Dalbergia monetaria is an Amazonian plant whose bark is widely used to treat urinary tract infections. This paper describes a bio-guided study of ethanolic extracts from the bark and leaves of D. monetaria, in a search for metabolites active against human pathogenic bacteria. In vitro assays were performed against 10 bacterial strains, highlighting methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Fractioning of the extracts was performed using instrumental and classical techniques, and samples were characterized by UHPLC-HRMS/MS. Ethyl acetate fractions from bark and leaves showed similar antibacterial activities. EAFB is enriched in isoflavone C-glucosides and EAFL enriched in proanthocyanidins. Subfractions from EAFL presented higher activity and showed a complex profile of proanthocyanidins constructed by (epi)-cassiaflavan and (epi)-catechin units, including dimers, trimers and tetramers. The fragmentation pattern emphasized the neutral loss of cassiaflavan units by quinone-methide fission. Fraction SL7-6, constituted by (ent)-cassiaflavan-(ent)-cassiaflavan-(epi)-catechin isomers, showed the lowest MIC against the S. aureus and P. aeruginosa with values corresponding to 64 and 32 µg/mL, respectively. Cassiaflavan-proanthocyanidins have not been found previously in another botanical genus, except in Cassia, and the traditional medicinal use of D. monetaria might be related to the antibacterial activity of proanthocyanidins characterized in the species.

AbstractThree previously undescribed natural products, phomopsinin A – C (1 – 3), together with three known compounds, namely, cis-hydroxymellein (4), phomoxanthone A (5) and cytochalasin L-696,474 (6), were isolated from the solid culture of Phomopsis sp. CAM212, an endophytic fungus obtained from Garcinia xanthochymus. Their structures were determined on the basis of spectroscopic data, including IR, NMR, and MS. The absolute configurations of 1 and 2 were assigned by comparing their experimental and calculated ECD spectra. Acetylation of compound 1 yielded 1a, a new natural product derivative that was tested together with other isolated compounds on lipopolysaccharide-stimulated RAW 264.7 cells. Cytochalasin L-696,474 (6) was found to significantly inhibit nitric oxide production, but was highly cytotoxic to the treated cells, whereas compound 1 slightly inhibited nitric oxide production, which was not significantly different compared to lipopolysaccharide-treated cells. Remarkably, the acetylated derivative of 1, compound 1a, significantly inhibited nitric oxide production with an IC50 value of 14.8 µM and no cytotoxic effect on treated cells, thereby showing the importance of the acetyl group in the anti-inflammatory activity of 1a. The study of the mechanism of action revealed that 1a decreases the expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory cytokine IL-6 without an effect on IL-1β expression. Moreover, it was found that 1a exerts its anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells by downregulating the activation of ERK1/2 and by preventing the translocation of nuclear factor κB. Thus, derivatives of phomopsinin A (1), such as compound 1a, could provide new anti-inflammatory leads.

The growing interest in the efficacy of phytomedicines and herbal supplements but also the increase in legal requirements for safety and reliable contents of active principles drive the development of analytical methods for the quality control of complex, multicomponent mixtures as found in plant extracts of value for the pharmaceutical industry. Here, we describe an ultra-performance liquid chromatography method (UPLC) coupled with quadrupole time of flight mass spectrometry (qTOF-MS) measurements for the large scale analysis of H. perforatum plant material and its commercial preparations. Under optimized conditions, we were able to simultaneously quantify and identify 21 metabolites including 4 hyperforins, 3 catechins, 3 naphthodianthrones, 5 flavonoids, 3 fatty acids, and a phenolic acid. Principal component analysis (PCA) was used to ensure good analytical rigorousness and define both similarities and differences among Hypericum samples. A selection of batches from 9 commercially available H. perforatum products available on the German and Egyptian markets showed variable quality, particularly in hyperforins and fatty acid content. PCA analysis was able to discriminate between various preparations according to their global composition, including differentiation between various batches from the same supplier. To the best of our knowledge, this study provides the first approach utilizing UPLC-MS-based metabolic fingerprinting to reveal secondary metabolite compositional differences in Hypericum extract.

