Structural Diversification of Pyrazinone Metabolites via Spontaneous Oxa-Michael Addition in Staphylococcus xylosus.

A family of pyrazinone metabolites (1-11) were characterized from Staphylococcus xylosus ATCC 29971. Six of them were hydroxylated or methoxylated, which were proposed to be produced by the rare noncatalytic oxa-Michael addition reaction with a water or methanol molecule. It was confirmed that isopropyl alcohol can also be the Michael donor of the reaction. 1-7 and the synthetic precursor 2a showed significant inhibition of breast cancer cell migration.

Characterization of Chemical Interactions between Clinical Drugs and the Oral Bacterium, Corynebacterium matruchotii, via Bioactivity-HiTES.

In the field of natural product research, the rediscovery of already-known compounds is one of the significant issues hindering new drug development. Recently, an innovative approach called bioactivity-HiTES has been developed to overcome this limitation, and several new bioactive metabolites have been successfully characterized by this method. In this study, we applied bioactivity-HiTES to Corynebacterium matruchotii, the human oral bacterium, with 3120 clinical drugs as potential elicitors. As a result, we identified two cryptic metabolites, methylindole-3-acetate (MIAA) and indole-3-acetic acid (IAA), elicited by imidafenacin, a urinary antispasmodic drug approved by the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). MIAA showed weak antibacterial activity against a pulmonary disease-causing Mycobacterium conceptionense with an IC50 value of 185.7 µM. Unexpectedly, we also found that C. matruchotii metabolized fludarabine phosphate, a USFDA-approved anticancer drug, to 2-fluoroadenine which displayed moderate antibacterial activity against both Bacillus subtilis and Escherichia coli, with IC50 values of 8.9 and 20.1 µM, respectively. Finally, acelarin, a prodrug of the anticancer drug gemcitabine, was found to exhibit unreported antibacterial activity against B. subtilis with an IC50 value of 33.6 µM through the bioactivity-HiTES method as well. These results indicate that bioactivity-HiTES can also be applied to discover biotransformed products in addition to finding cryptic metabolites in microbes.

Neurotrophic phenolic glycosides from the roots of Armoracia rusticana.

Armoracia rusticana P. G. Gaertner. belongs to the Brassicaceae family and has aroused scientific interest for its anti-inflammatory and anticancer activities. In a continuing investigation to discover bioactive constituents from A. rusticana, we isolated 19 phenolic glycosides including three undescribed flavonol glycosides and one undescribed neolignan glycoside from MeOH extract of this plant. Their structures were elucidated based on NMR spectroscopic analysis (1H, 13C, 1H-1H COSY, HSQC, and HMBC), HRESIMS, and chemical methods. The determination of their absolute configuration was accomplished by ECD and LC-MS analysis. All the compounds were assessed for their potential neurotrophic activity through induction of nerve growth factor in C6 glioma cell lines and for their anti-neuroinflammatory activity based on the measurement of inhibition levels of nitric oxide production and pro-inflammatory cytokines (i.e., IL-1ß, IL-6, and TNF-α) in lipopolysaccharide-activated microglia BV-2 cells.

Neurotrophic neolignans of Pinus koraiensis twigs.

Four undescribed neolignan analogs, together with eight known compounds, were isolated from the twigs of Pinus koraiensis (Korean pine). The chemical structure of the isolated compounds was determined through extensive spectroscopic analysis and chemical method. Their relative and absolute configurations were assigned through a well-established empirical rule and electronic circular dichroism (ECD) analysis, respectively. Four compounds (3 and 9-11) at 20 µM concentration showed significant neurotrophic effect by inducing nerve growth factor (NGF) secretion in C6 cells with the stimulation levels a range of 140.82 ± 4.62% to 160.04 ± 11.04%. Additionally, the result indicated that the glycosylation of neolignan led to an improvement in neurotrophic activity compared to their aglycone form. A compound (7) inhibited nitric oxide production with an IC50 value of 31.74 µM in LPS-activated BV2 cells.

