Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,ß-dehydrolovastatin (DHLV), α,ß-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 µM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 µM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 µM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.

Cholesterol-Lowering Effects of Asperidine B, a Pyrrolidine Derivative from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178: A Potential Cholesterol Absorption Inhibitor.

Isolated secondary metabolites asperidine B (preussin) and asperidine C, produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, were found to exhibit inhibitory effects against 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and oxidative stress in an in vitro assay. Whether or not the known pyrrolidine asperidine B and the recently isolated piperidine asperidine C have lipid-lowering effects remains unknown. Thus, this study aimed to investigate the hypocholesterolemic effects of asperidines B and C and identify the mechanisms involved in using in vitro, ex vivo, and in vivo models. The results show that both compounds interfered with cholesterol micelle formation by increasing bile acid binding capacity, similar to the action of the bile acid sequestrant drug cholestyramine. However, only asperidine B, but not asperidine C, was found to inhibit cholesterol uptake in Caco-2 cells by up-regulating LXRα without changing cholesterol transporter NPC1L1 protein expression. Likewise, reduced cholesterol absorption via asperidine-B-mediated activation of LXRα was also observed in isolated rat jejunal loops. Asperidine B consistently decreases plasma cholesterol absorption, similar to the effect of ezetimibe in rats. Therefore, asperidine B, the pyrrolidine derivative, has therapeutic potential to be developed into a type of cholesterol absorption inhibitor for the treatment of hypercholesterolemia.

Antispasmodic Effect of Asperidine B, a Pyrrolidine Derivative, through Inhibition of L-Type Ca2+ Channel in Rat Ileal Smooth Muscle.

Antispasmodic agents are used for modulating gastrointestinal motility. Several compounds isolated from terrestrial plants have antispasmodic properties. This study aimed to explore the inhibitory effect of the pyrrolidine derivative, asperidine B, isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, on spasmodic activity. Isolated rat ileum was set up in an organ bath. The contractile responses of asperidine B (0.3 to 30 µM) on potassium chloride and acetylcholine-induced contractions were recorded. To investigate its antispasmodic mechanism, CaCl2, acetylcholine, Nω-nitro-l-arginine methyl ester (l-NAME), nifedipine, methylene blue and tetraethylammonium chloride (TEA) were tested in the absence or in the presence of asperidine B. Cumulative concentrations of asperidine B reduced the ileal contraction by ~37%. The calcium chloride and acetylcholine-induced ileal contraction was suppressed by asperidine B. The effects of asperidine B combined with nifedipine, atropine or TEA were similar to those treated with nifedipine, atropine or TEA, respectively. In contrast, in the presence of l-NAME and methylene blue, the antispasmodic effect of asperidine B was unaltered. These results suggest that the antispasmodic property of asperidine B is probably due to the blockage of the L-type Ca2+ channel and is associated with K+ channels and muscarinic receptor, possibly by affecting non-selective cation channels and/or releasing intracellular calcium.

Microcystis Sp. Co-Producing Microcystin and Saxitoxin from Songkhla Lake Basin, Thailand.

The Songkhla Lake Basin (SLB) located in Southern Thailand, has been increasingly polluted by urban and industrial wastewater, while the lake water has been intensively used. Here, we aimed to investigate cyanobacteria and cyanotoxins in the SLB. Ten cyanobacteria isolates were identified as Microcystis genus based on16S rDNA analysis. All isolates harbored microcystin genes, while five of them carried saxitoxin genes. On day 15 of culturing, the specific growth rate and Chl-a content were 0.2-0.3 per day and 4 µg/mL. The total extracellular polymeric substances (EPS) content was 0.37-0.49 µg/mL. The concentration of soluble EPS (sEPS) was 2 times higher than that of bound EPS (bEPS). The protein proportion in both sEPS and bEPS was higher than the carbohydrate proportion. The average of intracellular microcystins (IMCs) was 0.47 pg/cell on day 15 of culturing, while extracellular microcystins (EMCs) were undetectable. The IMCs were dramatically produced at the exponential phase, followed by EMCs release at the late exponential phase. On day 30, the total microcystins (MCs) production reached 2.67 pg/cell. Based on liquid chromatograph-quadrupole time-of-flight mass spectrometry, three new MCs variants were proposed. This study is the first report of both decarbamoylsaxitoxin (dcSTX) and new MCs congeners synthesized by Microcystis.

