Four new beilschmiedic acid derivatives, cryptobeilic acids A–D (1–4), and tsangibeilin B (5) have been isolated from the bark of Beilschmiedia cryptocaryoides collected from Madagascar. Their structures were elucidated using detailed spectroscopic and spectrometric methods. Cryptobeilic acid A (1) and tsangibeilin B (5) structures were confirmed by single-crystal X-ray diffraction analysis. Compounds 1–5 displayed moderate antibacterial activity against Escherichia coli 6r3, Acinetobacter calcoaceticus DSM 586, and Pseudonamas stutzeri A1501, with the minimum inhibitory concentrations ranging from 10 to 50 μM, respectively. In addition, the compounds exhibited antiplasmodial activity against erythrocytic stages of chloroquine-resistant Plasmodium falciparum strain NF54 and weak cytotoxicity against L6 cell lines.

The biosynthetic potential of endophytic fungi has gained impetus in recent times owing to the continual discovery of fungal endophytes capable of synthesizing plant compounds. However, the sustained production of the desired plant compounds has not yet been achieved using endophytes. It is thus imperative to investigate the diverse interactions that endophytes have with coexisting endophytes, host plants, insect pests, and other specific herbivores. The precise role of these associations on the endophytic production of host plant compounds is mostly overlooked and open to future discoveries. Here, highlighted are the implications of the poorly investigated links and molecular mechanisms that might trigger similar chemical responses in both plants and endophytes. Elucidating such connections can not only enhance the understanding of evolution of complex defense mechanisms in plants and associated organisms, but also help in the sustained production of plant compounds using endophytes harbored within them.Graphical abstractHighlighted are poorly investigated interactions that endophytes have with host plants, insect herbivores and with each other, and the diverse molecular mechanisms that might trigger similar chemical responses in both plants and endophytes. Elucidating such networks can enhance discovery of desirable endophytes and further sustain production of host plant compounds using the isolated endophytes.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Plants and endophytic fungi can occasionally produce similar bioactive compounds. ► Endophytes interact with coexisting endophytes, host plant, and insect herbivores. ► Early signaling mechanism influencing such endophyte interactions should be studied. ► Elucidating the molecular basis of such multifaceted interactions is also important. ► These distinct interactions can trigger similar chemical responses in the partners.

Four acyclic triterpene derivatives named sapelenins G–J (1–4), along with eight known compounds, sapelenins A–D, ekeberin D2 (5), (+)-catechin and epicatechin, and anderolide G, were isolated from the stem bark of the Cameroonian medicinal plant, Entandrophragma cylindricum Sprague, on the basis of bioassay-guided fractionation. Their structures were determined by means of high-resolution mass spectrometry and NMR spectroscopic data, as well as by comparison with the literature values of their analogs. The absolute configurations of the compounds (1–4) were assigned by the modified Mosher’s method in conjunction with NOESY experiments and chemical modifications. The anti-inflammatory activities of the sapelenins were evaluated by assessing their ability to suppress or inhibit the secretion of cytokine interleukin-17 (IL-17) by human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA). The cytotoxicity of these compounds on PMBCs was further assessed for correctly interpreting their anti-inflammatory responses. The tested compounds demonstrated moderate to significant anti-inflammatory activities by suppressing the secretion of IL-17 by PHA-stimulated human PBMCs. One of them, sapelenin G (1), showed high potency in suppressing the secretion of IL-17 by PBMCs comparable to reference cyclosporine A, without causing any cytotoxic effects (negligible), and deserves further considerations towards developing an effective anti-inflammatory drug.Graphical abstractIn this manuscript, we report the isolation and characterization of sapelenins G–J (1–4), from the Cameroonian medicinal plant, Entandrophragma cylindricum, along with their anti-inflammatory and cytotoxic activities.Highlights► Acyclic triterpenes,sapelenins G–J (1–4) isolated from Entandrophragma cylindricum. ► Eight known compounds isolated in parallel. ► Anti-inflammatory activities evaluated by IL-17 ELISA. ► Cytotoxicity towards PBMCs also evaluated. ► Potent anti-inflammatory compound discovered.

