Anti-tyrosinase and antimelanogenic effect of cinnamic acid derivatives from Prunus mahaleb L.: Phenolic composition, isolation, identification and inhibitory activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional use of Prunus species against skin diseases and especially for skin lightning cosmeceutical purposes is widespread in many cultures. Prunus mahaleb L. is a well known food plant and used in the baking industry for flavoring. The fruit kernels (endocarp) are used in India for hyperpigmentation. AIM OF THE STUDY: To investigate the chemical composition with the antimelanogenesis effect of P. mahaleb seed and kernel extracts and isolated compounds. MATERIALS AND METHODS: Isolation studies performed from the methanol extracts obtained from kernels and structures were determined using NMR and MS analysis. Antimelanogenesis effect was determined by mushroom tyrosinase assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells. RESULTS: Five cinnamic acid derivatives were isolated and their structures (2-O-ß-glucopyranosyloxy-4-methoxy-hydrocinnamic acid (1), cis-melilotoside (2), dihydromelilotoside (3), trans-melilotoside (4), 2-O-ß-glucosyloxy-4-methoxy trans-cinnamic acid (5)) were elucidated using advanced spectroscopic methods. Mushroom tyrosinase enzyme inhibition of extracts, fractions and pure compounds obtained from P. mahaleb kernels were investigated and structure-activity relationship revealed. According to a detailed, comprehensive and validated LC-MS/MS technique analysis, vanilic acid (41.407 mg/g), protocatechuic acid (8.992 mg/g) and ferulic acid (4.962 mg/g) in the kernel ethylacetate fraction; quinic acid (14.183 mg/g), fumaric acid (8.349 mg/g) and aconitic acid (5.574 mg/g) were found as major phenolic compounds in the water fraction. The correlation of trace element copper content in extracts and fractions with mushroom enzyme activity was determined. By examining the enzyme kinetics of the compounds with effective cinnamic acid derivatives, inhibition types and enzyme binding constants Ki were calculated. Compounds 1,3 and 5 exhibited high noncompetitive tyrosinase inhibitory activity against L-tyrosine substrates, with IC50 values of 0.22, 0.31 and 0.37 mM respectively. In addition compounds 1, 3 and 5 showed dose-dependent inhibitory effects on intracellular tyrosinase and melanin levels in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 melanoma cells. CONCLUSIONS: Potent tyrosinase inhibitory compounds and extracts of P. mahaleb kernels suggest that it could be a new, non-toxic and inexpensive resource for the cosmeceutical industry and in skin diseases associated with hyperpigmentation.

The effect of Aspalathus linearis (Burm.f.) R.Dahlgren and its compounds on tyrosinase and melanogenesis.

Pigmentation, a process controlled by melanogenesis, plays a vital role in protecting the skin against harmful ultraviolet rays. The level of protection is compromised in case of hypopigmentation. This study aimed to evaluate an Aspalathus linearis extract, fractions and phytoconstituents, for their efficacy on melanogenesis stimulation. Fifteen compounds were kinetically assessed against tyrosinase; the rate-limiting enzyme of melanogenesis. Aspalathin and catechin significantly (p value < 0.001) increased the enzymatic rate, showing 50% stimulatory effects at 119.70 ± 2.06 µg/mL and 143.30 ± 2.74 µg/mL, respectively, by acting as subversive substrates. Five compounds inhibited the enzyme's activity, of which four exhibited competitive inhibition. To investigate the molecular interactions between the compounds and the active site, molecular docking was done, using tyrosinase (PBD: 2Y9X) and tyrosinase related protein 1 (PBD: 5M8P). All the compounds docked successfully with acceptable docking scores. Further quantitative structure-activity relationship analysis identified potential functional groups, linked to the specific activity. The crude extract, its fractions, and compounds exhibited low antiproliferative activity with 50% cell viability at concentrations higher than 100 µg/mL. Finally, both aspalathin and catechin exhibited a significant increase (4.5%) in melanin production at 119.82 µg/mL and 76.92 µg/mL, respectively. This is the first report of A. linearis' compounds on skin re-pigmentation.

Halogenated aromatic thiosemicarbazones as potent inhibitors of tyrosinase and melanogenesis.

