Molecular networking-guided isolation strategy of a new C-glycosyl flavone rotamer from Stellaria alsine and evaluation of anti-inflammatory and antioxidant activities.

INTRODUCTION: Stellaria alsine has traditionally been used as both a famine relief food and an alternative medicine in East Asia. Modern pharmacological studies have revealed that S. alsine has various biological effects such as anticancer, anti-hepatoma, anti-inflammatory, and antioxidative effects. However, the anti-inflammatory properties of chemical constituents derived from this plant have not been studied well. OBJECTIVES: To identify potential therapeutic candidate for treating inflammatory diseases such as inflammatory bowel disease (IBD). METHODS: The distribution of chemical compounds was investigated by Global Natural Product Social (GNPS)-based molecular networking (MN) analysis using UPLC-Orbitrap tandem mass spectrometry. The anti-inflammatory and antioxidative effects of S. alsine extracts and fractions were evaluated by measuring interleukin (IL)-8 and reactive oxygen species (ROS) productions. RESULTS: The active EA layer of S. alsine showed the highest percentage of major compounds by feature-based molecular networking. The top candidate structures of EA fraction were rapidly annotated as flavone C- or O-glycosides via an advanced analysis tool, Network Annotation Propagation (NAP). With the GNPS molecular networking-guided isolation strategy, a new C-glycosyl flavone rotamer (1) was isolated. The structures of the major (1a) and minor (1b) rotational isomers were determined by extensive NMR analysis and MS/MS fragmentation. Finally, the anti-inflammatory activity of 1 was predicted by molecular docking simulations with IL-8 protein. CONCLUSION: These results suggested that the compound 1 is a potential therapeutic candidate for inflammatory bowel disease (IBD).

Molecular Networking-Guided Isolation of a Phenolic Constituent from Prunus mume Seed and Its Antioxidant and Anti-Inflammatory Activities.

Prunus mume (Maesil) is used in health foods and alternative medicine in Korea. In the present study, the anti-inflammatory and antioxidant effects of phenolics from P. mume seed extracts were examined. First, the biological activities of various P. mume extracts were evaluated, and the profiles of their chemical compounds were investigated by Global Natural Products Social (GNPS)-molecular networking. Among these extracts, fermented Maesil seed extract (FMSE) showed potent anti-inflammatory and antioxidant activity, and demonstrated the presence of phenolic clusters in GNPS-based studies. Thus, the chemical constituents of this extract were further investigated. Subsequently, the chemical composition of the active CH2Cl2 fraction of FMSE was explored using an advanced GNPS analysis tool, MolNetEnhancer. In addition, the molecular structure of compound 1 from the CH2Cl2 fraction was similarly predicted with Network Annotation Propagation (NAP). Finally, the anti-inflammatory and antioxidant effects of compound 1 were confirmed by lipopolysaccharide (LPS)-induced nitric oxide production and DPPH assay. Western blot analysis revealed that compound 1 downregulated the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. The molecular docking simulation additionally confirmed significant interactions of 1 with iNOS and COX-2 proteins. Our findings suggested that an integrated GNPS-based approach could prioritize samples in the early fractionation process and improve the accuracy of target compound prediction.

Iridoid derivatives from Vitex rotundifolia L. f. with their anti-inflammatory activity.

Three undescribed iridoid glucosides and nine known compounds were isolated from Vitex rotundifolia L. f. Their structural elucidation was performed based on their spectroscopic data or acid hydrolysis followed by HPLC analysis and comparison of their NMR data with those reported in the literature. These iridoids were then evaluated for inflammatory effects through inhibition on NO production level in LPS-stimulated RAW264.7 cells. The active compounds, rotundifoliin C, isonishindacin A, agnuside, and eurostoside, were further investigated for their anti-inflammatory mechanisms of action on expression levels of iNOS and COX-2 proteins. In addition, V. rotundifolia fractions also significantly inhibited LPS-induced IL-8 production, with IC50 values ranging from 9.81 to 54.31 µg/mL. Rotundifoliin A, agnuside, VR-I (10-O-vanilloyl aucubin), and eurostoside showed inhibition rates of 55.5%, 94.6%, 55.6%, and 81.9% on IL-8 production at concentrations of 100 µM, respectively, compared to those of control without sample addition. The therapeutic properties of the plant might give rise to develop the functional products to treat inflammatory diseases.

