Secondary metabolites and transcriptomic analysis of novel pulcherrimin producer Metschnikowia persimmonesis KIOM G15050: A potent and safe food biocontrol agent.

Metschnikowia persimmonesis, a novel endophytic yeast strain isolated from Diospyros kaki calyx, possesses strong antimicrobial activity. We investigated its potential use as an environmentally safe food biocontrol agent through genomics, transcriptomics, and metabolomics. Secondary metabolites were isolated from M. persimmonesis, followed by chemical structure elucidation, PUL gene cluster identification, and RNA sequencing. Pulcherrimin was isolated using 2 M NaOH, its structure was confirmed, and the yield was quantified. Biocontrol efficacy of M. persimmonesis on persimmon fruits and calyx was evaluated by assessing lesion diameter and disease incidence. Following compounds were isolated from M. persimmonesis co-culture with Botrytis cinerea and Fusarium oxysporum: fusaric acid, benzoic acid, benzeneacetic acid, 4-hydroxybenzeneacetic acid, 4-(-2-hydoxyethyl)-benzoic acid, cyclo (Leu-Leu), benzenemethanol, 4-hydroxy-benzaldehide, 2-hydroxy-4-methoxy-benzoic acid, 4-hydroxy-benzoic acid, lumichrome, heptadecanoic acid, and nonadecanoic acid. Exposing M. persimmonesis to different growth media conditions (with or without sugar) resulted in the isolation of five compounds: Tyrosol, Cyclo (Pro-Val), cyclo(L-Pro-L-Tyr), cyclo(Leu-Leu), and cyclo(l-tyrosilylicine). Differentially expressed gene analysis revealed 3264 genes that were significantly expressed (fold change ≥2 and p-value ≤0.05) during M. persimmonesis growth in different media, of which only 270 (8.27%) showed altered expression in all sample combinations with Luria-Bertani Agar as control. Minimal media with ferric ions and tween-80 triggered the most gene expression changes, with the highest levels of PUL gene expression and pulcherrimin yield (262.166 mg/L) among all media treatments. M. persimmonesis also produced a higher amount of pulcherrimin (209.733 mg/L) than Metschnikowia pulcherrima (152.8 mg/L). M. persimmonesis inhibited the growth of Fusarium oxysporum in persimmon fruit and calyx. Toxicity evaluation of M. persimmonesis extracts showed no harmful effects on the liver and mitochondria of zebrafish, and no potential risk of cardiotoxicity in hERG-HEK293 cell lines. Thus, M. persimmonesis can be commercialized as a potent and safe biocontrol agent for preserving food products.

Anti-Fibrosis Effect of Panax ginseng and Inula japonica Formula in Human Pulmonary Fibroblasts.

Panax ginseng Meyer and Inula japonica Thunb. are well established in traditional medicine and are known for their therapeutic properties in managing a range of ailments such as diabetes, asthma, and cancer. Although P. ginseng and I. japonica can alleviate pulmonary fibrosis (PF), the anti-fibrosis effect on PF by the combination of two herbal medicines remains unexplored. Therefore, this study explores this combined effect. In conditions that were not cytotoxic, MRC-5 cells underwent treatment using the formula combining P. ginseng and I. japonica (ISE081), followed by stimulation with transforming growth factor (TGF)-ß1, to explore the fibroblast-to-myofibroblast transition (FMT). After harvesting the cells, mRNA levels and protein expressions associated with inflammation and FMT-related markers were determined to evaluate the antiinflammation activities and antifibrosis effect of ISE081. Additionally, the anti-migratory effects of ISE081 were validated through a wound-healing assay. ISE081 remarkably reduced the mRNA levels of interleukin (IL)-6, IL-8, α-smooth muscle actin (SMA), and TGF-ß1 in MRC-5 cells and suppressed the α-SMA and fibronectin expressions, respectively. Furthermore, ISE081 inhibited Smad2/3 phosphorylation and wound migration of MRC-5 cells. Under the same conditions, comparing those of ISE081, P. ginseng did not affect the expression of α-SMA, fibronectin, and Smad2/3 phosphorylation, whereas I. japonica significantly inhibited them but with cytotoxicity. The results indicate that the synergistic application of P. ginseng and I. japonica enhances the anti-fibrotic properties in pulmonary fibroblasts and concurrently diminishes toxicity. Therefore, ISE081 has the potential as a prevention and treatment herbal medicine for PF.

