Mechanism of the fungal-like particles in the inhibition of adipogenesis in 3T3-L1 adipocytes.

The dynamic ability of adipocytes in adipose tissue to store lipid in response to changes in the nutritional input and inflammatory elicitors has a major impact on human health. Previously, we established laminarin-coated beads or LCB as an inflammatory elicitor for adipocytes. However, it was not clear whether LCB inhibits lipid accumulation in adipocytes. Here, we show that LCB acts in the early stage of adipogenesis through both interleukin-1 receptor-associated kinases (IRAK) and spleen tyrosine kinase (SYK) pathways, resulting in the activation of the AMP-activated protein kinase (AMPK) and nuclear factor-κB (NF-κB) complexes, which subsequently cause cell cycle arrest, downregulation of the key transcription factors and enzymes responsible for adipogenesis, inhibition of adipogenesis, and stimulation of an inflammatory response. While LCB could effectively block lipid accumulation during the early stage of adipogenesis, it could stimulate an inflammatory response at any stage of differentiation. Additionally, our results raise a possibility that toll-like receptor 2 (TLR2) and C-type lectin domain family 7 member A (CLEC7A/Dectin-1) might be potential ß-glucan receptors on the fat cells. Together, we present the mechanism of LCB, as fungal-like particles, that elicits an inflammatory response and inhibits adipogenesis at the early stage of differentiation.

Fungal-like particles and macrophage-conditioned medium are inflammatory elicitors for 3T3-L1 adipocytes.

Adipocytes from white-adipose tissue are known to produce inflammatory cytokines, which play a major role in energy balance and metabolism. While they can respond to pathogen-associated molecular pattern (PAMPs) such as lipopolysaccharide (LPS) from bacteria, it is not known whether adipocytes can be stimulated by fungal cells. Previously, adipocytes were shown to produce toll-like receptor 2 (TLR2), a ß-glucan receptor, suggesting that they could respond to ß-glucan on the fungal cell wall. In this study, we show that heat-killed yeast induce an inflammatory response in adipocytes. Using fungal-like particles, namely laminarin-coated beads (LCB), we find that these particles trigger the expression of many key inflammatory genes in dose- and time-dependent fashions in adipocytes. These results suggest that ß-glucan on the fungal cell wall is sufficient to elicit an inflammatory response in adipocytes. In addition, we show that both LCB and LCB-treated conditioned medium from RAW 264.7 murine macrophages (LCB-RM) induce the expression of those inflammatory genes through IKKß-IκBα proteins. Together, we conclude that the fungal-like particles and the conditioned medium elicit an inflammatory response in adipocytes through the canonical or classical NF-κB pathway.

Phytoconstituents and Biological Activities of Garcinia dulcis (Clusiaceae): A Review.

Garcinia dulcis (Roxb.) Kurz is a tropical fruit tree native to Southeast Asia where it has a long history of use as a traditional medicine for the treatment of ailments such as lymphatitis, parotitis, struma, scurvy, cough, and sore throat. Despite its medicinal values, this plant is not well known and rarely found nowadays. Research on the phytochemical constituents and biological activities of G. dulcis have demonstrated that various parts of the plant contain an. abundance of bioactive compounds mainly xanthones and flavonoids, with significant pharmacological properties such as anti-atherosclerosis, anti-bacterial, anti-cancer, anti-hypertension, and anti-malarial. In the present review, current knowledge of the phytochemistry of G. dulcis and biological activities of its active constituents based on the available literature are summarized in order to explore application potentials and prospective research works on this plant.

A new phenalenone derivative from the soil fungus Penicillium herquei PSU-RSPG93.

One new phenalenone derivative, peniciherqueinone (1), together with five known phenalenone derivatives (2-6), one known anthraquinone (7) and two known acetophenones (8 and 9) were isolated from the soil fungus Penicillium herquei PSU-RSPG93. Their structures were established by spectroscopic evidence. The absolute configuration of 1 was determined by anisotropic effect and electronic circular dichroism spectroscopy. Compound 2 exhibited mild antioxidant activity and is noncytotoxic to Vero (African green monkey kidney fibroblasts) cell lines.