Potential function of loliolide as a novel blocker of epithelial-mesenchymal transition in colorectal and breast cancer cells.

Loliolide (LL), a naturally occurring monoterpenoid lactone isolated from Vicia tenuifolia Roth, can exhibit numerous pharmacological effects such as those related to anti-Parkinson, anti-oxidant, anti-cholinesterase, and anti-depressant. Epithelial-mesenchymal transition (EMT) plays a pivotal role in regulating tumor metastasis. CXCR4 and CXCR7 are G-protein-coupled receptors (GPRs), which can be stimulated by CXCL12. CXCL12/CXCR4/CXCXR7 axis can cause activation of multiple pathways including MAPKs, JAK/STAT pathway, and manganese superoxide dismutase (MnSOD) signaling. These events can initiate EMT process and induce cell invasion and migration. Here, we investigated whether LL can modulate the CXCR4 and CXCR7 and EMT process in colon cancer and breast cancer cells. We found that LL suppressed levels of CXCR4 and CXCR7, and exerted an inhibitory effect on these chemokines even after stimulation by CXCL12. LL suppressed expression of MnSOD and mesenchymal markers, whereas induced epithelial markers. In addition, LL significantly attenuated cellular invasion, migration, and metastasis. We noted that LL inhibited CXCR4/7 and EMT process even after stimulation of CXCL12 and MnSOD overexpression. Therefore, in this study, we provide evidences that targeting CXCR4/7 and MnSOD could inhibit the invasion, migration, and metastasis of cancer cells as well as negatively regulate the EMT process. Overall, our study suggested that LL might act as a potent suppressor of EMT process against colon and breast cancer cells.

Anti-inflammatory activity of caffeic acid derivatives from Ilex rotunda.

Ilex rotunda Thunb. has been used in traditional medicine for treating rheumatoid arthritis, relieving pain and indigestion. In the present study, we isolated three new caffeic acid benzyl ester (CABE) analogs (1-3) along with eight known compounds (4-11) from the extract of I. rotunda. The absolute configuration of α-hydoxycarboxylic acid in 1 was assigned with the phenylglycine methyl ester (PGME) method. We further investigated their anti-inflammatory activities in lipopolysaccharide (LPS)-induced macrophages (RAW 264.7) cells. Among them, compounds 2-4, 7, 8, 10, and 11 suppressed the production of nitric oxide (NO), pro-inflammatory mediators. It was additionally confirmed that the anti-inflammatory effect of active compound 2 was through significant suppression of cytokines, including interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, and IL-8 in LPS-stimulated RAW 264.7 cells and colon epithelial (HT-29) cells. Western blot analysis revealed that compound 2 decreased the LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and phosphorylated extracellular regulated kinase (pERK)1/2. The following molecular docking simulations showed the significant interactions of compound 2 with the iNOS protein. These results suggested that the compound 2 can be used as potential candidate for treating inflammatory diseases such as inflammatory bowel disease (IBD).

Analysis of Antioxidant Constituents from Ilex rotunda and Evaluation of Their Blood-Brain Barrier Permeability.

