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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Amurensin G, a potent natural SIRT1 inhibitor, rescues doxorubicin responsiveness via down-regulation of multidrug resistance 1.

The transition from a chemotherapy-responsive cancer to a chemotherapy-resistant one is accompanied by increased expression of multidrug resistance 1 (MDR1, p-glycoprotein), which plays an important role in the efflux from the target cell of many anticancer agents. We recently showed that a Forkhead box-containing protein of the O subfamily 1 (FoxO1) is a key regulator of MDR1 gene transcription. Because nuclear localization of FoxO1 is regulated by silent information regulator two ortholog 1 (SIRT1) deacetylase, we wondered whether SIRT1 dominates MDR1 gene expression in breast cancer cells. Overexpression of SIRT1 enhanced both FoxO reporter activity and nuclear levels of FoxO1. Protein expression of MDR1 and gene transcriptional activity were also up-regulated by SIRT1 overexpression. In addition, SIRT1 inhibition reduced both nuclear FoxO1 levels and MDR1 expression in doxorubicin-resistant breast cancer cells (MCF-7/ADR) cells. A potent SIRT1 inhibitor, amurensin G (from Vitis amurensis), was identified by screening plant extracts and bioassay-guided fractionation. The compound suppressed FoxO1 activity and MDR1 expression in MCF-7/ADR cells. Moreover, pretreatment of MCF-7/ADR cells with 1 µg/ml amurensin G for 24 h increased cellular uptake of doxorubicin and restored the responsiveness of MCF-7/ADR cells to doxorubicin. In xenograft studies, injection of 10 mg/kg i.p. amurensin G substantially restored the ability of doxorubicin to inhibit MCF-7/ADR-induced tumor growth. These results suggest that SIRT1 is a potential therapeutic target of MDR1-mediated chemoresistance and that it may be possible to develop amurensin G as a useful agent for chemoresistance reversal.

New stilbenoid with inhibitory activity on viral neuraminidases from Erythrina addisoniae.

Influenza occurs with seasonal variations and reaches the peak prevalence in winter causing the death of many people worldwide. A few inhibitors of viral neuraminidase, including amantadine, rimantadine, zanamivir, and oseltamivir, have been used as influenza therapy. However, as drug-resistant influenza viruses are generated rapidly, there is a need to identify new agents for chemotherapy against influenza. Therefore, research on more effective drugs has been given high priority. During the course of an anti-influenza screening program on natural products, two new compounds (1 and 2) along with seven known flavonoid derivatives (3-9) were isolated as active principles from an EtOAc-soluble extract of the root bark of Erythrina addisoniae. The stilbenoid (2) and chalcone (3, 4, and 6) compounds of the isolates exhibited stronger activity than the isoflavone ones. Compound 2, which is a formylated stilbenoid derivative, exhibited strong inhibition of both influenza H1N1 and H9N2 neuraminidases with IC(50) values of 8.80±0.34 µg/mL and 7.19±0.40 µg/mL, respectively.

C-Methylated Flavonoids from Cleistocalyx operculatus and Their Inhibitory Effects on Novel Influenza A (H1N1) Neuraminidase.

As part of an ongoing study focused on the discovery of anti-influenza agents from plants, four new (1-4) and 10 known (5-14) C-methylated flavonoids were isolated from a methanol extract of Cleistocalyx operculatus buds using an influenza H1N1 neuraminidase inhibition assay. Compounds 4, 7, 8, and 14, with a chalcone skeleton, showed significant inhibitory effects on the viral neuraminidases from two influenza viral strains, H1N1 and H9N2. Compound 4 showed the strongest inhibitory activity against the neuraminidases from novel influenza H1N1 (WT) and oseltamivir-resistant novel H1N1 (H274Y mutant) expressed in 293T cells with IC50 values of 8.15 ± 1.05 and 3.31 ± 1.34 µM, respectively. Compounds 4, 7, 8, and 14 behaved as noncompetitive inhibitors in the kinetic studies. These results indicate that C-methylated flavonoids from C. operculatus have the potential to be developed as neuraminidase inhibitors for novel influenza H1N1.

AMP-activated protein kinase (AMPK) activation by benzofurans and coumestans isolated from Erythrina abyssinica.

AMP-activated protein kinase (AMPK) has been proposed as a therapeutic target for the treatment of metabolic syndrome including obesity and type-2 diabetes. The bioassay-guided fractionation of an EtOAc-soluble extract of the stem bark of Erythrina abyssinica led to the isolation of a new coumestan, erythribyssin N (1), and two new benzofurans, erythribyssin F (2) and erythribyssin H (3), along with five known compounds (4-8). When tested for their stimulatory effects on AMPK activity at a concentration of 10 muM, compounds 4 and 5 showed potent activation, while compounds 1, 2, and 7 had moderate effects. These results suggest that benzofurans and coumestans may be new lead compounds for regulating the AMPK enzyme.

SIRT1 inhibitory diterpenoids from the Vietnamese medicinal plant Croton tonkinensis.

Silent information regulator two ortholog 1 (SIRT1) is a member of the sirtuin deacetylase family of enzymes that removes acetyl groups from the lysine residues in histones and other proteins. It has been suggested that SIRT1 inhibitors might be beneficial in the treatment of cancer and neurodegenerative diseases. Bioassay-guided fractionation of the MeOH extract of the leaves of CROTON TONKINENSIS resulted in the isolation of a new ENT-kaurane diterpenoid (1) along with 11 known compounds (2- 12). The structure of the new compound 1 was determined to be ENT-11 alpha-acetoxy-7 beta-hydroxykaur-16-en-15-one based on spectroscopic analyses. Compounds 3, 4, 6- 9, 11, and 12 exhibited SIRT1 inhibitory activity in an IN VITRO assay, with IC (50) values ranging from 16.08 +/- 0.11 to 44.34 +/- 2.32 microM. This is the first report showing the potential of ENT-kaurane diterpenoids as a new class of natural SIRT1 inhibitors.

Cytotoxic and PTP1B inhibitory activities from Erythrina abyssinica.

Bioassay-guided fractionation of the EtOAc extract of the stem bark of Erythrina abyssinica (Leguminosae) resulted in the isolation of three new (1-3), along with 12 known (4-15) pterocarpan derivatives. Their chemical structures were determined by physicochemical and spectroscopic data analysis (IR, UV, [alpha](D), CD, 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, tamoxifen-resistant MCF7 (MCF7/TAMR), adriamycin-resistant MCF7 (MCF7/ADR) and MDA-MB-231 breast cancer cell lines. Compounds which exhibited PTP1B inhibitory activity (IC(50) values ranging from 4.2+/-0.2 to 19.3+/-0.3 microM) showed strong cytotoxic activity (IC(50) values from 5.6+/-0.7 to 28.0+/-0.2 microM). Our data suggested that pterocarpans could be considered as new anticancer materials by PTP1B inhibition.

Crotonkinensins A and B, diterpenoids from the Vietnamese medicinal plant Croton tonkinensis.

Two new diterpenoids, crotonkinensins A (1) and B (2), were isolated from the leaves of the Vietnamese endemic medicinal plant Croton tonkinensis. Their structures were determined to be 7alpha,10alpha-epoxy-14beta-hydroxygrayanane-1(5),16(17)-dien-2,15-dione (1) and 7alpha,10alpha-epoxy-14beta-hydroxygrayanane-1(2),16(17)-dien-15-one (2) by spectroscopic analysis. Compounds 1 and 2 showed strong anti-inflammatory effects on the LPS-induced COX-2 promoter activity and COX-2 expression in Raw 264.7 cells.

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.