Lovastatin Production by Aspergillus sclerotiorum Using Agricultural Waste.

RESEARCH BACKGROUND: Lovastatin is a well-known drug used to reduce hypercholesterolaemia. However, the cost of lovastatin production is still high. Therefore, alternative low-cost carbon sources for the production of lovastatin are desirable. EXPERIMENTAL APPROACH: Four different agricultural wastes, namely corn trunks, rice husks, wild sugarcane, and soya bean sludge, were tested separately as substrates to produce lovastatin using a new fungal strain, Aspergillus sclerotiorum PSU-RSPG 178, under both submerged and solid-state fermentation (SSF). RESULTS AND CONCLUSIONS: Of these substrates and cultivation systems, soya bean sludge gave the highest lovastatin yield on dry mass basis of 0.04 mg/g after 14 days of SSF at 25 °C. Therefore, the soya bean sludge was separately supplemented with glucose, wheat flour, trace elements, palm oil, urea and molasses. The addition of the palm oil enhanced the lovastatin yield to 0.99 mg/g. In addition, the optimum conditions, which gave a lovastatin yield of (20±2) mg/g after 18 days of SSF, were soya bean sludge containing 80% moisture (dry basis) at a ratio of soya bean sludge (g) to mycelial agar plugs of 1:4, and a ratio of soya bean sludge (g) to palm oil (mL) of 1:2. Besides, the lovastatin yields obtained from SSF using fresh or dry soya bean sludge were not significantly different. NOVELTY AND SCIENTIFIC CONTRIBUTION: We conclude that A. sclerotiorum PSU-RSPG 178 has a good potential as an alternative strain for producing lovastatin using soya bean sludge supplemented with palm oil as a carbon source.

(-)-Kusunokinin inhibits breast cancer in N-nitrosomethylurea-induced mammary tumor rats.

Natural and synthetic (-)-kusunokinin inhibited breast cancer, colon cancer and cholangiocarcinoma cells at the G2/M phase and induced apoptosis. However, there is no report on the action and adverse effects of (-)-kusunokinin in animal models. In this study, we investigated the cytotoxic effect of (-)-kusunokinin from Piper nigrum on cancer cells. NMU-induced rat mammary tumors, an ER positive breast cancer model, were treated with (-)-kusunokinin. Proteins of interest related to cell cycle, angiogenesis, migration and signaling proteins were detected in tumor tissues. Results showed that (-)-kusunokinin exhibited strong cytotoxicity against breast, colon and lung cancer cells and caused low toxicity against normal fibroblast cells. For in vivo study, 7.0 mg/kg and 14.0 mg/kg of (-)-kusunokinin reduced tumor growth without side effects on body weight, internal organs and bone marrow. Combination of (-)-kusunokinin with a low effective dose of doxorubicin significantly inhibited tumor growth and provoked cell death in cancer tissues. Mechanistically, 14.0 mg/kg of (-)-kusunokinin decreased cell proliferation (c-Src, PI3K, Akt, p-Erk1/2 and c-Myc), cell cycle (E2f-1, cyclin B1 and CDK1), and metastasis (E-cadherin, MMP-2 and MMP-9) proteins in tumor tissues, which supports its anticancer effect. We further confirmed the antimigration effect of (-)-kusunokinin; the results show that this compound inhibited breast cancer cell (MCF-7) migration in a dose-dependent manner. In conclusion, the results suggest that 14 mg/kg of (-)-kusunokinin inhibited tumors through the reduction of signaling proteins and their downstream molecules. Therefore, (-)-kusunokinin becomes an intriguing candidate for cancer treatment as it provides a strong potency in cancer inhibition.

Anticancer activity of synthetic (±)-kusunokinin and its derivative (±)-bursehernin on human cancer cell lines.