Enhanced Expression of p53 and Suppression of PI3K/Akt/mTOR by Three Red Sea Algal Extracts: Insights on Their Composition by LC-MS-Based Metabolic Profiling and Molecular Networking.

Brown algae comprise up to 2000 species with wide dissemination in temperate zones. A comprehensive untargeted metabolic profiling guided by molecular networking of three uninvestigated Red-Sea-derived brown algae, namely Sirophysalis trinodis, Polycladia myrica, and Turbinaria triquetra, led to the identification of over 115 metabolites categorized as glycerolipids, fatty acids, sterol lipids, sphingolipids, and phospholipids. The three algae exhibited low-to-moderate antioxidant capacity using DPPH and ABTS assays. Preliminary in vitro antiproliferative studies showed that the algal extracts displayed high cytotoxic activity against a panel of cancer cell lines. The most potent activity was recorded against MCF-7 with IC50 values of 51.37 ± 1.19, 63.44 ± 1.13, and 59.70 ± 1.22 µg/mL for S. trinodis, P. myrica, and T. triquetra, respectively. The cytotoxicity of the algae was selective to MCF-7 without showing notable effects on the proliferation of normal human WISH cells. Morphological studies revealed that the algae caused cell shrinkage, increased cellular debris, triggered detachment, cell rounding, and cytoplasmic condensation in MCF-7 cancer cells. Mechanistic investigations using flow cytometry, qPCR, and Western blot showed that the algae induced apoptosis, initiated cell cycle arrest in the sub-G0/G1 phase, and inhibited the proliferation of cancer cells via increasing mRNA and protein expression of p53, while reducing the expression of PI3K, Akt, and mTOR.

Structure Revision of Anti-Inflammatory Indole Alkaloids with a 1,2-Benzisoxazole Ring.

(R)- and (S)-2-(benzo[d]isoxazol-3-yl)-2-ethylindolin-3-one [(±)-1] were previously isolated from NIRAM, a natural blue dye from Polygonum tinctorium, and their structures were initially proposed to possess a 1,2-benzisoxazole ring. In this study, the structures of (±)-1 were revised to have an indole-anthranilic acid fused tetracyclic ring rather than the 1,2-benzisoxazole ring by reanalysis of one-dimensional (1D) and two-dimensional (2D) NMR followed by density functional theory (DFT) chemical shift calculation, DP4+ technique, and ECD simulation.

Discovery and Biosynthesis of Imidazolium Antibiotics from the Probiotic Bacillus licheniformis.

Antibiotic resistance is one of the world's most urgent public health problems, and novel antibiotics to kill drug-resistant bacteria are needed. Natural product-derived small molecules have been the major source of new antibiotics. Here we describe a family of antibacterial metabolites isolated from a probiotic bacterium, Bacillus licheniformis. A cross-streaking assay followed by activity-guided isolation yielded a novel antibacterial metabolite, bacillimidazole G, which possesses a rare imidazolium ring in the structure, showing MIC values of 0.7-2.6 µg/mL against human pathogenic Gram-positive and Gram-negative bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and a lipopolysaccharide (LPS)-lacking Acinetobacter baumannii ΔlpxC. Bacillimidazole G also lowered MICs of colistin, a Gram-negative antibiotic, up to 8-fold against wild-type Escherichia coli MG1655 and A. baumannii. We propose a biosynthetic pathway to the characterized metabolites based on precursor-feeding studies, a chemical biological approach, biomimetic total synthesis, and a biosynthetic gene knockout method.

Anti-Inflammatory, Neurotrophic, and Cytotoxic Oxylipins Isolated from Chaenomeles sinensis Twigs.