Morphine is present in varying amounts as an endogenous product in human urine. Derivatization of morphine contained in urine with dansyl chloride yields a known product, which can be quantified by liquid chromatography mass spectrometry with high selectivity and sensitivity. Urine samples of 51 healthy individuals were spiked with stable-isotope labeled morphine, hydrolyzed and subjected to solid phase extraction followed by derivatization of morphine with dansyl chloride. The dansyl derivatives of naturally occurring morphine and deuterated internal standard were then detected by liquid chromatography–triple quadrupole mass spectrometry. Using the [N-CD3]-labeled internal standard and solid-phase extraction, a limit of detection of 35 fmol/ml (10 pg/ml) and a limit of quantification of 87.5 fmol/ml (25 pg/ml) was determined for morphine in human urine. This new LC–MS/MS method allowed the detection of endogenous morphine in human urine of 51 volunteers with an average value of 156.4 fmol/ml (44.7 ng/ml).

The pentacyclic quinoline alkaloid camptothecin (1) is a potent antineoplastic agent. Two of its analogues, 9-methoxycamptothecin (2) and 10-hydroxycamptothecin (3), exhibit similar potency but do not have the potential therapeutic drawbacks produced by unmodified 1. We have established methodology for the isolation and unequivocal identification and characterization of a novel endophytic fungus isolated from the inner bark of the medicinal plant Camptotheca acuminata, which produced 1−3 in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified by its morphology and authenticated by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Camptothecin (1) and its analogues were identified by 1H NMR spectroscopy and LC-HRMS and confirmed by comparison with authentic standards. The production pattern of the metabolites over seven successive subculture generations of this endophyte was studied. A sharp attenuation in the production of 1 and 2 was observed from the first- through to the seventh-generation subculture. Therefore, these results offer a caution as to the possibility of using endophytic fungi as alternate sources of plant secondary metabolite production. Further studies have been initiated on the analysis of the upstream metabolic intermediates to understand the steps at which the production of the metabolites in question is constrained.

The possible microbial mechanism of hypericin (1) and emodin (2) biosynthesis was studied in axenic submerged culture conditions in the endophytic fungus Thielavia subthermophila, isolated from Hypericum perforatum. The growth and secondary metabolite production of the endophyte remained independent of the illumination conditions. This production remained unaltered on spiking the medium with 3 or 5 mM 2, although the biomass accumulation was reduced. Neither emodin anthrone (3) nor protohypericin (4) could be detected at any stage of fermentation, irrespective of either spiking or illumination conditions. The endophytic metabolites exhibited photodynamic cytotoxicity against the human acute monocytic leukemia cell line (THP-1), at 92.7 vs 4.9%, and 91.1 vs 1.0% viability by resazurin and ATPlite assays, in light and in the dark, respectively. In trying to ascertain the presence/expression of the candidate hyp-1 gene in the endophyte, it was revealed that the hyp-1 gene was absent in T. subthermophila, indicating that the biosynthetic pathway in the endophytic fungus might be different and/or governed by a different molecular mechanism than the host plant or host cell suspension cultures. We have discussed the biosynthetic principles and evolutionary implications relating to endophytic T. subthermophila based on the results obtained.

For the first time, an endophytic fungus has been isolated from the stems of the medicinal herb Hypericum perforatum (St. John’s Wort). The fungus produced the napthodianthrone derivative hypericin (1) in rich mycological medium (potato dextrose broth) under shake flask and bench scale fermentation conditions. Emodin (2) was also produced simultaneously by the fungus under the same culture conditions. We propose 2 as the main precursor in the microbial metabolic pathway to 1. The fungus was identified by morphology and authenticated by 28S (LSU) rDNA sequencing. Compounds 1 and 2 were identified by LC-HRMS, LC-MS/MS, and LC-HRMS/MS and confirmed by comparison with authentic standards. In bioassays with a panel of laboratory standard pathogenic control strains, including fungi and bacteria, both fungal 1 and 2 possessed antimicrobial activity comparable to authentic standards. This endophytic fungus has significant scientific and industrial potential to meet the pharmaceutical demands for 1 in a cost-effective, easily accessible, and reproducible way.

The content of hypericins (hypericin and pseudohypericin), hyperforin, and flavonoids (rutin, hyperoside, quercitrin, and quercetin) and genetic profiles of eight accessions of Hypericum perforatum L., collected from different locations in India, have been determined. The secondary metabolite content was determined using a highly selective LC/MS/MS method. Pearson and Spearman's correlation coefficient were used to investigate the relationships between the secondary metabolites and a significant positive correlation was found between hypericin and pseudohypericin contents. Genetic profiling was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) methods. Among the 49 random primers used for the initial screening, only nine yielded polymorphic RAPD profiles. The SSR analysis shows that seven out of the 11 primers were polymorphic. There exists only a partial correlation between the chemical content and genetic profiling data among the accessions under study.

