Construction of an Artificial Biosynthetic Pathway for Zingerone Production in Escherichia coli Using Benzalacetone Synthase from Piper methysticum.

Zingerone (vanillylacetone; 4-hydroxy-3-methoxyphenylethyl methyl ketone) is a key component responsible for the pungency of ginger (Zingiber officinale). In this study, it was confirmed that a type III polyketide synthase (PKS) gene (pmpks) from Piper methysticum exhibits feruloyl-CoA-preferred benzalacetone synthase (BAS) activity. Based on these results, we constructed an artificial biosynthetic pathway for zingerone production from supplemented ferulic acid with 4-coumarate CoA ligase (4CL), PmPKS, and benzalacetone reductase (BAR). Furthermore, a de novo pathway for the production of zingerone was assembled using six heterologous genes, encoding tyrosine ammonia-lyase (optal), cinnamate-4-hydroxlase (sam5), caffeic acid O-methyltransferase (com), 4CL (4cl2nt), BAS (pmpks), and BAR (rzs1), in Escherichia coli. Using the engineered l-tyrosine-overproducing E. coli ΔCOS4 strain as a host, a maximum yield of 24.03 ± 2.53 mg/L zingerone was achieved by complete de novo synthesis.

Production of Bioactive 3'-Hydroxystilbene Compounds Using the Flavin-Dependent Monooxygenase Sam5.

The flavin-dependent monooxygenase Sam5 was previously reported to be a bifunctional hydroxylase with a coumarte 3-hydroxylase and a resveratrol 3'-hydroxylase activity. In this article, we showed the Sam5 enzyme has 3'-hydroxylation activities for methylated resveratrol (pinostilbene and pterostilbene), hydroxylated resveratrol (oxyresveratrol) and glycosylated resveratrol (piceid) as substrates. However, the use of piceid, a glycone type stilbene, as a substrate for bioconversion experiments with the Sam5 enzyme expressed in, Escherichia coli does not convert to the hydroxylated compound astringin, but it has converted in vitro enzyme reactions. Finally, we report a novel catalytic activity of Sam5 monooxygenase for the synthesis of piceatannol derivatives, 3'-hydroxylated stilbene compounds. Development of this bioproduction method for the hydroxylation of stilbenes is challenging because of the difficulty in expressing P450-type hydroxylase in E. coli and regionspecific chemical synthesis.

Structures and biological activities of azaphilones produced by Penicillium sp. KCB11A109 from a ginseng field.

Twelve metabolites, including five highly oxygenated azaphilones, geumsanols A-E, along with seven known analogues were isolated from Penicillium sp. KCB11A109, a fungus derived from a ginseng field. Their structures were assigned by spectroscopic means (NMR and MS), and stereochemistries were determined by extensive spectroscopic analyses ((1)H-(1)H coupling constants, NOESY, and HETLOC) and chemical derivatizations (modified Mosher's method and acetonide formation). The isolates were evaluated for their anticancer, antimicrobial, antimalarial activities, and phenotypic effects in zebrafish development. Of these compounds possessing no pyranoquinone core, only geumsanol E exhibited cytotoxic activities and toxic effects on zebrafish embryos, suggesting that a double bond at C-11 and C-12 is important for biological activity.

Enzymatic synthesis of novel isobavachalcone glucosides via a UDP-glycosyltransferase.

Glycosylation is often used to improve a natural product's properties such as water solubility, chemical stability, pharmacological potency, and structure diversification. In this study, we studied the enzymatic synthesis of novel isobavachalcone glucosides using a UDP-glycosyltransferase (YjiC) from Bacillus licheniformis DSM-13. The chemical structures of compounds 1 and 2 were elucidated by spectroscopic techniques, including LC-MS, MS, and NMR. Meanwhile, the parameters of glycosylation reaction such as incubation time, UDP-glucose concentration, and pH of buffer were also optimized during this study. Furthermore, the compounds 1 and 2 exhibited weak anti-proliferative activities against five human cancer cell lines, with IC50 values ranging from 58.6 to 86.6 µM.