A set of 21 halogenated thiosemicarbazones (TSCs) have been synthesized and its inhibitory properties toward activity diphenolase of mushroom tyrosinase and their ability to inhibition of melanogenesis in B16F10 murine, melanoma cell line have been investigated. The molecular docking to the active site of the enzyme has been also performed to investigate the nature of enzyme-inhibitor interactions. The obtained outcomes allowed us to perform SAR analysis. TSC 6, 12 and 21 exhibited the most potent inhibitory properties showing IC50 of 0.5, 0.9 and 0.8 µM, respectively. They revealed reversible and competitive manner of tyrosinase inhibition. According to SAR analysis, para-substituted acetophenone derivatives of thiosemicarbazones have the highest affinity to the enzyme among the investigated compounds. Melanin production in B16F10 cells was inhibited by all investigated compounds at the micromolar level. Suggested inhibition mechanism is based on the interaction between a sulfur atom of thiourea moiety of the thiosemicarbazones, and copper ions in the active site of the enzyme. These results might be useful in searching novel inhibitors of melanogenesis which could be used in the cosmetic and food industry.

N-Alkylamides from Piper longum L. and their stimulative effects on the melanin content and tyrosinase activity in B16 melanoma cells.

Piper longum L., known as long pepper, is an edible and medicinal plant used as spice and for the treatment of stomach disease and analgesia in traditional Chinese medicine. N-Alkylamides are the major secondary metabolites in this plant. Sixteen known N-alkylamides were isolated from P. longum. Their structures were established on the basis of spectroscopic data and comparison to reported literatures. Among them, five compounds were isolated from this plant for the first time. Ethanol extract, compounds 1, 2, 3, 7 and 11 exhibited potent ability to increase the melanin content and weak stimulative effect on the tyrosinase activity in a concentration-dependent manner. Moreover, compound 2 also presented strong capacity to increase the tyrosinase activity in a concentration-dependent manner. These results indicated that P. longum might be a good natural source of lead compound for skin disorder diseases.

Pharmacological and polyphenolic profiles of Phyllanthus phillyreifolius var. commersonii Müll. Arg: An unexplored endemic species from Mauritius.

Phyllanthus phillyreifolius var. commersonii Müll. Arg is an endemic plant of Mauritius. To date, no study has been performed concerning its polyphenolic profile and pharmacological properties. In this study, a decoction (water), ethyl acetate and methanol extracts of the aerial parts of P. phillyreifolius, obtained from different extraction procedures (maceration and Soxhlet), were studied for antibacterial, antioxidant, anticancer, and enzyme inhibitory properties along with their polyphenolic profile. The ethyl acetate macerated extract showed high antibacterial activity against B. cereus (MIC = 0.293 mg/mL) and E. coli (MIC = 0.417 mg/mL) while S. epidermidis was most susceptible to the ethyl acetate-Soxhlet extract (MIC = 0.521 mg/mL). The methanol-Soxhlet extract displayed the most potent cupric and ferric reducing power, and metal chelating effect, while the macerated methanolic extract was the most effective DPPH and ABTS scavenger, and BChE inhibitor. Only the ethyl acetate-Soxhlet extract exhibited α-glucosidase inhibition. All extracts exhibited a strong anti-tyrosinase activity, which was further investigated by molecular docking and molecular dynamic. After 48 h exposure to the extracts for HeLa cell lines, the ethyl acetate-Soxhlet extract showed the highest inhibition (IC50 = 533.1 µg/mL) while the decoction extract was more cytotoxic to MDA-MB-231 cells (IC50 = 337.4 µg/mL). Treatment of cancer cell lines with all P. phillyreifolius extracts resulted in a time-dependent reduction of cell viability for HeLa and dose-and time-dependent reduction for MDA-MB-231. Gene expression ratio of Bcl-2 to Bax was higher for all Soxhlet-extracts. Total phenolics (TPC) and flavonoids (TFC) content were highest in the decoction and methanol-Soxhlet extract, respectively (122.43 mg GAE/g extract and 31.28 mg RE/g extract, respectively). The extracts were abundant in ellagitannins, although phenolic acids and flavonoids were also detected. Granatin B was detected for the first time in Phyllanthus species. Overall, the aerial parts of P. phillyreifolius exemplify a potent reservoir of bioactive phytochemicals for therapeutic applications.

l-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation.

