Neutralization of CX3CL1 Attenuates TGF-ß-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis.

BACKGROUND: Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-ß. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-ß-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). METHODS: CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-ß-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay. RESULTS: An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-ß-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-ß-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-ß-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-ß-treated NHLFs. TP213 alleviated TGF-ß-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-ß-induced p65 and α-SMA expression in NHLFs. CONCLUSIONS: These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.

5ß,19-epoxycucurbitane triterpenoids from Momordica charantia and their anti-inflammatory and cytotoxic activity.

Five new 5ß,19-epoxycucurbitane triterpenoids, taikugausins A-E (1-5), together with 5ß,19-epoxy-25-methoxycucurbita-6,23-diene-3ß,19-diol (6), have been isolated and characterized from the 70 % EtOH extract of the fresh fruits of Momordica charantia. The chemical structures of compounds 1-6 were elucidated on the basis of extensive spectroscopic analyses, especially 2D NMR (HMQC, HMBC, and NOESY) experiments and HRESIMS data. The relationship between NMR chemical shifts and the configuration of C-19 with an OMe group in 5ß,19-epoxycucurbitane are described. Among them, compounds 3 and 4 exhibited remarkable anti-inflammatory activities by the inhibition of nitric oxide production at the concentration of 10 µg/mL. In addition, 3 and 4 also showed moderate cytotoxicity against WiDr, Hep G2, MCF-7, and HEp-2 human tumor cell lines.

Chemical constituents analysis and antidiabetic activity validation of four fern species from Taiwan.

Pterosins are abundant in ferns, and pterosin A was considered a novel activator of adenosine monophosphate-activated protein kinase, which is crucial for regulating blood glucose homeostasis. However, the distribution of pterosins in different species of ferns from various places in Taiwan is currently unclear. To address this question, the distribution of pterosins, glucose-uptake efficiency, and protective effects of pterosin A on ß-cells were examined. Our results showed that three novel compounds, 13-chloro-spelosin 3-O-ß-d-glucopyranoside (1), (3R)-Pterosin D 3-O-ß-d-(3'-p-coumaroyl)-glucopyranoside (2), and (2R,3R)-Pterosin L 3-O-ß-d-(3'-p-coumaroyl)-glucopyranoside (3), were isolated for the first time from four fern species (Ceratopteris thalictroides, Hypolepis punctata, Nephrolepis multiflora, and Pteridium revolutum) along with 27 known compounds. We also examined the distribution of these pterosin compounds in the mentioned fern species (except N. multiflora). Although all pterosin analogs exhibited the same effects in glucose uptake assays, pterosin A prevented cell death and reduced reactive oxygen species (ROS) production. This paper is the first report to provide new insights into the distribution of pterosins in ferns from Taiwan. The potential anti-diabetic activity of these novel phytocompounds warrants further functional studies.

New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes.

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2'-O-ß-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver-Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

First-in-Class Dual EZH2-HSP90 Inhibitor Eliciting Striking Antiglioblastoma Activity In Vitro and In Vivo.

Structural analysis of tazemetostat, an FDA-approved EZH2 inhibitor, led us to pinpoint a suitable site for appendage with a pharmacophoric fragment of second-generation HSP90 inhibitors. Resultantly, a magnificent dual EZH2/HSP90 inhibitor was pinpointed that exerted striking cell growth inhibitory efficacy against TMZ-resistant Glioblastoma (GBM) cell lines. Exhaustive explorations of chemical probe 7 led to several revelations such as (i) compound 7 increased apoptosis/necrosis-related gene expression, whereas decreased M phase/kinetochore/spindle-related gene expression as well as CENPs protein expression in Pt3R cells; (ii) dual inhibitor 7 induced cell cycle arrest at the M phase; (iii) compound 7 suppressed reactive oxygen species (ROS) catabolism pathway, causing the death of TMZ-resistant GBM cells; and (iv) compound 7 elicited substantial in vivo anti-GBM efficacy in experimental mice xenografted with TMZ-resistant Pt3R cells. Collectively, the study results confirm the potential of dual EZH2-HSP90 inhibitor 7 as a tractable anti-GBM agent.

