Hirocidins, Cytotoxic Metabolites from Streptomyces hiroshimensis, Induce Mitochondrion-mediated Apoptosis.

Recent advances in whole genome sequencing have revealed an immense microbial potential for the production of therapeutic small molecules, even from well-known producers. To access this potential, we subjected prominent antimicrobial producers to alternative antiproliferative assays using persistent cancer cell lines. Described herein is our discovery of hirocidins, novel secondary metabolites from Streptomyces hiroshimensis with antiproliferative activities against colon and persistent breast cancer cells. Hirocidin A is an unusual nine-membered carbocyclic maleimide and hirocidins B and C are relatives with an unprecedented, bridged azamacrocyclic backbone. Mode of action studies show that hirocidins trigger mitochondrion-dependent apoptosis by inducing expression of the key apoptotic effector caspase-9. The discovery of new cytotoxins contributes to scaffold diversification in anticancer drug discovery and the reported modes of action and concise total synthetic route for variant A set the stage for unraveling specific targets and biochemical interactions of the hirocidins.

Regulatory role of Echinochrome A in cancer-associated fibroblast-mediated lung cancer cell migration.

Echinochrome A (Ech A), a marine biosubstance isolated from sea urchins, is a strong antioxidant, and its clinical form, histochrome, is being used to treat several diseases, such as ophthalmic, cardiovascular, and metabolic diseases. Cancer-associated fibroblasts (CAFs) are a component of the tumor stroma and induce phenotypes related to tumor malignancy, including epithelial-mesenchymal transition (EMT) and cancer stemness, through reciprocal interactions with cancer cells. Here, we investigated whether Ech A modulates the properties of CAFs and alleviates CAF-induced lung cancer cell migration. First, we observed that the expression levels of CAF markers, Vimentin and fibroblast-activating protein (FAP), were decreased in Ech A-treated CAF-like MRC5 cells. The mRNA transcriptome analysis revealed that in MRC5 cells, the expression of genes associated with cell migration was largely modulated after Ech A treatment. In particular, the expression and secretion of cytokine and chemokine, such as IL6 and CCL2, stimulating cancer cell metastasis was reduced through the inactivation of STAT3 and Akt in MRC5 cells treated with Ech A compared to untreated MRC5 cells. Moreover, while conditioned medium from MRC5 cells enhanced the migration of non-small cell lung cancer cells, conditioned medium from MRC5 cells treated with Ech A suppressed cancer cell migration. In conclusion, we suggest that Ech A might be a potent adjuvant that increases the efficacy of cancer treatments to mitigate lung cancer progression.

Molecular networking and computational NMR analyses uncover six polyketide-terpene hybrids from termite-associated Xylaria isolates.

Fungi constitute the Earth's second most diverse kingdom, however only a small percentage of these have been thoroughly examined and categorized for their secondary metabolites, which still limits our understanding of the ecological chemical and pharmacological potential of fungi. In this study, we explored members of the co-evolved termite-associated fungal genus Xylaria and identified a family of highly oxygenated polyketide-terpene hybrid natural products using an MS/MS molecular networking-based dereplication approach. Overall, we isolated six no yet reported xylasporin derivatives, of which xylasporin A (1) features a rare cyclic-carbonate moiety. Extensive comparative spectrometric (HRMS2) and spectroscopic (1D and 2D NMR) studies allowed to determine the relative configuration across the xylasporin family, which was supported by chemical shift calculations of more than 50 stereoisomers and DP4+ probability analyses. The absolute configuration of xylasporin A (1) was also proposed based on TDDFT-ECD calculations. Additionally, we were able to revise the relative and absolute configurations of co-secreted xylacremolide B produced by single x-ray crystallography. Comparative genomic and transcriptomic analysis allowed us to deduce the putative biosynthetic assembly line of xylasporins in the producer strain X802, and could guide future engineering efforts of the biosynthetic pathway.

AM-18002, a derivative of natural anmindenol A, enhances radiosensitivity in mouse breast cancer cells.