BACKGROUND: The mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2(p)) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (p(cas)/Oca2(p-cas)) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. OBJECTIVE: The aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. METHODS: The characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). RESULTS: The proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. CONCLUSION: These results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines.

Activities of different types of Thai honey on pathogenic bacteria causing skin diseases, tyrosinase enzyme and generating free radicals.

BACKGROUND: Honey is a natural product obtained from the nectar that is collected from flowers by bees. It has several properties, including those of being food and supplementary diet, and it can be used in cosmetic products. Honey imparts pharmaceutical properties since it has antibacterial and antioxidant activities. The antibacterial and antioxidant activities of Thai honey were investigated in this study. RESULTS: The honey from longan flower (source No. 1) gave the highest activity on MRSA when compared to the other types of honey, with a minimum inhibitory concentration of 12.5% (v/v) and minimum bactericidal concentration of 25% (v/v). Moreover, it was found that MRSA isolate 49 and S. aureus were completely inhibited by the 50% (v/v) longan honey (source No. 1) at 8 and 20 hours of treatment, respectively. Furthermore, it was observed that the honey from coffee pollen (source No. 4) showed the highest phenolic and flavonoid compounds by 734.76 mg gallic/kg of honey and 178.31 mg quercetin/kg of honey, respectively. The antioxidant activity of the honey obtained from coffee pollen was also found to be the highest, when investigated using FRAP and DPPH assay, with 1781.77 mg FeSO4•7H2O/kg of honey and 86.20 mg gallic/kg of honey, respectively. Additionally, inhibition of tyrosinase enzyme was found that honey from coffee flower showed highest inhibition by 63.46%. CONCLUSIONS: Honey demonstrates tremendous potential as a useful source that provides anti-free radicals, anti-tyrosinase and anti-bacterial activity against pathogenic bacteria causing skin diseases.

Activities of different types of Thai honey on pathogenic bacteria causing skin diseases, tyrosinase enzyme and generating free radicals

BACKGROUND: Honey is a natural product obtained from the nectar that is collected from flowers by bees. It has several properties, including those of being food and supplementary diet, and it can be used in cosmetic products. Honey imparts pharmaceutical properties since it has antibacterial and antioxidant activities. The antibacterial and antioxidant activities of Thai honey were investigated in this study. RESULTS: The honey from longan flower (source No. 1) gave the highest activity on MRSA when compared to the other types of honey, with a minimum inhibitory concentration of 12.5% (v/v) and minimum bactericidal concentration of 25% (v/v). Moreover, it was found that MRSA isolate 49 and S. aureus were completely inhibited by the 50% (v/v) longan honey (source No. 1) at 8 and 20 hours of treatment, respectively. Furthermore, it was observed that the honey from coffee pollen (source No. 4) showed the highest phenolic and flavonoid compounds by 734.76 mg gallic/kg of honey and 178.31 mg quercetin/kg of honey, respectively. The antioxidant activity of the honey obtained from coffee pollen was also found to be the highest, when investigated using FRAP and DPPH assay, with 1781.77 mg FeSO4•7H2O/kg of honey and 86.20 mg gallic/kg of honey, respectively. Additionally, inhibition of tyrosinase enzyme was found that honey from coffee flower showed highest inhibition by 63.46%. CONCLUSIONS: Honey demonstrates tremendous potential as a useful source that provides anti-free radicals, anti-tyrosinase and anti-bacterial activity against pathogenic bacteria causing skin diseases.

Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice.

BACKGROUND: Many natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet. METHODS: In the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin. RESULTS: The result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis. CONCLUSION: The present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study.

Ssanghwa-tang, an oriental herbal cocktail, exerts anti-melanogenic activity by suppression of the p38 MAPK and PKA signaling pathways in B16F10 cells.