The antiviral activity of Thuja orientalis folium against Influenza A virus.

Thuja orientalis Folium (TOF) has been prescribed traditionally as an expectorant for inflammatory airway disease. In this study, we evaluated the anti-influenza A virus (IAV) activity of TOF by detecting GFP expressed by influenza A virus (A/PR/8/34-GFP) infection. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that TOF potently inhibited IAV infection, dose-dependently. Consistently, immunofluorescence and Q-PCR analysis results confirmed TOF significantly represses IAV protein and RNA expression. TOF inhibited IAV infection at the binding and entry step upon viral infection and interferes with HA protein. Further, TOF exhibited a virucidal effect and inhibited the neuraminidase activity of IAV. Additionally, TOF prevented the cytopathic effect caused by H1N1 and H3N2 IAV infection. Amentoflavone among the constituents in TOF exerted the strongest anti-IAV effect. Myricetin, quercetin, and quercitrin also inhibited IAV infection. However, the potent anti-IAV effect of TOF may be related to the synergistic effect of constituents, not by a single specific compound. Our results suggest TOF exhibits a significant inhibitory effect against IAV infection at multi-stages via the blockage of viral attachment and entry, inhibition of neuraminidase, and induction of virucidal effects.

In vitro propagation of Codonopsis pilosula (Franch.) Nannf. using apical shoot segments and phytochemical assessments of the maternal and regenerated plants.

BACKGROUND: Codonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC-MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared. RESULTS: Full-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L-1) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L-1) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants. CONCLUSIONS: The proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand.

Therapeutic Effects of Acer palmatum Thumb. Leaf Extract (KIOM-2015E) on Benzalkonium Chloride-Induced Dry Eye in a Mouse Model.

We determined the effects of two extracts from Acer palmatum Thumb. leaves (hot water extract KIOM-2015EW and 25% ethanol extract KIOM-2015EE) in a benzalkonium chloride (BAC)-induced dry eye mouse model. Dry eye was induced by 0.2% BAC for 2 weeks, followed by treatment three times (eye drop) or once (oral administration) daily with KIOM-2015E for 2 weeks. Treatment with both KIOM-2015EE and KIOM-2015EW resulted in a marked increase in tear volume production for the 4 days of treatment. The Lissamine Green staining score, TUNEL-positive cells, and inflammatory index were significantly decreased after 2 weeks. Topical KIOM-2015EE administration exhibited a greater improvement in decreasing the ocular surface staining scores, inflammation, dead cells, and increasing tear production in a dose-dependent manner compared with the other groups. Furthermore, KIOM-2015E significantly reduced the phosphorylation of NF-κB, which was activated in the BAC-treated cornea. Topical administration was much more effective than oral administration for KIOM-2015E and KIOM-2015EE was more effective than KIOM-2015EW. Application of KIOM-2015E resulted in clinical improvement, inhibited the inflammatory response, and alleviated signs of dry eye. These results indicate that KIOM-2015E has potential as a therapeutic agent for the clinical treatment of dry eye.

Broccoli Leaves Attenuate Influenza A Virus Infection by Interfering With Hemagglutinin and Inhibiting Viral Attachment.

