Protective Effects of Withagenin A Diglucoside from Indian Ginseng (Withania somnifera) against Human Dermal Fibroblast Damaged by TNF-α Stimulation.

Human skin is constructed with many proteins such as collagen and elastin. Collagen and elastin play a key role in providing strength and elasticity to the human skin and body. However, damage to collagen causes various symptoms such as wrinkles and freckles, which suggests that they are important to maintain skin condition. Extrinsic or intrinsic skin aging produces an excess of skin destructive factors such as tumor necrosis factor (TNF)-α, which is a major mediator of the aging process. In aged skin, TNF-α provokes the generation of intracellular ROS (reactive oxygen species). It triggers the excessive secretion of MMP-1, which is a collagen-degrading enzyme that causes the collapse of skin collagen. Therefore, we aimed to search for a natural-product-derived candidate that inhibits the skin damage caused by TNF-α in human dermal fibroblasts. In this study, the protective effect of withagenin A diglucoside (WAD) identified from Withania somnifera against TNF-α-stimulated human dermal fibroblasts is investigated. W. somnifera (Solanaceae), well-known as 'ashwagandha', is an Ayurvedic medicinal plant useful for promoting health and longevity. Our experimental results reveal that WAD from W. somnifera suppresses the generation of intercellular ROS. Suppressing intracellular ROS generation inhibits MMP-1 secretion and the collapse of type 1 collagen. The effect of WAD is shown to depend on the inhibition of MAPK phosphorylation, Akt phosphorylation, c-Jun phosphorylation, COX-2 expression, and NF-κB phosphorylation. Further, WAD-depressed expression of the pro-inflammatory cytokines IL-6 and IL-8 triggers various inflammatory reactions in human skin. These findings suggest that WAD has protective effects against skin damage. Accordingly, our study provides experimental evidence that WAD can be a potential agent that can be applied in various industrial fields, such as cosmetics and pharmaceuticals related to skin aging.

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.

Selenium Nanoparticles as Candidates for Antibacterial Substitutes and Supplements against Multidrug-Resistant Bacteria.

In recent years, multidrug-resistant (MDR) bacteria have increased rapidly, representing a major threat to human health. This problem has created an urgent need to identify alternatives for the treatment of MDR bacteria. The aim of this study was to identify the antibacterial activity of selenium nanoparticles (SeNPs) and selenium nanowires (SeNWs) against MDR bacteria and assess the potential synergistic effects when combined with a conventional antibiotic (linezolid). SeNPs and SeNWs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), zeta potential, and UV-visible analysis. The antibacterial effects of SeNPs and SeNWs were confirmed by the macro-dilution minimum inhibitory concentration (MIC) test. SeNPs showed MIC values against methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant enterococci (VRE) at concentrations of 20, 80, 320, and >320 µg/mL, respectively. On the other hand, SeNWs showed a MIC value of >320 µg/mL against all tested bacteria. Therefore, MSSA, MRSA, and VRSA were selected for the bacteria to be tested, and SeNPs were selected as the antimicrobial agent for the following experiments. In the time-kill assay, SeNPs at a concentration of 4X MIC (80 and 320 µg/mL) showed bactericidal effects against MSSA and MRSA, respectively. At a concentration of 2X MIC (40 and 160 µg/mL), SeNPs showed bacteriostatic effects against MSSA and bactericidal effects against MRSA, respectively. In the synergy test, SeNPs showed a synergistic effect with linezolid (LZD) through protein degradation against MSSA and MRSA. In conclusion, these results suggest that SeNPs can be candidates for antibacterial substitutes and supplements against MDR bacteria for topical use, such as dressings. However, for use in clinical situations, additional experiments such as toxicity and synergistic mechanism tests of SeNPs are needed.

Phytochemical Analysis of the Fruits of Sea Buckthorn (Hippophae rhamnoides): Identification of Organic Acid Derivatives.

