Cladobotric Acids: Metabolites from Cultures of Cladobotryum sp., Semisynthetic Analogues and Antibacterial Activity.

Three new polyketide-derived natural products, cladobotric acids G-I (1-3), and six known metabolites (4, 5, 8-11) were isolated from fermentation of the fungus Cladobotryum sp. grown on rice. Their structures were elucidated by extensive spectroscopic methods. Two metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of new products (12-20) by semisynthesis. The antibacterial activities of all these compounds were investigated against the Gram-positive pathogen Staphylococcus aureus including methicillin-susceptible (MSSA), methicillin-resistant and vancomycin-intermediate (MRSA/VISA), and heterogeneous vancomycin-intermediate (hVISA) strains. Results of these antibacterial assays revealed structural features of the unsaturated decalins important for biological activity.

The Biogenetic Origin of the Biologically Active Naematolin of Hypholoma Species Involves an Unusual Sesquiterpene Synthase.

Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5ß,6α,8ß-trihydroxycariolan, 5ß,8ß-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.

Further sesquiterpenoids from the rhizomes of Homalomena occulta and their anti-inflammatory activity.

The rhizomes of Homalomena occulta are called Qian-nian-jian in Traditional Chinese Medicine (TCM), which is widely consumed in China owing to its health benefits for the treatment of rheumatoid arthritis and for strengthening tendons and bones. A phytochemical investigation on this famous TCM yielded 19 sesquiterpenoids (1-19) with various carbocyclic skeletons including isodaucane (2, 8, and 9), guaiane (3), eudesmane (4 and 10-15), oppositane (5, 16, and 17), and aromadendrane (18 and 19) types. The structures of new compounds, Homalomenins A-E (1-5), were determined by diverse spectroscopic data. Compound 1 possessed a rare sesquiterpenoid skeleton and compound 5 represented the first example of 1,4-oxa-oppositane sesquiterpenoid. These isolates were evaluated for their inhibitory effects on COX-2 mRNA, COX-2 protein expression, and prostaglandin E2 (PGE2) production in Raw264.7 cells, which demonstrated that compounds 5, 18, 19 showed potent anti-inflammatory activity by suppressing LPS-induced COX-2 expression and PGE2 production in a dose-dependent manner.

Fusaric acid and analogues as Gram-negative bacterial quorum sensing inhibitors.

Taking advantage of microwave-assisted synthesis, efficient and expedite procedures for preparation of a library of fusaric acid and 39 analogues are reported. The fusaric acid analogues were tested in cell-based screening assays for inhibition of the las and rhl quorum sensing system in Pseudomonas aeruginosa and the lux quorum sensing system in Vibrio fischeri. Eight of the 40 compounds in the library including fusaric acid inhibited lux quorum sensing and one compound inhibited activity of the las quorum sensing system. To our delight, none of the compounds showed growth inhibitory effects in the tested concentration ranges.

Demethoxycurcumin Is A Potent Inhibitor of P-Type ATPases from Diverse Kingdoms of Life.

P-type ATPases catalyze the active transport of cations and phospholipids across biological membranes. Members of this large family are involved in a range of fundamental cellular processes. To date, a substantial number of P-type ATPase inhibitors have been characterized, some of which are used as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells. We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site of these pumps. Future research on biological effects of commercial preparations of curcumin should consider the heterogeneity of the material.

Antiviral phenolics from the leaves of Cleistocalyx operculatus.

During the screening program for anti-influenza agents from medicinal plants, the ethanolic extract of Cleistocalyx operculatus leaves was found to exhibit potential neuraminidase (NA) inhibitory activity. Bioassay-directed fractionation led to the isolation of two new acetophenones (1 and 2) and one new flavanone (3), along with six known compounds (4-9). The structures of all isolated compounds were elucidated using various spectroscopic methods and through comparison with the previous literature. Compounds 6 and 8 exhibited strong enzymatic inhibition on various neuraminidases from different influenza viruses, including H1N1, H9N2, novel H1N1, and oseltamivir-resistant novel H1N1 (H274Y mutation) expressed in HEK293 cells (IC50 values ranging from 5.07 ± 0.94 µM to 9.34 ± 2.52 µM, respectively). Kinetic experiments revealed the non-competitive inhibitory mode of both compounds 6 and 8. Furthermore, these flavonoids reduced the cytopathic effect of the H1N1 virus in MDCK cells. The present study suggests the potential of two flavonoids (6 and 8) as new lead compounds for the development of novel NA inhibitors in the future.