Broccoli (Brassica oleracea L. var. Italica) leaves are a byproduct of broccoli and could be used as a food source. The study aimed to evaluate the effect of broccoli leaves on influenza A virus (IAV) infection. We investigated the effect of ethanol extract of Broccoli leaves (EBL) on IAV infection using green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus (PR8-GFP IAV). When EBL and PR8-GFP IAV were cotreated to RAW 264.7 cells, the fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis showed that EBL significantly reduced the levels of GFP expression by influenza viral infection dose-dependently. Immunofluorescence (IF) analysis confirmed that EBL decreased the expression of IAV proteins. EBL exhibited a strong inhibitory effect of IAV binding on the cells and moderate virucidal impact. Consistently, EBL potently suppressed the hemagglutination by IAV infection. These results indicate that EBL prevents IAV attachment via the inhibition of HA upon viral infection. Finally, EBL as an HA inhibitor of IAV could be used as the natural antiviral source to protect against influenza viral infection.

GC-MS and LC-TOF-MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark.

Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and ß-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1ß secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and ß-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p < 0.001) TRAP activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited significant (p < 0.05) BMM cell viability. NO production was significantly inhibited (all p < 0.0001) by the sample. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and ß-sitosterol (p < 0.0001) and further, chlorogenic acid and naringenin remarkably inhibited IL-1ß production. The P. africana methanolic extract significantly inhibited RANKL-induced TRAP activity. The phytochemical study of P. africana stem bark revealed a number of chemical compounds with anti-osteoporosis activity. There was no observed hepatocyte apoptosis in the liver of zebrafish larvae. In conclusion, the stem bark of P. africana is non-toxic to the liver and its inhibition of TRAP activity makes it an important source for future anti-osteoporosis drug development.

Root Extract of a Micropropagated Prunus africana Medicinal Plant Induced Apoptosis in Human Prostate Cancer Cells (PC-3) via Caspase-3 Activation.

Prostate cancer is one of the major causes of cancer-related deaths among men globally. Medicinal plants have been explored as alternative treatment options. Herein, we assessed the in vitro cytotoxic effects of 70% ethanolic root extracts of six-month-old micropropagated Prunus africana (PIR) on PC-3 prostate cancer cells as an alternative to the traditionally used P. africana stem-bark extract (PWS) treatment. In vitro assays on PC-3 cells included annexin-V and propidium iodide staining, DAPI staining, and caspase-3 activity analysis through western blotting. PC-3 cells were exposed to PWS and PIR at different concentrations, and dose-dependent antiprostate cancer effects were observed. PC-3 cell viability was determined using CCK-8 assay, which yielded IC50 values of 52.30 and 82.40 µg/mL for PWS and PIR, respectively. Annexin-V and PI staining showed dose-dependent apoptosis of PC-3 cells. Significant (p < 0.001) percent of DAPI-stained apoptotic PC-3 cells were observed in PWS, PIR, and doxorubicin treatment compared with the negative control. PWS treatment substantially elevated cleaved caspase-3 levels in PC-3 cells compared with the PIR treatment. These results provide evidence for the antiprostate cancer potential of PIR and sets a basis for further research to enhance future utilization of roots of young micropropagated P. africana for prostate cancer treatment as an alternative to stem bark. Moreover, micropropagation approach may help provide the required raw materials and hence reduce the demand for P. africana from endangered wild population.

Association of CYP26C1 Promoter Hypomethylation with Small Vessel Occlusion in Korean Subjects.

The risk factors for stroke, a fatal disease, include type two diabetes, hypertension, and genetic influences. Small vessel occlusion (SVO) can be affected by epigenetic alterations, but an association between SVO and the methylation of cytochrome P450 family 26 subfamily C member 1 (CYP26C1) has not been identified. In this study, we measured the level of DNA methylation in the CYP26C1 promoter and the 5' untranslated region of 115 normal subjects and 56 patients with SVO in Korea. The DNA methylation level of each subject was measured by bisulfite amplicon sequencing, and statistical analysis was performed using the general linear model or Pearson's correlation. The average level of DNA methylation was markedly lower in patients with SVO than in normal subjects (20.4% vs. 17.5%). We found that the methylation of CYP26C1 has a significant positive correlation with blood parameters including white blood cells, hematocrit, lactate dehydrogenase, and Na+ in subjects with SVO. We predicted that binding of RXR-α and RAR-ß might be affected by CYP26C1 methylation at CpG sites -246-237 and -294-285. These findings suggest that CYP26C1 methylation in the promoter region may be a predictor of SVO.