Natural anmindenol A isolated from the marine-derived bacteria Streptomyces sp. caused potent inhibition of inducible nitric oxide synthase without any significant cytotoxicity. This compound consists of a structurally unique 3,10-dialkylbenzofulvene skeleton. We previously synthesized and screened the novel derivatives of anmindenol A and identified AM-18002, an anmindenol A derivative, as a promising anticancer agent. The combination of AM-18002 and ionizing radiation (IR) improved anticancer effects, which were exerted by promoting apoptosis and inhibiting the proliferation of FM3A mouse breast cancer cells. AM-18002 increased the production of reactive oxygen species (ROS) and was more effective in inducing DNA damage. AM-18002 treatment was found to inhibit the expansion of myeloid-derived suppressor cells (MDSC), cancer cell migration and invasion, and STAT3 phosphorylation. The AM-18002 and IR combination synergistically induced cancer cell death, and AM-18002 acted as a potent anticancer agent by increasing ROS generation and blocking MDSC-mediated STAT3 activation in breast cancer cells.

Determination of the Absolute Configuration of Secondary Alcohols in a Compound Mixture via the Application of Competing Enantioselective Acylation Coupled with LC/MS Analysis.

The determination of natural product stereochemistry plays a significant role in drug discovery and development. Understanding the stereochemistry of natural products is essential for predicting and optimizing their interactions with biological targets, which, in turn, influences their therapeutic efficacy, safety, and overall impact on living organisms. Here, we present the first application of competitive enantioselective acylation (CEA) reactions in conjunction with LC/MS analysis for determining the absolute configuration of secondary alcohols in natural products which were purified as a mixture. This approach utilizes the enantiomeric pair of HBTM (homobenzotetramisole) catalysts, demonstrating sufficient kinetic resolution for the acylation of secondary alcohols. The rapid reaction kinetics were quantitatively estimated with LC/MS analysis as the characterization technique for the enantioselective transformations. Our study has expanded the application of the CEA reaction coupled with LC/MS analysis to mixtures. Utilizing LC/MS analysis, the CEA reaction offers a sensitive and simple method for stereochemistry determination. Additionally, the application of the CEA reaction is cost/time-effective since only small quantities of substrates and a short reaction time are required for characterizing the absolute configuration of secondary alcohols in natural products compared to other conventional methods.

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.

Targeted Discovery of Cryptic Enediyne Natural Products via FRET-Coupled High-Throughput Elicitor Screening.

Enediyne antibiotics are a striking family of DNA-cleaving natural products with high degrees of cytotoxicity and structural complexity. Microbial genome sequences, which have recently accumulated, point to an untapped trove of "cryptic" enediynes. Most of the cognate biosynthetic gene clusters (BGCs) are sparingly expressed under standard growth conditions, making it difficult to characterize their products. Herein, we report a fluorescence-based DNA cleavage assay coupled with high-throughput elicitor screening for the rapid, targeted discovery of cryptic enediyne metabolites. We applied the approach to Streptomyces clavuligerus, which harbors two such BGCs with unknown products, identified steroids as effective elicitors, and characterized 10 cryptic enediyne-derived natural products, termed clavulynes A-J with unusual carbonate and terminal olefin functionalities, with one of these congeners matching the recently reported jejucarboside. Our results contribute to the growing repertoire of enediynes and provide a blueprint for identifying additional ones in the future.

Estrogenic Activity of 4-Hydroxy-Benzoic Acid from Acer tegmentosum via Estrogen Receptor α-Dependent Signaling Pathways.

Acer tegmentosum, a deciduous tree belonging to Aceraceae, has been used in traditional oriental medicine for treating hepatic disorders, such as hepatitis, cirrhosis, and liver cancer. We evaluated the estrogen-like effects of A. tegmentosum using an estrogen receptor (ER)-positive breast cancer cell line, namely MCF-7, to identify potential phytoestrogens and found that an aqueous extract of A. tegmentosum promoted cell proliferation in MCF-7 cells. Five phenolic compounds (1-5) were separated and identified from the active fraction using bioassay-guided fractionation of crude A. tegmentosum extract and phytochemical analysis. The chemical structures of the compounds were characterized as vanillic acid (1), 4-hydroxy-benzoic acid (2), syringic acid (3), isoscopoletin (4), and (E)-ferulic acid (5) based on the analysis of their nuclear magnetic resonance spectra and liquid chromatography-mass spectrometry data. All five compounds were evaluated using an E-screen assay for their estrogen-like effects on MCF-7 cells. Among the tested compounds, only 4-hydroxy-benzoic acid (2) promoted the proliferation of MCF-7 cells, which was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of 4-hydroxy-benzoic acid (2) was evaluated via western blotting analysis to determine the expression levels of extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), serine/threonine kinase (AKT), and ERα. Our results demonstrated that 4-hydroxy-benzoic acid (2) induced the increase in the protein expression levels of p-ERK, p-AKT, p-PI3K, and p-Erα, concentration dependently. Collectively, these experimental results suggest that 4-hydroxy-benzoic acid (2) is responsible for the estrogen-like effects of A. tegmentosum and may potentially aid in the control of estrogenic effects during menopause.