NCI677397 targeting USP24-mediated induction of lipid peroxidation induces ferroptosis in drug-resistant cancer cells.

Cancer represents a profound challenge to healthcare systems and individuals worldwide. The development of multiple drug resistance is a major problem in cancer therapy and can result in progression of the disease. In our previous studies, we developed small-molecule inhibitors targeting ubiquitin-specific peptidase 24 (USP24) to combat drug-resistant lung cancer. Recently, we found that the USP24 inhibitor NCI677397 induced ferroptosis, a type of programmed cell death, in drug-resistant cancer cells by increasing lipid reactive oxygen species (ROS) levels. In the present study, we investigated the molecular mechanisms and found that the targeting of USP24 by NCI677397 increased gene expression of most lipogenesis-related genes, such as acyl-CoA synthetase long-chain family member 4 (ACSL4), and activated autophagy. In addition, the activity of several antioxidant enzymes, such as glutathione peroxidase 4 (GPX4) and dihydrofolate reductase (DHFR), was inhibited by NCI677397 treatment via an increase in protein degradation, thereby inducing lipid ROS production and lipid peroxidation. In summary, we demonstrated that NCI677397 induced a marked increase in lipid ROS levels, subsequently causing lipid peroxidation and leading to the ferroptotic death of drug-resistant cancer cells. Our study provides new insights into the clinical use of USP24 inhibitors as ferroptosis inducers (FINs) to block drug resistance during chemotherapy.

Metabolism of (2S)-pterosin A: identification of the phase I and phase II metabolites in rat urine.

The metabolic profile of the potent hypoglycemic agent, (2S)-pterosin A (1), in rat urine via intragastrical oral administration was investigated. In total, 19 metabolites (M1-M19) were identified. Among these, 16 metabolites were characterized by high-performance liquid chromatography solid-phase extraction-tube transfer-NMR, and seven metabolites were further isolated from the treated urine to enable further structural determination. Twelve of these are new compounds. The phase I metabolites of 1 were formed via various oxidations at positions C-3, C-10, C-12, C-13, or C-1 followed by decarboxylation of C-10 or C-14, and lactonization at C-12/C-14 or C-14/C-12. The phase II metabolites were glucuronide conjugates from the parent compound or phase I metabolites. The major metabolites were found to be (2S)-14-O-glucuronylpterosin A (M9), (2S)-2-hydroxymethylpterosin E (M14), and (±)-pterosin B (M19). Quantitative HPLC analysis of metabolites, based on similar UV absorption and use of the regression equation of 1, indicated that ∼71% 1 was excreted as metabolites in rat urine.

Tanshinone IIA attenuates cyclic strain-induced endothelin-1 expression in human umbilical vein endothelial cells.

1. Tanshinone IIA, one of the active components of the Radix of Salvia miltiorrhiza, is used in traditional Chinese medicine to treat cardiovascular diseases. However, the intracellular mechanism of action of tanshinone IIA remain to be determined. The aims of the present study were to test the hypothesis that tanshinone IIA alters strain-induced endothelin (ET)-1 expression and nitric oxide (NO) production, as well as to identify the putative signalling pathways involved, in human umbilical vein endothelial cells (HUVEC). 2. Cultured HUVEC were exposed to cyclic strain in the presence of 1-10 µmol/L tanshinone IIA. Expression of ET-1 was examined by reverse transcription-polymerase chain reaction and ELISA. Phosphorylation of endothelial NO synthase (eNOS) and activating transcription factor (ATF) 3 was assessed by western blot analysis. 3. Tanshinone IIA (3 and 10 µmol/L) inhibited strain-induced ET-1 expression. In contrast, NO production, eNOS phosphorylation and ATF3 expression were enhanced by tanshinone IIA. The eNOS inhibitor N(G) -nitro-L-arginine methyl ester (l-NAME; 100 µmol/L), the phosphatidylinositol 3-kinase inhibitor LY294002 (5 µmol/L) and the soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; 10 µmol/L) inhibited tanshinone IIA-induced increases in ATF3 expression. Moreover, treatment of HUVEC with either an NO donor (3,3-bis [aminoethyl]-1-hydroxy-2-oxo-1-triazene; 500 µmol/L) or an ATF3 activator (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; 5 µmol/L) resulted in the repression of strain-induced ET-1 expression. The inhibitory effect of tanshinone IIA on strain-induced ET-1 expression was significantly attenuated by l-NAME, ODQ and the transfection of small interfering RNA for ATF3. 4. In conclusion, tanshinone IIA inhibits strain-induced ET-1 expression by increasing NO and upregulating ATF3 in HUVEC. The present study provides important new insights into the molecular pathways that may contribute to the beneficial effects of tanshinone IIA in the cardiovascular system.