In Vitro Antiosteoporosis Activity and Hepatotoxicity Evaluation in Zebrafish Larvae of Bark Extracts of Prunus jamasakura Medicinal Plant.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 µg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 µM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p < 0.001) at all concentrations of P. jamasakura extracts compared to the control treatment. At 50 µg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p < 0.01) BMM cell viability compared to the control treatment. P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p < 0.01) effects on lipopolysaccharide (LPS)-induced NO production at 200 µg/ml and exhibited significant (p < 0.01) and (p < 0.05) stimulative effects, respectively, on RAW 264.7 cell viability. No overt hepatotoxicity was observed in the liver of zebrafish larvae in any of the treatments. Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Neuroprotective effects of Acer palmatum thumb. leaf extract (KIOM-2015E) against ischemia/reperfusion-induced injury in the rat retina.

Purpose: The present study aimed to determine whether the administration of Acer palmatum thumb. leaf extract (KIOM-2015E) protects against the degeneration of rat retinal ganglion cells after ischemia/reperfusion (I/R) induced by midbrain cerebral artery occlusion (MCAO). Methods: Sprague-Dawley rats were subjected to 90 min of MCAO, which produces transient ischemia in both the retina and brain due to the use of an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. This was followed by reperfusion under anesthesia with isoflurane. The day after surgery, the eyes were treated three times (eye drop) or one time (oral administration) daily with KIOM-2015E for five days. Retinal histology was assessed in flat mounts and vertical sections to determine the effect of KIOM-2015E on I/R injury. Results: A significant loss of brain-specific homeobox/POU domain protein 3A (Brn3a) and neuron-specific class III beta-tubulin (Tuj-1) fluorescence and a marked increase in glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) expression were observed after five days in the PBS-treated MCAO group compared to the sham-operated control group. However, KIOM-2015E treatment reduced (1) MCAO-induced upregulation of GFAP and GS, (2) retinal ganglion cell loss, (3) nerve fiber degeneration, and (4) the number of TUNEL-positive cells. KIOM-2015E application also increased staining for parvalbumin (a marker of horizontal cell associated calcium-binding protein and amacrine cells) and recoverin (a marker of photoreceptor expression) in rats subjected to MCAO-induced retinal damage. Conclusions: Our findings indicated that KIOM-2015E treatment exerted protective effects against retinal damage following MCAO injury and that this extract may aid in the development of novel therapeutic strategies for retinal diseases, such as glaucoma and age-related macular disease.

Inhibition of Programmed Death Receptor-1/Programmed Death Ligand-1 Interactions by Ginsenoside Metabolites.

Evidence suggests that programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) targeted inhibitors act as an immune checkpoint blockade, indicating that these compounds may be useful in cancer immunotherapy by inhibiting the immune response between T-cells and tumors. Previous studies have shown that ginsenosides can regulate the expression of PD-1 and PD-L1 in target diseases; however, it remains unknown whether ginsenosides act as a blockade of PD-1/PD-L1 interactions. In this study, we used competitive ELISA to investigate 12 ginsenosides for their ability to block PD-1/PD-L1 interactions. In addition, we performed a protein-ligand docking simulation and examined the hydrophobic interactions and hydrogen bonds formed at the interfaces between the ginsenosides and PD-L1/PD-1. Eight out of the 12 ginsenosides studied showed inhibition of PD-1/PD-L1 interactions at 35% at the maximum concentration (1 µM). Among them, Rg3 and Compound K (C-K) demonstrated the highest inhibitory effects. Rg3 and C-K were further identified for their interaction efficacy with PD-1/PD-L1, which supported our results demonstrating the blocking activity of these compounds against PD-1/PD-L1 binding interactions. Collectively, our findings suggest that some ginsenosides, including Rg3 and C-K, inhibit PD-1/PD-L1 binding interactions. Therefore, these compounds may prove useful as part of an overall immuno-oncological strategy.

