Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of ...highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.
Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its ...hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-α-induced NF-κB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 μM, except for isosilybin B, which was toxic to cells above 10 μM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 μM. Silymarin suppressed TNF-α activation of NF-κB dependent transcription, which involved partial inhibition of IκB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.
Two separate commercial products of kratom Mitragyna speciosa (Korth.) Havil. Rubiaceae were used to generate reference standards of its indole and oxindole alkaloids. While kratom has been studied ...for over a century, the characterization data in the literature for many of the alkaloids are either incomplete or inconsistent with modern standards. As such, full 1H and 13C NMR spectra, along with HRESIMS and ECD data, are reported for alkaloids 1–19. Of these, four new alkaloids (7, 11, 17, and 18) were characterized using 2D NMR data, and the absolute configurations of 7, 17, and 18 were established by comparison of experimental and calculated ECD spectra. The absolute configuration for the N(4)-oxide (11) was established by comparison of NMR and ECD spectra of its reduced product with those for compound 7. In total, 19 alkaloids were characterized, including the indole alkaloid mitragynine (1) and its diastereoisomers speciociliatine (2), speciogynine (3), and mitraciliatine (4); the indole alkaloid paynantheine (5) and its diastereoisomers isopaynantheine (6) and epiallo-isopaynantheine (7); the N(4)-oxides mitragynine-N(4)-oxide (8), speciociliatine-N(4)-oxide (9), isopaynantheine-N(4)-oxide (10), and epiallo-isopaynantheine-N(4)-oxide (11); the 9-hydroxylated oxindole alkaloids speciofoline (12), isorotundifoleine (13), and isospeciofoleine (14); and the 9-unsubstituted oxindoles corynoxine A (15), corynoxine B (16), 3-epirhynchophylline (17), 3-epicorynoxine B (18), and corynoxeine (19). With the ability to analyze the spectroscopic data of all of these compounds concomitantly, a decision tree was developed to differentiate these kratom alkaloids based on a few key chemical shifts in the 1H and/or 13C NMR spectra.
Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, ...consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40-85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43-73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases.
Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the ...current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA‐dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti‐HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV‐infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell‐to‐cell spread of virus. Conclusion: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell. HEPATOLOGY 2010
Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than ...$100 million annually. In an attempt to shed light on Echinacea's mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.
Green tea is a popular botanical natural product, and its consumption has been associated with pharmacokinetic natural product‐drug interactions (NP‐DIs). Unpublished clinical data show that acute ...and chronic oral administration of green tea decreased the systemic exposure of raloxifene in healthy subjects. These clinical data indicate an intestinal interaction, although the mechanism for reduced absorption and systemic exposure is not known. NP‐mediated modulation of drug solubility in the intestinal lumen is an area of increasing interest and concern. Intestinal bile acids can enhance the absorption of hydrophobic compounds by micellar solubilization, and raloxifene is a Biopharmaceutic Drug Disposition Classification System (BDDCS) class 2 drug with poor water solubility. We hypothesize that green tea reduces intestinal absorption of raloxifene through decreased micellar solubility. To investigate this interaction, bile acid mixed micelles were prepared and incubated with raloxifene, green tea extract (GTE), (‐)‐epigallocatechin gallate EGCG, and a combination of raloxifene with GTE or EGCG. The size of the micelles was determined by dynamic light scattering and raloxifene quantification was determined by UPLC‐MS/MS. Raloxifene did not affect micelle diameter (micelle alone: 63.2 ± 5.6 nm, micelle with raloxifene 76.0 ± 5.8). In contrast, micelle diameter increased after incubation with GTE (416 ± 150.3 nm), EGCG (300.9 ± 173 nm), raloxifene + GTE (451 ± 164.2 nm), and raloxifene + EGCG (294.8 ± 303 nm). The micellar solubility of raloxifene was decreased by ~80% after incubation with GTE, whereas EGCG produced inconsistent results. These data suggest GTE decreased the micellar solubility of raloxifene and may play a role in the observed green tea‐raloxifene intestinal interaction.