Dammarane triterpenes from the leaves of Panax ginseng enhance cellular immunity.

In our search for immune stimulating materials from natural source, bioassay-guided fractionation of a methanol extract of Panax ginseng leaves led to the isolation of three dammarane triterpenes (1-3), including two previously unknown compounds 27-demethyl-(E,E)-20(22),23-dien-3ß,6α,12ß-trihydroxydammar-25-one (1) and 3ß,20(S)-dihydroxydammar-24-en-12ß,23ß-epoxy-20-O-ß-D-glucopyranoside (2). Their structures were elucidated on the basis of spectroscopic methods, chemical transformation, and by the comparison with those of literature data. Compounds 1-3 significantly increased interleukin-12 expression in LPS-activated mouse peritoneal macrophage at a concentration of 100 ng/mL. Furthermore, compound 1 strongly increased the Th1 response-mediated cytokine IL-2, and decreased Th2 response-mediated cytokines IL-4 and IL-6 expression at 100 ng/mL on ConA-activated splenocytes. This study indicated that compound 1 showed a better effect on cellular immunity, and provided new chemical entities as promising lead compounds for the treatment of cellular immunity-related diseases.

In vitro and in vivo hepatoprotective effect of ganodermanontriol against t-BHP-induced oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (Fr.) Karst. (Ganodermataceae) is a mushroom which is used as a traditional remedy in the treatment of human diseases such as hepatitis, liver disorders, hypercholesterolemia, arthritis, bronchitis and tumorigenic diseases. This study targets the evaluation of hepatoprotective activity of ganodermanontriol, a sterol isolated from Ganoderma lucidum, and the investigation of its mechanism of action in Hepa1c1c7 and murine liver cells upon tert-butyl hydroperoxide (t-BHP)-induced inflammation. t-BHP was utilized to stimulate an anti-inflammatory reaction in the hepatic cell lines and murine hepatic tissue examined. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were used to estimate the expression of ganodermanontriol (GDT)-induced proteins, including heme oxidase-1 (HO-1) and mitogen-activated protein kinases (MAPKs) as well as the corresponding mRNA. Luciferase assays were conducted to evaluate the interaction between NF-E2-related factor-2 (Nrf-2), the antioxidant response element (ARE), and the promoter region of the HO-1 gene and subsequent gene expression. Biochemical markers for hepatotoxicity were monitored to assess whether GDT protected the cells from the t-BHP-mediated oxidative stimuli. RESULTS: GDT induced HO-1 expression via the activation of Nrf-2 nuclear translocation and the subsequent transcription of the HO-1 gene in vitro and in vivo, which seemed to be regulated by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p38 signaling pathways. GDT exhibited in vitro and in vivo hepatoprotective activity as determined by the lowered levels of hepatic enzymes and malondialdehydes and the elevated glutathione levels. CONCLUSIONS: This study validates the ethnopharmacological application of Ganoderma lucidum as a treatment for hepatic disorders. GDT induced in vitro and in vivo anti-inflammatory activity in t-BHP-damaged hepatic cells through the expression of HO-1, and in which PI3K/Akt and p38 kinases are involved. Our study motivates further research in the exploration of potent hepatoprotective agents from Ganoderma lucidum.

Ku70 acetylation and modulation of c-Myc/ATF4/CHOP signaling axis by SIRT1 inhibition lead to sensitization of HepG2 cells to TRAIL through induction of DR5 and down-regulation of c-FLIP.