A new dimeric neolignan from Magnolia grandiflora L. seeds.

Bioassay-guided fractionation of the MeOH extract of Magnolia grandiflora seeds resulted in the isolation of a new dimeric neolignan, named bishonokiol A (1), as well as two known neolignans magnolol (2) and honokiol (3). The structures of the compounds were determined on the basis of data obtained using NMR and MS. Bishonokiol A (1) showed potent anti-proliferative activities in four human cancer cell lines, with IC50 values ranging from 5.1 to 7.5 µM. Additionally, bishonokiol A (1) induced apoptosis, as well as down-regulated the expression of the anti-apoptotic protein Bcl-2 and caspase-3 cleavage in HepG2 cell line.

An isoaurone and other constituents from Trichosanthes kirilowii seeds inhibit hypoxia-inducible factor-1 and nuclear factor-kappaB.

Hypoxia-inducible factor-1 and nuclear factor-kappaB have become important targets in cancer treatment due to their critical role in the regulation of genes involved in tumorigenesis. Bioassay-guided fractionation of the methanol extract of Trichosanthes kirilowii seeds led to the isolation of a naturally rare isoaurone, 4',6-dihydroxy-4-methoxyisoaurone (1), together with three known compounds, cucurbitacin B (2), 6-(3-hydroxy-4-methoxystyryl)-4-methoxy-2H-pyran-2-one (3), and blumenol A (4). All compounds inhibited HIF-1 and NF-kappaB activities in reporter assays. Compounds 1-3 potently inhibited HIF-1alpha accumulation and VEGF secretion under hypoxic condition. These results suggest that the tumor cell growth inhibitory activity of T. kirilowii is likely associated with the inhibition of HIF-1 and NF-kappaB activities.

A peroxisome proliferator-activated receptor-gamma agonist and other constituents from Chromolaena odorata.

Peroxisome proliferator-activated receptors (PPARs) are key regulators of lipid and glucose metabolism and have become important therapeutic targets for various diseases. The phytochemical investigation of the chloroform-soluble extract of Chromolaena odorata led to the isolation of a PPAR-gamma agonist, (9 S,13 R)-12-oxo-phytodienoic acid (1), together with 12 other compounds. The structures of chromomoric acid G (2), a new dehydrogenated derivative of 1, and chromolanone (3) were elucidated based on spectroscopic methods. Compound 1 showed a significant effect on PPAR-gamma activation in comparison with rosiglitazone. However, compound 2 was inactive, suggesting that the dehydrogenation of the prostaglandin-like structure in 1 abrogates its PPAR-gamma agonistic activity.

Hypoxia-inducible factor-1 inhibitory benzofurans and chalcone-derived diels-alder adducts from Morus species.

Hypoxia-inducible factor-1 (HIF-1) is the central mediator of cellular responses to low oxygen concentrations and vital to many aspects of cancer biology. Bioassay-guided fractionation of the chloroform-soluble extracts of Morus species using a hypoxia response element (HRE)-dependent reporter assay led to identification of six benzofurans (1-6) and two chalcone-derived Diels-Alder adducts (7, 8) from Mori Cortex Radicis and three prenylated benzofurans (9-11) and four chalcone-derived Diels-Alder adducts (12-15) from Morus bombycis. The structure of the new 2-arylbenzofuran-type compound, moracin Q (3), was elucidated by spectroscopic methods, and the absolute configuration of 2 was determined for the first time. The selected compounds (1-3, 5, 7, 9, 10, and 12) from the cell-based reporter assay were found to inhibit hypoxia-induced HIF-1alpha accumulation in a dose-dependent manner in human hepatocelluar carcinoma cell-line Hep3B cells. Furthermore, these compounds were also active against hypoxia-induced vascular endothelial growth factor (VEGF) secretion in Hep3B cells.