Hippophae rhamnoides L. (Elaeagnaceae), commonly known as "Sea buckthorn" and "Vitamin tree", is a spiny deciduous shrub whose fruit is known for its nutritional composition, such as vitamin C, and is consumed as a dietary supplement worldwide. As part of our ongoing efforts to identify structurally new and bioactive constituents from natural resources, the phytochemical investigation of the extract of H. rhamnoides fruits led to the isolation of one malate derivative (1), five citrate derivatives (2-6), and one quinate derivative (7). The structures of the isolated compounds were elucidated by analysis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data and high-resolution electrospray ionization (HR-ESI) liquid chromatography-mass spectrometry (LC/MS) data. Three of the citrate derivatives were identified as new compounds: (S)-1-butyl-5-methyl citrate (3), (S)-1-butyl-1'-methyl citrate (4), and (S)-1-methyl-1'-butyl citrate (6), which turned out to be isolation artifacts. The absolute configurations of the new compounds were established by quantum chemical electronic circular dichroism (ECD) calculation, which is an informative tool for verifying the absolute configuration of organic acid derivatives. The isolated compounds 1-7 were evaluated for their stimulatory effects on osteogenesis. Compounds 1, 3, 4, 6, and 7 stimulated osteogenic differentiation up to 1.4 fold, compared to the negative control. These findings provide experimental evidence that active compounds 1, 3, 4, 6, and 7 induce the osteogenesis of mesenchymal stem cells and activate bone formation.

Identification of Bioactive Natural Product from the Stems and Stem Barks of Cornus walteri: Benzyl Salicylate Shows Potential Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

Cornus walteri (Cornaceae), known as Walter's dogwood, has been used to treat dermatologic inflammation and diarrheal disease in traditional oriental medicine. As part of an ongoing research project to discover natural products with biological activities, the anti-inflammatory potential of compounds from C. walteri in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages were explored. Phytochemical analysis of the methanol extract of the stem and stem bark of C. walteri led to the isolation of 15 chemical constituents. These compounds were evaluated for their inhibitory effects on the production of the proinflammatory mediator nitric oxide (NO) in LPS-stimulated macrophages, as measured by NO assays. The molecular mechanisms underlying the anti-inflammatory activity were investigated using western blotting. Our results demonstrated that among 15 chemical constituents, lupeol and benzyl salicylate inhibited NO production in LPS-activated RAW 264.7 macrophages. Benzyl salicylate was more efficient than NG-monomethyl-L-arginine mono-acetate salt (L-NMMA) in terms of its inhibitory effect. In addition, the mechanism of action of benzyl salicylate consisted of the inhibition of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKß), inhibitor of kappa B alpha (IκBα), and nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. Furthermore, benzyl salicylate inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Taken together, these results suggest that benzyl salicylate present in the stem and stem bark of C. walteri has potential anti-inflammatory activity, supporting the potential application of this compound in the treatment of inflammatory diseases.

Ulmusakidian, a new coumarin glycoside and antifungal phenolic compounds from the root bark of Ulmus davidiana var. japonica.

Bioactivity-driven LC/MS-based phytochemical analysis of the root bark extract of Ulmus davidiana var. japonica led to the isolation of 10 compounds including a new coumarin glycoside derivative, ulmusakidian (1). The structure of the new compound was elucidated using extensive spectroscopic analyses via 1D and 2D NMR spectroscopic data interpretations, HR-ESIMS, and chemical transformation. The isolated compounds 1-10 were tested for their antifungal activity against human fungal pathogens Cryptococcus neoformans and Candida albicans. Compounds 9 and 10 showed antifungal activity against C. neoformans, with the lowest minimal inhibitory concentration (MIC) of 12.5-25.0 µg/mL, whereas none of the compounds showed antifungal activity against C. albicans.

Grapefruit Seed Extract as a Natural Derived Antibacterial Substance against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria are increasing due to the abuse and misuse of antibiotics, and nosocomial infections by MDR bacteria are also increasing. The aim of this study was to identify new substances that can target MDR bacteria among 12 plant extracts that are known to have antibacterial effects. The experiments were performed by the disk diffusion test and microdilution minimum inhibitory concentration (MIC) test, as described by the Clinical and Laboratory Standards Institute (CLSI). By screening against methicillin-sensitive Staphylococcus aureus (MSSA), grapefruit seed extract (GSE) was selected from 12 plant extracts for subsequent experiments. GSE showed antibacterial effects against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) in the disk diffusion test. Even at the lowest concentration, GSE showed antibacterial activity in the microdilution MIC test. As a result, we can conclude that GSE is a naturally derived antibacterial substance that exhibits a favorable antibacterial effect even at a very low concentration, so it is a good candidate for a natural substance that can be used to prevent or reduce nosocomial infections as coating for materials used in medical contexts or by mixing a small amount with other materials.

Nanoscale Calcium Salt-Based Formulations As Potential Therapeutics for Osteoporosis.