Ilex rotunda Thunb., has been used to treat common cold, tonsillitis, and eczema. It is also a source of antioxidants. However, information regarding its antioxidative phytochemical composition is still incomplete and limited. In this present study, we initially determined DPPH radical scavenging activity of the extracts of I. rotunda fruits, twigs, and leaves. Among them, the twig extract exhibited a potential of antioxidant capacity. Based on antioxidant effect guided experiments, extraction condition using 80% EtOH was then optimized. DPPH and ABTS radical scavenging assays were also performed for fractions. The n-butanol fraction showed the highest antioxidant effect. Using chromatographic methods, eight marker compounds (1-8) were further isolated. Their structures were determined by spectroscopic and mass data. Method validation was employed to quantitate contents of these eight marker compounds. Subsequently, the HPLC-DPPH method was used to evaluate the contribution of certain compounds to total antioxidant activity of the extract. Lastly, parallel artificial membrane permeability assay for blood-brain barrier (PAMPA-BBB) was applied to investigate brain-penetrable antioxidants from I. rotunda extract. As a result, compound 7 (4,5-dicaffeoylquinic acid) showed significant antioxidant activity and penetration across the BBB via transcellular passive diffusion. Our findings suggested that compound 7 can be used as a therapeutic potential candidate in natural product-based central nervous system (CNS) drug discovery.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

Analysis of Antioxidant Phytochemicals and Anti-Inflammatory Effect from Vitex rotundifolia L.f.

An extraction method using 80% EtOH was selected and applied to obtain the total extracts from leaves, flowers, fruits, twigs, and roots of Vitex rotundifolia L.f. based on the antioxidant activity-guided experiments. Subsequently, total extract from each part of V. rotundifolia was successfully partitioned into fractions, which were evaluated for their antioxidant and anti-inflammatory properties via DPPH, ABTS, and NO assays, respectively. Among them, EtOAc (E) and n-butanol (B) fractions showed the potent antioxidant activity and the methylene chloride (MC) fractions of roots, leaves, and fruits that exhibited strong scavenging activity on DPPH and ABTS radicals. In the anti-inflammatory assay, n-hexane (H) and MC fractions of leaves potently inhibited NO production in LPS-stimulated RAW264.7 cells, followed by E fractions derived from fruits, flowers, twigs, and roots, along with B fractions from flowers and twigs. Additionally, a comprehensive HPLC-decoupled MS profiling was established and validated using seven isolated marker compounds (1-7), which were identified by analysis of their UV, NMR, and MS data. The established method was also applied for quantification of these marker compounds in each organ collected from different locations, and to assess their antioxidant capacity by a screening DPPH-HPLC method. Principal component analysis suggested the botanical organs from this plant correlated with the marker compound contents in association with bioactivity. The study results are a prelude to further studies involving the active fractions and provide a comprehensive insight into the functional products of this plant against oxidative diseases.

Identification of natural product modulators of Merkel cell carcinoma cell growth and survival.

Merkel cell carcinoma (MCC) is a rare, but aggressive skin cancer the incidence of which has increased significantly in recent years. The majority of MCCs have incorporated Merkel cell polyomavirus (VP-MCC) while the remainder are virus-negative (VN-MCC). Although a variety of therapeutic options have shown promise in treating MCC, there remains a need for additional therapeutics as well as probes for better understanding MCC. A high-throughput screening campaign was used to assess the ability of > 25,000 synthetic and natural product compounds as well as > 20,000 natural product extracts to affect growth and survival of VN-MCC and VP-MCC cell lines. Sixteen active compounds were identified that have mechanisms of action reported in the literature along with a number of compounds with unknown mechanisms. Screening results with pure compounds suggest a range of potential targets for MCC including DNA damage, inhibition of DNA or protein synthesis, reactive oxygen species, and proteasome inhibition as well as NFκB inhibition while also suggesting the importance of zinc and/or copper binding. Many of the active compounds, particularly some of the natural products, have multiple reported targets suggesting that this strategy might be a particularly fruitful approach. Processing of several active natural product extracts resulted in the identification of additional MCC-active compounds. Based on these results, further investigations focused on natural products sources, particularly of fungal origin, are expected to yield further potentially useful modulators of MCC.

Roseabol A, a New Peptaibol from the Fungus Clonostachys rosea.

