Antimicrobial activity in the common seawhip, Leptogorgia virgulata (Cnidaria: Gorgonaceae)

Antimicrobial activity was examined in the gorgonian Leptogorgia virgulata (common seawhip) from South Carolina waters. Extraction and assay protocols were developed to identify antimicrobial activity in crude extracts of L. virgulata. Detection was determined by liquid growth inhibition assays using Escherichia coli BL21, Vibrio harveyii, Micrococcus luteus, and a Bacillus sp. isolate. This represents the first report of antimicrobial activity in L. virgulata, a temperate/sub-tropical coral of the western Atlantic Ocean. Results from growth inhibition assays guided a fractionation scheme to identify active compounds. Reverse-phase HPLC, HPLC-mass spectrometry, and 1H and 13C NMR spectroscopy were used to isolate, purify, and characterize metabolites in antimicrobial fractions of L. virgulata. Corroborative HPLC-MS/NMR evidence validated the presence of homarine and a homarine analog, well-known emetic metabolites previously isolated from L. virgulata, in coral extracts. In subsequent assays, partially-purified L. virgulata fractions collected from HPLC-MS fractionation were shown to contain antimicrobial activity using M. luteus and V. harveyii. This study provides evidence that homarine is an active constituent of the innate immune system in L. virgulata. We speculate it may act synergistically with cofactors and/or congeners in this octocoral to mount a response to microbial invasion and disease.     

Sensitive fluorescence HPLC assay for AQ-13, a candidate aminoquinoline antimalarial, that also detects chloroquine and N-dealkylated metabolites

A sensitive, specific and reproducible fluorescence high performance liquid chromatography (HPLC) assay has been developed for the separate or simultaneous measurement of AQ-13 (a candidate 4-aminoquinoline antimalarial), chloroquine (CQ), and their metabolites in whole blood. After liquid-solid extraction using commercially available extraction cartridges, these two aminoquinolines (AQs) and their metabolites were separated on C18 (Xterra RP18) columns using a mobile phase containing 60% borate buffer (20 mM, pH 9.0) and 40% acetonitrile with isocratic elution at a flow-rate of 1.0 ml/min. The assay uses a biologically inactive 8-chloro-4-aminoquinoline (AQ-18) as its internal standard (IS). There is a linear relationship between the concentrations of these AQs and the peak area ratio (ratio between the peak area of the AQ or metabolite and the peak area of the IS) on the chromatogram. Linear calibration curves with correlation coefficients > or = 0.997 (r2 > or = 0.995, p < 0.001) were obtained for AQ-13, CQ and their N-dealkylated metabolites. Reproducibility of the assay was excellent with coefficients of variation (CVs) < or = 3.8% for AQ-13 and its metabolites, and < or =2.5% for CQ and its metabolites. The sensitivity of the assay is 5 nM using 1.0 ml of blood and a 20 microl injection volume, and can be increased by using 5.0 ml of blood with an injection volume of 40 microl.     

Fluorometric high-performance liquid chromatographic analysis of 10-deazaaminopterin, 10-ethyl-10-deazaaminopterin, and known metabolites

The antifolate compounds 10-deazaaminopterin (10-dAM) and 10-ethyl-10-deazaaminopterin (10-EdAM) are therapeutically superior to methotrexate in transplanted murine tumor systems and in human tumor xenografts growing in immunodeficient "nude" mice. The increased therapeutic index of these analogs correlates with their selective uptake, retention, and polyglutamation within neoplastic cells. We have developed a fluorescence high-performance liquid chromatographic assay applicable to 10-dAM, 10-EdAM, their polyglutamate anabolites, and their 7-hydroxy (7-OH) and deglutamate catabolites. The assay is based upon the high native fluorescence of pteridine-containing compounds which contain carbon in the 10 position. The assay employs a reverse-phase C-18 column and an ascending acetonitrile gradient in 50 mM phosphate, pH 7.0. The compounds are extracted from plasma and urine with 95 +/- 7% and 98 +/- 2% recoveries, respectively, using C-18 Sep-Paks. The linear range of the assay is, for 10-dAM, 2-100 nM, and for 10-EdAM, 1-100 nM. Polyglutamated metabolites of [3H]10-EdAM isolated from L1210 cells have been separated by HPLC with identification of five derivatives (Glu 1-5) confirmed by enzymatic peak shift using serum conjugase and by quantitative correlation of fluorescence intensity, radioactivity, and titration inhibition of dihydrofolate reductase. The assay has been used successfully in pharmacokinetic analyses of plasma and urine samples from patients receiving 10-dAM and 10-EdAM. In patients who had received 10-EdAM, 7-OH-10-EdAM, and the deglutamate catabolite were also detected. This HPLC fluorescence assay is superior to the dihydrofolate reductase inhibition and binding assays with regard to specificity and precision; moreover, it can provide a means for simultaneous assay of the physiologically important anabolites and catabolites of these new antifolates.     

