM) when L-tyrosine was used as the substrate.     

Effects on Tyrosinase Activity by the Extracts of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> and Related Mushrooms

The inhibitory effects on tyrosinase activity by extracts of several mushrooms belonging to Basidiomycetes were evaluated. Among the tested mushrooms (Ganoderma lucidum, Antrodia camphorata, Agaricus brasiliensis, and Cordyceps militaris), G. lucidum exhibited significant inhibition of tyrosinase activity (IC(50) value 0.32 mg/ml), compared to those prepared from other Basidiomycetes. Tyrosinase inhibitors are effective components of skin-lightening compounds and other cosmetics; currently many of the facial mask cosmetics in the market contain Ganoderma extracts in their ingredients. The finding that mushroom extracts contain tyrosinase activity inhibition will contribute to better understanding of how their 'healing' properties in various Chinese traditional herbal on skin care products.     

Keratinocytes Suppress TRP‐1 Expression and Reduce Cell Number of Co‐cultured Melanocytes – Implications For Grafting of Patients with Vitiligo

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well‐integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co‐cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down‐regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase‐related protein‐1 (TRP‐1) negative in confluent well‐integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP‐1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP‐1 expression.     

Lipid peroxidation inhibitory compounds from daylily (Hemerocallis fulva) leaves

Daylilies (Hemerocallis spp.) have been used as food and in traditional medicine for thousands of years in eastern Asia. The leaves of the plant are used in the treatment of inflammation and jaundice. In studies of the aqueous methanol extracts of fresh Hemerocallis fulva leaves, 1',2',3',4'-tetrahydro-5'-deoxy-pinnatanine (1), pinnatanine (2), roseoside (3), phlomuroside (4), lariciresinol (5), adenosine (6), quercetin 3-O-beta-D-glucoside (7), quercetin 3,7-O-beta-D-diglucopyranoside (8), quercetin 3-O-alpha-L-rhamnopyransol-(1-->6)-beta-D-glucopyranosol-7-O-beta-D-glucopyranoside (9), isorhamnetin-3-O-beta-D-6'-acetylglucopyranoside (10) and isorhamnetin-3-O-beta-D-6'-acetylgalactopyranoside (11) were isolated. All of these compounds were tested for their in vitro lipid peroxidation inhibitory activities. Compounds 3-5 and 7-11 were found to possess strong antioxidant properties, inhibiting lipid oxidation by 86.4, 72.7, 90.1, 79.7, 82.4, 89.3, 82.2, and 93.2%, respectively at 50 microg/mL. Compound 1 is novel and compounds 3-6 and 8-11 described here in are isolated for the first time from daylily leaves.     

Synthesis and evaluation of 2',4',6'-trihydroxychalcones as a new class of tyrosinase inhibitors

In this study, we synthesized a series of hydroxychalcones and examined their tyrosinase inhibitory activity. The results showed that 2',4',6'-trihydroxychalcone (1), 2,2',3,4',6'-pentahydroxychalcone (4), 2',3,4,4',5,6'-hexahydroxychalcone (5), 2',4',6'-trihydroxy- 3,4-dimethoxychalcone (9) and 2,2',4,4',6'-pentahydroxychalcone (15) exhibited high inhibitory effects on tyrosinase with respect to l-tyrosine as a substrate. By the structure-activity relationship study, it was suggested that the 2',4',6'-trihydroxyl substructure in the chalcone skeleton were efficacious for the inhibition of tyrosinase activity. And also, the catechol structure on B-ring of chalcones was not advantageous for the inhibitory potency. Furthermore, 15 (IC(50)=1microM) was found to show the highest activity out of a set of 15 hydroxychalcones, even better than both 2,2',4,4'-tetrahydroxychalcone (13, IC(50)=5microM) and kojic acid (16, IC(50)=12microM), which were known as potent tyrosinase inhibitors. Kinetic study revealed that 15 acts as a competitive inhibitor of tyrosinase with K(i) value of 3.1microM.     