Evaluation of antioxidant and free radical scavenging capacities of polyphenolics from pods of Caesalpinia pulcherrima.

Thirteen polyphenolics were isolated from fresh pods of Caesalpinia pulcherrima using various methods of column chromatography. The structures of these polyphenolics were elucidated as gallic acid (1), methyl gallate (2), 6-O-galloyl-d-glucoside (3), methyl 6-O-galloyl-ß-d-glucoside (4), methyl 3,6-di-O-galloyl-α-d-glucopyranoside (5), gentisic acid 5-O-α-d-(6'-O-galloyl)glucopyranoside (6), guaiacylglycerol 4-O-ß-d-(6'-O-galloyl)glucopyranoside (7), 3-methoxy-4-hydroxyphenol 1-O-ß-d-(6'-O-galloyl) glucopyranoside (8), (+)-gallocatechin (9), (+)-catechin (10), (+)-gallocatechin 3-O-gallate (11), myricetin 3-rhamnoside (12), and ampelopsin (13). All isolated compounds were tested for their antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and peroxynitrite radicals scavenging assays. Among those compounds, 11, 12, and 2 exhibited the best DPPH-, hydroxyl-, and peroxynitrite radical-scavenging activities, respectively. Compound 7 is a new compound, and possesses better scavenging activities towards DPPH but has equivalent hydroxyl radical scavenging activity when compared to BHT. The paper is the first report on free radical scavenging properties of components of the fresh pods of Caesalpinia pulcherrima. The results obtained from the current study indicate that the free radical scavenging property of fresh pods of Caesalpinia pulcherrima may be one of the mechanisms by which this herbal medicine is effective in several free radical mediated diseases.

Transformation of isosteviol lactam by fungi and the suppressive effects of its transformed products on LPS-induced iNOS expression in macrophages.

From the screening of 21 microbial strains, Absidia pseudocylindrospora ATCC 24169 and Aspergillus niger BCRC 32720 were found to reproducibly transform isosteviol lactam (4α-carboxy-13α-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam) (3) into various compounds. Preparative-scale transformation of 3 with Abs. pseudocylindrospora yielded two new hydroxylated compounds (4 and 5), with conservation of the lactam ring. Preparative-scale transformation of 3 with Asp. niger afforded seven new compounds, 6 and 9-14, together with the known compounds 7 and 8. A single-crystal X-ray diffraction experiment confirmed the structure of 14. The suppressive effects of compounds 1-14 on the lipopolysaccharide-induced expression of the inducible nitric oxide synthase gene in RAW 264.7 macrophages were examined by a reverse-transcription real-time PCR analysis. With the exception of 7, all other compounds significantly reduced levels of iNOS mRNA relative to control cells, which were induced by LPS alone. Compounds 2, 3, and 5 were similar in activity to dexamethasone, while 9 was more potent.

Synthesis and evaluation of antibacterial activities of 5,7-dihydroxycoumarin derivatives.

This study examines the synthesis and antibacterial activities of 5,7-dihydroxycoumarin derivatives, whose structures were confirmed using analytical and spectral data. Twenty compounds were tested for their antibacterial activities against five microbial species such as E. coli, S. aureus, K. pneumonia, P. aeruginosa, and S. typhimurium were studied. Compounds 5 and 12 exhibited the most potent activity against Staphylococcus aureus with a MIC value of 2.5 µg/mL for each of the compounds.