BACKGROUND: Ssanghwa-tang (SHT) is a widely used medication for the treatment of fatigue, pain, inflammation, hypothermia, erectile dysfunction, cancer, and osteoporosis in Asia, however, role of SHT on the melanin synthesis has not been checked previously. Thus, the present study was designed to determine the effect of SHT on α-melanocyte stimulating hormone (α-MSH)-induced melanogensis and its mechanisms of action in murine B16F10 melanoma cells. METHOD: Cellular melanin content and tyrosinase activity in murine B16F10 melanoma cells were determined after α-MSH stimulation with or without pre-treatment of SHT at the concentration of 250 and 500 µg/ml. Expression level of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, microphthalmia-associated transcription factor (MITF), and activation of c-AMP-dependent protein kinase (PKA), c-AMP-related element binding protein (CREB), and mitogen-activated protein kinases (MAPKs) were examined by Western blot analysis. RESULTS: SHT significantly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of MITF, tyrosinase, and TRP-1. In addition, SHT remarkably suppressed tyrosinase, CRE, and MITF luciferase reporter activity in a resting state as well as in α-MSH-stimulating condition. Phosphorylation of p38 MAPK by α-MSH stimulation was efficiently blocked by SHT pre-treatment. Moreover, SHT as an herbal cocktail showed synergistic anti-melanogenic effect compared with that of each single constituent herb. CONCLUSION: SHT efficiently inhibited c-AMP-induced melanin synthesis in B16F10 cells via suppression of PKA and p38 MAPK signaling pathways and subsequently decreased the level of CREB phosphorylation, MITF, and melanogenic enzymes. These results indicate that SHT may be useful as herbal medicine for treating hyperpigmentation and cosmetics as a skin-whitening agent.

Inhibitory effects of salidroside and paeonol on tyrosinase activity and melanin synthesis in mouse B16F10 melanoma cells and ultraviolet B-induced pigmentation in guinea pig skin.

Salidroside, the major active component of Rhodiola rosea, a herb with antioxidant, free radical scavenging and tyrosinase inhibitory effects, has been recently reported in protecting the kerationcytes from the UV radiation, suggesting the potential of this component in depigmentation. Paeonol is isolated from Moutan Cortex Radicis with anti-inflammation/microbial activities, was reported to induce the down-regulation of microphthalmia-associated transcription factor and subsequently tyrosinase. To testify the potential of these compounds as melanin formation inhibitors for hyperpigmentation therapy, the influence of salidroside and paeonol on pigmentation was investigated. With arbutin as a positive control, salidroside and paeonol were evaluated for their inhibitory effect on the cell viability, tyrosinase activity and melanin synthesis in B16F10 melanoma cells, as well as their effects in UVB-induced hyperpigmentation in brown guinea pig skins. It was demonstrated that the significant inhibition of salidroside (33.0%) and paeonol (22.2-30.9%) on the tyrosinase activity is slightly lower than that of arbutin (18.4-44.7%). However, salidroside exhibited the dose-dependent inhibition (30.6-42.0%) in melanin synthesis at a low concentration of 100 µM, paeonol and arbutin expressed inhibition rates of 27.4-37.2% and 25.8-45.6% within 500-1000 µM. The in vivo topical application of these compounds was demonstrated to obviously decrease the hyperpigmentation on UVB stimulated guinea pig skin. This study provided the original evidence for the salidroside and paeonol as therapeutic agents for pigmentation disorder and skin lightening, with further clinical investigation of these compounds in the field of depigmentation was suggested.

Dodoviscin a inhibits melanogenesis in mouse b16-f10 melanoma cells.

Nowadays, abnormal hyperpigmentation in human skin such as melasma, freckles, and chloasma has become a serious esthetic problem. Cutaneous depigmenting agents could be used to treat these hyperpigmentation-associated dieseases. Dodoviscin A is a natural product isolated from the aerial parts of Dodonaea viscosa. In the present study, we evaluated the effect of dodoviscin A on melanin production in B16-F10 melanoma cells for the first time. We found that dodoviscin A inhibited melanin biosynthesis induced by 3-isobutyl-1-methylxanthine and PD98059 significantly, and there was no obvious effect on the viability of dodoviscin A-treated B16-F10 cells. Meanwhile, dodoviscin A could suppress the activity of mushroom tyrosinase in the cell-free assay system and also decrease 3-isobutyl-1-methylxanthine-induced tyrosinase activity and expression of mature tyrosinase protein in B16-F10 cells. Western blotting analysis showed that dodoviscin A inhibited 3-isobutyl-1-methylxanthine and forskolin-induced phosphorylation of the cAMP response element binding protein in B16-F10 cells. These results indicate that dodoviscin A may be a new promising pigmentation-altering agent for cosmetic and therapeutic applications.