Chemical transformations of oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene) into a potent tyrosinase inhibitor and a strong cytotoxic agent

From oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene 1), seven derivatives were prepared, including trans-2-methoxy-4,3',5'-trihydroxystilbene (2), trans-2,3'-dimethoxy-4,5'-dihydroxystilbene (3), trans-4,3'-dimethoxy-2,5'-dihydroxystilbene (4), trans-2,4,3',5'-tetramethoxystilbene (5) and cis-2,4,3',5'-tetramethoxystilbene (6), 2,4,3',5'-tetrahydroxybibenzyl (7), and 2,4,3',5'-tetramethoxybibenzyl (8). The tetrahydroxybibenzyl 7, a hydrogenation product of 1, exhibited more potent tyrosinase inhibitory activity than the parent compound, without cytotoxicity. A kinetic study revealed that 7 was a reversible and non-competitive inhibitor of mushroom tyrosinase with l-dopa as the substrate. Analysis of the K(i) values indicated that 7 has a slightly higher affinity to the enzyme than 1. Compound 6, a tetra-O-methylated analogue of 1 with cis-configuration, was deprived of inhibitory effect on the enzyme tyrosinase, but showed very strong cytotoxicity against the human cancer cells KB, BC, and NCI-H187, with potency comparable to those of the anticancer agents ellipticine and doxorubicin. Data on the tyrosinase inhibitory activity and cytotoxicity of 1-8 indicated that O methylation on stilbene 1 destroyed anti-tyrosinase activity but generated cytotoxicity. Thus, facile preparations of a potent tyrosinase inhibitor (7) and a strong cytotoxic agent (6) from the natural product 1 were achieved through simple chemical reactions.     

Possible involvement of proteolytic degradation of tyrosinase in the regulatory effect of fatty acids on melanogenesis.

The purpose of this study was to investigate the mechanism of fatty acid-induced regulation of melanogenesis. An apparent regulatory effect on melanogenesis was observed when cultured B16F10 melanoma cells were incubated with fatty acids, i.e., linoleic acid (unsaturated, C18:2) decreased melanin synthesis while palmitic acid (saturated, C16:0) increased it. However, mRNA levels of the melanogenic enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2), were not altered. Regarding protein levels of these enzymes, the amount of tyrosinase was decreased by linoleic acid and increased by palmitic acid, whereas the amounts of TRP1 and TRP2 did not change after incubation with fatty acids. Pulse-chase assay by [35S]methionine metabolic labeling revealed that neither linoleic acid nor palmitic acid altered the synthesis of tyrosinase. Further, it was shown that linoleic acid accelerated, while palmitic acid decelerated, the proteolytic degradation of tyrosinase. These results suggest that modification of proteolytic degradation of tyrosinase is involved in regulatory effects of fatty acids on melanogenesis in cultured melanoma cells.     

Geranylated phenolic constituents from the fruits of Artocarpus nobilis

Chemical investigation of the combined dichloromethane and ethyl acetate extracts of the fruits of Artocarpus nobilis, furnished four new geranylated phenolic constituents, 2,4,4'-trihydroxy-3-[(2E)-5-methoxy-3,7-dimethylocta-2,6-dienyl]chalcone (4), 1-(3,4-dihydro-3,5-dihydroxy-2-methyl-2-(3-methyl-2-butenyl)-2H-1-benzopyran-6-yl-3-(4-hydroxyphenyl)-2(E)-propen-1-one (5), 8-geranyl-3',4',7-trihydroxyflavone (8), 3'-geranyl-4',5,7-trihydroxyflavanone (9), together with known related compounds, xanthoangelol (1), xanthoangelol B (2), 3-geranyl-2,3',4,4'-tetrahydroxychalcone (3), lespeol (6), 8-geranyl-4',7-dihydroxyflavanone (7), and isonymphaeol-B (10). Compounds 3, 8 and 10 showed strong antioxidant activity against DPPH radical by spectrophotometric method.     