Anti-UVC irradiation and metal chelation properties of 6-benzoyl-5,7-dihydroxy-4-phenyl-chromen-2-one: an implications for anti-cataract agent.

Coumarin derivative 1, 5,7-dihydroxy-6-(3-methyl-1-butyryl)-4-phenyl-chromen- 2-one, has been reported to possess radical scavenging activity and DNA protection. We have synthesized a series of coumarins with structural modifications at positions C4, C5, C6 and C7 and evaluated them for their anti-UVC properties. Coumarin 7, 6-benzoyl-5,6-dihydroxy-4-phenyl-chromen-2-one, was found to have the most potent activity in protecting porcine γ-crystallin against UVC insults. Results of fluorescence assays indicated that compound 7 was capable of decreasing the loss of intensity while lens crystallins and DNA PUC19 were irradiated with UVC. Presence of compound 7 decreased hydroxyl radical levels determined by probe 1b and the free iron concentrations determined by Ferrozine reagent. The chelation assay showed that compound 7 was chelated to metal via 6-CO and 5-OH on the benzopyrone ring. The observed protective effects of compound 7 towards crystallins from insults of UVC and free radicals may be due to its iron-chelating activity and its peak absorption at 254 nm.

Metabolomics investigation of exercise-modulated changes in metabolism in rat liver after exhaustive and endurance exercises.

Exhaustive exercise and endurance exercise training modify the physiological status of the body differently. The present study aimed to evaluate the alteration in biochemical composition with exhaustive and endurance exercises in rats using metabolomics strategy. The metabolite profile of liver tissue was investigated on gas chromatography-mass spectrometry (GC-MS). Data further underwent partial least-squares-discriminant analysis (PLS-DA) to compare the effects on metabolites in sedentary control, exhaustively exercised and endurance trained rats. GC-MS detected 115 highly reproducible peaks in chromatograms from individual liver tissue extracts, and we identified 55 of them. The three groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids, and carbohydrates. Endurance training elevated the greater rate of tricarboxylic acid cycle and antioxidant activity, and exhaustive exercise led to accumulated urea markers and an inflammation response in liver. In addition, GC-MS-based metabolomic analysis is a promising tool to investigate a pathological status with different exercise programs.

Caffeic acid improves the bioavailability of L-dopa in rabbit plasma.

The impacts of caffeic acid (3,4-dihydroxycinnamic acid, CA) on the pharmacokinetics of levodopa (L-dopa) were studied in rabbits. A single dose of 5/1.25 mg kg(-1) L-dopa/carbidopa was administered alone or was co-administered with three different doses of caffeic acid (2.5, 5, and 10 mg kg(-1)), or a single dose of 5 mg kg(-1) caffeic acid was administered alone via an intramuscular route to six rabbits each in a crossover treatment protocol. Plasma levels of L-dopa, 3-O-methyldopa (3-OMD), caffeic acid, and ferulic acid were determined and subsequently used to calculate their pharmacokinetic parameters. The results indicated that caffeic acid administered at a dose of 10 mg kg(-1) decreased about 22% of the peripheral formation of 3-OMD and about 31% of the C(max) of 3-OMD. In addition, the metabolic ratios (MR, AUC of 3-OMD/AUC of L-dopa) decreased by about 22%. Results also indicated that caffeic acid significantly decreased the proportion of 3-OMD (p < 0.05). In contrast, the parameters of neither caffeic acid nor ferulic acid were significantly affected by L-dopa/carbidopa. In conclusion, caffeic acid at a dose of 10 mg kg(-1) can significantly affect the COMT metabolic pathway of L-dopa.

Human monoclonal antibody GNX-8 directed to extended type 1 chain: Specific binding to human colorectal cancer.

We observed previously that two carbohydrate epitopes, extended type 1 chain Le(a)-Le(a) and Le(b)-Le(a), are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of "KM mice" with colonic cancer cell line Colo205, and with purified Le(b)-Le(a) glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le(b)-Le(a), bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le(b)-Le(a), whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-alpha-GalNAc, indicating that these epitopes are O-linked glycans.Immunohistological reactivity of GNX-8 at 1 mug/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 mug/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le(b) antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le(b)-Le(a) antigen, provides a novel direction of immunotherapy for human colorectal cancer. (c) 2009 UICC.

