MALDI-HRMS Imaging Maps the Localization of Skyrin, the Precursor of Hypericin, and Pathway Intermediates in Leaves of Hypericum Species.

Hypericum perforatum and related species (Hypericaceae) are a reservoir of pharmacologically important secondary metabolites, including the well-known naphthodianthrone hypericin. However, the exact biosynthetic steps in the hypericin biosynthetic pathway, vis-à-vis the essential precursors and their localization in plants, remain unestablished. Recently, we proposed a novel biosynthetic pathway of hypericin, not through emodin and emodin anthrone, but skyrin. However, the localization of skyrin and its precursors in Hypericum plants, as well as the correlation between their spatial distribution with the hypericin pathway intermediates and the produced naphthodianthrones, are not known. Herein, we report the spatial distribution of skyrin and its precursors in leaves of five in vitro cultivated Hypericum plant species concomitant to hypericin, its analogs, as well as its previously proposed precursors emodin and emodin anthrone, using MALDI-HRMS imaging. Firstly, we employed HPLC-HRMS to confirm the presence of skyrin in all analyzed species, namely H. humifusum, H. bupleuroides, H. annulatum, H. tetrapterum, and H. rumeliacum. Thereafter, MALDI-HRMS imaging of the skyrin-containing leaves revealed a species-specific distribution and localization pattern of skyrin. Skyrin is localized in the dark glands in H. humifusum and H. tetrapterum leaves together with hypericin but remains scattered throughout the leaves in H. annulatum, H. bupleuroides, and H. rumeliacum. The distribution and localization of related compounds were also mapped and are discussed concomitant to the incidence of skyrin. Taken together, our study establishes and correlates for the first time, the high spatial distribution of skyrin and its precursors, as well as of hypericin, its analogs, and previously proposed precursors emodin and emodin anthrone in the leaves of Hypericum plants.

Isolation, Characterization and Targeted Metabolic Evaluation of Endophytic Fungi Harbored in 14 Seed-Derived Hypericum Species.

Medicinal plants of the genus Hypericum are rich sources of bioactive naphthodianthrones, which are unique in the plant kingdom, but quite common in fungal endophytes. Cultivable endophytic fungi were isolated from 14 different Hypericum spp. originating from seeds grown under in vitro conditions and further acclimated to outdoor conditions. Among 37 fungal isolates yielded from the aerial and underground plant organs, 25 were identified at the species level by the fungal barcode marker internal transcribed spacer rDNA and protein-coding gene region of tef1α. Ten of them were isolated from Hypericum spp. for the first time. The axenic cultures of the isolated endophytes were screened for the production of extracellular enzymes, as well as bioactive naphthodianthrones and their putative precursors by Bornträger's test and HPLC-HRMS. Traces of naphthodianthrones and their intermediates, emodin, emodin anthrone, skyrin, or pseudohypericin, were detected in the fungal mycelia of Acremonium sclerotigenum and Plectosphaerella cucumerina isolated from Hypericum perforatum and Hypericum maculatum, respectively. Traces of emodin, hypericin, and pseudohypericin were released in the broth by Scedosporium apiospermum, P. cucumerina, and Fusarium oxysporum during submerged fermentation. These endophytes were isolated from several hypericin-producing Hypericum spp. Taken together, our results reveal the biosynthetic potential of cultivable endophytic fungi harbored in Hypericum plants as well as evidence of the existence of remarkable plant-endophyte relationships in selected non-native ecological niches. A possible role of the extracellular enzymes in plant secondary metabolism is discussed.

Harnessing the Phytotherapeutic Treasure Troves of the Ancient Medicinal Plant Azadirachta indica (Neem) and Associated Endophytic Microorganisms.