A new 11 amino acid linear peptide named roseabol A (1) and the known compound 13-oxo-trans-9,10-epoxy-11(E)-octadecenoic acid (2) were isolated from the fungus Clonostachys rosea. Combined NMR and MS analysis revealed that roseabol A (1) contained amino acid residues characteristic of the peptaibol family of peptides such as isovaline, α-aminoisobutyric acid, hydroxyproline, leucinol, and an N-terminal isovaleric acid moiety. The amino acid sequence was established by a combination of NMR studies and tandem MS fragmentation analyses, and the absolute configurations of the constituent amino acids of 1 were determined by the advanced Marfey's method. Compound 2 showed inhibitory activity against Merkel cell carcinoma, a rare and difficult-to-treat type of skin cancer, with an IC50 value of 16.5 µM.

Neopetrothiazide: An Intriguing Pentacyclic Thiazide Alkaloid from the Sponge Neopetrosia sp.

Neopetrothiazide (1), a pentacyclic isoquinoline quinone, was isolated from a Neopetrosia sp. sponge. The structure elucidation was facilitated by utilizing long-range heteronuclear single quantum multiple bond correlation (LR-HSQMBC) and heteronuclear multiple bond correlation (HMBC) nuclear magnetic resonance (NMR) pulse sequences optimized to detect four- and five-bond 1H-13C heteronuclear correlations. These NMR experiments can help assign proton-deficient structural motifs like neopetrothiazide (1), which has 14 contiguous nonprotonated centers (C, N, and S). Neopetrothiazide (1), with an unprecedented thiazide-fused structural scaffold, is the first natural product containing a thiazide moiety.

Denigrins and Dactylpyrroles, Arylpyrrole Alkaloids from a Dactylia sp. Marine Sponge.

Seven new arylpyrrole alkaloids (1-7), along with four known compounds, were isolated from an extract of a Dactylia sp. nov. marine sponge, and their structures were elucidated by interpretation of NMR and MS spectroscopic data. Denigrins D-G (1-4) have highly substituted pyrrole or pyrrolone rings in their core structures, while dactylpyrroles A-C (5-7) have tricyclic phenanthrene cores. Due to the proton-deficient nature of these scaffolds, key heteronuclear correlations from 1H-15N HMBC and LR-HSQMBC NMR experiments were used in the structure assignment of denigrin D (1). Dictyodendrin F (8), a previously described co-metabolite, inhibited transcription driven by the oncogenic PAX3-FOXO1 fusion gene with an IC50 value of 13 µM.

Suberitamides A-C, Aryl Alkaloids from a Pseudosuberites sp. Marine Sponge that Inhibit Cbl-b Ubiquitin Ligase Activity.

Three new aryl alkaloids named suberitamides A-C (1-3), were isolated from an extract of the marine sponge Pseudosuberites sp. collected along the coast of North Carolina. Their planar structures were established by extensive nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. To assign the challenging relative configuration of the saturated five-membered ring in suberitamide A (1), a simple and efficient NMR protocol was applied that is based on the analysis of 2- and 3-bond 1H-13C spin-spin coupling constants using a PIP (pure in-phase) HSQMBC (heteronuclear single quantum multiple bond correlation) IPAP (in-phase and anti-phase) experiment. Suberitamides A (1) and B (2) inhibited Cbl-b, an E3 ubiquitin ligase that is an important modulator of immune cell function, with IC50 values of approximately 11 µM.

Cytotoxic Scalarane Sesterterpenes from the Sponge Hyrtios erectus.

Twelve new sesterterpenes along with eight known sesterterpenes were isolated from the marine sponge Hyrtios erectus collected off the coast of Chuuk Island, the Federated State of Micronesia. Based upon a combination of spectroscopic and computational analyses, these compounds were determined to be eight glycine-bearing scalaranes (1-8), a 3-keto scalarane (9), two oxidized-furan-bearing scalaranes (10 and 11), and a salmahyrtisane (12). Several of these compounds exhibited weak antiproliferation against diverse cancer cell lines as well as moderate anti-angiogenesis activities. The antiproliferative activity of new compound 4 was found to be associated with G0/G1 arrest in the cell cycle.