Ivy gourd (Coccinia grandis L. Voigt) root suppresses adipocyte differentiation in 3T3-L1 cells.

BACKGROUND: Ivy gourd (Coccinia grandis L. Voigt) is a tropical plant widely distributed throughout Asia, Africa, and the Pacific Islands. The anti-obesity property of this plant has been claimed but still remains to be scientifically proven. We therefore investigated the effects of ivy gourd leaf, stem, and root on adipocyte differentiation by employing cell culture model. METHODS: Dried roots, stems, and leaves of ivy gourd were separately extracted with ethanol. Each extract was then applied to 3T3-L1 pre-adipocytes upon induction with a mixture of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone, for anti-adipogenesis assay. The active extract was further fractionated by a sequential solvent partitioning method, and the resulting fractions were examined for their abilities to inhibit adipogenesis in 3T3-L1 cells. Differences in the expression of adipogenesis-related genes between the treated and untreated cells were determined from their mRNA and protein levels. RESULTS: Of the three ivy gourd extracts, the root extract exhibited an anti-adipogenic effect. It significantly reduced intracellular fat accumulation during the early stages of adipocyte differentiation. Together with the suppression of differentiation, expression of the genes encoding PPARγ, C/EBPα, adiponectin, and GLUT4 were down-regulated. Hexane-soluble fraction of the root extract also inhibited adipocyte differentiation and decreased the mRNA levels of various adipogenic genes in the differentiating cells. CONCLUSIONS: This is the first study to demonstrate that ivy gourd root may prevent obesity based mainly on the ability of its active constituent(s) to suppress adipocyte differentiation in vitro. Such an inhibitory effect is mediated by at least down-regulating the expression of PPARγ-the key transcription factor of adipogenesis in pre-adipocytes during their early differentiation processes.

Benzene, coumarin and quinolinone derivatives from roots of Citrus hystrix.

Two coumarins, hystrixarin (1) and (+)-hopeyhopin (2); a benzenoid derivative, hystroxene-I (3) and a quinolinone alkaloid, hystrolinone (4), along with 33 known compounds were isolated from the crude acetone extract of the roots of Citrus hystrix. Their structures were determined by analysis of 1D and 2D NMR spectroscopic data. The antioxidant, anti-HIV and antibacterial activities of the isolated compounds were also evaluated.

Morelloflavone, a biflavonoid inhibitor of migration-related kinases, ameliorates atherosclerosis in mice.

While macrophages take up modified LDL to form foam cells and multiply to develop fatty streaks, vascular smooth muscle cells (VSMC) migrate from the media to intima, secrete extracellular matrix, and increase the volume of atherosclerotic lesions. A medicinal plant Garcinia dulcis has been used in traditional Thai medicine for centuries to treat various chronic human diseases. Morelloflavone, a biflavonoid and an active ingredient of the plant, has been shown to inhibit VSMC migration through its inhibition of multiple migration-related kinases such as focal adhesion kinase, c-Src, ERK, and RhoA. However, the exact role of morelloflavone in atherosclerogenesis was unknown. We fed Ldlr(-/-)Apobec1(-/-) mice with either normal chow or chow containing 0.003% morelloflavone for 8 mo and assessed the extent of atherosclerosis by the en face and cross-sectional analyses. A cell composition analysis of atherosclerotic tissue was carried out using immunohistochemical staining. Oral morelloflavone therapy significantly reduced the atherosclerotic areas of the mouse aortas (a 26% reduction), without changing plasma lipid profiles or weights. Immunohistochemical analyses showed that morelloflavone reduced the number of VSMC in the atherosclerotic lesion while it did not change the density of macrophages in the lesion or the percentages of proliferating and apoptotic cells. Oral, low-dose, morelloflavone therapy retards atherosclerogenesis by limiting the migration of VSMC into the intima in the mouse model of human atherosclerosis. Upon further investigation, morelloflavone may be found to be a novel oral antiatherosclerotic agent and a viable addition to the conventional therapies such as statins in humans.