In this study, we investigated the role of c-Myc/ATF4/CHOP signaling pathway in sensitization of human hepatoma HepG2 cells to TRAIL. Knockdown of SIRT1 or treatment with SIRT1 inhibitor caused the up-regulation of DR5 and down-regulation of c-FLIP through modulation of c-Myc/ATF4/CHOP pathway, and subsequently enhanced the cytotoxic and apoptotic effects of TRAIL on HepG2 cells. Interestingly, SIRT1 interacted directly with c-FLIP(L) and Ku70, and treatment with SIRT1 inhibitor enhanced acetylation of Ku70 and subsequently decreased its binding to c-FLIP. And this was followed by degradation of c-FLIP. Moreover, Ku70(-/-) MEF and Ku70-knockdown HepG2 cells showed the increased levels of c-Myc, ATF4, CHOP, and DR5 and decreased level of c-FLIP. These results were followed by increased sensitivity of Ku70(-/-) MEF cells and Ku70-knockdown HepG2 cells to TRAIL compared with their control cells. These findings reveal for the first time that SIRT1 inhibition increases Ku70 acetylation, and the acetylated Ku70 with a decreased function mediates the induction of DR5 and the down-regulation of c-FLIP by up-regulating c-Myc/ATF4/CHOP pathway, and consequently promotes the TRAIL-induced apoptosis of HepG2 cells. This study provides important mechanistic insight of the synergism exhibited by SIRT1 inhibition and TRAIL.

New prenylated isoflavonoids as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Erythrina addisoniae.

Bioassay-guided fractionation of the EtOAc extract of the root of Erythrina addisoniae (Leguminosae) resulted in the isolation of four new (1-4), along with 2 known prenylated isoflavonoids (5-6). The structures of the isolates were assigned on the basis of spectroscopic data analysis, focusing on interpretation of 1D and 2D NMR, and MS data. All the isolates were evaluated for their inhibitory effects on protein tyrosine phosphatase 1B (PTP1B), as well as their growth inhibition on MCF7, adriamycin-resistant MCF7 (MCF7/ADR), and MDA-MB-231 breast cancer cell lines. Compounds which exhibited PTP1B inhibitory activity (IC(50) values ranging from 4.6 ± 0.3 to 24.2 ± 2.1 µM) showed potential cytotoxic activity (IC(50) values ranging from 3.97 ± 0.17 to 11.4 ± 1.9 µM). Taken together, our data suggest that prenylated isoflavonoids, especially the isoflavone-type skeleton could be considered as new lead compounds against breast cancer via PTP1B inhibition.

Potent vasodilation effect of amurensin G is mediated through the phosphorylation of endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) has important regulatory functions in vascular tone, and impaired endothelium-dependent vasodilatation is a key event in diabetes and atherosclerosis. Vitis amurensis grapes containing resveratrol oligomers are consumed as wine and fruit and have antioxidative and neuroprotective effects. In this study, our goal was identify the most potent eNOS-activating compound among six stilbenes and oligostilbenes found in V. amurensis and to clarify its molecular mechanism. Among the six tested compounds, amurensin G most potently relaxed endothelium-intact aortic rings and increased eNOS phosphorylation and nitric oxide (NO) production. Amurensin G increased both estrogen receptor (ER) phosphorylation and ER-dependent gene transcription, and ERα or ERß inhibition suppressed amurensin G-mediated eNOS phosphorylation. Amurensin G enhanced the activities of phosphatidylinositol 3-kinase (PI3K) and Src and their chemical inhibitors suppressed amurensin G-stimulated eNOS phosphorylation. Moreover, amurensin G activated AMP-activated protein kinase (AMPK), and amurensin G-stimulated eNOS phosphorylation and PI3K activation were reversed by AMPK inhibition. ER inhibition reversed AMPK-dependent PI3K activation in response to amurensin G. Amurensin G-mediated endothelium-dependent relaxation was blocked by inhibition of AMPK, ER, Src, or PI3K. These results suggest that amurensin G enhances NO production via eNOS phosphorylation in endothelial cells, and ER-dependent AMPK/PI3K pathways are required. Amurensin G would be applicable to prevent atherosclerosis.

Terpenylated coumarins as SIRT1 activators isolated from Ailanthus altissima.