Azadirachta indica, commonly known as neem, is an evergreen tree of the tropics and sub-tropics native to the Indian subcontinent with demonstrated ethnomedicinal value and importance in agriculture as well as in the pharmaceutical industry. This ancient medicinal tree, often called the "wonder tree", is regarded as a chemical factory of diverse and complex compounds with a plethora of structural scaffolds that is very difficult to mimic by chemical synthesis. Such multifaceted chemical diversity leads to a fantastic repertoire of functional traits, encompassing a wide variety of biological activity and unique modes of action against specific and generalist pathogens and pests. Until now, more than 400 compounds have been isolated from different parts of neem including important bioactive secondary metabolites such as azadirachtin, nimbidin, nimbin, nimbolide, gedunin, and many more. In addition to its insecticidal property, the plant is also known for antimicrobial, antimalarial, antiviral, anti-inflammatory, analgesic, antipyretic, hypoglycaemic, antiulcer, antifertility, anticarcinogenic, hepatoprotective, antioxidant, anxiolytic, molluscicidal, acaricidal, and antifilarial properties. Notwithstanding the chemical and biological virtuosity of neem, it has also been extensively explored for associated microorganisms, especially a class of mutualists called endophytic microorganisms (or endophytes). More than 30 compounds, including neem "mimetic" compounds, have been reported from endophytes harbored in the neem trees in different ecological niches. In this review, we provide an informative and in-depth overview of the topic that can serve as a point of reference for an understanding of the functions and applications of a medicinal plant such as neem, including associated endophytes, within the overall theme of phytopathology. Our review further exemplifies the already-noted current surge of interest in plant and microbial natural products for implications both within the ecological and clinical settings, for a more secure and sustainable future.

Chemometric evaluation of hypericin and related phytochemicals in 17 in vitro cultured Hypericum species, hairy root cultures and hairy root-derived transgenic plants.

OBJECTIVES: The objective of this study was to ascertain the presence and correlations among eight important secondary metabolites viz. hypericin, pseudohypericin, emodin, hyperforin, rutin, hyperoside, quercetin and quercitrin in different organs of 17 in vitro cultured Hypericum species, along with H. tomentosum and H. tetrapterum hairy root cultures, and hairy root-derived transgenic plants of H. tomentosum. METHODS: Samples were extracted and analysed by LC-MS. The LC-MS data were subjected to chemometric evaluations for metabolite profiling and correlating the phytochemical compositions in different samples. KEY FINDINGS: Hypericin, pseudohypericin and their proposed precursor emodin were detected in various levels in the leaves of eight Hypericum species. The highest content of hypericins and emodin was found in H. tetrapterum, which contains the studied secondary metabolites in all plant organs. A significant positive correlation between hypericins and emodin was observed both by principal component analysis (PCA) and multidimensional scaling (MDS), indicating the role of emodin as a possible precursor in the biosynthetic pathway of hypericins. Flavonoids were found in all tested plant organs except roots of H. pulchrum. The hairy roots lacked hypericin, pseudohypericin, emodin, hyperforin and rutin. However, the hairy root-derived transgenic plants showed a significant increase in flavonoids. CONCLUSIONS: This study broadens knowledge about the phytochemical composition of selected in vitro cultured Hypericum species, compared to that of hairy root cultures and hairy root-derived transgenic plants.

Cardenolides and dihydro-ß-agarofuran sesquiterpenes from the seeds of Salacia staudtiana.

Phytochemical studies of the seeds of the Cameroonian medicinal plant, Salacia staudtiana, resulted in the isolation and identification of five new cardenolides (1-5) as well as a new dihydro-ß-agarofuran (9), along with eight known compounds. The structures of all compounds were elucidated by 1D/2D NMR, ESI-HRMS data and comparison with literature data. The relative configurations of the new compounds were defined by X-ray crystallography analysis, NOESY correlations and coupling constants. We evaluated their antibacterial efficacy against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, and two pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa, compared to the standard antibiotics, streptomycin and gentamicin. Moreover, we assessed the antibacterial activity of the crude extract of the seeds in parallel to evaluate the plausible synergistic effects of the compounds in chemical defense of the seeds during germination and plant reproduction. The isolated compounds showed moderate antibacterial activities against the tested organisms. Compounds 1 and 3 and the crude extract exhibited distinct antibacterial activities against B. subtilis and S. aureus. The isolated compounds showed weak DPPH radical scavenging properties compared to the reference standard (Trolox). Our study lends evidence to the antibacterial chemical defense of S. staudtiana seeds by seed-borne compounds.

Cardenolides from the stem bark of Salacia staudtiana.