Sesquiterpene and xanthone derivatives from the sea fan-derived fungus Aspergillus sydowii PSU-F154.

Three new sesquiterpenes, named aspergillusenes A and B and (+)-(7S)-7-O-methylsydonic acid, and two new hydrogenated xanthone derivatives, named aspergillusones A and B, were isolated from the sea fan-derived fungus Aspergillus sydowii PSU-F154 together with 10 known compounds. Their structures were identified on the basis of spectroscopic data. The isolated compounds were evaluated for their antioxidant activity.

Morelloflavone from Garcinia dulcis as a novel biflavonoid inhibitor of HMG-CoA reductase.

Morelloflavone, a biflavonoid from Garcinia dulcis previously shown to have hypocholesterolemic activity, was examined for its effect on HMG-CoA reductase, the rate-limiting enzyme of the cholesterol biosynthetic pathway. By using the catalytic domain of house mouse HMG-CoA reductase, morelloflavone was found to inhibit the enzyme activity by competing with HMG-CoA whereas it was non-competitive towards NADPH. The inhibition constants (K(i)) with respect to HMG-CoA and NADPH were 80.87 ± 0.06 µm and 103 ± 0.07 µm, respectively. Both flavonoid subunits of this compound, naringenin and luteolin, equally competed with HMG-CoA with K(i) of 83.58 ± 4.37 µm and 83.59 ± 0.94 µm, respectively, and were also non-competitive with NADPH (K(i) of 182 ± 0.67 µm and 188 ± 0.14 µm, respectively). Due to these findings, we suggest that each subunit of morelloflavone would occupy the active site of the enzyme, thereby blocking access of its substrate. The present study thus demonstrates the ability of morelloflavone from G. dulcis to inhibit HMG-CoA reductase in vitro. As a result, this biflavonoid might serve as a new candidate for the future development of hypocholesterolemic agents.

Antioxidant, antibacterial and antigiardial activities of Walsura robusta Roxb.

Walsura robusta Roxb. (Family: Meliaceae) is a well-known multi-purpose medicinal plant, and has been employed for a wide range of disease conditions without documented scientific data. In the current study, four pure isolated compounds, 3,4,5-trimethoxyphenyl beta-D-glucopyranoside (1), turpinionoside A (2), (+)-lyoniresinol 3alpha-O-beta-D-glucopyranoside (3) and (-)-lyoniresinol 3alpha-O-beta-D-glucopyranoside (4), were isolated from the leaves and twigs of W. robusta. Biological evaluation for free radical scavenging, antibacterial and antigiardial activities was performed. We investigated antioxidant effects of the crude extracts as well as the isolated compounds using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), hydroxyl radical (OH), and superoxide anion (O(2)) scavenging assays. Three phenolic glucosides (1, 3 and 4) were found to possess strong antioxidant activity. They scavenged DPPH(*) with IC(50) values in the range of 51.5-86.6 microM. We also detected the superoxide dismutase-like activities in compounds 3 and 4 which are lignan glucosides, demonstrating potent superoxide scavenging activity with IC(50) values in the range of 0.8 and 0.7 microM, respectively. Other biological activities including antibacterial and antigiardial assays were carried out. Preliminary results demonstrated that most extracts, except the diethyl ether extract, exhibited inhibition zones against all Gram-positive bacteria including Bacillus cereus, Staphylococcus aureus, Streptococcus mutans, and S. pyogenes. Aqueous extracts of this plant species could inhibit Gram-positive and some Gram-negative bacteria such as Escherichia coli, Salmonella typhi and Shigella sonnei. However, the determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of W. robusta on all tested bacterial strains showed only weak activity, and their MBCs were greater than 25 mg mL(-1). For antigiardial activity, incubation with 2 x 10(5) trophozoites mL(-1) of the culture medium with the crude extracts at concentration ranged from 31.25 to 1000 microg mL(-1) demonstrated no activity (MIC > 1000 microg mL(-1)).

