Antibacterial Flavonoids and Other Compounds from the Aerial Parts of Vernonia guineensis Benth. (Asteraceae)

An extensive phytochemical study of the aerial parts of Vernonia guineensis Benth. (Asteraceae) led to the isolation of a new flavone, vernoguinoflavone and a naturally isolated glycerol ester, eicosanoic acid 2‐hydroxy‐1,3‐propanediyl ester, together with eighteen known secondary metabolites including quercetin, luteolin, vernopicrin, vernomelitensin, β‐amyrin, oleanolic acid, ursolic acid, lupeol, betulinic acid, β‐carotene, a mixture of stigmasterol and β‐sitosterol, β‐sitosterol‐3‐O‐β‐D‐glucoside, 2,3‐dihydroxypropyl heptacosanoate, pentacosanoic acid, docosan‐1‐ol, tritriacontan‐1‐ol, and heptatriacontan‐1‐ol. Eleven compounds are reported herein for the first time from this species. The structures of these compounds were elucidated on the basis of extensive spectroscopic analyses, particularly 1D and 2D NMR, and HR‐ESI‐MS and by comparison of their data with those reported in the literature. The crude extract, fractions and some isolated compounds were evaluated for their antibacterial activity against Gram‐negative bacteria: Escherichia coli (ATCC 25922), Shigella flexineri (NR 518), Salmonella muenchen, Salmonella typhimurium and Salmonella typhi (ATCC 19430). All the tested compounds demonstrated inhibitory activities against the tested enteric bacteria with MIC values ranging from 3.12 to 100 μg/ml. Three flavonoids isolated from the most active fraction demonstrated the best bioactivities against Escherichia coli, Salmonella muenchen and Salmonella typhimurium with MIC values ranging from 3.12 to 25 μg/mL.     

Protective effects of luteolin-7-O-β-D-glucuronide methyl ester from the ethyl acetate fraction of Lycopi Herba against pro-oxidant reactive species and low-density lipoprotein peroxidation

In this study the potent scavenging activity of “Lycopi Herba” (LH) extract was studied using the following: evaluation of the total phenolics, measuring the antioxidant activity by Trolox equivalent antioxidant concentration, measuring the scavenging effects on reactive oxygen species, on reactive nitrogen species, and measuring the inhibitory effect on Cu²⁺ induced human low-density lipoprotein oxidation in vitro. The ethyl acetate fraction from the LH extracts were found to have a potent scavenging activity against all of the reactive species tested, as well as an inhibitory effect on LDL oxidation. Therefore, we isolated and identified luteolin-7-O-β-D-glucuronide methyl ester as the major compound from the ethyl acetate fraction of LH and their antioxidant activities were evaluated.     

Sol-gel powders and supported sol-gel polymers for immobilization of lipase in ester synthesis

Candida rugosa lipase was entrapped in hybrid organic–inorganic sol-gel powder prepared by acid-catalyzed polymerization of tetramethoxysilane (TMOS) and alkyltrimethoxysilanes, and used in catalyzing esterification reactions between ethanol and butyric acid in hexane. Optimum preparation conditions were studied, which are gels made from propyltrimethoxysilane (PTMS)/TMOS molar ratio=4:1, hydrolysis time of silane precursor=30 min, water/silane molar ratio=24, enzyme loading=6.25% (w/w) of gel, and 1 mg PVA/mg lipase. The percentage of protein immobilization was 95% and the resulting lipase specific activity was 59 times higher than that of a non-immobilized lyophilized lipase. To prepare magnetic lipase-immobilized sol-gel powder (MLSP) for easier recovery of the biocatalyst, Fe₃O₄ nanoparticles were prepared and co-entrapped with lipase during gel formation. This procedure induced surface morphological change of the sol-gel powder and showed adverse effect on enzyme activity. Hence, although only 9% decrease in protein immobilization efficiency was observed, the corresponding reduction in enzyme activity could be up to 45% when sol-gel powder was doped with 25% (v/v) Fe₃O₄ magnetic nanoparticles solution. Lipase-immobilized sol-gel polymer was also formed within the pores of different porous supports to improve its mechanical stability. Non-woven fabric, with a medium pore size of all the supports tested, was found to be the best support for this purpose. The thermal stability of lipase increased 55-fold upon entrapment in sol-gel materials. The half-lives of all forms of sol-gel-immobilized lipase were 4 months at 40 °C in hexane.     

Aspergillus melleus protease-catalyzed peptide synthesis using the carbamoylmethyl ester as an acyl donor in 1,1,1,3,3,3-hexafluoro- 2-propanol/N,N-dimethylformamide

The coupling between the carbamoylmethyl ester of an N-protected amino acid or dipeptide (at 25 mM) and an amino acid amide (at 100 mM) was achieved using Aspergillus melleus protease in 1,1,1,3,3,3-hexafluoro-2-propanol/N,N-dimethylformamide (1:1, v/v); the coupling efficiencies were dependent largely on the combination of amino acid residues: e.g. the dipeptide yields after 48 h were for l-Ala + Gly, 100% and for l-Leu + l-Leu, 16%.     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.     

Isolation of a Novel Carotenoid,OH-chlorobactene Glucoside Hexadecanoate,and Related Rare Carotenoids from Rhodococcus sp. CIP and Their Antioxidative Activities

In the course of screening for antioxidative carotenoids from bacteria, we isolated and identified a novel carotenoid, OH-chlorobactene glucoside hexadecanoate (4), and rare carotenoids, OH-chlorobactene glucoside (1), OH-γ-carotene glucoside (2) and OH-4-keto-γ-carotene glucoside hexadecanoate (3) from Rhodococcus sp. CIP. The singlet oxygen (¹O₂) quenching model of these carotenoids showed potent antioxidative activities IC₅₀ 14.6 μM for OH-chlorobactene glucoside hexadecanoate (4), 6.5 μM for OH-chlorobactene glucoside (1), 9.9 μM for OH-γ-carotene glucoside (2) and 7.3 μM for OH-4-keto-γ-carotene glucoside hexadecanoate (3).     

The Effect of Chitosan and Bion on Resistance to Pink Snow Mould in Perennial Ryegrass and Winter Wheat

The effects of chitosan on resistance to pink snow mould (Microdochium nivale) were studied in young winter wheat (Triticum aestivum L.) and perennial ryegrass (Lolium perenne L.) under controlled environmental conditions. In perennial ryegrass, the putative defence activator Bion was also tested. Resistance was measured as regrowth of plants after inoculation with M. nivale and incubation in darkness at 2°C. In winter wheat, pre‐treatment with chitosan at 1000 μg per plant increased resistance to subsequent infection by M. nivale, but this effect was less significant in a replicate experiment. Chitosan‐treated winter wheat plants expressed the gene for the pathogenesis‐related protein chitinase at higher levels than non‐treated plants. Chitinase gene expression was also stimulated by M. nivale infection in winter wheat. Perennial ryegrass pre‐treated with Bion or chitosan and inoculated with M. nivale did not display better regrowth after incubation than non‐treated, inoculated plants. Rather, regrowth was reduced in some of the Bion‐treated plants after incubation. We speculate that the cost or the mechanism of induced resistance makes Bion non‐effective in plants that are not actively growing. Bion at concentrations of 10, 100 and 1000 μg active ingredient per ml, and the highest concentration of chitosan used (2000 μg per ml) reduced in vitro growth of the pathogen, suggesting that both defence activators possess antifungal activity.     

Subsite specificities of granzyme M: a study of inhibitors and newly synthesized thiobenzyl ester substrates

Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.