Microwave-assisted xylanase reaction: impact in the production of prebiotic xylooligosaccharides.

The enzymatic production of prebiotic xylooligosaccharides (XOS) has become an attractive way to valorise lignocellulosic biomass. However, despite numerous xylanases reported for potential use in the production of XOS, most of the family GH10 also produce xylose. This monosaccharide can negatively affect the selectivity to stimulate the growth of intestinal microorganisms beneficial to human health. In this work, thermostable alkali-tolerant xylanase (BhXyn10A) from Bacillus halodurans S7 has been used to produce XOS under conventional convective heat transfer and microwave radiation. The microwave-assisted reaction markedly decreases the xylose content in the hydrolysates and significantly increases the yield of XOS, compared to conventional heating. Molecular dynamics simulations of BhXyn10A have shown increased fluctuations of the amino acids of the aglycone subsites suggesting that these subsites can determine the production of xylose. Thus, microwave heating could affect the amino acid fluctuations in the aglycone subsites reducing the xylose formation. These findings open up new avenues in enzyme technology for the production of XOS.

Sesquiterpene lactones from Ambrosia arborescens Mill. inhibit pro-inflammatory cytokine expression and modulate NF-κB signaling in human skin cells.

BACKGROUND: Ambrosia arborescens has been used in Andean traditional medicine to reduce problems associated with various inflammatory diseases and conditions, although the underlying mechanism is unknown. HYPOTHESIS/PURPOSE: The sesquiterpene lactones (SLs) coronopilin and damsin, which are major secondary metabolites of A. arborescens, have anti-inflammatory activity by attenuation of IL-6 and MCP-1 expression and inhibition of NF-κB in human dermal fibroblasts (HDFa) and human keratinocytes (HaCaT). STUDY DESIGN: In order to confirm a high concentration of damsin and coronopilin in the plant material, a quantitative method was developed. The effect of the pure compounds on cytokine and NF-κB expression was examined, as well as their effects on HDFa and HaCaT cell morphology and viability. METHODS: Coronopilin and damsin were quantified by HPLC-DAD analysis, from EtOAc extracts of the aerial parts of A. arborescens. Cell morphology was investigated by phase-contrast microscopy and cell viability by the MTT assay. IL-6 and MCP-1 cytokine gene expression was assessed by quantitative real-time RT-PCR in LPS stimulated cells. The NF-κB pathway was studied through western blotting of the phosphorylated forms of p65 and p50/p105, as well as the non-phosphorylated IκB. Dexamethasone was used as positive control. RESULTS: Dry aerial parts contained 12.3  mg/g and 13.4  mg/g of coronopilin and damsin, respectively. Treatment with either compound (1-10 µM) for 24  h attenuated LPS-induced mRNA expression of the pro-inflammatory cytokine IL-6 and the chemokine MCP-1 in HDFa cells. The down-regulation of MCP-1 mRNA induced by coronopilin and damsin was confirmed on the protein level. Damsin reduced phosphorylated p65 and p105 subunits in HDFa cells. Neither coronopilin nor damsin affected HDFa cell morphology and viability within the used concentration range (1-10 µM). Also, in HaCaT cells, treatment with damsin (1-10 µM) for 24  h inhibited the MCP-1 expression, and damsin thereby attenuated cytokine expression both in HDFa and HaCaT cells. CONCLUSION: We show that coronopilin and damsin from A. arborescens inhibit pro-inflammatory IL-6 and MCP-1 expression in human skin cells via NF-κB inhibition, suggesting that they may be useful for antagonizing inflammatory conditions of the human skin.

Anti-cancer stem cell activity of a sesquiterpene lactone isolated from Ambrosia arborescens and of a synthetic derivative.

New regimens are constantly being pursued in cancer treatment, especially in the context of treatment-resistant cancer stem cells (CSCs) that are assumed to be involved in cancer recurrence. Here, we investigated the anti-cancer activity of sesquiterpene lactones (SLs) isolated from Ambrosia arborescens and of synthetic derivatives in breast cancer cell lines, with a specific focus on activity against CSCs. The breast cancer cell lines MCF-7, JIMT-1, and HCC1937 and the normal-like breast epithelial cell line MCF-10A were treated with the SLs damsin and coronopilin, isolated from A. arborescens, and with ambrosin and dindol-01, synthesized using damsin. Inhibitory concentration 50 (IC50) values were obtained from dose-response curves. Based on IC50 values, doses in the µM range were used for investigating effects on cell proliferation, cell cycle phase distribution, cell death, micronuclei formation, and cell migration. Western blot analysis was used to investigate proteins involved in cell cycle regulation as well as in the NF-κB pathway since SLs have been shown to inhibit this transcription factor. Specific CSC effects were investigated using three CSC assays. All compounds inhibited cell proliferation; however, damsin and ambrosin were toxic at single-digit micromolar ranges, while higher concentrations were required for coronopilin and dindol-01. Of the four cell lines, the compounds had the least effect on the normal-like MCF-10A cells. The inhibition of cell proliferation can partly be explained by downregulation of cyclin-dependent kinase 2. All compounds inhibited tumour necrosis factor-α-induced translocation of NF-κB from the cytoplasm to the nucleus. Damsin and ambrosin treatment increased the number of micronuclei; moreover, another sign of DNA damage was the increased level of p53. Treatment with damsin and ambrosin decreased the CSC subpopulation and inhibited cell migration. Our results suggest that these compounds should be further investigated to find efficient CSC-inhibiting compounds.