Targeted Discovery of Cryptic Metabolites with Antiproliferative Activity.

Microorganisms have provided a rich source of therapeutically valuable natural products. Recent advances in whole genome sequencing and bioinformatics have revealed immense untapped potential for new natural products in the form of silent or "cryptic" biosynthetic genes. We herein conducted high-throughput elicitor screening (HiTES) in conjunction with cytotoxicity assays against selected cancer cell lines with the goal of uncovering otherwise undetectable cryptic metabolites with antiproliferative activity. Application to Streptomyces clavuligerus facilitated identification of clavamates A and B, two bioactive metabolites with unusual structural features, as well as facile activation of a gene cluster coding for tunicamycin, which exhibited strong growth-inhibitory activity. The elicitor we identified was pleiotropic, additionally leading to the discovery of a modified, bicyclic pentapeptide natural product. Our results highlight the utility of this approach in identifying new molecules with antiproliferative activity from even overexploited microbial strains.

Antitumor Potential of Withanolide Glycosides from Ashwagandha (Withania somnifera) on Apoptosis of Human Hepatocellular Carcinoma Cells and Tube Formation in Human Umbilical Vein Endothelial Cells.

Hepatocellular carcinoma (HCC) is the fastest-growing tumor capable of spreading to other organs via blood vessels formed by endothelial cells. Apoptosis and angiogenesis-targeting therapies are attractive for cancer treatment. In this study, we aimed to study the in vitro cytotoxicity of Withania somnifera against human HCC (HepG2) cells, identify potential antitumoral withanolide glycosides from the active fraction, and elucidate cytotoxic molecular mechanisms of identified bioactive compounds. W. somnifera (Solanaceae), well-known as 'ashwagandha', is an Ayurvedic medicinal plant used to promote health and longevity, and the MeOH extract of W. somnifera root exhibited cytotoxicity against HepG2 cells during initial screening. Bioactivity-guided fractionation of the MeOH extract and subsequent phytochemical investigation of the active n-BuOH-soluble fraction resulted in the isolation of five withanolide glycosides (1-5), including one new metabolite, withanoside XIII (1), aided by liquid chromatography-mass spectrometry-based analysis. The new compound structure was determined by 1D and 2D nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectroscopy, electronic circular dichroism, and enzymatic hydrolysis. In addition, withanoside XIIIa (1a) was identified as the new aglycone (1a) of 1. Isolated withanolide glycosides 1-5 and 1a were cytotoxic toward HepG2 cells; withagenin A diglucoside (WAD) (3) exhibited the most potent cytotoxicity against HepG2 cells, with cell viability less than 50% at 100 µM. WAD cytotoxicity was mediated by both extrinsic and intrinsic apoptosis pathways. Treatment with WAD increased protein expression levels of cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, Bcl-2-associated X protein (Bax), and cleaved poly(ADP-ribose) polymerase (cleaved PARP) but decreased expression levels of B-cell lymphoma 2 (Bcl-2). Moreover, WAD inhibited tubular structure formation in human umbilical vein endothelial cells (HUVECs) by inhibiting the protein expression of vascular endothelial growth factor receptor 2 and its downstream pathways, including extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). These effects were also enhanced by co-treatment with ERK and PI3K inhibitors. Overall, these results indicate that WAD (3) induced HepG2 apoptosis and inhibited HUVEC tube formation, suggesting its potential application in treating liver cancers.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus.

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.

Identification of anti-adipogenic withanolides from the roots of Indian ginseng (Withania somnifera).