DHICA Oxidase Activity of TRP1 and Interactions With Other Melanogenic Enzymes

Tyrosinase-related protein 1 (TRP1) maps to the brown locus in mice. Although the specific function of TRP1 has been in dispute, mutations in its structural gene result in the formation of brown rather than black melanin. We have investigated the melanogenic function of TRP1 by using immune-affinity purification of the protein and also by using transfection of its gene into fibroblasts to study its characteristics. We show that TRP1 has the ability to oxidize DHICA, a melanogenic intermediate derived from DOPAchrome. In addition, TRP1 has the ability to interact with tyrosinase and significantly stabilize the latter's catalytic function.     

Effects of hydroxystilbene derivatives on tyrosinase activity

Synthesis of melanin starts from the conversion of L-tyrosine to 3,4-dihydroxyphenylalanine (L-dopa) and then the oxidation of L-dopa yields dopaquinone by tyrosinase. Therefore, tyrosinase inhibitors have been established as important constituents of depigmentation agents. Recently, polyhydroxystilbene compounds, which are trans-resveratrol (3,4('),5-trihydroxy-trans-stilbene) analogs, have been demonstrated as potent tyrosinase inhibitors. However, their detailed inhibitory mechanisms are not clearly understood. In the present study, a variety of synthesized hydroxystilbene compounds were tested for their inhibitory effects against murine tyrosinase activity. The inhibitory potencies of the hydroxy-trans-stilbene compounds were remarkably elevated by increasing number of phenolic hydroxy substituents. Methylated hydroxy-trans-stilbene lost the inhibitory activity. Furthermore, hydrogenated hydroxystilbene or hydroxy-cis-stilbene exerted little or no inhibitory effect compared with hydroxy-trans-stilbene on tyrosinase activity. The structure-activity relationships demonstrated in the present study suggest that the phenolic hydroxy groups and trans-olefin structure of the parent stilbene skeleton contribute to the inhibitory potency of hydroxystilbene for tyrosinase activity.     

A comparison of human immunodeficiency virus type-1 protease inhibition activities by the aqueous and methanol extracts of Chinese medicinal herbs

The aqueous and methanol extracts of thirty-one herbs traditionally used as anti-fever remedies in China were screened for their in vitro inhibition on human immunodeficiency virus type-1 protease (HIV-1 PR). The activity of recombinant HIV-1 protease was determined by sequence-specific cleavage at the Tyr-Pro bond of the fluorogenic substrate (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg) or by HPLC anaylsis of the cleavage products after incubation of the enzyme with a synthetic peptide substrate (Acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide). Among the herbal extracts examined, the aqueous extracts of Prunella vulgaris and Scutellaria baicalensis and the methanol extracts of Woodwardia unigemmata, Paeonica suffruticosa and Spatholobus suberectus elicited significant inhibition (>90%) at a concentration of 200 microg/ml.     

Biological Insights and NMR Metabolic Profiling of Different Extracts of Spermacoce verticillata (L.) G. Mey.

Spermacoce verticillata (L.) G. Mey. is commonly used in the folk medicine by various cultures to manage common diseases. Herein, the chemical and biological profiles of S. verticillata were studied in order to provide a comprehensive characterization of bioactive compounds and also to highlight the therapeutic properties. The in vitro antioxidant activity using free-radical scavenging, phosphomolybdenum, ferrous-ion chelating and reducing power assays, and the inhibitory activity against key enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase of S. verticillata extracts (dichloromethane, ethyl acetate, methanol and water) were investigated. The highest total phenolic and flavonoid content were observed in the methanolic and aqueous extracts. Exhaustive 2DNMR investigation has revealed the presence of rutin, ursolic and oleanoic acids. The methanolic extract, followed by aqueous extract have showed remarkable free radical quenching and reducing ability, while the dichloromethane extract was the best source of metal chelators. The tested extracts showed notable inhibitory activity against cholinesterases (AChE: 1.63–4.99 mg GALAE/g extract and BChE: 12.40–15.48 mg GALAE/g extract) and tyrosinase (60.85–159.64 mg KAE/g extract). No inhibitory activity was displayed by ethyl acetate and aqueous extracts against BChE and tyrosinase, respectively. All the tested extracts showed modest α-amylase inhibitory activity, while only the ethyl acetate and aqueous extracts were potent against α-glycosidase. This study further validates the use of S. verticillata in the traditional medicine, while advocating for further investigation for phytomedicine development.     