Cyclopeptide alkaloids are an abundant class of plant cyclopeptides with over 200 analogs described and bioactivities ranging from analgesic to antiviral. While these natural products have been known ...for decades, their biosynthetic basis remains unclear. Using a transcriptome‐mining approach, we link the cyclopeptide alkaloids from Ceanothus americanus to dedicated RiPP precursor peptides and identify new, widely distributed split BURP peptide cyclase containing gene clusters. Guided by our bioinformatic analysis, we identify and isolate new cyclopeptides from Coffea arabica, which we named arabipeptins. Reconstitution of the enzyme activity for the BURP found in the biosynthesis of arabipeptin A validates the activity of the newly discovered split BURP peptide cyclases. These results expand our understanding of the biosynthetic pathways responsible for diverse cyclic plant peptides and suggest that these side chain cross‐link modifications are widely distributed in eudicots.
Cyclopeptide alkaloids are a diverse class of plants natural products, yet little is known of their biosynthesis. By combining transcriptomics and genome mining, we uncover a widespread biosynthetic cassette responsible for side chain cross‐linked cyclopeptides. We validate these observations by isolating a new cyclopeptide from Coffea arabica and reconstituting enzymatic activity for the family defining split BURP peptide cyclase.
Sixteen polyketides belonging to diverse structural classes, including monomeric/dimeric tetrahydroxanthones and resorcylic acid lactones, were isolated from an organic extract of a fungal culture ...Setophoma terrestris (MSX45109) by bioactivity‐directed fractionation as part of a search for anticancer leads from filamentous fungi. Of these, six were new: penicillixanthone B (5), blennolide H (6), 11‐deoxyblennolide D (7), blennolide I (9), blennolide J (10), and pyrenomycin (16). The known compounds were: secalonic acid A (1), secalonic acid E (2), secalonic acid G (3), penicillixanthone A (4), paecilin B (8), aigialomycin A (11), hypothemycin (12), dihydrohypothemycin (13), pyrenochaetic acid C (14), and nidulalin B (15). The structures were elucidated by a set of spectroscopic and spectrometric techniques: the absolute configurations of compounds 1–10 were determined by ECD spectroscopy combined with time‐dependent density functional theory (TDDFT) calculations, whereas a modified Mosher's ester method was used for compound 16. The cytotoxic activities of compounds 1–15 against the MDA‐MB‐435 (melanoma) and SW‐620 (colon) cancer cell lines were evaluated. Compounds 1, 4, and 12 were the most potent, with IC50 values ranging from 0.16 to 2.14 μM. When tested against a panel of bacteria and fungi, compounds 3 and 5 showed promising activity against the Gram‐positive bacterium Micrococcus luteus, with MIC values of 5 and 15 μg mL–1, respectively.
Sixteen polyketides, six of them new, were isolated from a fungal culture (Setophoma terrestris). Cytotoxicity assays were used to develop structure–activity relationships amongst the series.
Synthetic biological approaches, such as site-directed biosynthesis, have contributed to the expansion of the chemical space of natural products, making possible the biosynthesis of unnatural ...metabolites that otherwise would be difficult to access. Such methods may allow the incorporation of fluorine, an atom rarely found in nature, into complex secondary metabolites. Organofluorine compounds and secondary metabolites have both played pivotal roles in the development of drugs; however, their discovery and development are often via nonintersecting tracks. In this context, we used the biosynthetic machinery of Trichoderma arundinaceum (strain MSX70741) to incorporate a fluorine atom into peptaibol-type molecules in a site-selective manner. Thus, fermentation of strain MSX70741 in media containing ortho- and meta-F-phenylalanine resulted in the biosynthesis of two new fluorine-containing alamethicin F50 derivatives. The fluorinated products were characterized using spectroscopic (1D and 2D NMR, including 19F) and spectrometric (HRESIMS/MSn) methods, and their absolute configurations were established by Marfey’s analysis. Fluorine-containing alamethicin F50 derivatives exhibited potency analogous to the nonfluorinated parent when evaluated against a panel of human cancer cell lines. Importantly, the biosynthesis of fluorinated alamethicin F50 derivatives by strain MSX70741 was monitored in situ using a droplet–liquid microjunction–surface sampling probe coupled to a hyphenated system.
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