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.     

Resveratrol as a kcat type inhibitor for tyrosinase: potentiated melanogenesis inhibitor

Resveratrol exhibited the inhibitory activity against mushroom tyrosinase (EC1.14.18.1) through a k(cat) inhibition. Resveratrol itself did not inhibit tyrosinase but rather was oxidized by tyrosinase. In the enzymatic assays, resveratrol did not inhibit the diphenolase activity of tyrosinase when l-3,4-dihydroxyphenylalanin (L-DOPA) was used as a substrate; however, L-tyrosine oxidation by tyrosinase was suppressed in presence of 100 μM resveratrol. Oxidation of resveratrol and inhibition of L-tyrosine oxidation suggested the inhibitory effects of metabolites of resveratrol on tyrosinase. After the 30 min of preincubation of tyrosinase and resveratrol, both monophenolase and diphenolase activities of tyrosinase were significantly suppressed. This preincubational effect was reduced with the addition of L-cysteine, which indicated k(cat) inhibition or suicide inhibition of resveratrol. Furthermore, investigation was extended to the cellular experiments by using B16-F10 murine melanoma cells. Cellular melanin production was significantly suppressed by resveratrol without any cytotoxicity up to 200 μM. trans-Pinosylvin, cis-pinosylvin, dihydropinosylvin were also tested for a comparison. These results suggest that possible usage of resveratrol as a tyrosinase inhibitor and a melanogenesis inhibitor.     

Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid

This study describes a fluorescent HPLC assay for measuring 20-hydroxyeicosatetraenoic acid (20-HETE) and other cytochrome P-450 metabolites of arachidonic acid in urine, tissue, and interstitial fluid. An internal standard, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, was added to samples, and the lipids were extracted and labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. P-450 metabolites were separated on a C18 reverse-phase HPLC column. Coelution and gas chromatography-mass spectrometry studies confirmed the identity of the 20-HETE peak. The 20-HETE peak can be separated from those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatrienoic acids. Known amounts of 20-HETE were used to generate a standard curve (range 1-10 ng, r(2) = 0.98). Recovery of 20-HETE from urine averaged 95%, and the intra-assay variation was <5%. Levels of 20-HETE were measured in 100 microliter of urine and renal interstitial fluid or 0.1 mg of renal tissue. The assay was evaluated by studying the effects of 1-aminobenzotriazole (ABT) on the excretion of 20-HETE in rats. ABT reduced excretion of 20-HETE by >65% and inhibited the formation of 20-HETE by renal microsomes. The availability of this assay should facilitate work in this field.     

高速逆流色谱法分离凤尾草中的芹菜素和木犀草素

建立了高速逆流色谱分离纯化芹菜素和木犀草素的方法。两相溶剂系统为氯仿-甲醇-水（4：3：2）,上相为固定相,下相为流动相进行洗脱。从100mg粗提物中一步分离得到17．0mg芹菜素和22．5mg木犀草素,经高效液相色谱分析,纯度分别为92％和96％。其化学结构由1H和13CNMR鉴定。     

Quantitative analysis of mouse tyrosinase by enzyme-linked immunosorbent assay

A sensitive, specific, competitive enzyme-linked immunosorbent assay (ELISA) was developed for quantitative analysis of tyrosinase. Binding sites of anti-tyrosinase antibodies were competed for by purified tyrosinase adsorbed onto microtiter plates and a known (standard) or unknown (sample) amount of tyrosinase in solution. Adsorbed antibodies were detected by goat anti-rabbit IgG F(ab')2 labeled with peroxidase. A sensitivity range of 2.1 to 14 ng (30-200 fmol)/well was obtained. SDS was found to be the most suitable detergent for solubilizing the enzyme. Tyrosinase was extracted from B16 mouse melanoma and assayed by the ELISA. The tyrosinase content per mg melanoma protein was 505 +/- 106 (S.D.) ng. This assay is not only useful for measuring the content of normal tyrosinase in crude extracts but also is possibly applicable to detecting the unprocessed tyrosinases.     