Kusunokinin is a potent lignan compound with a several biological properties including antitrypanosomal and anticancer. In this study, (±)-kusunokinin and its derivative, (±)-bursehernin, were synthesized and investigated for their anticancer activities on cell viability, cell cycle arrest and apoptosis in cancer cell lines including breast cancer (MCF-7, MDA-MB-468 and MDA-MB-231), colon cancer (HT-29) and cholangiocarcinoma (KKU-K100, KKU-M213 and KKU-M055) cells. The result showed that (±)-kusunokinin and (±)-bursehernin represented the strongest growth inhibition against breast cancer (MCF-7) and cholangiocarcinoma (KKU-M213) cells with the IC50 values of 4.30 ±â€¯0.65 µM and 3.70 ±â€¯0.79 µM, respectively, both of which were lower than IC50 of normal fibroblast cells (L929). Etoposide was used as a positive control since this chemotherapeutic drug is in the lignan group same as (±)-kusunokinin. Surprisingly, etoposide showed less cytotoxicity than (±)-kusunokinin and its derivative on MCF-7, HT-29, KKU-M213 and KKU-K100. Moreover, (±)-bursehernin induced cell cycle arrest at G2/M phase, meanwhile (±)-kusunokinin tended to increased cell population at G2/M phase but did not show the significant difference compared with non-treated cells. Interestingly, protein levels related to cell proliferation pathway (topoisomerase II, STAT3, cyclin D1, and p21) were significantly decreased at 72 h. Both compounds induced apoptotic cell in time-dependent manner as confirmed by MultiCaspase assay. In conclusion, synthetic compound, (±)-kusunokinin and (±)-bursehernin, showed anticancer effects via the reduction of cell proliferation proteins and induction of apoptosis.

(-)-Kusunokinin and piperloguminine from Piper nigrum: An alternative option to treat breast cancer.

Several studies have reported that active compounds isolated from Piper nigrum possess anticancer properties. However, there are no data on anticancer activity of (-)-kusunokinin and piperlonguminine. The purposes of this study were to isolate active compounds from P. nigrum and identify the molecular mechanisms underlying growth and apoptosis pathway in breast cancer cells. Two bioactive compounds, (-)-kusunokinin and piperlonguminine, were isolated from P. nigrum. Cytotoxicity and the molecular mechanism were measured by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry and Western blot analysis. We found that the active compounds, which effect cancer cell lines were (-)-kusunokinin and piperlonguminine. These compounds have potent cytotoxic effects on breast cancer cells (MCF-7 and MDA-MB-468) and colorectal cells (SW-620). (-)-Kusunokinin demonstrated a cytotoxic effect on MCF-7 and MDA-MB-468 with IC50 values of 1.18 and 1.62µg/mL, respectively. Piperlonguminine had a cytotoxic effect on MCF-7 and MDA-MB-468 with IC50 values of 1.63 and 2.19µg/mL, respectively. Both compounds demonstrated lower cytotoxicity against normal breast cell lines with IC50 values higher than 11µg/mL. Cell cycle and apoptotic analysis using flow cytometry, showed that the (-)-kusunokinin and piperlonguminine induced cell undergoing apoptosis and drove cells towards the G2/M phase. Moreover, both compounds decreased topoisomerase II and bcl-2. The increasing of p53 levels further increased p21, bax, cytochrome c, caspase-8, -7 and -3 activities, except caspase-9. These results suggest that the (-)-kusunokinin and piperlonguminine have been shown to have potent anticancer activities through extrinsic pathway and G2/M phase arrest.

γ-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178.

Chromatographic separation of the broth extract of the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 resulted in isolation of four γ-butenolide-furanone dimers, aspersclerotiorones A-D, a furanone derivative, aspersclerotiorone E, and two γ-butenolide derivatives, aspersclerotiorones F and G, together with six known compounds, penicillic acid, dihydropenicillic acid, 5,6-dihydro-6-hydroxypenicillic acid, 6-methoxy-5,6-dihydropenicillic acid, coculnol and (4R,5R)-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-en-1-one. Their structures were determined by spectroscopic evidence. For aspersclerotiorones A and B, the structures were confirmed by single-crystal X-ray diffraction crystallography. Penicillic acid displayed weak antibacterial activity against Staphylococcus aureus and Escherichia coli with equal MIC values of 128 µg/mL, and it was noncytotoxic towards African green monkey kidney fibroblast cells.

The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of Vatica diospyroides symington type SS fruit include effects on mode of cell death.