Oxylipins are important biological molecules with diverse roles in human and plants such as pro-/anti-inflammatory, antimicrobial, and regulatory activity. Although there is an increasing number of plant-derived oxylipins, most of their physiological roles in humans remain unclear. Here, we describe the isolation, identification, and biological activities of four new oxylipins, chaenomesters A-D (1-4), along with a known compound (5), obtained from Chaenomeles sinensis twigs. Their chemical structures were determined by spectroscopic (i.e., NMR) and spectrometric (i.e., HRMS) data analysis including 1H NMR-based empirical rules and homonuclear-decoupled 1H NMR experiments. Chaenomester D (4), an omega-3 oxylipin, showed a potent inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells (NO production, 8.46 ± 0.68 µM), neurotrophic activity in C6 cells through the induction of the secretion of nerve growth factor (NGF, 157.7 ± 2.4%), and cytotoxicity in A549 human cancer cell lines (IC50 = 27.4 µM).

Triterpenoids from the leaves of Abies koreana and their biological activities.

Seven undescribed triterpenoids, abikoranes A-G, along with three known triterpenoids were isolated from the leaves of Abies koreana E. H. Wilson. The structures of compounds were elucidated by 1D and 2D NMR, HRMS, ECD, specific rotation, and DP4+ analysis. Abikorane A represents the second example of nor-3,4-seco-17,14-friedo-lanostane triterpenoid. Among the isolates, some compounds showed strong cytotoxic activities against some of four tested cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-116) with the values of IC50 0.89-9.62 µM, inhibited lipopolysaccharide-stimulated nitric oxide production with IC50 values of 11.57-15.16 µM, and exhibited significant nerve growth factor release effect (192.54 ± 12.33%) from C6 glioma cells.

Isolation, structure elucidation, total synthesis, and biosynthesis of dermazolium A, an antibacterial imidazolium metabolite of a vaginal bacterium Dermabacter vaginalis.

Dermabacter vaginalis is a human-derived bacterium isolated from vaginal fluid of a Korean female in 2016. Although several human-related species in Dermabacter genus have been reported there are few studies on their bioactive metabolites. Dermazolium A (1), a rare imidazolium metabolite, was isolated from D. vaginalis along with five known metabolites (2-6) and their chemical structures were determined by NMR, HRMS, and MS/MS data analysis. Feeding experiments using predicted precursors and biomimetic total synthesis of 1 corroborated its structure and led to suggestion of biosynthetic pathway of 1. Antibacterial tests on the isolated compounds showed that 1 is a mild antibacterial agent with MIC values of 41 µg/mL against methicillin-resistant Staphylococcus aureus (MRSA) USA300, Lacticaseibacillus paracasei subsp. paracasei KCTC 3510 and Brevibacterium epidermidis KCTC 3090.

Cellular Stress-Induced Metabolites in Escherichia coli.

Escherichia coli isolates commonly inhabit the human microbiota, yet the majority of E. coli's small-molecule repertoire remains uncharacterized. We previously employed erythromycin-induced translational stress to facilitate the characterization of autoinducer-3 (AI-3) and structurally related pyrazinones derived from "abortive" tRNA synthetase reactions in pathogenic, commensal, and probiotic E. coli isolates. In this study, we explored the "missing" tryptophan-derived pyrazinone reaction and characterized two other families of metabolites that were similarly upregulated under erythromycin stress. Strikingly, the abortive tryptophanyl-tRNA synthetase reaction leads to a tetracyclic indole alkaloid metabolite (1) rather than a pyrazinone. Furthermore, erythromycin induced two naphthoquinone-functionalized metabolites (MK-hCys, 2; and MK-Cys, 3) and four lumazines (7-10). Using genetic and metabolite analyses coupled with biomimetic synthesis, we provide support that the naphthoquinones are derived from 4-dihydroxy-2-naphthoic acid (DHNA), an intermediate in the menaquinone biosynthetic pathway, and the amino acids homocysteine and cysteine. In contrast, the lumazines are dependent on a flavin intermediate and α-ketoacids from the aminotransferases AspC and TyrB. We show that one of the lumazine members (9), an indole-functionalized analogue, possesses antioxidant properties, modulates the anti-inflammatory fate of isolated TH17 cells, and serves as an aryl-hydrocarbon receptor (AhR) agonist. These three systems described here serve to illustrate that new metabolic branches could be more commonly derived from well-established primary metabolic pathways.

