Complete NMR assignments of undegraded asterosaponins from Asterias amurensis.

Four asterosaponins, thornasteroside A (1), versicoside A (2), anasteroside B (3), and asteronylpentaglycoside sulfate (4), were isolated from the predatory starfish Asterias amurensis Lütken. Unlike previous studies focusing on structure elucidation by degradation of the complex saponin molecules, complete nuclear magnetic resonance (NMR) assignment for the intact molecules was accomplished using 600 MHz high magnetic field NMR. The complete set of NMR assignments can help in the structure elucidation of asterosaponins isolated in low yields without resorting to chemical degradation. Furthermore, this approach can be extended to other complex steroidal saponins, which may accelerate the discovery of bioactive secondary metabolites from this invasive starfish species.

Illudins C2 and C3 stimulate lipolysis in 3T3-L1 adipocytes and suppress adipogenesis in 3T3-L1 preadipocytes.

The secondary metabolites illudins C2 (1) and C3 (2), obtained from the culture broth of Coprinus atramentarius, have been shown to possess antimicrobial activity. In the present study, we discovered novel biological activities of 1 and 2 in lipolysis of differentiated 3T3-L1 adipocytes and adipogenesis of 3T3-L1 preadipocytes. Compounds 1 and 2 exhibit a dose-dependent increase in glycerol release and thereby reduce intracellular lipid accumulation. The stimulatory effects of 1 and 2 on lipolysis are prevented by cAMP-dependent protein kinase (PKA) and extracellular signal-regulated kinase (ERK) inhibitors. Compounds 1 and 2 down-regulated perilipin and also affected the mRNA and protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). However, 1 and 2 treatment leads to a significant increase in PKA-mediated phosphorylation of HSL at S563 and S660. In addition, 1 and 2 treatment in 3T3-L1 preadipocytes induces down-regulation of the critical transcription factors, CCAAT/enhancer binding protein α and ß (C/EBPα and C/EBPß), and peroxisome proliferator activated receptor γ (PPARγ), which are required for adipogenesis, and accordingly inhibits adipogenesis. These results suggest that 1 and 2 might be useful for treating obesity due to their modulatory effects on fat by affecting adipocyte differentiation and fat mobilization.

Boseongazepines A-C, pyrrolobenzodiazepine derivatives from a Streptomyces sp. 11A057.

Three new pyrrolobenzodiazepine derivatives, boseongazepines A-C (1-3), were isolated from a culture broth of Streptomyces sp. 11A057, together with the known compound usabamycin B (4). The structures of 1-4 were determined through the analysis of spectroscopic data including extensive 1D-, 2D-NMR, and MS techniques. Cell growth inhibition effects of these compounds were evaluated against Jurkat, K-562, HL-60, and HepG2 cell lines.

Hypo-pigmenting effect of sesquiterpenes from Inula britannica in B16 melanoma cells.

During the course of screens to identify anti-melanogenic agents from natural resources, we found that the methanol extract of the dried flower of Inula britannica L. inhibited melanin synthesis in cultured melanoma cells stimulated with 3-isobutyl-1-methylxanthine (IBMX). A bioassay-guided isolation of the chloroform fraction of the I. britannica using an in vitro melanogenesis inhibition assay led to the isolation of sesquiterpenes, 1-O-acetylbritannilactone (1), britannilactone (2) and neobritannilactone B (3). Compounds 1 and 2 significantly reduced melanin production in a dose-dependent manner with IC50 values of 13.3 and 15.5 µM, respectively, whereas compound 3 was found to be cytotoxic. Compound 1 also inhibited the tyrosinase activity only in cell based-systems. Western blot analysis indicated that compound 1 inhibited melanogenesis by activating extracellular signal-regulated kinase (ERK) and Akt signaling and also inhibiting cAMP related binding protein, which regulates its downstream pathway, including tyrosinase, tyrosinase related protein-1 and TRP-2. These results demonstrated that compound 1, a major sesquiterpene from the flowers of I. britannica, exhibited anti-melanogenic activity by suppression of tyrosinase expression via ERK and Akt signaling. Taken together, our results suggest that these compounds may act as potent natural skin-lightening agents.

Inhibitory effect of melanogenesis by 5-pentyl-2-furaldehyde isolated from Clitocybe sp.

In the continued search for melanogenesis inhibitors from microbial metabolites, we found that the culture broth of Clitocybe sp. MKACC 53267 inhibited melanogenesis in B16F10 melanoma cells. The active component was purified by solvent extraction, silica gel chromatography, Sephadex LH-20 column chromatography, and finally by preparative HPLC. Its structure was determined as 5- pentyl-2-furaldehyde on the basis of the UV, NMR, and MS spectroscopic analysis. The 5-pentyl-2-furaldehyde potently inhibited melanogenesis in B16F10 cells with an IC50 value of 8.4 microgram/ml, without cytotoxicity.