Four new terpenylated coumarins (1-4) were isolated from the stem bark of Ailanthus altissima by bioactivity-guided fractionation using an in vitro SIRT1 deacetylation assay. Their structures were identified as (2'R,3'R)-7-(2',3'-dihydroxy-3',7'-dimethylocta-6'-enyloxy)-6,8-dimethoxycoumarin (1), 6,8-dimethoxy-7-(3',7'-dimethylocta-2',6'-dienyloxy)coumarin (2), (2'R,3'R,6'R)-7-(2',3'-dihydroxy-6',7'-epoxy-3',7'-dimethyloctaoxy)-6,8-dimethoxycoumarin (3), and (2'R,3'R,4'S,5'S)-6,8-dimethoxy-7-(3',7'-dimethyl-4',5'-epoxy-2'-hydroxyocta-6'-enyloxy)coumarin (4). Compounds 1-4 strongly enhanced SIRT1 activity in an in vitro SIRT1-NAD/NADH assay and an in vivo SIRT1-p53 luciferase assay. These compounds also increased the NAD-to-NADH ratio in HEK293 cells. The present results suggest that terpenylated coumarins from A. altissima have a direct stimulatory effect on SIRT1 deacetylation activity and may serve as lead molecules for the treatment of some age-related disorders.

Xanthones from Polygala karensium inhibit neuraminidases from influenza A viruses.

The emergence of the H1N1 swine flu pandemic has the possibility to develop the occurrence of disaster- or drug-resistant viruses by additional reassortments in novel influenza A virus. In the course of an anti-influenza screening program for natural products, 10 xanthone derivatives (1-10) were isolated by bioassay-guided fractionation from the EtOAc-soluble extract of Polygala karensium. Compounds 1, 3, 5, 7, and 9 with a hydroxy group at C-1 showed strong inhibitory effects on neuraminidases from various influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In addition, these compounds reduced the cytopathic effect of H1N1 swine influenza virus in MDCK cells. Our results suggest that xanthones from P. karensium may be useful in the prevention and treatment of disease by influenza viruses.

Amurensin G, a novel SIRT1 inhibitor, sensitizes TRAIL-resistant human leukemic K562 cells to TRAIL-induced apoptosis.

Many types of cancer cells remain resistant towards TRAIL-induced cytotoxicity by the blockade of apoptotic signaling cascades. Thus, sensitizers are needed to enhance the effect of TRAIL-based cancer therapies. Although synergistic tumor cell death has been reported when various HDAC inhibitors were administered with TRAIL in a variety of human cancers, the effect of inhibitors of Class III HDAC such as SIRT1 have not been reported. We reported here for the first time that inhibition of SIRT1 augmented the cytotoxic and apoptotic effects of TRAIL on human leukemic K562 cells. Knockdown of SIRT1 or treatment with amurensin G, a potent new SIRT1 inhibitor, up-regulated the levels of DR5 and c-Myc and down-regulated the level of c-FLIP(L/S). Furthermore, knockdown of SIRT1 or treatment with amurensin G augmented the molecular responses to TRAIL, including activation of caspase-8, -9 and -3, PARP cleavage, up-regulation of Bax, and down-regulation of Bcl-2. Amurensin G-enhanced TRAIL-induced apoptosis was abrogated by caspase inhibitor Z-VAD-FMK. These findings suggest that the suppression of SIRT1 with siRNA or amurensin G sensitize the TRAIL-resistant K562 cell to TRAIL-induced apoptosis, possibly by the up-regulation of c-Myc and DR5 surface expression and the down-regulations of c-FLIP and Mcl-1. In addition, amurensin G, a potent new SIRT1 inhibitor, would be used as a sensitizer of TRAIL in TRAIL-resistant leukemic cells.

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and ß-catenin and hypoxic preconditioning effect of polyphenols.

SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and ß-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and ß-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and ß-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, ß-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and ß-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning.

Symmetric dimers of ent-kaurane diterpenoids with cytotoxic activity from Croton tonkinensis.

Breast cancer is the most common malignant tumor in women these days accounting for approximately 24% of all cancer. During our screening program searching for cytotoxic materials from natural products, two new symmetric dimers of ent-kaurane diterpenoid, crotonkinensins C (1) and D (2), with connectivity at C-17 were isolated from the leaves of the Vietnamese endemic medicinal plant Croton tonkinensis. Their structures were determined on the basis of physicochemical and spectroscopic data. Compound 2 showed a potent cytotoxic activity against MCF-7, tamoxifen-resistant MCF-7 (MCF-7/TAMR), adriamycin-resistant MCF-7 (MCF-7/ADR), and MDA-MB-231 breast cancer cell lines.

ent-Kaurane diterpenoids from Croton tonkinensis stimulate osteoblast differentiation.

Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11ß-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), ent-14ß-hydroxy-18-acetoxykaur-16-ene (3), and ent-7α-hydroxy-18-acetoxykaur-16-ene (4). Compounds 1-4 significantly increased alkaline phosphatase activity and osteoblastic gene promoter activity. Compounds 1-3 also increased the levels of ALP and collagen type I alpha mRNA in C2C12 cells in a dose-dependent manner. These results suggest that ent-kaurane diterpenoids from C. tonkinensis have a direct stimulatory effect on osteoblast differentiation and may be potential therapeutic molecules against bone diseases such as osteoporosis.

New dammarane-type glucosides as potential activators of AMP-activated protein kinase (AMPK) from Gynostemma pentaphyllum.

AMP-activated protein kinase (AMPK) is a key sensor and regulator of glucose, lipid, and energy metabolism throughout the body. Activation of AMPK improves metabolic abnormalities associated with metabolic diseases including obesity and type-2 diabetes. The oriental traditional medicinal herbal plant, Gynostemma pentaphyllum, has shown a wide range of beneficial effects on glucose and lipid metabolism. In this study, we found that G. pentaphyllum contains two novel dammarane-type saponins designated as damulin A (1), 2α,3ß,12ß-trihydroxydammar-20(22)-E,24-diene-3-O-[ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranoside], and damulin B (2), 2α,3ß,12ß-trihydroxydammar-20,24-diene-3-O-[ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranoside], that strongly activate AMPK in cultured L6 myotube cells. Damulins A and B also increased ß-oxidation and glucose uptake with increasing GluT4 translocation to the plasma membrane in L6 myotube cells. Taken together our results indicate that activation of AMPK by damulins A and B may contribute to beneficial effect of G. pentaphyllum on glucose and lipid metabolism.

New 5-deoxyflavonoids and their inhibitory effects on protein tyrosine phosphatase 1B (PTP1B) activity.

In the course of our program to search for protein tyrosine phosphatase 1B (PTPB) inhibitors, five new 5-deoxyflavonoids along with eight known derivatives were isolated from EtOAc layer of the root bark of Erythrina abyssinica. Their structures were elucidated on the basis of spectroscopic (IR, UV, MS, CD, 1D- and 2D-NMR) and physicochemical analyses. All isolates exhibited moderate inhibitory effects on the enzyme assay with IC50 values ranging from 14.9 ± 1.6 to 98.1 ± 11.3 µM. Compounds with prenyl and methoxy groups in the B ring (1, 2, 4, 8, and 13) possessed strong activity (IC(50) 14.9 ± 1.6 to 19.2 ± 1.1 µM), while compounds (3, 5, and 9) with 2,2-dimethylpyrano ring showed less inhibitory effect (IC50 22.6 ± 2.3 to 72.9 ± 9.7 µM). These results suggest that prenyl and methoxy groups may be responsible for the increase on the activity of 5-deoxyflavonoids against PTP1B, but the presence of 2,2-dimethylpyrano ring on the B ring may be induced the decrease of PTP1B inhibitory activity.

Chalcones as novel influenza A (H1N1) neuraminidase inhibitors from Glycyrrhiza inflata.

The emergence of highly pathogenic influenza A virus strains, such as the new H1N1 swine influenza (novel influenza), represents a serious threat to global human health. During our course of an anti-influenza screening program on natural products, one new licochalcone G (1) and seven known (2-8) chalcones were isolated as active principles from the acetone extract of Glycyrrhiza inflata. Compounds 3 and 6 without prenyl group showed strong inhibitory effects on various neuraminidases from influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In addition, the efficacy of oseltamivir with the presence of compound 3 (5 µM) was increased against H274Y neuraminidase. This evidence of synergistic effect makes this inhibitor to have a potential possibility for control of pandemic infection by oseltamivir-resistant influenza virus.