Structure Revision of Balsamisides A-D and Establishment of an Empirical Rule for Distinguishing Four Classes of Biflavonoids.

Balsamisides A-D (1-4) are anti-inflammatory and neurotrophic biflavonoidal glycosides originally proposed to possess an epoxide functionality at the C-2/C-3 position. However, there are inconsistencies in their 13C NMR chemical shift values with those of previously reported analogs, indicating that reanalysis of NMR data for structures of 1-4 is necessary. Computational methods aided by the DP4+ probability technique and ECD calculations enabled structural reassignment of 1-4 to have a 2,3-dihydro-3-hydroxyfuran (3-DHF) instead of an epoxide. Additionally, two new biflavonoidal glycosides, balsamisides E and F (14 and 18), possessing a 2,3-dihydro-2-hydroxyfuran (2-DHF) and a 1,4-dioxane ring, respectively, were characterized by conventional NMR and MS data analysis as well as DP4+ and ECD methods. Systematic 13C NMR analysis was performed on the four aforementioned classes of biflavonoids with a 2- or 3-DHF, epoxide, or 1,4-dioxane. As a result, diagnostic 13C NMR chemical shift values of C-2/C-3 for rapid determination of these four biflavonoid classes were formulated, and based on this first empirical rule for (bi)flavonoids eight previously reported ones were structurally revised.

Establishment of the Mouse Model of Social Avoidance Induced by Female-Directed Female Aggression.

Background: Most preclinical research on the effects of stress has been done on male subjects, even though women are more prone than men to experience stress-related problems. Chronic social defeat stress (CSDS) is a rodent model of psychosocial stress. However, this model has been challenged in female mouse studies since neither male nor female resident mice attack intruder females. A female-to-female CSDS model is needed to investigate the physiological and behavioral aspects. Methods: The intruders were either male or female C57BL/6J mice, whereas the residents were male or ovariectomized (OVX) female CD-1 mice. The CD-1 aggressor mice had direct physical contact with the C57BL/6J mice for 10 min before initiating sensory contact with them for 24 h. Jump escape and freezing were evaluated during the social defeat of days 1 and 12. Experimental C57BL/6J mice underwent a social interaction test after suffering social defeat for 12 days. Results: We found that the number of attack bites and attack latency had a significant negative correlation during the selection of aggressors. In the single-housed OVX mice, 34% of mice met the criterion of the selection of aggressors. However, single-housed OVX mice did not show sustained aggressive behavior (eg, attack bites) through the 12-day CSDS. As a result, we did not find susceptible mice during the social interaction test. In contrast, during the selection of aggressors, 42% of OVX mice housed with partners satisfied the criterion and displayed consistently aggressive behavior. CSDS produced susceptible (50%) and resilient (50%) phenotypes during the social interaction test. Notably, male and OVX female CD-1 mice housed with partners had similar amounts of attack bites and attack rates over the 12-day CSDS. Finally, we found that chronically socially defeated male and female mice displayed different coping behaviors (eg, active vs passive) with social defeat. Conclusions: Our study demonstrates that OVX CD-1 mice housed with mates exhibited territorial aggression toward female intruders, producing susceptibility and resilience to social avoidance. Additionally, socially defeated male and female mice displayed different behavioral susceptibility to social defeat.

Structures and antiosteoclastogenic activity of compounds isolated from edible lotus (Nelumbo nucifera Gaertn.) leaves and stems.