Swinhopeptolides A and B: Cyclic Depsipeptides from the Sponge Theonella swinhoei That Inhibit Ras/Raf Interaction.

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.

Elucidation of Spirodactylone, a Polycyclic Alkaloid from the Sponge Dactylia sp., and Nonenzymatic Generation from the Co-metabolite Denigrin B.

Spirodactylone (1), a hexacyclic indolizidone alkaloid possessing a novel spiro ring system, was isolated from the marine sponge Dactylia sp. The structure was elucidated by extensive spectroscopic methods including application of the LR-HSQMBC NMR pulse sequence. Oxidative cyclization of denigrin B (2), an aryl-substituted 2-oxo-pyrroline derivative that was also isolated from the sponge extract, provided material identical to spirodactylone (1). This confirmed the assigned structure and provides insight into the probable biogenesis of 1.

Cyclopeptides from the Sponge Stylissa flabelliformis.

Three new cyclopeptides, phakellistatins 20-22 (1-3), as well as 10 known cyclopeptides of the same structural class were isolated from the tropical sponge Stylissa flabelliformis. By a combination of chemical and spectroscopic methods, the structures of the new compounds were determined to be an epimeric mixture of cycloheptapeptides (1) and two epimeric cyclodecapeptides (2 and 3) related to the phakellistatins. The cyclopeptides were evaluated for in vitro cytotoxicity against a variety of cancer cell lines, and compounds 2 and 3 exhibited significant activity.

Inhibitory Effects of Flavonoids from Spatholobus suberectus on Sortase A and Sortase A-Mediated Aggregation of Streptococcus mutans.

Seven flavonoids were isolated from Spatholobus suberectus via repetitive column chromatography and high-performance liquid chromatography. The chemical structures of these compounds were identified by spectroscopic analysis and comparison with values reported in the literature. Among the flavonoids tested, 7-hydroxy-6-methoxyflavanone (1) and formononetin (4) exhibited strong inhibitory activity against Streptococcus mutans SrtA, with IC50 values of 46.1 and 41.8 µM, respectively, but did not affect cell viability. The onset and magnitude of inhibition of saliva-induced aggregation in S. mutans treated with compounds 1 and 4 were comparable to the behavior of a srtA-deletion mutant without treatment.

Manzamine Alkaloids from an Acanthostrongylophora sp. Sponge.

Five new manzamine alkaloids (1-5) and new salt forms of two known manzamines (6 and 7), along with seven known compounds (8-14) of the same structural class, were isolated from an Indonesian Acanthostrongylophora sp. sponge. On the basis of the results of combined spectroscopic analyses, the structure of kepulauamine A (1) was determined to possess an unprecedented pyrrolizine moiety, while others were functional group variants of known manzamines. These compounds exhibited weak cytotoxicity, moderate antibacterial activity, and mild inhibition against the enzyme isocitrate lyase.

Spatholobus suberectus Dunn. constituents inhibit sortase A and Staphylococcus aureus cell clumping to fibrinogen.

Sortases are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus (S. aureus), deletion of sortase isoform results in a significant reduction in virulence and infection potential. Twenty flavonoids were isolated from the stem of the folk medicinal plant Spatholobus suberectus Dunn. These compounds were tested against S. aureus-derived sortase A (SrtA), a key transpeptidase for bacterial virulence. Among these active flavonoids, 7-hydroxy-6-methoxy-flavanone (3) and formononetin (10) were identified as compounds with promising SrtA inhibitory activity. These compounds also exhibited inhibitory activity against S. aureus cell clumping to fibrinogen. The suppression of cell-clumping activity indicates the potential of these compounds in the treatment of S. aureus infections via the inhibition of SrtA.