Cyclohexene, diketopiperazine, lactone and phenol derivatives from the sea fan-derived fungi Nigrospora sp. PSU-F11 and PSU-F12.

Nigrosporanenes A (1) and B (2), two new cylohexene derivatives, and tyrosol (3) were isolated from the sea fan-derived fungus Nigrospora sp. PSU-F11, whereas five known compounds: 4-hydroxybenzoic acid (4), aplysiopsene D (5), 3-isochromanone (6), (-)-drimenin (7) and diketopiperazine derivative (8), were obtained from the fungus Nigrospora sp. PSU-F12. Their structures were established by spectroscopic evidence. We also tested their cytotoxic (on African green monkey kidney fibroblast and breast cancer cells), antioxidant (in the DPPH assay), and antibacterial (against the standard Staphylococcus aureus ATCC 25923 and methicillinresistant S. aureus) activities.

Antioxidant activities of four edible seaweeds from the southern coast of Thailand.

The aqueous extracts of four marine algae, Caulerpa racemosa var. macrophysa, Gracilaria tenuistipitata var. tenuistipitata, Sargassum sp., and Ulva lactuca, from the coastal areas in Southern Thailand, were prepared by boiling dried seaweed powder in water for 3 h, and by autoclaving each sample at 120 degrees C for 3 h. They were then freeze-dried and evaluated for their antioxidant activities using DPPH (1, 1-diphenyl-2-picrylhydrazyl), hydroxyl radical (OH(*)) and superoxide anion (O(2)(*-) ) scavenging assays. Boiling extracts of the seaweeds, except C. racemosa, were found to have higher total phenolic contents (TPC) than those obtained from the autoclave method. The antioxidant results also showed that O(2)(*-) scavenging activity existed only in the boiling extracts of C. racemosa, G. tenuistipitata, and U. lactuca. In DPPH and OH(*) assays, however, almost all the boiling extracts were less active than the autoclave ones. Among the four alga species, Sargassum sp. was the most active. Both extracts of this seaweed had the highest TPC and also displayed the strongest DPPH(*) and OH(*) inhibitory activities. A strong positive-correlation between the antioxidant potency and TPC of the autoclave extracts was found, while for the boiling extracts such relation was very weak. This result thus reflected that in addition to the phenolic compounds, there might be some other active components present in these extracts involved in the antioxidant activity.

Embryonic lethality of fortilin-null mutant mice by BMP-pathway overactivation.

BACKGROUND: Fortilin negatively regulates apoptosis and is overexpressed in cancer. However, the role of fortilin in mammalian development is not clear. METHODS AND RESULTS: In order to evaluate the physiological role of fortilin in vivo, we performed a targeted disruption of the fortilin gene in mice. Fortilin(+/-) mice have the ability to survive and exhibit normal growth, while fortilin(-/-) mice are embryonically lethal around the 3.5 days post-coital (dpc). Cultured blastocysts from fortilin(+/-) embryos undergo normal outgrowth to produce inner cell mass (ICM) and trophoblasts (TB), while ICM of fortilin(-/-) embryos either fails to outgrow or prematurely disintegrates. Mouse embryonic fibroblasts (MEF) derived from fortilin(+/-) embryos are more susceptible to noxious stimuli than are wild type embryos. It has been consistently shown in Xenopus embryos that the depletion of fortilin's message severely compromises the formation of neural tissue, even in the brain, while overexpression of fortilin induces the partial double body axis in embryos and is capable of blocking BMP4-induced transcription of Vent1, Vent2, and Msx1 genes. This suggests that fortilin is an inhibitor of the BMP pathway. Strikingly, when fortilin levels are reduced by siRNA, BMP4 causes MEF to undergo extensive DNA-fragmentation, while DNA fragmentation is minimal in the presence of fortilin. In addition, BMP4 induces more Msx2 in the absence of fortilin than in its presence. Furthermore, Msx2 overexpression causes MEF to undergo apoptotic cell death. CONCLUSION: We conclude that in early phase of development, fortilin functions as an inhibitor of the BMP pathway. The presence of fortilin in the very early stages of development is required for the survival of embryos. GENERAL SIGNIFICANCE: Abnormalities in the fortilin gene may be associated with early pregnancy loss.