陕西秦艽药源植物及秦艽药材HPLC的比较研究

目的:系统比较研究秦艽药材与其药原植物间主要化学成分的差异。方法:高效液相色谱法,色谱柱:Diamonsil C18(200 mm×4.6 mm,5um);洗脱条件:乙腈-水溶液梯度洗脱;检测波长:240 nm;柱温:30℃;流速:0.5 mL.min-1。结果:陇县、太白秦艽中龙胆苦苷含量明显高于市售秦艽药材;通过确定7个共有峰建立陕西秦艽HPLC指纹图谱,所测定的29批秦艽药材具有很高的相似度,而秦艽伪品相似度较低。采用HPLC各峰面积及其相对之比进行聚类分析结果显示,采自太白的10个样本和陇县的8个样本分别聚为两支,表明秦艽化学成分含量与其地理分布存在密切关系。结论:本实验建立的指纹图谱可用于秦艽及其药材鉴别和质量控制;聚类分析可以鉴别秦艽产地。     

金黄色葡萄球菌拮抗菌株的筛选鉴定及其抗菌物质分析

【背景】植物内生菌的次生代谢产物是新型天然活性物质的重要来源。【目的】从芍药内生细菌中筛选对金黄色葡萄球菌有抑菌活性的菌株和次生代谢产物。【方法】采用平板对峙法筛选拮抗菌株,根据形态学特征和分子生物学的方法鉴定菌株,PCR扩增检测合成脂肽类物质的功能基因;运用牛津杯法依次测定内生细菌发酵液和脂肽类粗提物的抑菌活性,利用Sephadex LH-20凝胶层析分离脂肽类物质,利用基质辅助激光解吸电离飞行时间质谱分析具有抑菌作用的分离组分。【结果】共筛选出13株对金黄色葡萄球菌具有不同程度抑制作用的内生菌株,其中菌株SY11的抑菌作用最为显著,其发酵液和脂肽类粗提物均具有较强的抑制作用。结合形态学鉴定以及16S r RNA基因序列分析,鉴定其为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。PCR扩增检测表明菌株SY11含有3个合成脂肽类物质的功能基因fenA、ituD和srfkn,推测该菌株可能具有合成脂肽类物质的能力。根据具有抑菌活性分离组分的质谱分析结果,推测其有效物质的主要成分为Bacillomycin D。【结论】解淀粉芽孢杆菌SY11对金黄色葡萄球菌有良好抑制效果,其脂肽类粗提物也具有较强的体外抑菌活性。本研究为芍药内生细菌的开发应用奠定了基础。     

Antifungal and antibacterial activities of indigenous <Emphasis Type="Italic">Streptomyces</Emphasis> isolates from saline farmlands: prescreening,ribotyping and metabolic diversity

A culture collection of 110 indigenous Streptomyces strains originally isolated from saline farmlands (Punjab, Pakistan) using stringent methods was screened biologically and chemically to investigate their potential for the production of bioactive secondary metabolites. In a biological screening the crude extracts obtained from the culture broth of selected strains were analysed for their activity against a set of test organisms, including Gram-positive, Gram-negative bacteria, fungi and microalgae using the disk diffusion bioassay method. Additionally a cytotoxicity test was performed by means of the brine shrimp microwell cytotoxicity assay. In a chemical screening each of the crude extracts was analysed by TLC using various staining reagents and by HPLC-MS/MS measurements. The results depicted an impressive chemical diversity of crude extracts produced by these strains. The taxonomic status of the selected strains was confirmed by preliminary physiological testing and 16S rRNA gene sequencing.     