Osteoporosis causes severe bone damage, posing potential risks to human health, patient quality of life, and society. Calcium has been widely shown to enhance bone density and prevent osteoporosis-related bone fractures. Here, we focused on calcium salt formulations containing natural substances and their possible therapeutic effects on osteoporosis. In particular, we developed a nanoscale calcium salt of natural origin and formulated nanocomposite tablets supplemented with vitamin D (Vit D), herb Rhodiola rosea (R. rosea) and natural mineral Shilajit that are known to be antiosteoporotic. The calcium salt nanocomposites exhibited no toxicity, and particularly the formulation containing R. rosea stimulated osteogenic differentiation. The calcium salt nanocomposites inhibited osteoclastic activity, including RANKL expression, as shown by a decrease in tartrate-resistant acid phosphatase (TRAP)-positive cells. When administered orally to osteoporotic rats for 45 days, the calcium salt nanocomposites reduced bone resorption, as evidenced by the significantly higher bone volume and density, increase in osteoblasts and decrease in osteoclasts compared to those in nontreated control rats. Systemic administration of the nanocomposites caused no severe stomach toxicity or damage over the test period, during which no renal stone growth was observed. On the basis of their significant bilateral effects in stimulating osteoblasts and inhibiting osteoclasts and the resultant efficacy in an osteoporotic model, the nanocomposite tablets composed of a calcium salt and natural products can be considered novel nanotherapeutics for osteoporosis treatment.

Identification of bioactive heterocyclic compounds from mulberry and their protective effect against streptozotocin-induced apoptosis in INS-1 cells.

A phytochemical investigation of the MeOH extracts from mulberries (the fruit of Morus alba L.) led to the identification of six heterocyclic compounds (1­6). These compounds were screened to detect whether they protected pancreatic INS­1 cells from streptozotocin (STZ)­induced cytotoxicity. Compound 3 was the most effective at preventing STZ­induced cytotoxicity and the production of reactive oxygen species (ROS) in INS­1 cells. In addition, compound 3 effectively prevented apoptosis induced by STZ in INS­1 cells. Compound 3 also prevented STZ­mediated cleavage of caspase­8, caspase­3 and poly (ADP­ribose) polymerase and increased the expression of B­cell lymphoma­2 (Bcl­2), an anti­apoptotic Bcl­2 family protein. In conclusion, the results of the present study indicate that compound 3 extracted from the fruit of M. alba was highly effective in preventing type 1 diabetes mellitus and may be a novel treatment option.

Antigastritis effects of Armillariella tabescens (Scop.) Sing. and the identification of its anti-inflammatory metabolites.

OBJECTIVES: This study demonstrates the biological and chemical analysis of the mushroom Armillariella tabescens (Scop.) Sing. (Tricholomataceae). METHODS: Chemical structures of the isolates were determined by 1D and 2D NMR, and ESI-MS, as well as comparison with previously reported data. All isolates were tested for anti-inflammatory effects based on their ability to inhibit LPS-stimulated nitric oxide (NO) production in RAW264.7 cells. KEY FINDINGS: We found that the MeOH extract of the fruiting bodies of A. tabescens showed antigastritis activity against ethanol-induced gastric damage in rats and notably reduced the gastric damage index compared to control in a concentration-dependent manner. Chemical investigation of the MeOH extract led to the isolation of four steroids (1-4), three alkaloids (5-7), two nucleic acids (8-9) and four fatty acids (10-13). This is the first study to report the identification of all isolates, except for compound 7, from A. tabescens. Compounds 1, 2, 3, 4 and 10 showed inhibition on LPS-stimulated NO production. Treatment with compound 10 inhibited expression of iNOS, COX-2, phospho-IKKα, IKKα, phospho-IκBα, IκBα and NF-kappa B in LPS-stimulated RAW264.7 cells. CONCLUSIONS: Compound 10 likely contributes to the health benefits of A. tabescens as an antigastritis agent through its anti-inflammatory effects.