Morelloflavone blocks injury-induced neointimal formation by inhibiting vascular smooth muscle cell migration.

BACKGROUND: In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon. METHODS: In endothelium-denudated mouse carotid arteries, oral morelloflavone-an active ingredient of the Thai medicinal plant Garcinia dulcis-significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis. RESULTS: These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest. GENERAL SIGNIFICANCE: We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.

Human fortilin is a molecular target of dihydroartemisinin.

Dehydroartemisinin (DHA) is an effective anti-malaria agent. Fortilin is an anti-apoptotic molecule overexpressed in many human cancers. Here, we show that DHA binds human fortilin, increases the ubiquitination of fortilin, shortens fortilin's half-life in a proteasome-dependent fashion, and reduces cellular levels of fortilin in varieties of cells. DHA induced DNA fragmentation in U2OS cells in a fortilin-dependent manner. The fortilin-knocked-down cells were less susceptible--and fortilin-overexpressing cells more susceptible--to DHA than were wild-type cells, suggesting that apoptotic effects of DHA are-at least partly-conferred through fortilin. Together, these data suggest that fortilin is a molecular target of DHA. DHA and its derivative may prove to be viable anti-cancer agents in fortilin-overexpressing cancers.

Biological activities of extracts from endophytic fungi isolated from Garcinia plants.

Sixty-five crude extracts from 51 selected endophytic fungi isolated from Garcinia species were tested for various bioactivities. Eighty per cent of the fungal extracts from fermentation broths and mycelia displayed bioactivities: antimycobacterial (76.9%), antimalarial (14.1%), antiviral (16.7%), antioxidant (22.2%), antiproliferation (11.1% against NCI-H187 and 12.7% against KB cells), and cytotoxicity to Vero cells (40.0%). Based on internal transcribed spacer rRNA sequence analysis, 15 bioactive isolates were identified as Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor, Penicillium, Pestalotiopsis, and Phomopsis spp. One isolate (N24) was matched with an unidentified fungal endophyte. These results indicate that endophytic fungi isolated from Garcinia plants in Thailand are potential sources of various bioactive natural products.

Metabolites from the xylariaceous fungus PSU-A80.

One hypoxylonol, xylarenol (1), one hexadienoic acid, xylarenoic acid (2), and one tetralone, xylarenone (3), were isolated from the xylariaceous fungus PSU-A80 together with ten known compounds. The structures were established by analysis of spectroscopic data. 8-Methoxy-1-naphthol, one of the known metabolites, displayed good radical scavenging potency with an IC(50) value of 30 microg/ml.

Inhibition of human lipoprotein oxidation by morelloflavone and camboginol from Garcinia dulcis.

A biflavonoids, morelloflavone (1) and a prenyltated xanthone, camboginol (2), isolated from the fruits of Garcinia dulcis (Roxb.) Kurz., exhibited strong antioxidation effects in both Fe2+ -mediated and non-metal induced human low-density lipoprotein (LDL) oxidations. However, a well-known antioxidant, alpha-tocopherol (vitamin E), was found less potent than both compounds based on the same test systems.