A sequential aerobic/microaerophilic decolorization of sulfonated mono azo dye Golden Yellow HER by microbial consortium GG-BL

This study is a part of efforts to develop new batch method with the help of prepared consortium GG-BL using two microbial cultures viz. Galactomyces geotrichum MTCC 1360 and Brevibacillus laterosporus NCIM 2298, varying oxidation conditions for the bio-treatment processes to produce reusable water by decolorization of Golden Yellow HER (GYHER) to less toxic metabolites. Consortium was found to be much faster for decolorization and degradation of GYHER as compared to the individual strains. The intensive metabolic activity of these strains led to 100% decolorization of GYHER (50 mg l⁻¹) within 24 h with significant reduction in chemical oxygen demand (84%) and total organic carbon (63%). The presence of veratryl alcohol oxidase, NADH-DCIP reductase and induction in laccase, tyrosinase, azo reductase and riboflavin reductase during decolorization suggests their role in decolorization process. Substrate staining of nondenaturing polyacrylamide electrophoresis gel (PAGE) also confirms induction of oxidative enzymes during GYHER degradation. The degradation of the GYHER into different metabolites by individual organism and in consortium was confirmed using High Performance Thin Layer Chromatography (HPTLC), High Performance Liquid Chromatography (HPLC), Fourier Transform Infra Red Spectroscopy (FTIR), Gas Chromatography Mass Spectroscopy (GC–MS) analysis. Phytotoxicity studies revealed nontoxic nature of the metabolites of GYHER.     

Casein fermentate of Lactobacillus animalis DPC6134 contains a range of novel propeptide angiotensin-converting enzyme inhibitors

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (+/-15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (+/-15), 83.71 (+/-19), and 42.36 (+/-11), respectively, where ACE inhibition was determined with 80 microl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from alpha-, beta-, and kappa-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to beta-casein f(73-89); peptide IGSENSEKTTMP, corresponding to alpha(s1)-casein f(201212); peptide SQSKVLPVPQ, corresponding to beta-casein f(166-175); peptide MPFPKYPVEP, corresponding to beta-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to beta-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 microM to 790 microM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.     

Applications of thin layer chromatography,high performance liquid chromatography and mass spectrometry in the fermentation and isolation of the antibiotic nybomycin

Summary Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrometry (MS) methods have been developed for the analysis of the antibiotic nybomycin, its derivatives deoxynybomycin and nybomycin acetate, during the fermentation and isolation of nybomycin. Using a quantitative HPLC based assay, the time course of nybomycin production (nybomycin titers) in 1000 liter fermentations was determined. Desorption chemical ionization mass spectrometry (DCI/MS) of standard nybomycin samples, fermentation broth samples and purified fractions suggested the co-production of deoxynybomycin which was not reported previously from this organism. TLC and HPLC were used to confirm the presence of deoxynybomycin in the crude extracts of fermentation broths.     

Biodegradation of Green HE4B: Co-substrate effect,biotransformation enzymes and metabolite toxicity analysis

A high exhaust reactive dye, Green HE4B (GHE4B) was 98% degraded in nutrient medium by Pseudomonas desmolyticum NCIM 2112 (pd2112) within 72 h at static condition. Decolorization time in synthetic 10 g/l molasses. Addition of 5 g/l peptone to NaCl medium had reduced decolorization time from 108 to 72 h. Beef extract do not contribute more to the inducing effect of peptone, however it is a good co-substrate in sucrose or urea containing NaCl medium. Intracellular lignin peroxidase (Lip), laccase and tyrosinase activities were induced by 150, 355 and 212%, respectively till maximum dye removal took place. Aminopyrine N-demethylase (AND) and dichlorophenol indophenol reductase (DCIP-reductase) activities in pd2112 were induced by 130 and 20%, respectively at 72 h of incubation during GHE4B decolorization. By high performance liquid chromatography (HPLC) analysis, 4-hydroxybenzene sulfonic acid and 4-amino, 6-hydroxynaphthalene 2-sulfonic acids were identified as metabolites formed during 24–72 h incubation. Fourier transform infrared spectroscopy (FTIR) analysis supports the formation of these aromatic amines. pd2112, aerobically degraded GHE4B metabolites (formed at static condition) showing stationary phase of 6 days. There was no germination inhibition of Sorghum bicolor and Triticum aestivum by GHE4B metabolites at 3,000 ppm concentration however untreated dye showed germination inhibition at the same concentration. GHE4B metabolites did not show any microbial toxicity at 10,000 ppm concentration.     