Curcuzedoalide contributes to the cytotoxicity of Curcuma zedoaria rhizomes against human gastric cancer AGS cells through induction of apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma zedoaria Roscoe (Zingiberaceae), also known as white turmeric or zedoaria, has been used in Ayurveda and traditional Chinese medicine to treat various cancers, and it possesses several sesquiterpenoid compounds. OBJECTIVE: This study aimed to evaluate the therapeutic effects of a methanolic (MeOH) extract of C. zedoaria rhizomes, as well as its active constituents, against gastric cancer, which is a frequently diagnosed cancer in South Korea. MATERIALS AND METHODS: Repeated column chromatography, together with semi-preparative HPLC purification, was used to separate the bioactive constituents from the C. zedoaria MeOH extract. The cytotoxic effects of the C. zedoaria MeOH extract and its active compounds were measured in human gastric cancer AGS cells. Expression of proteins related to apoptosis was evaluated using Western blotting analysis. RESULTS: The MeOH extract of C. zedoaria rhizomes exerted a cytotoxic effect on AGS cells (IC50: 96.60 ± 4.87µg/mL). Based on the bioactivity-guided fractionation for antiproliferative activity, a chemical investigation of the MeOH extract led to the isolation of five sesquiterpenes including isoprocurcumenol (1), germacrone (2), curzerenone (3), curcumenol (4), and curcuzedoalide (5). Among these, curcuzedoalide demonstrated the strongest effect in suppressing gastric cancer cell proliferation in a dose-dependent manner with an IC50 value of 125.11±2.77µM. Western blotting analysis showed that curcuzedoalide inhibited AGS human gastric cancer cell viability by activating caspase-8, caspase-9, caspase-3, and PARP, which contributed to apoptotic cell death in AGS human gastric cancer cells. CONCLUSION: These data indicate that curcuzedoalide contributed to the cytotoxicity of C. zedoaria by activating the cleavage of caspases and PARP, which are representative markers for apoptosis. Therefore, curcuzedoalide is a positive candidate for the development of novel chemotherapeutics.

In vitro assessment of selected Korean plants for antioxidant and antiacetylcholinesterase activities.

CONTEXT: Antiacetylcholinesterase (AChE) drugs have been a main therapeutic treatment for Alzheimer's disease because increased AChE levels play a key role in reducing neurotransmission. OBJECTIVES: Extracts from 35 Korean plants were selected and screened for antioxidant and anti-cholinesterase activity to explore new sources derived from Korean natural resources that could be used as AD therapeutic agents. MATERIALS AND METHODS: The antioxidant effect of extracts from 35 selected Korean plants was determined using two most common free radical scavenging assays using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS). Additionally, the effect of extracts, identified as antioxidants, on acetylcholinesterase inhibition was assessed by an acetylcholinesterase assay kit. RESULTS: Out of 36 extracts of 35 plants tested, Oenothera biennis L. (9.09 µg/mL), Saururus chinensis (Lour.) Baill. (9.52 µg/mL) and Betula platyphylla var. japonica (9.85 µg/mL) showed strong DPPH scavenging activity. Twelve other extracts also exerted moderate free radical scavenging activities with IC50 values ranging from 10 to 50 µg/mL. Antioxidant capacity detected by ABTS assay was only significant in O. biennis (23.40 µg/mL), while the other extracts were weak or unable to reduce the production of ABTS. Based on the antioxidant activities of these plant extracts, 19 extracts with IC50 values less than 100 µg/mL in DPPH assay were selected for further AChE inhibition assay. Among the extracts tested, the IC50 value for Prunella vulgaris var. lilacina NAKAI (18.83 µg/mL) in AChE inhibitory activity was the lowest, followed by O. biennis (20.09 µg/mL) and Pharbitis nil Chosy (22.79 µg/mL). CONCLUSIONS: Considering complex multifactorial etiology of AD, the extracts of P. vulgaris var. lilacina (aerial part), O. biennis (seed) and P. nil (seed) may be safe and ideal candidates for future AD modifying therapies.

Caffeoyl glucosides from Nandina domestica inhibit LPS-induced endothelial inflammatory responses.

Endothelial dysfunction is a key pathological feature of many inflammatory diseases, including sepsis. In the present study, a new caffeoyl glucoside (1) and two known caffeoylated compounds (2 and 3) were isolated from the fruits of Nandina domestica Thunb. (Berberidaceae). The compounds were investigated for their effects against lipopolysaccharide (LPS)-mediated endothelial inflammatory responses. At 20 µM, 1 and 2 inhibited LPS-induced hyperpermeability, adhesion, and migration of leukocytes across a human endothelial cell monolayer in a dose-dependent manner suggesting that 1 and 2 may serve as potential scaffolds for the development of therapeutic agents to treat vascular inflammatory disorders.