Seven new cardenolides, staudtianoside A-F (1-6) and staudtianogenin A (8), were isolated along with six known compounds from the stem bark of the Cameroonian medicinal plant Salacia staudtiana Loes. ex Fritsch. The structures were elucidated by means of ESI-HRMS and NMR spectroscopic methods and by comparison with literature data. The relative configurations of the new compounds were determined by X-ray diffraction analysis, NOESY correlation and coupling constants. We evaluated the antibacterial efficacy of the isolated compounds against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, as well as against two human pathogenic clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa. Compounds 1, 2 and 8 exhibited marked antibacterial potencies against the clinically relevant P. aeruginosa that were comparable to the standard antibiotics. Compound 2 was also active against S. aureus and E. coli. Further, compounds 5 and 8 demonstrated efficacy against E. coli as well as B. subtilis. The structure-activity relationship of the tested compounds is discussed.

Spatial profiling of maytansine during the germination process of Maytenus senegalensis seeds.

The ecological role of maytansine, an important antineoplastic and antimicrobial compound with high cytotoxicity, particularly as a chemical defense compound has remained elusive since its discovery in the 1970s in Maytenus and Putterlickia plants. In the present study, we have used MALDI-imaging-HRMS to visualize the occurrence as well as spatial and temporal distribution of maytansine in a Maytenus senegalensis plant, seeds obtained from the mother plant during seeding stage, through the germination of the seeds, and finally up to the establishment of seedlings (or daughter plants). Although the mother plant was devoid of maytansine, the bioactive compound was found to be distributed in the cotyledons and the endosperm of the seeds with an augmented accretion towards the seed coat. Furthermore, maytansine was always detected in the emerging seedlings, particularly the cortex encompassing the radicle, hypocotyl, and epicotyl. The typical pattern of accumulation of maytansine not only in the seeds but also during germination provides a proof-of-concept that M. senegalensis is ecologically primed to trigger the production of maytansine in vulnerable tissues such as seeds during plant reproduction. By utilizing maytansine as chemical defense compound against predators and/or pathogens, the plant can ensure viability of the seeds and successful germination, thus leading to the next generation of daughter plants.

Antibacterial secondary metabolites from an endophytic fungus, Fusarium solani JK10.

Extensive chemical investigation of the endophytic fungus, Fusarium solani JK10, harbored in the root of the Ghanaian medicinal plant Chlorophora regia, using the OSMAC (One Strain Many Compounds) approach resulted in the isolation of seven new 7-desmethyl fusarin C derivatives (1-7), together with five known compounds (8-12). The structures of the new compounds were elucidated by analysis of their spectroscopic data including 1D, 2D NMR, HRESI-MSn and IR data. The relative configuration of compounds 1/2 was deduced by comparison of their experimental electronic circular dichroism (ECD) and optical rotation data with those reported in literature. The absolute configuration of solaniol (10), a known compound with undefined absolute stereochemistry, was established for the first time by X-ray diffraction analysis of a single-crystal structure using Cu-Kα radiation. The antibacterial activities of the crude fungal extract and the compounds isolated from the fungus were evaluated against some clinically important bacterial strains such as Staphylococcus aureus and Bacillus subtilis, as well as an environmental strain of Escherichia coli and the soil bacterium Acinetobacter sp. BD4. Compounds 3/4 and 6 exhibited antibacterial efficacies against the soil bacterium Acinetobacter sp., comparable to the reference standard streptomycin. All the tested compounds (1-9) demonstrated antibacterial activity against the environmental strain of E. coli, whereas no antibacterial activity was observed against S. aureus and B. subtilis. The antibacterial activity of the isolated compounds typically against E. coli and Acinetobacter sp. provides further insight into the possible involvement of root-borne endophytes in chemical defense of their host plants in selected ecological niches.

Epigenetic Modulation of Endophytic Eupenicillium sp. LG41 by a Histone Deacetylase Inhibitor for Production of Decalin-Containing Compounds.