Microbial transformation of isosteviol oxime and the inhibitory effects on NF-kappaB and AP-1 activation in LPS-stimulated macrophages.

Microbial transformation of isosteviol oxime (ent-16-E-hydroxyiminobeyeran-19-oic acid) (2) with Aspergillus niger BCRC 32720 and Absidia pseudocylindrospora ATCC 24169 yielded several compounds. In addition to bioconverting the d-ring to lactone and lactam moieties, 4alpha-carboxy-13alpha-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (7) and 4alpha-carboxy-13alpha-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam (10), one known compound, ent-1beta,7alpha-dihydroxy-16-oxo-beyeran-19-oic acid (6), and five new compounds, ent-7alpha-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (3), ent-1beta,7alpha-dihydroxy-16-E-hydroxyiminobeyeran-19-oic acid (4), ent-1beta-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (5), ent-8beta-cyanomethyl-13-methyl-12-podocarpen-19-oic acid (8), and ent-8beta-cyanomethyl-13-methyl-13-podocarpen-19-oic acid (9), were isolated from the microbial transformation of 2. Elucidation of the structures of these isolated compounds was primarily based on 1D and 2D NMR, and HRESIMS data, and 3-5 were further confirmed by X-ray crystallographic analyses. Additionally, the inhibitory effects of all of these compounds were evaluated on NF-kappaB and AP-1 activation in LPS-stimulated RAW 264.7 macrophages. Among the compounds tested, 5 and 10 significantly inhibited NF-kappaB activation, with 5 showing equal potency to dexamethasone; 3 and 6-9 significantly inhibited AP-1 activation, particularly 8, which showed more inhibitory activity than dexamethasone.

Anemonin is a natural bioactive compound that can regulate tyrosinase-related proteins and mRNA in human melanocytes.

BACKGROUND: Melanin is the pigment responsible for skin color. Melanin synthesis occurs with the participation of the tyrosinase (TYR) family of proteins including TYR, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2(TRP2/DCT). OBJECTIVE: The effect of a newly isolated natural compound that inhibits hyperpigmentation on the regulation of the TYR family of proteins was examined. METHODS: The natural compound, anemonin, was isolated from Clematis crassifolia Benth and was used to inhibit cellular TYR activity; it was found to have a low cytotoxicity (cell viability > 80%) in human melanocytes. RESULTS: In human melanocytes, anemonin showed both time- and dose-dependent inhibition (IC(50) 43.5 microM) of TYR. Western blot analysis and immunocytochemical staining revealed that expression of TYR, TRP1, and TRP2 was decreased in anemonin-treated melanocytes. Additionally, reverse transcription and quantitative real-time polymerase chain reaction analyses revealed that expression of mRNAs for MITF, TYR, TYRP1, and TYRP2 was also suppressed by anemonin. CONCLUSION: The natural compound, anemonin, an active compound of C. crassifolia, inhibits pigmentation synthesis in human melanocytes. Anemonin inhibits melanin synthesis by inhibiting the transcription of the genes encoding MITF, TYR, TRP1, and TRP2. This natural compound may be a candidate for cosmetic use.

Role of sympathetic tone in the loss of syringin-induced plasma glucose lowering action in conscious Wistar rats.

Syringin is an active principle purified from the rhizome and root parts of Eleutherococcus senticosus (Araliaceae). The present study is designed to clarify the role of sympathetic activation in the insulinotropic effect of syringin. Plasma glucose lowering effect accompanying with the increase of plasma insulin and C-peptide were obtained in pentobarbital anesthetized Wistar rats 60min after an intravenous (i.v.) injection of syringin (100 microg/kg). However, neither the plasma glucose lowering action, nor the raised plasma levels of insulin and C-peptide can be obtained in conscious rats received same syringin treatment. Otherwise, the insulin-releasing and plasma glucose lowering actions of syringin (100 microg/kg, i.v.) were appeared in conscious rats under chemical sympathectomy using an intraperitoneal injection of guanethidine. In addition, plasma glucose lowering action of syringin (100 microg/kg, i.v.) was observed in conscious rats with alpha1-adrenoceptor blockade by prazosin. The stimulatory actions of syringin on the secretion of plasma insulin and C-peptide were also obtained in prazosin-treated conscious rats. The obtained results suggest that insulinotropic effect of syringin on the plasma glucose regulation is impaired in conscious rats with a regular sympathetic tone; decrease of sympathetic tone as observed in anesthetized animal might be helpful in the therapeutic benefit of syringin.