Hypermethylation of the TSPOAP1-AS1 Promoter May Be Associated with Obesity in Overweight/Obese Korean Subjects.

Obesity is a major chronic disease associated with the risk of serious cardiovascular or endocrinal diseases, such as hypertension, diabetes, atherosclerosis and stroke. Considerable interest has been directed towards the potential effects of epigenetic variations in obesity. In this study, we evaluated DNA methylation level at the promoter region of the gene encoding TSPO-associated protein 1 antisense RNA 1 (TSPOAP1-AS1) in 80 overweight/obese subjects (body mass index (BMI) > 25) and 104 non-obese subjects who participated in the SOPI-Stroke study in Korea. DNA methylation was measured using bisulfite amplicon sequencing (BSAS). A general linear model or relative correlation was used to determine the effects of DNA methylation on obesity and obese phenotypes. Notably, the mean level of DNA methylation was significantly higher in the overweight/obese group than in the non-obese group (18.62% vs. 17.18%). Further analyses revealed significant positive correlations of the BMI, the serum total cholesterol and low-density lipoprotein cholesterol levels with the DNA methylation level (p = 0.0493, p = 0.003, and p = 0.0094, respectively). The study findings suggest an association between DNA methylation at the TSPOAP1-AS1 promoter and overweight/obesity. Accordingly, methylation in this promoter region might be a potential predictor of obesity.

Hoveniae Semen Seu Fructus Ethanol Extract Exhibits Anti-Inflammatory Activity via MAPK, AP-1, and STAT Signaling Pathways in LPS-Stimulated RAW 264.7 and Mouse Peritoneal Macrophages.

Hoveniae semen seu fructus (HSF, fruit and seed of Hovenia dulcis Thunb) is an important traditional herbal medicine and food supplement in East Asia for the treatment of liver diseases, alcohol poisoning, obesity, allergy, and cancer. HSF has also been reported to have anti-inflammatory activity, but the cellular mechanism of action is not fully understood. We assessed the anti-inflammatory properties of an HSF ethanol (HSFE) extract and explored its precise mechanism. The ability of HSFE to suppress inflammatory responses was investigated in a murine macrophage cell line, RAW 264.7, and mouse primary macrophages. Secretions of NO, proinflammatory cytokines, inflammatory factors, and related proteins were measured using the Griess assay, ELISA, Western blot analysis, and real-time PCR, respectively. In addition, the main components of HSFE were analyzed by HPLC, and their anti-inflammatory activity was confirmed. Our results showed that pretreatment of HSFE markedly reduced the expression of NO and iNOS without causing cytotoxicity and significantly attenuated secretion of proinflammatory cytokines, including TNF-α, IL-6, and IL-1ß. In addition, HSFE strongly suppressed phosphorylation of MAPK and decreased the activation of AP-1, JAK2/STAT, and NF-κB in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner. Furthermore, HSFE strongly suppressed the inflammatory cytokine levels in mouse peritoneal macrophages. Also, as a result of HPLC analysis, three main components, ampelopsin, taxifolin, and myricetin, were identified in the HSFE extract, and each compound effectively inhibited the secretion of inflammatory mediators induced by LPS. These findings show that HSFE exerts anti-inflammatory effects by suppressing the activation of MAPK, AP-1, JAK2/STAT, and NF-κB signaling pathways in LPS-stimulated macrophages. In addition, the anti-inflammatory efficacy of HSFE appears to be closely related to the action of the three main components. Therefore, HSFE appears to be a promising candidate for the treatment of inflammatory diseases.