The essential oils of the aerial parts of nine species of Hypericum (Hypericum barbatum, Hypericum hirsutum, Hypericum linarioides, Hypericum maculatum, Hypericum olympicum, Hypericum perforatum, Hypericum richeri, Hypericum rumeliacum and Hypericum tetrapterum), collected from different locations in Southeast Serbia, were obtained by steam distillation and analyzed by GC and GC–MS. The essential oils investigated were characterized by a high content of non-terpene compounds and a low content of monoterpenes. The contents of non-terpenes, monoterpenes and sesquiterpenes in oils of the species H. barbatum, H. richeri and H. rumeliacum (section Drosocaprium) were similar and these oils were characterized by high contents of fatty acids. The oils of H. hirsutum and H. linarioides (section Taeniocarpium) contained a high percentage of n-nonane. There were similarities in contents of non-terpenes and sesquiterpenes in oils of species that belong to the section Hypericum (H. maculatum, H. perforatum and H. tetrapterum). The oil of H. olympicum differed from others by higher terpene content. A comparison was also carried out of the chemical composition of the essential oils from flower, leaf and stem of H. perforatum and it revealed that the highest concentration of non-terpene compounds was found in the flower and stem oil, while a high concentration of sesquiterpenes was characteristic for leaf oil. There were significant differences in the concentrations of the same compounds in the essential oils of H. maculatum, H. olympicum and H. perforatum, collected in different years from the same location which could be explained by seasonal differences. All data were statistically processed with principal component analysis and cluster analysis. The main conclusion from the above data is that genetic and environmental factors both play a role in determining the composition of essential oils of the Hypericum species studied.

The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum Jacq., H. hirsutum L., H. linarioides Bosse, H. maculatum Crantz, H. rumeliacum Boiss. and H. tetrapterum Fries), collected from different locations in Serbia, have been analyzed. Methanol extracts of the aerial parts of the plants were obtained by accelerated solvent extraction (ASE) at 40 °C and 100 bar, and analyzed for five pharmacologically important standard constituents (hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin) by LC–MS/MS. The highest content of hypericin and pseudohypericin was observed in the H. barbatum extract, while the highest content of hyperforin and quercitrin was found in the H. tetrapterum extract and the highest content of hyperoside in the H. maculatum extract. A literature survey shows that the above six Hypericum species, with the exception of H. maculatum, have not been previously genetically profiled. In order to correlate the chemical constituents of the species under investigation with their genetic factors, genetic profiling of these species was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) profiles of the above selected plants. Among the 52 random primers used for the initial screening, only 10 yielded polymorphic RAPD profiles. A total of 111 polymorphic markers were generated using these primers. The SSR analysis shows that 8 out of the 10 primers used were polymorphic. The correlation among the species under investigation using the two genetic markers was performed using Jaccuard’s coefficients of similarity and a high correlation (r = 0.99) was obtained. The main conclusion from the above data is that there exists a stronger correlation for secondary metabolite contents with RAPD data than with SSR data among the six Hypericum species from Serbia.The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum, H. hirsutum, H. linarioides, H. maculatum, H. rumeliacum and H. tetrapterum), collected from different locations in Serbia, were analyzed. There exists a strong correlation of secondary metabolite contents and RAPD data among the species under study.

Fluoroquinolones are amongst the most important antibiotics used in veterinary medicine. On this account the behavior of difloxacin (DIF) and its metabolites was investigated by administering the 14C-labeled and non-labeled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Sarafloxacin (SAR) was found to be the major metabolite, three further trace metabolites were also recovered, applying high-resolution (HR) mass spectrometric technique. The identification of DIF and SAR was confirmed by comparison with the spectroscopic and chromatographic data of the authentic references. The identification of the three trace metabolites was performed by HR-MS/MS. Only 8.1% of the administered radioactivity remained in the pig after 10 days and DIF accounted for 95.9% of the radioactivity excreted. More than 99% of the labeled compounds were detected and identified in the manure. The mean recoveries for all single electrolytes were ⩾94%. Linearity was established over concentration range 10–10,000 μg/kg manure with a correlation coefficient ⩾0.99. By using in vitro antimicrobial activity tests against a group of standard pathogenic control strains, the results showed that the residual antibiotic concentrations in the manure of pigs are high enough to exhibit antibacterial activity.

The phenolic constituents and antioxidant activity of ethanol and water extract of the flowers of Hypericum venustum, collected in Turkey, were investigated. The extracts possess strong reducing power, free radicals and hydrogen peroxide scavenging activity, as well as metal chelating ability.