Constituents from the Formosan apple reduce tyrosinase activity in human epidermal melanocytes.

Tyrosinase is a copper-containing monooxygenase that catalyzes melanin synthesis in skin melanocytes. Herein, 13 compounds from the Formosan apple (Malus doumeri var. formosana), an indigenous Taiwanese plant, were isolated and identified. The active constituents were identified as 3-hydroxyphloretin (7) and catechol (9); they exhibited potent hydroxyl radical-scavenging (IC(50) values, 0.6 and 1.1 microM) and cellular tyrosinase-reducing activities (IC(50) values, 32 and 22 microM) in human epidermal melanocytes. In addition, we evaluated the level of several tyrosinase-related proteins by Western blot analysis. In contrast to 3-hydroxyphloretin (7), which showed no effect on the level of these proteins, catechol (9) reduced their activity and the expression of the respective genes, as determined by quantitative real-time PCR. In a kinetic analysis of mushroom tyrosinase, 3-hydroxyphloretin (7) was a competitive inhibitor. These two constituents exhibited metal-coordinating interactions with copper ions in a virtual model of molecular docking with human tyrosinase. Thus, 3-hydroxyphloretin (7) and catechol (9) were the most active constituents from the Formosan apple; they exhibited anti-oxidant and tyrosinase reducing activities, suggesting their possible use as cosmetic agents.

Microbial metabolism of steviol and steviol-16alpha,17-epoxide.

Steviol (2) possesses a blood glucose-lowering property. In order to produce potentially more- or less-active, toxic, or inactive metabolites compared to steviol (2), its microbial metabolism was investigated. Incubation of 2 with the microorganisms Bacillus megaterium ATCC 14581, Mucor recurvatus MR 36, and Aspergillus niger BCRC 32720 yielded one new metabolite, ent-7alpha,11beta,13-trihydroxykaur-16-en-19-oic acid (7), together with four known related biotransformation products, ent-7alpha,13-dihydroxykaur-16-en-19-oic acid (3), ent-13-hydroxykaur-16-en-19-alpha-d-glucopyranosyl ester (4), ent-13,16beta,17-trihydroxykauran-19-oic acid (5), and ent-13-hydroxy-7-ketokaur-16-en-19-oic acid (6). The preliminary testing of antihyperglycemic effects showed that 5 was more potent than the parent compound (2). Thus, the microbial metabolism of steviol-16alpha,17-epoxide (8) with M. recurvatus MR 36 was continued to produce higher amounts of 5 for future study of its action mechanism. Preparative-scale fermentation of 8 yielded 5, ent-11alpha,13,16alpha,17-tetrahydroxykauran-19-oic acid (10), ent-1beta,17-dihydroxy-16-ketobeyeran-19-oic acid (11), and ent-7alpha,17-dihydroxy-16-ketobeyeran-19-oic acid (13), together with three new metabolites: ent-13,16beta-dihydroxykauran-17-acetoxy-19-oic acid (9), ent-11beta,13-dihydroxy-16beta,17-epoxykauran-19-oic acid (12), and ent-11beta,13,16beta,17-tetrahydroxykauran-19-oic acid (14). The structures of the compounds were fully elucidated using 1D and 2D NMR spectroscopic techniques, as well as HRFABMS. In addition, a GRE (glucocorticoid responsive element)-mediated luciferase reporter assay was used to initially screen the compounds 3-5, and 7 as glucocorticoid agonists. Compounds 4, 5 and 7 showed significant effects.