An endophytic fungus, Eupenicillium sp. LG41, isolated from the Chinese medicinal plant Xanthium sibiricum, was subjected to epigenetic modulation using an NAD+-dependent histone deacetylase (HDAC) inhibitor, nicotinamide. Epigenetic stimulation of the endophyte led to enhanced production of two new decalin-containing compounds, eupenicinicols C and D (3 and 4), along with two biosynthetically related known compounds, eujavanicol A (1) and eupenicinicol A (2). The structures and stereochemistry of the new compounds were elucidated by extensive spectroscopic analysis using LC-HRMS, NMR, optical rotation, and ECD calculations, as well as single-crystal X-ray diffraction. Compounds 3 and 4 exist in chemical equilibrium with two and three cis/trans isomers, respectively, as revealed by LC-MS analysis. Compound 4 was active against Staphylococcus aureus with an MIC of 0.1 µg/mL and demonstrated marked cytotoxicity against the human acute monocytic leukemia cell line (THP-1). We have shown that the HDAC inhibitor, nicotinamide, enhanced the production of compounds 3 and 4 by endophytic Eupenicillium sp. LG41, facilitating their isolation, structure elucidation, and evaluation of their biological activities.

Occurrence and spatial distribution of maytansinoids in Putterlickia pyracantha, an unexplored resource of anticancer compounds.

Maytansinoids possess remarkable antibiotic activities along with high cytotoxicity, many of which are currently used (or in clinical trials) in the treatment of breast cancer. Celastraceous plants and their associated microorganisms serve as an important resource of maytansinoids. Here, we report the occurrence and structural elucidation of several maytansinoids in Putterlickia pyracantha plants bioprospected in South Africa. In addition to maytansine, which is already known to be present in this species, we show the presence of maytanprine, maytanbutine, maytanvaline, normaytancyprine and an abundant maytansine precursor in different tissues using high-resolution mass spectrometry. Furthermore, we identified two new hydroxylated maytansinoids by HRMS(2) analyses. We also employed MALDI-imaging-HRMS to study the spatial distribution and localization of the maytansinoids within the different plant tissues. On the one hand, the fragmentation pathways of the maytansinoids we report herein using HRMS(n) will allow quick identification of these compounds in the future without isolating from the natural resources. On the other hand, MALDI-imaging-HRMS revealed insights into the plausible ecological roles and biosynthetic pathways of these compounds in P. pyracantha plants.

Antibacterial Azaphilones from an Endophytic Fungus, Colletotrichum sp. BS4.

Three new compounds, colletotrichones A-C (1-3), and one known compound, chermesinone B (4a), were isolated from an endophytic fungus, Colletotrichum sp. BS4, harbored in the leaves of Buxus sinica, a well-known boxwood plant used in traditional Chinese medicine (TCM). Their structures were determined by extensive spectroscopic analyses including 1D and 2D NMR, HRMS, ECD spectra, UV, and IR, as well as single-crystal X-ray diffraction, and shown to be azaphilones sharing a 3,6a-dimethyl-9-(2-methylbutanoyl)-9H-furo[2,3-h]isochromene-6,8-dione scaffold. Owing to the remarkable antibacterial potency of known azaphilones coupled to the usage of the host plant in TCM, we evaluated the antibacterial efficacy of the isolated compounds against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, as well as against two human pathogenic clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa. Compound 1 exhibited marked antibacterial potencies against the environmental strains that were comparable to the standard antibiotics. Compound 3 was also active against E. coli. Finally, compound 2a exhibited the same efficacy as streptomycin against the clinically relevant bacterium S. aureus. The in vitro cytotoxicity of these compounds on a human acute monocytic leukemia cell line (THP-1) was also assessed. Our results provide a scientific rationale for further investigations into endophyte-mediated host chemical defense against specialist and generalist pathogens.

Flavanones of Erythrina livingstoniana with antioxidant properties.

Six new flavanones (1-6), together with six known compounds were isolated from Erythrina livingstoniana. Their structures were elucidated on the basis of NMR data and HRMS(n) fragmentation pathway and by comparison with literature data. Compounds 5, 7 and 8 showed remarkable DPPH free radical scavenging efficacies. The compounds, however, did not demonstrate an anti-inflammatory potential when tested using a PGE2 (prostaglandin E2) competitive enzyme immunoassay. The plausible biosynthetic pathways of the isolated compounds are described.

Prenylated 2-arylbenzofuran derivatives with potent antioxidant properties from Chlorophora regia (Moraceae).