Extraction of Glucuronoarabinoxylan from Quinoa Stalks (Chenopodium quinoa Willd.) and Evaluation of Xylooligosaccharides Produced by GH10 and GH11 Xylanases.

Byproducts from quinoa are not yet well explored sources of hemicellulose or products thereof. In this work, xylan from milled quinoa stalks was retrieved to 66% recovery by akaline extraction using 0.5 M NaOH at 80 °C, followed by ethanol precipitation. The isolated polymer eluted as a single peak in size-exclusion chromatography with a molecular weight of >700 kDa. Analysis by Fourier transform infrared spectroscopy and nuclear magnetic resonance (NMR) combined with acid hydrolysis to monomers showed that the polymer was built of a backbone of ß(1 → 4)-linked xylose residues that were substituted by 4-O-methylglucuronic acids, arabinose, and galactose in an approximate molar ratio of 114:23:5:1. NMR analysis also indicated the presence of α(1 → 5)-linked arabinose substituents in dimeric or oligomeric forms. The main xylooligosaccharides (XOs) produced after hydrolysis of the extracted glucuronoarabinoxylan polymer by thermostable glycoside hydrolases (GHs) from families 10 and 11 were xylobiose and xylotriose, followed by peaks of putative substituted XOs. Quantification of the unsubstituted XOs using standards showed that the highest yield from the soluble glucuronoarabinoxylan fraction was 1.26 g/100 g of xylan fraction, only slightly higher than the yield (1.00 g/100 g of xylan fraction) from the insoluble fraction (p < 0.05). No difference in yield was found between reactions in buffer or water (p > 0.05). This study shows that quinoa stalks represent a novel source of glucuronoarabinoxylan, with a substituent structure that allowed for limited production of XOs by GH10 or GH11 enzymes.

Crystal structure of ß-glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides.

The ß-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal ß-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4' and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-ß-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872-884. © 2016 Wiley Periodicals, Inc.

Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB:DNA complex studied by agarose gel electrophoresis.

The nuclear factor κB (NF-κB) is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB - DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB-DNA complex. The most active compounds are substituted at C-3 (α-carbonyl), in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

Multiple anticancer effects of damsin and coronopilin isolated from Ambrosia arborescens on cell cultures.

Terpenoids in plants are important sources for drug discovery. In this study, we extracted damsin and coronopilin, two sesquiterpene lactones, from Ambrosia arborescens and examined their anticancer effects on cell cultures. Damsin and coronopilin inhibited cell proliferation, DNA biosynthesis and formation of cytoplasmic DNA histone complexes in Caco-2 cells, with damsin being more potent than coronopilin. Further studies using the luciferase reporter system showed that damsin and coronopilin also inhibited expressions of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription-3 (STAT3), indicating that these sesquiterpenes can interfere with NF-κB and STAT3 pathways. Finally, we examined the effects of two synthetic dibrominated derivatives of damsin, 11α,13-dibromodamsin and 11ß,13-dibromodamsin. While bromination appeared to weaken the antiproliferative effects of damsin, the ß epimer had strong inhibitory effects on STAT3 activation. In conclusion, the sesquiterpene lactones damsin and coronopilin have inhibitory effects on cell proliferation, DNA biosynthesis and NF-κB and STAT3 pathways, thus being potentially important for discovery of drugs against cancer.

A method for rapid screening of ketone biotransformations: detection of whole cell Baeyer-Villiger monooxygenase activity.

A method for screening of ketone biotransformations was developed and applied to the identification of Baeyer-Villiger monooxygenase (BVMO) activity. The method was based on the formation of a purple coloured product on reaction between an enolizable ketone and 3,5-dinitrobenzoic acid in an alkaline solution. Absorbance of the colour decreased with the size of the cycloketone ring. Stoichiometric ratio between cycloketone and 3,5-dinitrobenzoic acid was 1:1 at maximum absorbance. The method was applied for monitoring the consumption of cyclohexanone by bacteria under aerobic conditions, and was found to be potentially useful for both screening assays and quantitative measurements of BVMO activity. Compared to other existing methods, this method is faster, cheaper and amenable for whole cell assays.

Antioxidant and antimutagenic polyisoprenylated benzophenones and xanthones from Rheedia acuminata.

Dichloromethane extract of the stem bark of Rheedia acuminata yielded three benzophenones with antioxidant activity, the new one named acuminophenone A (1), guttiferone K (2) and isoxanthochymol (3), along with the known xanthones formoxanthone C (4) and macluraxanthone (5). The structures were established through interpretation of their spectroscopic data, the stereochemistry of compounds (1) and (2) were resolved by experimental and computational experiments and their antioxidant activities were measured using the DPPH, ABTS and TEAC assays. The antioxidant results showed that metabolites 1, 4 and 5 had a better antioxidant activity than the reference compound quercetin. In addition, we evaluate the mutagenicity and antimutagenicity of the CH2Cl2 extract as well as of the free radical scavenger compounds 1, 4 and 5 by the AMES Salmonella/microsomal test. No mutagenicity was found in the CH2Cl2 extract using Salmonella typhimurium strains TA98, TA100, TA102, TA1537 and TA1538, with or without S9 metabolic activation. The pure compounds neither showed mutagenicity in TA 102 strain and the most important result was the strong reduction of mutagenic effect induced by hydrogen peroxide in S. typhimurium TA102, with or without S9, showed by the compounds 1 (more than 93%) and 4 (more than 88%) at 0.02 microg/plate.