Characterization of tyrosinase inhibitory constituents from the aerial parts of Humulus japonicus using LC-MS/MS coupled online assay

In the screening of natural products for the development as cosmetic ingredients, the EtOAc-soluble fraction of Humulus japonicus showed tyrosinase inhibitory activity. HPLC-MS/MS coupled online tyrosinase assay of EtOAc-soluble fraction of H. japonicus characterized the twenty-eight constituents including two unknown ones and their tyrosinase inhibitory activity. Fractionation of H. japonicus using various chromatographic techniques yielded thirty-eight compounds. The chemical structures of isolated compounds were identified by spectroscopic analysis. As characterized by HPLC-MS/MS analysis, we isolated twenty-four predicted compounds and further identified two unknown ones, named humulusides A (1) and B (2). Additional ten compounds were also identified by purification. Tyrosinase inhibitory activity of isolated compounds were evaluated, which was closely correlated with the results from HPLC-MS/MS coupled online tyrosinase assay. Consistent with predicted data, two major compounds, trans-N-coumaroyltyramine (14) and cis-N-coumaroyltyramine (15) showed tyrosinase inhibition with IC₅₀ values of 40.6 and 36.4?μM. Taken together, H. japonicus is suggested as whitening ingredient in cosmetic products. In addition, HPLC-MS/MS coupled tyrosinase assay is powerful tool for predicting active compounds with short time and limited amounts, although identification of new compounds and verification of predicted data are also needs to be demonstrated by further experiment.     

Tyrosinase inhibition due to interaction of homocyst(e)ine with copper: the mechanism for reversible hypopigmentation in homocystinuria due to cystathionine beta-synthase deficiency.

Deficiency of cystathionine beta-synthase (CBS) is a genetic disorder of transsulfuration resulting in elevated plasma homocyst(e)ine and methionine and decreased cysteine. Affected patients have multisystem involvement, which may include light skin and hair. Reversible hypopigmentation in treated homocystinuric patients has been infrequently reported, and the mechanism is undefined. Two CBS-deficient homocystinuric patients manifested darkening of their hypopigmented hair following treatment that decreased plasma homocyst(e)ine. We hypothesized that homocyst(e)ine inhibits tyrosinase, the major pigment enzyme. The activity of tyrosinase extracted from pigmented human melanoma cells (MNT-1) that were grown in the presence of homocysteine was reduced in comparison to that extracted from cells grown without homocysteine. Copper sulfate restored homocyst(e)ine-inhibited tyrosinase activity when added to the culture cell media at a proportion of 1.25 mol of copper sulfate per 1 mol of DL-homocysteine. Holo-tyrosinase activity was inhibited by adding DL-homocysteine to the assay reaction mixture, and the addition of copper sulfate to the reaction mixture prevented this inhibition. Other tested compounds, L-cystine and betaine did not affect tyrosinase activity. Our data suggest that reversible hypopigmentation in homocystinuria is the result of tyrosinase inhibition by homocyst(e)ine and that the probable mechanism of this inhibition is the interaction of homocyst(e)ine with copper at the active site of tyrosinase.     

Design,synthesis and anti-melanogenic effect of cinnamamide derivatives

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25?µM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-β-phenyl-α,β-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.     

Inhibition of melanogenesis by dioctyl phthalate isolated from Nigella glandulifera Freyn

Although a number of melanogenesis inhibitors have recently been reported and used as cosmetic additives, none is completely satisfactory, leaving a need for novel skin-depigmenting agents. Thus, to develop a novel skin-depigmenting agent from natural sources, the inhibition of melanogenesis by Chinese plants was evaluated. A methanolic extract of Nigella glandulifera Freyn was found to inhibit the melanin synthesis of murine B16F10 melanoma cells by 43.7% and exhibited a low cytotoxicity (8.1 %) at a concentration of 100 microg/ml. Thus, to identify the melanogenesis-inhibiting mechanism, the inhibitory activity towards tyrosinase, the key enzyme of melanogenesis, was further evaluated, and the results showed inhibitory effects on the activity of intracellular tyrosinase yet not on mushroom tyrosinase. Finally, to isolate the compounds with a hypopigmenting capability, activity-guided isolation was performed, and Dioctyl phthalate identified as inhibiting melanogenesis.