Background: Withania somnifera (Solanaceae), generally known as Indian ginseng, is a medicinal plant that is used in Ayurvedic practice for promoting health and longevity. This study aims to identify the bioactive metabolites from Indian ginseng and elucidate their structures. Methods: Withanolides were purified by chromatographic techniques, including HPLC coupled with LC/MS. Chemical structures of isolated withanolides were clarified by analyzing the spectroscopic data from 1D and 2D NMR, and HR-ESIMS experiment. Absolute configurations of the withanolides were established by the application of NMR chemical shifts and ECD calculations. Anti-adipogenic activities of isolates were evaluated using 3T3-L1 preadipocytes with Oil Red O staining and quantitative real-time PCR (qPCR). Results: Phytochemical examination of the roots of Indian ginseng afforded to the isolation of six withanolides (1-6), including three novel withanolides, withasilolides G-I (1-3). All the six compounds inhibited adipogenesis and suppressed the enlargement of lipid droplets, compared to those of the control. Additionally, the mRNA expression levels of Fabp4 and Adipsin, the adipocyte markers decreased noticeably following treatment with 25 µM of 1-6. The active compounds (1-6) also promoted lipid metabolism by upregulating the expression of the lipolytic genes HSL and ATGL and downregulating the expression of the lipogenic gene SREBP1. Conclusion: The results of our experimental studies suggest that the withasilolides identified herein have anti-adipogenic potential and can be considered for the development of therapeutic strategies against adipogenesis in obesity. Our study also provides a mechanistic rationale for using Indian ginseng as a potential therapeutic agent against obesity and related metabolic diseases.

Induction of Diverse Cryptic Fungal Metabolites by Steroids and Channel Blockers.

Fungi offer a deep source of natural products but remain underutilized. Most biosynthetic gene clusters (BGCs) that can be detected are silent or "cryptic" in standard lab cultures and their products are thus not interrogated in routine screens. As genetic alterations are difficult and some strains can only be grown on agar, we have herein applied an agar-based high-throughput chemical genetic screen to identify inducers of fungal BGCs. Using R. solani and S. sclerotiorum as test cases, we report 13 cryptic metabolites in four compound groups, including sclerocyclane, a natural product with a novel scaffold. Steroids were the best elicitors and follow-up studies showed that plant-steroids trigger sclerocyclane synthesis, which shows antibiotic activity against B. plantarii, an ecological competitor of S. sclerotiorum. Our results open new paths to exploring the chemical ecology of fungal-plant interactions and provide a genetics-free approach for uncovering cryptic fungal metabolites.

GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.

Structural Elucidation of Cryptic Algaecides in Marine Algal-Bacterial Symbioses by NMR Spectroscopy and MicroED.

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal-bacterial symbiotic interactions.

Non-peptide secondary metabolites from poisonous mushrooms: overview of chemistry, bioactivity, and biosynthesis.

Covering: up to June 2021A wide variety of mushrooms have traditionally been recognized as edible fungi with high nutritional value and low calories, and abundantly produce structurally diverse and bioactive secondary metabolites. However, accidental ingestion of poisonous mushrooms can result in serious illnesses and even death. Chemically, mushroom poisoning is associated with secondary metabolites produced in poisonous mushrooms, causing specific toxicity. However, many poisonous mushrooms have not been fully investigated for their secondary metabolites, and the secondary metabolites of poisonous mushrooms have not been systematically summarized for details such as chemical composition and biosynthetic mechanisms. The isolation and identification of secondary metabolites from poisonous mushrooms have great research value since these compounds could be lethal toxins that contribute to the toxicity of mushrooms or could provide lead compounds with remarkable biological activities that can promote advances in other related disciplines, such as biochemistry and pharmacology. In this review, we summarize the structures and biological activities of secondary metabolites identified from poisonous mushrooms and provide an overview of the current information on these metabolites, focusing on their chemistry, bioactivity, and biosynthesis.

Identification of Renoprotective Phytosterols from Mulberry (Morus alba) Fruit against Cisplatin-Induced Cytotoxicity in LLC-PK1 Kidney Cells.

The aim of this study was to explore the protective effects of bioactive compounds from the fruit of the mulberry tree (Morus alba L.) against cisplatin-induced apoptosis in LLC-PK1 pig kidney epithelial cells. Morus alba fruit is a well-known edible fruit commonly used in traditional folk medicine. Chemical investigation of M. alba fruit resulted in the isolation and identification of six phytosterols (1-6). Their structures were determined as 7-ketositosterol (1), stigmast-4-en-3ß-ol-6-one (2), (3ß,6α)-stigmast-4-ene-3,6-diol (3), stigmast-4-ene-3ß,6ß-diol (4), 7ß-hydroxysitosterol 3-O-ß-d-glucoside (5), and 7α-hydroxysitosterol 3-O-ß-d-glucoside (6) by analyzing their physical and spectroscopic data as well as liquid chromatography/mass spectrometry data. All compounds displayed protective effects against cisplatin-induced LLC-PK1 cell damage, improving cisplatin-induced cytotoxicity to more than 80% of the control value. Compound 1 displayed the best effect at a relatively low concentration by inhibiting the percentage of apoptotic cells following cisplatin treatment. Its molecular mechanisms were identified using Western blot assays. Treatment of LLC-PK1 cells with compound 1 decreased the upregulated phosphorylation of p38 and c-Jun N-terminal kinase (JNK) following cisplatin treatment. In addition, compound 1 significantly suppressed cleaved caspase-3 in cisplatin-induced LLC-PK1 cells. Taken together, these findings indicated that cisplatin-induced apoptosis was significantly inhibited by compound 1 in LLC-PK1 cells, thereby supporting the potential of 7-ketositosterol (1) as an adjuvant candidate for treating cisplatin-induced nephrotoxicity.

