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.

Fungal peroxygenases represent an exciting new enzyme class for stereo-selective hydroxylation reactions. They are capable of the oxyfunctionalisation of a large, diverse scope of substrates including alkanes and steroids as well as the heteroatoms sulfur and nitrogen. The outstanding activities and stabilities as well as their reliance on hydrogen peroxide as co-substrate renders it a highly interesting biocatalyst.

The screening effort of large protein variant libraries renders the probability of coincidental discovering a new enzyme with non-natural activity to almost zero - the so-called numbers problem. Insights into the origin of life, evolution and enzymatic promiscuity, combined with the inspiration of methods from organic chemistry, offer solutions for this problem. With the newly discovered enzymes synthetic micro production units shall be established in a Leibniz Research Cluster where engineering and biotechnology are combined.

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Late stage enzymatic prenylation and methylation are means to diversify (natural) compounds and to specify their functions. In eukaryotes and microbes, these steps are performed by large enzyme families, the prenyl and methyl transferases, which modify various types of small molecules, like isoprenoids, phenolics or alkaloids, but also DNA and proteins. We investigate the theoretical basis of these processes and possible commercial applications in synthetic chemistry.

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.

In Arabidopsis, deactivation of cyclin-dependent kinases via phosphorylation has no function in cell proliferation, growth, and stress response. In other eukaryotes, however, this is mandatorily required for maintaining genomic integrity.

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.

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.