BACKGROUND: Vatica diospyroides type LS is a known source of valuable compounds for cancer treatment, however, in contrast little is known about therapeutic efficacy of type SS. OBJECTIVE: This study focused on in vitro cytotoxicity of these fruit extracts, and the cell death mode they induce in breast cancer cells. MATERIALS AND METHODS: Acetone extracts of fruit were tested for cytotoxicity against MCF-7 and MDA-MB-231 cell lines. The apoptosis and necrosis of these cells were quantified by fluorescence activated cell sorter (FACS) and western blot analyses. RESULTS: After 72 h of treatment, the 50% growth inhibition concentrations (IC50) levels were 16.21 ± 0.13 µg/mL against MCF-7 and 30.0 ± 4.30 µg/mL against MDA-MB-231, indicating high and moderate cytotoxicity, respectively. From the FACS results, we estimate that the cotyledon extract at half IC50 produced 11.7% dead MCF-7 cells via apoptosis, whereas another concentrations both apoptosis and necrosis modes co-existed in a dose-dependent manner. In MDA-MB-231 cell line, only the apoptosis was induced by the pericarp extract in a dose-dependent manner. With the extracts at half IC50 concentration, in both cells, the expression of p21 decreased while that of Bax increased within 12-48 h of dosing, confirming apoptosis induced by time-dependent responses. Apoptosis dependent on p53 was found in MCF-7, whereas the mutant p53 of MDA-MB-231 cells was expressed. CONCLUSION: The results indicate that fruit extracts of V. diospyroides have cytotoxic effects against MCF-7 and MDA-MB-231 cells via apoptosis pathway in a dose-dependent manner. This suggests that the extracts could provide active ingredients for the development, targeting breast cancer therapy.

Cytotoxic activity of Piper cubeba extract in breast cancer cell lines.

This study aimed to evaluate the cytotoxicity of a crude extract of Piper cubeba against normal and breast cancer cell lines. To prepare the extract, P. cubeba seeds were ground, soaked in methanol and dichloromethane and isolated by column chromatography. Fractions were tested for cytotoxicity effects on normal fibroblast (L929), normal breast (MCF-12A) and breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). The most effective fraction was selected for DNA fragmentation assay to detect apoptotic activity. The results showed that the methanolic crude extract had a higher cytotoxic activity against MDA-MB-468 and MCF-7 than a dichloromethane crude extract. Then, the methanolic crude extract was separated into six fractions, designated A to F. Fraction C was highly active against breast cancer cell lines with an IC50 value less than 4 µg/mL. Therefore, Fraction C was further separated into seven fractions, CA to CG. The 1H-NMR profile showed that Fraction CE was long chain hydrocarbons. Moreover, Fraction CE demonstrated the highest activity against MCF-7 cells with an IC50 value of 2.69 ± 0.09 µg/mL and lower cytotoxicity against normal fibroblast L929 cells with an IC50 value of 4.17 ± 0.77 µg/mL. Finally, DNA fragmentation with a ladder pattern characteristic of apoptosis was observed in MCF-7, MDA-MB-468, MDA-MB-231 and L929 cells, but not in MCF-12A cells.

Extracts from Vatica diospyroides type SS fruit show low dose activity against MDA-MB-468 breast cancer cell-line via apoptotic action.

Very strong antiproliferative action of V. diospyroides type SS fruit extracts (IC50 range of 1.60-17.45 µg/mL) in MDA-MB-468 cell-line was observed in an MTT assay. After dosing of an extract concentration at half IC50 to cell line for 24 to 72 hours, treated cells were subjected to Annexin V-FITC/PI binding assay, followed by FACS and western blot analyses. Significant apoptotic death was observed with all extract treatments and both exposure times. Dosing with acetone extract of pericarp and cotyledon induced the highest apoptotic populations (33 and 32%, resp.), with the lowest populations of viable cells (65 and 67%, resp.). During 24 to 72 hours of dosing with methanolic extract of pericarp, the populations of viable and early apoptotic cells decreased significantly from 72.40 to 71.32% and from 12.00 to 6.36%, respectively, while the late apoptotic and nonviable cell populations continuously increased from 15.30 to 19.18% and from 0.30 to 3.14%, respectively. The expression of Bax increased within 12-48 hours of dosing, confirming apoptosis induced by time-dependent responses. The mutant p53 of MDA-MB-468 cells was expressed. Our results indicate that apoptosis and time-dependent therapeutic actions contribute to the cytotoxic effects of V. diospyroides type SS fruit on MDA-MB-468 cell.