Extracts of Chlorophora regia are frequently used in Ghana in traditional medicine. There is, however, no reported data on the chemical composition of the plant. Comprehensive phytochemical investigation of the stem bark of C. regia resulted in the isolation of three new prenylated 2-arylbenzofuran derivatives, regiafuran A-C (1-3), and one new prenylated flavonol (4), together with fifteen known compounds (5-19). Their structures were elucidated by combined spectroscopic analysis of their NMR and HRESI-MS(n) data. Compounds 1, 2, 5, 9 and 15 exhibited remarkable free radical scavenging properties with IC50 values of 1.9 µg/ml, 2.4 µg/ml, 2.2 µg/ml, 2.1 µg/ml and 1.8 µg/ml, respectively, compared to the standard trolox (IC50 1.1 µg/ml). The isolated compounds did not, however, show any anti-inflammatory potential when tested using a PGE2 (prostaglandin E2) competitive enzyme immunoassay.

Anti-inflammatory tirucallane triterpenoids from Anopyxis klaineana Pierre (Engl.), (Rhizophoraceae).

Phytochemical investigation of the stem bark extract of Anopyxis klaineana was carried out by various chromatographic techniques resulting in the isolation and characterization of three new tirucallane triterpenoids, namely 3,23-dioxotirucalla-7,24-dien-21-oic acid (1), 3,4-secotirucalla-23-oxo-4(28)7,24-trien-3,21-dioic acid (2) and 3,4-secotirucalla-4-hydroxy-23-oxo-7,24-diene-3,21-dioic acid-21-methyl ester (3), along with nine known compounds (4-12). The structural elucidation of the compounds was performed by means of high-resolution mass spectrometry (HRMS(n)), nuclear magnetic resonance (NMR), and by comparison to literature data. Although none of the isolated compounds showed antibacterial efficacy against selected environmental and clinically important pathogenic Gram-positive and -negative bacteria, they demonstrated moderate DPPH free radical scavenging properties. Furthermore, compounds 1 and 7 exhibited remarkable anti-inflammatory potential in a prostaglandin E2 (PGE2) competitive enzyme immunoassay with IC50 values of 3.63 µM and 10.23 µM, respectively.

Endophytic Diaporthe sp. LG23 Produces a Potent Antibacterial Tetracyclic Triterpenoid.

A new lanostanoid, 19-nor-lanosta-5(10),6,8,24-tetraene-1α,3ß,12ß,22S-tetraol (1), characterized by the presence of an aromatic B ring and hydroxylated at C-1, C-3, C-12, and C-22, was isolated from an endophytic fungus, Diaporthe sp. LG23, inhabiting leaves of the Chinese medicinal plant Mahonia fortunei. Six biosynthetically related known steroids were also isolated in parallel. Their structures were confirmed on the basis of detailed spectroscopic analysis in conjunction with the published data. Compound 1, an unusual fungus-derived 19-nor-lanostane tetracyclic triterpenoid with an aromatic B-ring system, exhibited pronounced antibacterial efficacy against both Gram-positive and -negative bacteria, especially the clinical isolates of Streptococcus pyogenes and Pseudomonas aeruginosa as well as a human pathogenic strain of Staphylococcus aureus. Our results reveal the potential of endophytes not only in conferring host fitness but also in contributing toward traditional host plant medicines.

Erylivingstone A-C with antioxidant and antibacterial activities from Erythrina livingstoniana.

The chemical study of Erythrina livingstoniana has led to the isolation of three new flavanones namely 5,7,3'-trihydroxy-4'-methoxy-5'-formylflavanone (erylivingstone A) (1), 5,7,3'-trihydroxy-5'-(2-hydroxy-3-methylbut-3-enyl)-4'-methoxyflavanone (erylivingstone B) (2) and 5,7,3'-trihydroxy-5'-(3-hydroxy-3-methyl-trans-but-1-enyl)-4'-methoxyflavanone (erylivingstone C) (3) together with three known compounds (4-6). Their structures were elucidated on the basis of NMR data, HRMS(n) fragmentation pathway and by comparison with literature data. We evaluated the antibacterial efficacies and free-radical scavenging potential of the isolated compounds (1-6). The typical environmental strains of Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, as well as against the clinically important Staphylococcus aureus, Streptococcus pyogenes and E. coli (risk-group 2) were used for the antibacterial assay. Compounds 5 and 6 exhibited the most pronounced efficacy against tested environmental bacteria as well as against the pathogenic strain of E. coli. Compound 3 was also quite active against these three bacterial strains. The isolated compounds showed weak radical scavenging properties with compound 6 being the most active, followed by compounds 2, 3 and 5.