Eudesmanolides from Taraxacum mongolicum and their inhibitory effects on the production of nitric oxide.

A new eudesmanolide, 1ß,3ß-dihydroxy-eudesman-11(13)-en-6α,12-olide (1) was isolated and identified from Taraxacum mongolicum, together with two known compounds, 1ß,3ß-dihydroxyeudesman-6α,12-olide (2) and loliolide (3). The structure of 1 was established by analysis of its physical and spectroscopic data. 1 was found to have an inhibitory activity on nitric oxide production with an IC(50) of 38.9 µM in activated RAW 264.7 cells.

Synthesis of (S)-(+)-decursin and its analogues as potent inhibitors of melanin formation in B16 murine melanoma cells.

We report the synthesis of a novel series of highly potent melanin inhibitors which were obtained through structural modification of an anticancer compound S-(+)-decursinol. The in vitro inhibitory potencies of the newly synthesized compounds were evaluated against α-MSH induced melanin production in B16 murine melanoma cells. Among the compounds evaluated, compounds 2, 3, 6b, 7a, 7b, 8a and 8b emerged as highly potent inhibitors of melanin production. Besides, these compounds demonstrated significantly low cytotoxicity.

Hypopigmentary action of dihydropyranocoumarin D2, a decursin derivative, as a MITF-degrading agent.

In this study, the decursin derivative dihydropyranocoumarin D2 (1) was selected for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). The results showed that 1 effectively inhibited melanin synthesis in a concentration-dependent manner, but that it did not inhibit tyrosinase in a cell-free system. In addition, the changes in ERK, Akt, and microphthalmia-associated transcription factor (MITF) in response to treatment with 1 were assessed. The results revealed that ERK was dramatically up-regulated and MITF was down-regulated in response to treatment with 1, but that Akt was unchanged. Therefore, the effects of 1 on melanogenesis were examined in the absence or presence of PD98059 (a specific inhibitor of the ERK pathway). PD98059 restored hypopigmentation and the down-regulation of MITF induced by 1. Finally, MITF down-regulation by 1 was clearly restored by both chloroquine, a lysosomal proteolysis inhibitor, and MG132, a proteasome inhibitor.

Two acetylated megastigmane glycosides from the leaves of Ilex paraguariensis.

Two acetylated megastigmane glycosides, matenosides A (1) and B (2), have been isolated from the MeOH extract of Ilex paraguariensis leaves, and their structures were elucidated on the basis of spectroscopic analysis. Compounds 1 and 2 exhibited human neutrophil elastase (HNE) inhibitory activity with IC(50) values of 50.4 muM and 11.1 microM, respectively.

Secondary metabolites of Volvariella bombycina and their inhibitory effects on melanogenesis.

Four compounds were isolated from the broth culture of Volvariella bombycina and they were identified as ergosta-4,6,8(14),22-tetraene-3-one (1), ergosterol peroxide (2), indole-3-carboxaldehyde (3) and indazole (4) by interpretation of spectroscopic data. Among them, compound 2 exhibited melanogenesis inhibitory effect in cultured B16 mouse melanoma cells.

Evaluation of human neutrophil elastase inhibitory effect of iridoid glycosides from Hedyotis diffusa.

Five iridoid glycosides were isolated from the MeOH extract of Hedyotis diffusa, and their structures were elucidated as E-6-O-p-methoxycinnamoyl scandoside methyl ester (1), Z-6-O-p-methoxycinnamoyl scandoside methyl ester (2), E-6-O-p-feruloyl scandoside methyl ester (3), E-6-O-p-coumaroyl scandoside methyl ester (4), and Z-6-O-p-coumaroyl scandoside methyl ester (5) by interpretation of their spectroscopic data. All the isolated compounds were evaluated for human neutrophil elastase inhibitory effect, and compound 1 showed potent activity with an IC(50) value of 18.0muM. The molecular docking simulation suggested a structural model for the inhibition of human neutrophil elastase by compound 1.

Clitocybin D, a novel human neutrophil elastase inhibitor from the culture broth of Clitocybe aurantiaca.

Clitocybin D, a novel human neutrophil elastase inhibitor, was isolated from the culture broth of Clitocybe aurantiaca. This compound was purified by solvent extraction, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. The compound was determined to be 4-(4,6-dihydroxy-3-methoxy-3H-isoindol-1-yl)-benzoic acid on the basis of 1D and 2D NMRs and MS spectroscopic analysis. Analysis of the human neutrophil elastase (HNE) inhibitory activity of the isolated compound revealed that it showed significant HNE inhibitory activity with an IC(50) value of 17.8 micronM.