Suppression of α-MSH and IBMX-induced melanogenesis by cordycepin via inhibition of CREB and MITF, and activation of PI3K/Akt and ERK-dependent mechanisms.

Cordycepin has been a traditional medicine in China and Korea for centuries. This study explored the inhibitory effect of cordycepin on melanogenesis and the relative molecular mechanisms. Cordycepin inhibited melanin synthesis-related enzymes, such as tyrosinase, tyrosinase-related protein-1 (TRP1) and tyrosinase-related protein-2 (TRP2). α-MSH and IBMX were reported as melanin synthesis enhancers. Both of them could increase intracellular melanin synthesis by activation of the microphthalmia-associated transcription factor (MITF) signaling pathway. In the MITF pathway, the phosphorylation of cAMP related binding protein (CREB) activated the transcription of MITF, resulting in increasing melanin synthesis. Cordycepin also decreased the phosphorylation of CREB induced by α-MSH and IBMX in B16F10 melanoma cells. Accordingly, cordycepin inhibited melanogenesis signaling pathways by activating ERK and AKT signaling pathways to regulate the suppression of MITF and its downstream pathways including tyrosinase, TRP1 and TRP2. These results indicate the role of cordycepin as a potent depigmenting agent for cosmetics.

[Effect of zibuganshenfang on the expression and activity of tyrosinase on B16 cells].

OBJECTIVE: ZiBuGanShenFang (ZBGSF) is a traditional herbal formula, which has showed an outstanding therapeutic effect on vitiligo clinically. Our aim is to investigate the influence of ZBGSF drug serum on the expression and activity of Tyrosinase in B16 cells, explore the mechanism of ZBGSF on Vitiligo treatment further. METHOD: tyrosinase activity was measured by zymological methods, western blotting and RT-PCR were used to measure the protein content and mRNA level of tyrosinase and related proteins, respectively. RESULT: ZBGSF drug serum had no effect on the proliferation of B16 cells. But it could promote Tyrosinase activity significantly and increase protein content and mRNA level of tyrosinase and related proteins in B16 cells. CONCLUSION: Promoting the expression of tyrosinase protein and mRNA may be the elementary basis of ZBGSF on Vitiligo treatment.

Inhibitory effect of Cucumis sativus on melanin production in melanoma B16 cells by downregulation of tyrosinase expression.

We compared the inhibitory effects on melanogenesis of six plant parts (leaves, stems, roots, whole fruits, calyxes, and fruits without calyxes) of Cucumis sativus. MeOH extracts of leaves and stems inhibited melanin production in B16 cells. These extracts did not affect the activity of mushroom tyrosinase or crude enzyme lysate from B16 cells. However, the extracts decreased tyrosinase expression at the protein level. These results suggest that the depigmenting mechanism of extracts from leaves and stems of C. SATIVUS involves the expression of tyrosinase. Of eight compounds isolated from the leaves, lutein ( 1) (IC (50) = 170.7 microM) and (+)-(1 R,2 S,5 R,6 S)-2,6-di-(4'-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane ( 2) (IC (50) = 270.8 microM) were found to suppress melanogenesis. Whereas 1 was found to markedly decrease the expression levels of tyrosinase, 2 only weakly reduced tyrosinase expression. This suggests that 1 is an active component in the leaves of C. sativus and is a potentially useful skin-whitening agent.

Melanogenesis inhibition by an oolong tea extract in b16 mouse melanoma cells and UV-induced skin pigmentation in brownish guinea pigs.