Phenolic constituents of Amorpha fruticosa that inhibit NF-kappaB activation and related gene expression.

NF-kappaB is known to play a crucial role in the regulation of genes controlling the immune system, apoptosis, tumor cell growth, and tissue differentiation. Bioassay-guided fractionation of the n-hexane-soluble fraction of a methanol extract of Amorpha fruticosa afforded four new compounds, 5, 7, 8, and 9, and eight known compounds. Their structures were elucidated by spectroscopic methods. All compounds inhibited NF-kappaB activity, and tephrosin (1), 11-hydroxytephrosin (2), and deguelin (3) were the most active, with IC50 values of 0.11, 0.19, and 0.22 microM, respectively, in TNF-alpha-stimulated HeLa cell-based reporter gene assays. Further investigations showed that compounds 1, 5, and 6 blocked NF-kappaB/DNA binding activity and suppressed the expression of NF-kappaB target genes.

Abietane diterpenes from Salvia miltiorrhiza inhibit the activation of hypoxia-inducible factor-1.

The hypoxia-inducible factor-1 (HIF-1) has been known to be correlated to the adaptation and proliferation of tumor cells; therefore HIF-1 has become an important target in the development of anticancer drugs. A phytochemical study of the CHCl3-soluble fraction of Salvia miltiorrhiza, which strongly inhibited hypoxia-induced reporter gene expression, led to the isolation of 12 abietane-type diterpenes. Of these compounds, sibiriquinone A (1), sibiriquinone B (2), cryptotanshinone (3), and dihydrotanshinone I (4) potently inhibited hypoxia-induced luciferase expression with IC50 values of 0.34, 3.36, 1.58, and 2.05 microM on AGS cells, a human gastric cancer cell line, and 0.28, 3.18, 1.36, and 2.29 microM on Hep3B cells, a human hepatocarcinoma cell line, respectively. Consistently, 1 and 4 dose-dependently suppressed the HIF-1alpha accumulation and 1 inhibited mRNA expression of vascular endothelial growth factor (VEGF) under hypoxia. These results suggest that the anticancer activity of tanshinones is likely at least in part associated with their inhibition of HIF-1 accumulation.

Inhibition of NF-kappa B activation through targeting I kappa B kinase by celastrol, a quinone methide triterpenoid.

Celastrol, a quinone methide triterpenoid, was isolated as an inhibitor of NF-kappaB from Celastrus orbiculatus. This compound dose-dependently inhibited a variety of stimuli-induced NF-kappa B-regulated gene expression and the DNA-binding of NF-kappa B in different cell lines without affecting DNA-binding activity of AP-1. Preincubation of celastrol completely blocked the LPS-, TNF-alpha-, or PMA-induced degradation and phosphorylation of I kappa B alpha. Importantly, celastrol inhibited IKK activity and the constitutively active IKK beta activity in a dose-dependent manner without either affecting the NF-kappa B activation induced by RelA over-expression or directly suppressing the DNA-binding of activated NF-kappa B. However, mutation of cysteine 179 in the activation loop of IKK beta abolished sensitivity towards to celastrol, suggesting that celastrol suppressed the NF-kappa B activation by targeting cysteine 179 in the IKK. To verify that celastrol is a NF-kappa B inhibitor, we investigated its effect on some NF-kappa B target genes expressions. Celastrol prevented not only LPS-induced mRNA expression of iNOS and TNF-alpha, but also TNF-alpha-induced Bfl-1/A1 expression, a prosurvival Bcl-2 homologue. Consistent with these results, celastrol significantly suppressed the production of NO and TNF-alpha in LPS-stimulated RAW264.7 cells, and increased the cytotoxicity of TNF-alpha in HT-1080 cells. We also demonstrated that celastrol showed anti-inflammatory and anti-tumor activities in animal models. Taken together, this study extends our understanding on the molecular mechanisms underlying the anti-inflammatory and anti-cancer activities of celastrol and celastrol-containing medicinal plant, which would be a valuable candidate for the intervention of NF-kappa B-dependent pathological conditions.