Steroidal Alkaloids from Veratrum nigrum Enhance Glucose Uptake in Skeletal Muscle Cells.

Veratrum nigrum is recognized as a medicinal plant used for the treatment of hypertension, stroke, and excessive phlegm. Chemical investigation of the roots and rhizomes led to the isolation of five new steroidal alkaloids, jervine-3-yl formate (1), veramarine-3-yl formate (2), jerv-5,11-diene-3ß,13ß-diol (3), (1ß,3ß,5ß)-1,3-dihydroxyjervanin-12(13)-en-11-one (4), and veratramine-3-yl acetate (5). Compounds 1 and 5 exhibited potent inhibitory activity (11.3 and 4.7 µM, respectively) against protein tyrosine phosphatase 1B (PTP1B), which has emerged as a viable target for treatment of type 2 diabetes mellitus. On the basis of their PTP1B inhibitory activity, the compounds were evaluated for their potential to enhance glucose uptake in C2C12 skeletal muscle cells. The insulin-stimulated glucose uptake was enhanced upon treatment with compounds 1 and 5 (10 µM) by 49.9 ± 6.5% and 56.0 ± 9.7%, respectively, in a more potent manner than that with the positive control rosiglitazone (47.3 ± 3.4% at 30 µM). These results suggest that steroidal alkaloids serve as practical antidiabetes mellitus leads capable of enhancing glucose uptake.

In vitro and in vivo hepatoprotective effect of ganodermanontriol against t-BHP-induced oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (Fr.) Karst. (Ganodermataceae) is a mushroom which is used as a traditional remedy in the treatment of human diseases such as hepatitis, liver disorders, hypercholesterolemia, arthritis, bronchitis and tumorigenic diseases. This study targets the evaluation of hepatoprotective activity of ganodermanontriol, a sterol isolated from Ganoderma lucidum, and the investigation of its mechanism of action in Hepa1c1c7 and murine liver cells upon tert-butyl hydroperoxide (t-BHP)-induced inflammation. t-BHP was utilized to stimulate an anti-inflammatory reaction in the hepatic cell lines and murine hepatic tissue examined. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were used to estimate the expression of ganodermanontriol (GDT)-induced proteins, including heme oxidase-1 (HO-1) and mitogen-activated protein kinases (MAPKs) as well as the corresponding mRNA. Luciferase assays were conducted to evaluate the interaction between NF-E2-related factor-2 (Nrf-2), the antioxidant response element (ARE), and the promoter region of the HO-1 gene and subsequent gene expression. Biochemical markers for hepatotoxicity were monitored to assess whether GDT protected the cells from the t-BHP-mediated oxidative stimuli. RESULTS: GDT induced HO-1 expression via the activation of Nrf-2 nuclear translocation and the subsequent transcription of the HO-1 gene in vitro and in vivo, which seemed to be regulated by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p38 signaling pathways. GDT exhibited in vitro and in vivo hepatoprotective activity as determined by the lowered levels of hepatic enzymes and malondialdehydes and the elevated glutathione levels. CONCLUSIONS: This study validates the ethnopharmacological application of Ganoderma lucidum as a treatment for hepatic disorders. GDT induced in vitro and in vivo anti-inflammatory activity in t-BHP-damaged hepatic cells through the expression of HO-1, and in which PI3K/Akt and p38 kinases are involved. Our study motivates further research in the exploration of potent hepatoprotective agents from Ganoderma lucidum.

Anti-listerial compounds from Asari Radix.

Asari Radix, the roots of Asarum heterotropoides F. Maekawa var. manshuricum F. Maekawa or A. sieboldii F. Maekawa, has traditionally been used for the treatment of various infectious diseases. Since its MeOH extract inhibited the growth of Listeria monocytogenes in a preliminary test, the aim of this study was to isolate and identify the anti-listerial compounds from the plant. Activity-guided fractionation led to the isolation of seven compounds 1-7 from the MeOH extract, and their chemical structures were identified by comparison of the spectroscopic data with those in the literature. Compounds 1-7 exhibited inhibitory activity against all five tested strains of L. monocytogenes with diameter of inhibition zones ranging from 7 to 11 mm in the agar disc diffusion method. Compounds 1-3 and 7 demonstrated potent antimicrobial effects on the L. monocytogenes strains, with MICs between 62.5 and 125 µg/mL. This is the first report that AR possesses inhibitory activity against L. monocytogenes.