To investigate the new physiological functions of oolong tea, the effects on melanogenesis were studied. An oolong tea extract inhibited melanogenesis without affecting cell growth in B16 mouse melanoma cells. However, the oolong tea extract hardly showed any inhibitory effect on mushroom tyrosinase in a cell-free system. The effects of an oolong tea extract on the intracellular tyrosinase level in B16 cells were therefore studied. All the levels of activity, protein and mRNA were decreased in the oolong tea extract-treated cells. We also investigated the inhibitory effects of oolong tea on the pigmentation induced by ultraviolet B (UVB) by using brownish guinea pigs in vivo. The number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes increased by UVB was repressed by an oral administration of oolong tea. These results imply that oolong tea might be effective in whitening and that its inhibitory effect on melanogenesis was involved in the decrease of intracellular tyrosinase at the mRNA level.

The effect of Sargassum fusiforme polysaccharide extracts on vibriosis resistance and immune activity of the shrimp, Fenneropenaeus chinensis.

Immunostimulants are valuable for control of shrimp diseases and the immunostimulatory effects of some polysaccharide additives for shrimp have been reported. In this study, the Sargassum fusiforme polysaccharide extract (SFPSE) was assessed as a feed additive when supplemented in the diet (0%, 0.5%, 1.0%, and 2.0%) for juvenile shrimp, Fenneropenaeus chinensis, in order to study the effects of SFPSE on vibriosis resistance and immune activity. Shrimp were cultured in the same pond with cages. The body weight, survival, the cumulative mortality after injection with Vibrio harveyi (30 microl V. harveyi suspension at 9.3 x 10(7) CFU ml(-1) per shrimp), the total haemocyte counts (THCs), the protein concentration and the phenoloxidase (PO) activity in supernatant of haemolymph, the lysozyme (LSZ) and superoxide dismutase (SOD) activity in muscle of the shrimp were assayed after 14 days feeding period. The results indicated that shrimp survival under the stress of V. harveyi was affected by the dietary SFPSE. The shrimp treated with 1.0% and 0.5% SFPSE displayed significantly lower cumulative mortalities after being injected with V. harveyi suspension 24 and 30 h later, respectively, compared with that of the control. However, cumulative mortality of 2.0% SFPSE treatment was not significantly different from that of the control. There was no significant difference of cumulative mortality between 0.5% and 1.0% SFPSE treatment groups. The immune activities of the shrimp also were affected by dosage of dietary SFPSE. The THCs of the shrimp rose with increasing SFPSE dosage. The protein concentration and PO activity in supernatant of haemolymph as well as muscular LSZ activity first rose then dropped with increasing SFPSE dosage. The protein concentration in supernatant of haemolymph appeared a maximum of 167.46 mg ml(-1) in 1.0% SFPSE treatment. The PO activity and LSZ activity reached the peaks as 13.20 U and 3.21 U mgprot(-1) in 0.5% SFPSE treatment, respectively. SOD activity of the shrimp was not significantly affected by dietary SFPSE. It is therefore suggested that oral administration of SFPSE at an optimal level of 0.5% and 1.0% for 14 days effectively improved vibriosis resistance and enhanced immune activity of the shrimp in general.

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.

In vitro anti-tyrosinase activity of 5-(hydroxymethyl)-2-furfural isolated from Dictyophora indusiata.

The inhibition of mushroom tyrosinase by methanolic extract of Dictyophora indusiata was evaluated and the bioactive component was characterized and identified as 5-(hydroxymethyl)-2-furfural (HMF) by chromatographic and spectroscopic means. Kinetic studies revealed it to be a noncompetitive inhibitor for the oxidation of L-DOPA. On the basis of these findings some related analogues were also tested for their anti-tyrosinase activity, in order to gain more insight into structure and activity relationship among these heterocyclic compounds.

Structural criteria for depigmenting mechanism of arbutin.

Arbutin, hydroquinone-O-beta-D-glucopyranoside (1) was found to inhibit the oxidation of l-tyrosine (monophenolase activity) catalyzed by mushroom tyrosinase. However, arbutin itself was oxidized as a monophenol substrate at an extremely slow rate, and this oxidation was accelerated as soon as catalytic amounts (0.01 mM) of l-3,4-dihydroxyphenylalanine (L-DOPA) became available as a cofactor. The result observed was supported by monitoring oxygen consumption. The depigmenting mechanism of arbutin previously reported is supportable if a cofactor is not available in the melanocytes. The combination with L-ascorbic acid is a useful application, particularly when oxygen is limited.