One new 1,4-bis-phenyl-1,4-butanedione glycoside (14), one new eudesmane-type sesquiterpenoid (16), and 16 known compounds were isolated from the leaves and stems of Nelumbo nucifera Gaertn. The structures of the isolated compounds were elucidated by interpretation of their 1D and 2D NMR spectroscopic and HRESIMS data. Time-dependent density functional theory calculations and Electronic Circular Dichroism (ECD) spectroscopy was used to determine absolute configurations of the new eudesmane-type sesquiterpenoid (16). All the isolated compounds were examined for their antiosteoclastogenic activity. Preliminarily results of the TRAP staining on RAW 264.7 cells indicated that compounds 1 and 11 possess potential inhibitory effects on RANKL-induced osteoclast formation. Further bioassay investigation was carried out to reveal that compounds 1 and 11 suppressed RANKL-induced osteoclast formation in a concentration-dependent manner with the inhibition up to 55% and 78% at the concentration of 10 µM, respectively. In addition, the structure-activity relationship analysis showed that the 1,3-dioxole substitute and the double bond at C-6a/C-7 in the aporphine skeleton may be responsible for the antiosteoclastogenic activity. The findings provided valuable insights for the discovery and structural modification of aporphine alkaloids as the antiosteoclastogenic lead compounds.

Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons.

While chronic stress increases hyperpolarization-activated current (Ih) in dorsal hippocampal CA1 neurons, the underlying molecular mechanisms are entirely unknown. Following chronic social defeat stress (CSDS), susceptible mice displayed social avoidance and impaired spatial working memory, which were linked to decreased neuronal excitability, increased perisomatic hyperpolarization-activated cyclic nucleotide-gated (HCN) 1 protein expression, and elevated Ih in dorsal but not ventral CA1 neurons. In control mice, bath application of corticosterone reduced neuronal excitability, increased tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and HCN1 protein expression, and elevated Ih in dorsal but not ventral CA1 region/neurons. Corticosterone-induced upregulation of functional Ih was mediated by the glucocorticoid receptor (GR), HCN channels, and the protein kinase A (PKA) but not the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Three months after the end of CSDS, susceptible mice displayed persistent social avoidance when exposed to a novel aggressor. The sustained behavioral deficit was associated with lower neuronal excitability and higher functional Ih in dorsal CA1 neurons, both of which were unaffected by corticosterone treatment. Our findings show that corticosterone treatment mimics the pathophysiological effects of dorsal CA1 neurons/region found in susceptible mice. The aberrant expression of HCN1 protein along the somatodendritic axis of the dorsal hippocampal CA1 region might be the molecular mechanism driving susceptibility to social avoidance.

Terpenoids from Glechoma hederacea var. longituba and their biological activities.

Glechoma hederacea var. longituba (common name: ground ivy) has been used for the treatment of asthma, bronchitis, cholelithiasis, colds, and inflammation. In the present study, three new sesquiterpene glycosides (1-3), two new diterpene glycosides (4 and 5), and four known compounds (6-9) were isolated from its MeOH extract. A structure elucidation was performed for the five new compounds (1-5) using 1D and 2D NMR, HRESIMS, DP4+ and ECD calculations, and chemical methods. All the isolates (1-9) were assessed for their antineuroinflammatory activities on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells, nerve growth factor (NGF) secretion stimulation activities in C6 glioma cells, and cytotoxic activities against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15). Compounds 2 and 5-7 exhibited inhibitory effects on the NO production with IC50 values of 52.21, 47.90, 61.61, and 25.35 µM, respectively. Compound 5 also exhibited a significant stimulating effect on NGF secretion (122.77 ± 8.10%). Compound 9 showed potent cytotoxic activity against SK-OV-3 (IC50 = 3.76 µM) and SK-MEL-2 (IC50 = 1.48 µM) cell lines, while 7 displayed a strong cytotoxic activity against the SK-MEL-2 (IC50 = 9.81 µM) cell line.

Procyanidins and Phlobatannins from the Twigs of Rosa multiflora and Their Neuroprotective Activity.