The present paper describes the phytochemical and anti-staphylococcal activity investigation of the dichloromethane extract of the Brazilian plant Zizyphus joazeiro Mart. The purification steps were guided by bioassays against 17 bacterial strains of clinical sources, including methicillin-resistant (MRSA) and ‐sensitive (MSSA) Staphylococcus aureus as well as MRSA (ATCC 33591) and MSSA (ATCC 29213) reference strains. One of the more active fractions is comprised of three lupane-type triterpenes, the methylbetulinate (1) as well as the known betulinic (2) and alphitolic (3) acids and, for the first time in the Z. joazeiro, two ceanothane type triterpenes, the methylceanothate (4) and the epigouanic acid A (5). These substances were assayed against one clinical (PVL+) and the reference strains of S. aureus as well as the ATTC 12228 strain of S. epidermidis, in concentrations that varied from 128 to 0.125 µg/mL in order to establish the minimum inhibitory concentration (MIC) of the drugs. The minimum bactericide concentration (MBC) was also evaluated to distinguish the bactericidal from bacteriostatic activity of the crude fractions and single compounds. Compounds 3 and 4 possess the highest antibacterial activity. They inhibit all bacteria tested at 32 µg/mL and 16 µg/mL, respectively, while the other compounds showed no activity at 128 µg/mL. In contrast to single compounds, the triterpenoid fraction showed bactericidal activity at 256 µg/mL. Structural elucidations are based on 1D and 2D NMR spectroscopy as well as HR‐FT‐ICR‐MS experiments.

Two new triterpenoids, named gouanic acid A (1) and gouanic acid B (2), were isolated from the aerial parts of Gouania ulmifolia, along with six known compounds. The structures of the new compounds were determined by spectroscopic methods, mainly NMR (1D and 2D) and mass spectrometry. The new compounds did not show significant antimicrobial activities.

Geranylgeraniol (GGOH) is an acyclic diterpene that posesses apoptotic activity to cancer cells [1]. It has been proposed to be the main intermediate of the biosynthetic pathway of plaunotol, an antipeptic ulcer drug from Croton stellatopilosus [2]. Our enzymological studies showed that GGOH is formed from the dephosphorylation of geranylgeranyl pyrophosphate (GGPP), through sequential monodephosphorylation [3], by the action of GGPP phosphatase enzyme [4]. As part of our interest in manipulating the gene of GGPP phosphatase for the production of GGOH in Escherichia coli system, we began with cloning of cDNA encoding prenyl diphosphate phosphatase from C. stellatopilosus. The degenerated primers were designed from the alignment of amino acid sequences of prenyl diphosphate phosphatase in database. The full-length gene was obtained by RACE-PCR. The cDNA contained an open reading frame encoding 888 amino acids with a calculated molecular mass of 33.6 kDa. The phosphatase motif [5] was included in the deduced amino acid sequence consisting of KX6RP, PSGH, and SRX5HX3D. Its amino acid sequence showed 71% identity to phosphatidic acid phosphatase from Vigna unguiculata. The topology prediction of the enzyme indicated that it was a transmembrane protein with 6 transmembrane regions. The recombinant prenyl diphosphate phosphatase and its 4 designed truncated genes were expressed in Escherichia coli BL21(DE3)RIL. Detection of their phosphatase activities by using [1-3H]GGPP and farnesyl pyrophosphate ([1-3H]FPP) as substrates showed that their enzymatic products of [1-3H]GGOH and [1-3H]FOH, respectively, were formed in the assay mixture. The results suggested the potential of GGOH production by the recombinant E. coli although the expression of the recombinant gene was still in low level.

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The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL , encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A1, i.e. between the central 3‐methylpyrrole‐2,4‐dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mn2+ or Mg2+. The apparent K m values were determined as 26 µm for the 3‐methylpyrrole‐2,4‐dicarboxylic acid and 44 µm for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3‐Dimethylallyl‐4‐hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference from the genuine acyl substrate of CouL.

Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus‐1 (HIV‐1) Tat protein and the N‐terminal peptide Tat(1–9) inhibit DP IV activity and T cell proliferation. Therefore, the N‐terminal amino acid sequence of HIV‐1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2‐R(1–9), bearing the N‐terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell‐expressed DP IV [Wrenger, S., Faust, J., Mrestani‐Klaus, C., Fengler, A., Stöckel‐Maschek, A., Lorey, S., Kähne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem. 275 , 22180–22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1–9) can result in a change of the inhibition type. Certain Tat(1–9)‐related peptides are found to be competitive, and others linear mixed‐type or parabolic mixed‐type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed‐type mechanism, attributed to both non‐mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide‐based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.

Recent cell culture experiments indicated that extracts of Vitex agnus-castus (VAC) may contain yet unidentified phytoestrogens. Estrogenic actions are mediated via estrogen receptors (ER). To investigate whether VAC compounds bind to the currently known isoforms ERα or ERß, ligand binding assays (LBA) were performed. Subtype specific ER-LBA revealed a binding of VAC to ERß only. To isolate the ERß-selective compounds, the extract was fractionated by bio-guidance. The flavonoid apigenin was isolated and identified as the most active ERß-selective phytoestrogen in VAC. Other isolated compounds were vitexin and penduletin. These data demonstrate that the phytoestrogens in VAC are ERß-selective.