Melanogenesis inhibitors from the desert plant Anastatica hierochuntica in B16 melanoma cells

The methanolic extract from the whole plants of Anastatica hierochuntica, an Egyptian herbal medicine, was found to inhibit melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. Among the constituents isolated, anastatin A, silybin A, isosilybins A and B, eriodictyol, luteolin, kaempferol, quercetin, hierochins A and B, (2R,3S)-2,3-dihydro-2-(3,4-dimethoxyphenyl)-3-hydroxymethyl-5-(2-formylvinyl)-7-hydroxybenzofuran, (+)-dehydrodiconiferyl alcohol, (+)-balanophonin, 1-(4-hydroxy-3-methoxyphenyl)-2-{4-[(E)-3-hydroxy-1-propenyl]-2-methoxyphenoxy}-1,3-propanediol, and 3,4-dihydroxybenzaldehyde substantially inhibited melanogenesis with IC₅₀ values of 6.1–32 μM. With regard to the mechanism of action of silybins and isosilybins, the inhibition of tyrosinase activity suggested to be important. In addition, isosilybins A and B inhibited the mRNA expression of TRP-2, but silybins A and B oppositely enhanced the mRNA expression of tyrosinase and TRP-1 and -2 at 10 and/or 30 μM, and the inhibition of phosphorylation of extracellular signal-regulated kinases (ERK1/2) is involved in the enhanced expression of mRNA, at least in part, similar to that of PD98059.     

Anticancer activity and mechanism of Scutellaria barbata extract on human lung cancer cell line A549

Scutellaria barbata (S. barbata), a traditional Chinese herbal medicine native to southern China, is widely used as an anti-inflammatory and a diuretic in China. Several studies have indicated that extracts of S. barbata have growth inhibitory effects on a number of human cancers. Treatment of lung cancer, digestive system cancers, hepatoma, breast cancer, and chorioepithelioma by S. barbata extracts was reported. However, the mechanism underlying the antitumor activity was unclear. In this study, we studied the growth inhibitory effect of S. barbata and determined its mechanism of antitumor activity using human lung cancer cell line A549. Our results showed that ethanol extracts of S. barbata greatly inhibited A549 cell growth, with IC50 of 0.21 mg/ml. The major mechanisms of inhibition included cell apoptosis and cytotoxic effects. cDNA microarray analysis showed that 16 genes, involved in DNA damage, cell cycle control, nucleic acid binding and protein phosphorylation, underwent more than 5-fold change. These data indicated that these processes are involved in S. barbata-mediated killing of cancer cells. A surprising finding is that CD209, related to dendritic cell (DC) function, was dramatically downregulated by 102-fold. Further functional studies are needed to assess the role of the array-identified genes in S. barbata mediated anticancer activity.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.     

Constituents with tyrosinase inhibitory activities from branches of Ficus erecta var. sieboldii King

Phytochemical investigation of the branches of Ficus erecta var. sieboldii King resulted in the isolation of eight constituents: p-hydroxybenzoic acid (1), methyl p-hydroxybenzoate (2), vanillic acid (3), methyl vanillate (4), syringic acid (5), β-sitosterol (6), α-amyrin acetate (7), and ethyl linoleate (8). Their chemical structures were identified via spectroscopic means as well as by comparing their data with literature values. Studies on tyrosinase inhibition activities were conducted for the isolated compounds. Among them, p-hydroxybenzoic acid (1) and methyl p-hydroxybenzoate (2) were identified as active tyrosinase inhibitors with IC(50) values of 0.98?±?0.042 and 0.66?±?0.025?mM, respectively, showing comparable activities to that of arbutin (IC(50)?=?0.32?±?0.015?mM), a standard control. Inhibition kinetics, as analyzed by Lineweaver-Burk plots, indicated that compounds 1 and 2 were competitive inhibitors of diphenolase of mushroom tyrosinase. Notably, isolated compounds 1-8 were reported for the first time as constituents of F. erecta.