Friedolanostanes and xanthones from the twigs of Garcinia hombroniana.

The twigs of Garcinia hombroniana yielded six compounds: two 17,14-friedolanostanes (garcihombronanes K-L, 1-2) and four xanthones (garcihombronones A-D, 3-6) together with 14 known compounds including four friedolanostanes, one lanostane, six xanthones, two benzoic acid derivatives and one biflavonoid. Their structures were elucidated by analysis of spectroscopic data and comparison of the NMR data with those reported previously. Their antibacterial activity against methicillin-resistant Staphylococcus aureus and S. aureus was evaluated.

Metabolites from the mangrove-derived fungus Xylaria cubensis PSU-MA34.

Two new succinic acid derivatives, xylacinic acids A (1) and B (2), along with seven known compounds, including one succinic acid derivative (3), three mellein derivatives (4-6), cytochalasin D (7), 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (8) and isosclerone (9), were isolated from the mangrove-derived fungus Xylaria cubensis PSU-MA34. Their structures were established by spectroscopic evidence. They were evaluated for cytotoxicity against KB cells and antibacterial activity against Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus.

Phthalide and isocoumarin derivatives produced by an Acremonium sp. isolated from a mangrove Rhizophora apiculata.

Nine new fungal metabolites, one phthalide derivative, acremonide (1), and eight isocoumarin derivatives, acremonones A-H (2-9), were isolated from the mangrove-derived fungus Acremonium sp. PSU-MA70 together with 10 known compounds. Their structures were determined by NMR analysis. The known 8-deoxytrichothecin and trichodermol exhibited moderate antifungal activity against Candida albicans and Cryptococcus neoformanns, respectively.

A ß-resorcylic macrolide from the seagrass-derived fungus Fusarium sp. PSU-ES73.

A new ß-resorcylic macrolide, 5'-hydroxyzearalenone (1), and six known ß-resorcylic macrolides were isolated from the seagrass-derived fungus Fusarium sp. PSU-ES73. Their structures were established by analysis of spectral data. All of the isolated compounds were evaluated for their antibacterial activity against Staphylococcus aureus, both standard and methicillin-resistant strains, as well as their antifungal activity against Cryptococcus neoformans. Only the known compound zearalenone (2) displayed weak antibacterial and antifungal activities.

Azaphilone and isocoumarin derivatives from the endophytic fungus Penicillium sclerotiorum PSU-A13.

Two new azaphilone derivatives, penicilazaphilones A (1) and B (2), and one new isocoumarin, penicilisorin (3), together with six known compounds were isolated from the endophytic fungus Penicillium sclerotiorum PSU-A13. Their structures were identified by analysis of spectroscopic data. The antimicrobial activity against Staphylococcus aureus, Candida albicans and Cryptococcus neoformans as well as the inhibitory effect on human immunodeficiency virus (HIV)-1 integrase and protease were examined.

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.

An [11]cytochalasin derivative from the marine-derived fungus Xylaria sp. PSU-F100.

A new [11]cytochalasin derivative, xylarisin (1), was isolated from the marine-derived fungus Xylaria sp. PSU-F100 along with six known metabolites: three mellein derivatives (2-4), one pyrone derivative (5) and two carboxylic acids (6,7). The structure and stereochemistry of 1 were determined by NMR and X-ray diffraction analyses. All isolated compounds showed mild antibacterial activity against standard Staphylococcus aureus ATCC 25923 and methicillin-resistant strain.

Lactone derivatives from the marine-derived fungus Penicillium sp. PSU-F44.

Two new fungal metabolites, penicipyrone (1), and penicilactone (2), were isolated from the marine-derived fungus Penicillium sp. PSU-F44 along with three known macrolides, (+)-brefeldin A (3), (+)-brefeldin C (4), and 7-oxobrefeldin A (5). Their antimicrobial activities against methicillin-resistant Staphylococcus aureus SK1 and Microsporum gypseum SH-MU-4 were examined.