Phenanthroquinolizidine alkaloids from the roots of Boehmeria pannosa potently inhibit hypoxia-inducible factor-1 in AGS human gastric cancer cells.

A bioassay-guided phytochemical investigation on the methanol extract of Boehmeria pannosa, using a HIF-1-mediated reporter gene assay, led to the isolation of two phenanthroquinolizidine alkaloids, (-)-cryptopleurine (1) and (-)-(15R)-hydroxycryptopleurine (2). The structure of the new compound 2 was determined by spectroscopic methods. Compounds 1 and 2 potently inhibited the hypoxia-induced expression of a reporter gene under the control of a hypoxia response element (HRE) with IC(50) values of 8.7 and 48.1 nM, respectively. Furthermore, 1 and 2 suppressed the accumulation of HIF-1alpha protein in a dose-dependent manner, but not the HIF-1beta protein and inhibited expression of vascular endothelial growth factor (VEGF) by hypoxia.

New sesquiterpene dimers from Inula britannica inhibit NF-kappaB activation and NO and TNF-alpha production in LPS-stimulated RAW264.7 cells.

A bioassay-guided isolation of an ethyl acetate-soluble extract of the aerial parts of Inula britannica var. chinensis (Rupr.) Regel, using an in vitro NF-kappaB reporter gene assay, led to the isolation of four new sesquiterpene dimers bearing a norbornene moiety, inulanolides A-D, and three known sesquiterpenes, 1,6alpha-dihydroxyeriolanolide, 1-acetoxy-6alpha-hydroxyeriolanolide, and eupatolide. The structures of the new compounds were elucidated by spectroscopic methods. Among these compounds, inulanolides B and D and eupatolide, exhibited potent inhibitory activity on the LPS-induced NF-kappaB activation with IC50 values of 0.49 microM, 0.48 microM, and 1.54 microM, respectively. Consistent with their inhibitory effect on NF-kappaB activation, compounds and also strongly inhibited the production of NO and TNF-alpha in the LPS-stimulated RAW264.7 cells with IC50 values in the range of 2 microM.

Inhibitors of the LPS-induced NF-kappaB activation from Artemisia sylvatica.

Three guaianolide sesquiterpene lactones, 3alpha,4alpha-epoxyrupicolins C-E, together with six known sesquiterpenes, artemisolide, 3-methoxytanapartholide, deacetyllaurenobiolide, moxartenolide as well as arteminolides B and D were isolated by bioassay-guided fractionation from the methanol extract of the aerial parts of Artemisia sylvatica using the NF-kappaB mediated reporter gene assay. All isolated compounds displayed inhibitory activity on the LPS-induced NF-kappaB activation, NO production, and TNF-alpha production with IC50 values of 0.49-7.17, 1.46-6.16, and 3.19-27.76 microM, respectively, in RAW264.7 cells. It was also established that arteminolide B suppressed the expression of NF-kappaB target genes such as iNOS and COX-2. This is the first report of NF-kappaB inhibitory activities of these compounds and supports the pharmacological use of Artemisia sylvatica, which has been employed as an herbal medicine for the treatment of inflammation.

Anti-inflammatory effect of kamebakaurin in in vivo animal models.

We have identified kamebakaurin as an inhibitor of NF-KB and elucidated its molecular mechanism as a specific inhibitor in the DNA-binding activity of the p50 subunit of NF-KB. Here, we describe its anti-inflammatory activity in in vitro and in vivo models. Kamebakaurin dose-dependently inhibited not only the expression of inflammatory NF-KB target genes such as iNOS,COX-2, and TNF-x, but also the production of PGE2 and TNF-a in LPS-stimulated RAW264.7 cells. Moreover, in an air pouch model of inflammation, it suppressed the recruitment of neutrophils,production of TNF-a as well as PGE2 in the pouch exudates induced by carrageenan. In addition, kamebakaurin dose-dependently suppressed the inflammation in an adjuvant arthritis model. Oral administration of 20 mg/kg kamebakaurin resulted in the 75% decrease of paw volume. Taken together, kamebakaurin, a specific inhibitor of DNA-binding activity of the p50 subunit, is a valuable candidate for the intervention in NF-KB-dependent pathological conditions such as inflammation.