Three new procyanidins (1-3), two new phlobatannins (6 and 7), a new flavan-3,4-diol glycoside (9), and a new neolignan glycoside (10), along with three previously reported compounds (4, 5, and 8) were isolated from the twigs of Rosa multiflora. The chemical structures of the new compounds (1-3, 6, 7, 9, and 10) were characterized by spectroscopic data interpretation, including NMR (1H and 13C NMR, 1H-1H COSY, HSQC, HMBC, and NOESY) and HRESIMS analysis. Experimental ECD data analysis was conducted to assign the absolute configurations of the new compounds (1-3, 6, 7, 9, and 10). The absolute configuration of the sugar moieties was verified through a chiral derivatization method and LC-MS analysis. All the isolated compounds (1-10) were evaluated for their anti-neuroinflammatory activity based on inhibitory effects on nitric oxide production using a lipopolysaccharide-stimulated murine microglia BV-2 cell line and for their neurotrophic effects on nerve growth factor induction in C6 glioma.

Anticancer and Anti-Neuroinflammatory Constituents Isolated from the Roots of Wasabia japonica.

Wasabi (Wasabia japonica (Miq.) Matsum.) is a pungent spice commonly consumed with sushi and sashimi. From the roots of this plant, a new 2-butenolide derivative (1) and 17 previously reported compounds (2-18) were isolated and structurally characterized. Their chemical structures were characterized based on the conventional NMR (1H and 13C, COSY, HSQC, and HMBC) and HRESIMS data analysis. All of these phytochemicals (1-18) were evaluated for their antiproliferative effects on the four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and MKN-1), for their inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 microglia cells, and for their nerve growth factor (NGF)-releasing effect from C6 glioma cells. Among the isolated compounds, compound 15 showed powerful antiproliferative activities against A549 and SK-MEL-2 cell lines with IC50 values of 2.10 and 9.08 µM, respectively. Moreover, the new compound 1 exhibited moderate NO inhibition activity with IC50 value of 45.3 µM.

PTP1B and α-glucosidase inhibitory activities of the chemical constituents from Hedera rhombea fruits: Kinetic analysis and molecular docking simulation.

In this study, we present the first investigation of Hedera rhombea Bean fruit, which led to the isolation of six undescribed compounds including two megastigmane glucosides, two rare 1,4-dioxane neolignanes, and two quinic acid derivatives, together with 26 known compounds. Their structures and absolute configurations were elucidated by extensive analysis of NMR spectroscopic data, HRMS, and ECD calculations. This is the first report on the isolation of methyl 3-O-caffeoyl-5-O-p-coumaroylquinate from a natural source. Among the isolated compounds, falcarindiol and caffeoyltryptophan showed significant PTP1B inhibition with IC50 values of 7.32 and 16.99 µM, respectively, compared to those of the positive controls [sodium orthovanadate (IC50 = 17.96 µM) and ursolic acid (IC50 = 4.53 µM)]. These two compounds along with several other compounds displayed significant α-glucosidase inhibitions with IC50 values ranging from 12.88 to 91.89 µM, stronger than that of the positive control (acarbose, IC50 = 298.07 µM). Enzyme kinetic analysis indicated that caffeoyltryptophan and falcarindiol displayed competitive and mixed-type PTP1B inhibition, respectively, whereas the α-glucosidase inhibition type was mixed-type for caffeoyltryptophan and uncompetitive (rarely reported for a-glucosidase inhibitors) for falcarindiol. In addition, molecular docking results showed that these active compounds exhibited good binding affinities toward both PTP1B and α-glucosidase with negative binding energies. The results of the present study demonstrate that these active compounds might be beneficial in the treatment of type 2 diabetes.

Phenolic constituents from twigs of Aleurites fordii and their biological activities.

Three new neolignan glycosides (1-3), a new phenolic glycoside (15), and a new cyanoglycoside (16) were isolated and characterized from the twigs of Aleurites fordii together with 14 known analogues (4-14 and 17-19). The structural elucidation of the new compounds was performed through the analysis of their NMR, HRMS, and ECD spectra and by chemical methods. All isolated compounds were tested for their antineuroinflammatory and neuroprotective activities.