Spatial chemo-profiling of hypericin and related phytochemicals in Hypericum species using MALDI-HRMS imaging.

Advanced analytical imaging techniques, including matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) imaging, can be used to visualize the distribution, localization, and dynamics of target compounds and their precursors with limited sample preparation. Herein we report an application of MALDI-HRMS imaging to map, in high spatial resolution, the accumulation of the medicinally important naphthodianthrone hypericin, its structural analogues and proposed precursors, and other crucial phytochemical constituents in the leaves of two hypericin-containing species, Hypericum perforatum and Hypericum olympicum. We also investigated Hypericum patulum, which does not contain hypericin or its protoforms. We focused on both the secretory (dark glands, translucent glands, secretory canals, laminar glands, and ventral glands) and the surrounding non-secretory tissues to clarify the site of biosynthesis and localization of hypericin, its possible precursors, and patterns of localization of other related compounds concomitant to the presence or absence of hypericin. Hypericin, pseudohypericin, and protohypericin accumulate in the dark glands. However, the precursor emodin not only accumulates in the dark glands but is also present outside the glands in both hypericin-containing species. In hypericin-lacking H. patulum, however, emodin typically accumulates only in the glands, thereby providing evidence that hypericin is possibly biosynthesized outside the dark glands and thereafter stored in them. The distribution and localization of related compounds were also evaluated and are discussed concomitant to the occurrence of hypericin. Our study provides the basis for further detailed investigation of hypericin biosynthesis by gene discovery and expression studies.

Antibacterial secondary metabolites from an endophytic fungus, Eupenicillium sp. LG41.

Two new compounds containing the decalin moiety, eupenicinicols A and B (1 and 2), two new sirenin derivatives, eupenicisirenins A and B (3 and 4), and four known compounds, (2S)-butylitaconic acid (5), (2S)-hexylitaconic acid (6), xanthomegnin (7), and viridicatumtoxin (8), were isolated from an endophytic fungus, Eupenicillium sp. LG41, harbored in the roots of the Chinese medicinal plant Xanthium sibiricum. Their structures were confirmed through combined spectroscopic analysis (NMR and HRMS(n)), and their absolute configurations were deduced by ECD calculations or optical rotation data. Since the endophytic fungus was isolated from the roots, the antibacterial efficacies of the compounds 1-6 were investigated against Bacillus subtilis and Acinetobacter sp. BD4, which typically inhabit soil, as well as the clinically important Staphylococcus aureus and Escherichia coli. (2S)-Butylitaconic acid (5) and (2S)-hexylitaconic acid (6) exhibited pronounced efficacy against Acinetobacter sp., corroborating the notion that root-endophytes provide chemical defense to the host plants. Compound 2 was highly active against the clinically relevant S. aureus. By comparing 1 with 2, it was revealed that altering the substitution at C-11 could drastically increase the antibacterial efficacy of 1. Our study reveals plausible ecological roles of the endophyte and its potential pharmaceutical use as a source of antibacterial compounds.

Tramadol--a true natural product?

We have independently investigated the source of tramadol, a synthetic analgesic largely used for treating moderate to severe pain in humans, recently found in the roots of the Cameroonian medicinal plant, Nauclea latifolia. We found tramadol and its three major mammalian metabolites (O-desmethyltramadol, N-desmethyltramadol, and 4-hydroxycyclohexyltramadol) in the roots of N. latifolia and five other plant species, and also in soil and local water bodies only in the Far North region of Cameroon. The off-label administration of tramadol to cattle in this region leads to cross-contamination of the soil and water through feces and urine containing parent tramadol as well as tramadol metabolites produced in the animals. These compounds can then be absorbed by the plant roots and also leached into the local water supplies. The presence of tramadol in roots is, thus, due to an anthropogenic contamination with the synthetic compound.