Chemical constituents from the leaves of Ilex paraguariensis inhibit human neutrophil elastase.

Human neutrophil elastase (HNE), a serine protease with broad target specificity, is the only enzyme responsible for the degradation of elastin which is an insoluble elastic fibrous protein in animal connective tissue. Biologically, elastase activity significantly increased with age, which results in a reduced skin elasticity and in the appearance of wrinkles or stretchmarks. In the course of our screening program for HNE inhibitors from natural source, the MeOH extract of Ilex paraguariensis leaves showed strong HNE inhibitory effect. Bioassay-guided fractionation led to the isolation of a new pyrrole alkaloid (1), along with seventeen known compounds (2-18) from the MeOH extract of Ilex paraguariensis leaves, and their chemical structures were elucidated on the basis of spectroscopic analysis. All isolated compounds were evaluated for HNE inhibitory activity, and the result demonstrated that dicaffeoylquinic acid derivatives (12, 13, 14, 15 and 16) and flavonoids (8 and 17) exhibited potent HNE inhibitory activity with IC50 values ranging from 1.4 to 7.3 microM.

Silymarin inhibits melanin synthesis in melanocyte cells.

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

Aspochalasin I, a melanogenesis inhibitor from Aspergillus sp.

In the course of screening for the melanogenesis inhibitors, aspochalasin I was isolated from solid-state culture of Aspergillus sp. Fb020460. Its structure was determined by spectroscopic analysis including mass spectroscopy and NMR analysis. Aspochalasin I potently inhibited melanogenesis in Mel-Ab cells with an IC50 value of 22.4 microM without cytotoxicity.

Free radical scavenging and antielastase activities of flavonoids from the fruits of Thuja orientalis.

Bioassay-guided fractionation of the MeOH extract of Thuja orientalis fruits using a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay led to the isolation of 9 flavonoids: cupressuflavone (1), amentoflavone (2), robustaflavone (3), afzelin (4), (+)-catechin (5), quercitrin (6), hypolaetin 7-O-beta-xylopyranoside (7), isoquercitrin (8) and myricitrin (9). Their chemical structures were determined by spectroscopic analyses. The free radical scavenging and human neutrophil elastase (HNE) inhibitory activities were evaluated for the isolated compounds. By DPPH scavenging assay, compounds 5, 6, 7, 8 and 9 showed anti-oxidant activities with IC(50) values of 28.66, 31.19, 18.30, 26.63 and 15.10 microM, respectively. By ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] scavenging assay, these compounds also exhibited potent anti-oxidant activities with IC(50) values of 6.77, 13.96, 6.97, 22.79 and 9.96 microM, respectively. Of note, compounds 1, 2 and 3 showed significant HNE inhibitory activities with IC(50) values of 8.09, 1.27 and 1.33 microM, respectively.

Reticulone, a novel free radical scavenger produced by Aspergillus sp.

Bioassay-guided fractionation of the culture broth of Aspergillus sp. FN070449 (KCTC 26428) using a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay led to the isolation of two compounds: reticulone (1) and reticulol (2). Their chemical structures were elucidated on the basis of UV, IR, NMR, and MS spectroscopic analysis. Compound 1 exhibited more potent free radical scavenging activity on ABTS.+ (2,2'-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]) and DPPH radicals than did butylated hydroxyanisole (BHA) and caffeic acid.

Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to piericidin A, a GRP78 down-regulator.

Glucose deprivation, a pathophysiological cell condition, causes up-regulation of GRP78 and induction of etoposide resistance in human cancer cells. The induction of drug resistance can be partly explained by the fact that GRP78 can block activation of caspase-7 induced by treatment with etoposide. Therefore, downregulating GRP78 expression may be a novel strategy anticancer drug development. Based on that premise, we established a screening program for anticancer agents that exhibit preferential cytotoxic activity for etoposide-resistant cancer cells under glucose-deprived conditions. We recently isolated an active compound, AR-054, from the culture broth of Streptomyces sp., which prevents stress-induced etoposide resistance in vitro. AR-054 was identified as piericidin A, a prototypical compound, by ESI-MS analysis and various NMR spectroscopic methods. Here, we showed that piericidin A suppressed the accumulation of GRP78 protein and was also highly toxic to etoposide-resistant HT-29 cells, with IC50 values for colony formation of 6.4 and 7.7 nM under 2-deoxyglucose supplemented and glucose-deprived conditions, respectively. Interestingly, piericidin A had no effect under normal growth conditions. Therefore, we suggest that piericidin A prevents up-regulation of GRP78, and exhibits cytotoxicity in glucose-deprived HT-29 cells that are resistant to etoposide.