Withasomniferol D, a New Anti-Adipogenic Withanolide from the Roots of Ashwagandha (Withania somnifera).

Withania somnifera (Solanaceae), well-known as 'Indian ginseng' or 'Ashwagandha', is a medicinal plant that is used in Ayurvedic practice to promote good health and longevity. As part of an ongoing investigation for bioactive natural products with novel structures, we performed a phytochemical examination of the roots of W. somnifera employed with liquid chromatography-mass spectrometry (LC/MS)-based analysis. The chemical analysis of the methanol extract of W. somnifera roots using repeated column chromatography and high-performance liquid chromatography under the guidance of an LC/MS-based analysis resulted in a new withanolide, withasomniferol D (1). The structure of the newly isolated compound was elucidated by spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high-resolution (HR) electrospray ionization (ESI) mass spectroscopy, and its absolute configuration was established by electronic circular dichroism (ECD) calculations. The anti-adipogenic activities of withasomniferol D (1) were evaluated using 3T3-L1 preadipocytes with Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR). We found that withasomniferol D (1) inhibited adipogenesis and suppressed the enlargement of lipid droplets compared to the control. Additionally, the mRNA expression levels of adipocyte markers Fabp4 and Adipsin decreased noticeably following treatment with 25 µM of withasomniferol D (1). Taken together, these findings provide experimental evidence that withasomniferol D (1), isolated from W. somnifera, exhibits anti-adipogenic activity, supporting the potential application of this compound in the treatment of obesity and related metabolic diseases.

Bioactive Phytochemicals from Mulberry: Potential Anti-Inflammatory Effects in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

The fruits of the mulberry tree (Morus alba L.), known as white mulberry, have been consumed in various forms, including tea, beverages, and desserts, worldwide. As part of an ongoing study to discover bioactive compounds from M. alba fruits, the anti-inflammatory effect of compounds from M. alba were evaluated in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Phytochemical analysis of the ethanol extract of the M. alba fruits led to the isolation of 22 compounds. Among the isolated compounds, to the best of our knowledge, compounds 1, 3, 5, 7, 11, 12, and 14-22 were identified from M. alba fruits for the first time in this study. Inhibitory effects of 22 compounds on the production of the nitric oxide (NO) known as a proinflammatory mediator in LPS-stimulated RAW 264.7 macrophages were evaluated using NO assays. Western blot analysis was performed to evaluate the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5). We evaluated whether the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5) following LPS stimulation in RAW 264.7 macrophages occurred because of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKß), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB) and activations of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Cyclo(L-Pro-L-Val) (5) significantly suppressed phosphorylations of IKKα, IKKß, IκBα, and NF-κB and activations of iNOS and COX-2 in a concentration-dependent manner. Taken together, these results indicate that cyclo(L-Pro-L-Val) (5) can be considered a potential therapeutic agent for the treatment of inflammation-associated disorders.

Antioxidant and Anti-Inflammatory Effects of White Mulberry (Morus alba L.) Fruits on Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

In this study, the protective effects of white mulberry (Morus alba) fruits on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were investigated. The ethanol (EtOH) extract of white mulberry fruits and its derived fractions contained adequate total phenolic and flavonoid contents, with good in vitro antioxidant radical scavenging activity. The extract and fractions also markedly inhibited ROS generation and antioxidant activity. After treatment with the EtOH extract and its fractions, LPS stimulation-induced elevated nitric oxide (NO) production was restored, which was primarily mediated by downregulation of inducible NO synthase expression. A total of 20 chemical constituents including flavonoids, steroids, and phenolics were identified in the fractions using ultra-high-performance liquid chromatography (UHPLC)-quadrupole time-of-flight (QTOF) high-resolution mass spectrometry (HRMS). These findings provide experimental evidence of the protective effects of white mulberry fruit extract against oxidative stress and inflammatory responses, suggesting their nutraceutical and pharmaceutical potential as natural antioxidant and anti-inflammatory agents.