Quinolone alkaloids with inhibitory activity against nuclear factor of activated T cells from the fruits of Evodia rutaecarpa.

Nine quinolone alkaloids (1-9) from the fruits of Evodia rutaecarpa were investigated for their inhibitory activity on nuclear factor of activated T cells (NFAT)-dependent transcription in comparison with nuclear factor (NF)-kappa B-dependent transcription using a reporter gene assay. These alkaloids showed inhibitory effects against NFAT activity, with IC(50) values between 0.91 microM and 15.91 microM. Of the N-methylated quinolones, the longer aliphatic side chain at the quinolone ring showed stronger inhibition of NFAT activity. These N-methylated compounds showed comparable inhibitory effects against NF-kappa B activity. However, quinolone alkaloids without the N-methyl group showed a more selective inhibition of NFAT activity.

ent-Kaurane diterpenoids from croton tonkinensis inhibit LPS-induced NF-kappaB activation and NO production.

Four ent-kaurane diterpenoids including two known, ent-7alpha,14beta-dihydroxykaur-16-en-15-one (1) and ent-18-acetoxy-7alpha-hydroxykaur-16-en-5-one (2), and two new, ent-1beta-acetoxy-7alpha,14beta-dihydroxykaur-16-en-15-one (3) and ent-18-acetoxy-7alpha,14beta-dihydroxykaur-16-en-15-one (4), were isolated from the leaves of Croton tonkinensis in a search for inhibitors of NF-kappaB activation and nitric oxide production. These ent-kauranoids inhibited LPS-induced NF-kappaB activation in murine macrophage RAW264.7 cells at IC50 values between 0.07 and 0.42 microM. Consistently, the ent-kauranoids markedly reduced LPS-induced NO production in a comparable concentration-dependent manner.

Sauchinone, a lignan from Saururus chinensis, suppresses iNOS expression through the inhibition of transactivation activity of RelA of NF-kappaB.

Sauchinone, a known lignan, was isolated from the root of Saururus chinensis as an active principle responsible for inhibiting the production of NO in LPS-stimulated RAW264.7 cells by activity-guided fractionation. Sauchinone dose-dependently inhibited not only the production of NO, but also the expression of iNOS mRNA and protein in LPS-stimulated RAW 264.7 cells. Furthermore, sauchinone prevented LPS-induced NF-kappaB activation, which is known to play a critical role in iNOS expression, assessed by a reporter assay under the control of NF-kappaB. However, an electrophoretic mobility shift assay (EMSA) demonstrated that sauchinone did not suppress the DNA-binding activity of NF-kappaB or the degradation of IkappaB-alpha induced by LPS. Further analysis revealed that transactivation activity of RelA subunit of NF-kappaB was dose-dependently suppressed in the presence of sauchinone. Taken together, our results suggested that sauchinone could inhibit production of NO in LPS-stimulated RAW264.7 cells through the suppression of NF-kappaB by inhibiting transactivation activity of RelA subunit.

Two new furanoditerpenes from Saururus chinenesis and their effects on the activation of peroxisome proliferator-activated receptor gamma.

Two new acyclic furanoditerpene compounds, saurufuran A (1) and B (2), were obtained from the root of Saururus chinensis, and their structures were elucidated by means of 1D and 2D NMR spectroscopic analyses. Saurufuran A (1) is effective on the activation of peroxisome proliferator-activated receptor gamma (PPARgamma) with an EC(50) value of 16.7 microM; however, saurufuran B (2), with an EC(50) value of >100 microM, weakly activated the PPARgamma.