Antiplatelet Activity of Acylphloroglucinol Derivatives Isolated from Dryopteris crassirhizoma.

Platelets are an important component of the initial response to vascular endothelial injury; however, platelet dysfunction induces the acute clinical symptoms of thrombotic disorders, which trigger severe cardiovascular diseases such as myocardial infarction, ischemia, and stroke. In this study, we investigated the Dryopteris crassirhizoma's antiplatelet activity. A water extract of D. crassirhizoma (WDC) was partitioned into dichloromethane (DCM), ethyl acetate, n-butyl alcohol, and water. Among these four fractions, the DCM fraction potently inhibited the collagen-stimulated platelet aggregation in a concentration-dependent manner. From this fraction, five different acylphloroglucinol compounds and one flavonoid were isolated by activity-guided column chromatography. They were identified by comparing their mass, 1H-, and 13C-NMR spectral data with those reported in the literature. Quantifying the six compounds in WDC and its DCM fraction by high-performance liquid chromatography (HPLC) revealed that butyryl-3-methylphloroglucinol (compound 4) was the most abundant in these samples. Additionally, butyryl-3-methylphloroglucinol showed the strongest inhibitory activity in the collagen- and arachidonic acid (AA)-induced platelet aggregation, with inhibition ratios of 92.36% and 89.51% in the collagen and AA-induced platelet aggregation, respectively, without cytotoxicity. On the active concentrations, butyryl-3-methylphloroglucinol significantly suppressed the convulxin-induced platelet activation. Regarding the structure-activity relationships for the five acylphloroglucinol compounds, our results demonstrated that the functional butanonyl, methoxy, and hydroxy groups in butyryl-3-methylphloroglucinol play important roles in antiplatelet activity. The findings indicate that acylphloroglucinols, including butyryl-3-methylphloroglucinol from D. crassirhizom, possess an antiplatelet activity, supporting the use of this species for antiplatelet remedies.

Enhancement of neuroprotective activity of Sagunja-tang by fermentation with lactobacillus strains.

BACKGROUND: Sagunja-tang (SGT) is widely used in traditional herbal medicine to treat immune system and gastrointestinal disorders and reportedly has protective effects against inflammation, cancer, and osteoporosis. In this study, we fermented SGT with different Latobacillus strains and investigated the change in phytochemical compositions in SGT and enhancement of it neuroprotective effects in SH-SY5Y human neuroblastoma. METHODS: Marker components, including ginsenoside Rg1, glycyrrhizin, liquiritin, liquiritigenin, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid, in SGT, were qualitatively and quantitatively analyzed using high-performance liquid chromatography-diode array detection and liquid chromatography-mass spectrometry. SGT was fermented with eight different Lactobacillus strains to yield eight fermented SGTs (FSGTs). The conversion efficiencies of SGT marker components were determined in each FSGT. To detect the protective effect of SGT and FSGT, reactive oxygen species (ROS) assay and mitochondrial membrane potentials (MMPs) assay were performed in SH-SY5Y cells. RESULTS: Compared with the other FSGTs, SGT166, i.e., SGT fermented with L. plantarum 166, had high conversion efficiency, as indicated by increased amounts of glycyrrhizin, liquiritigenin, and atractylenolides I-III. In SH-SY5Y cells, protection against cell death induced by H2O2 and etoposide was high using SGT166 and very low using SGT. Furthermore, ROS production and mitochondrial membrane potential disruption in SH-SY5Y cells were markedly suppressed by SGT166 treatment, which demonstrated that inhibition of ROS generation may be one of the neuroprotective mechanisms of SGT166. CONCLUSIONS: This study demonstrated that fermentation of SGT with L. plantarum 166 enhanced suppression of oxidative stress and MMP loss. This enhanced neuroprotective effect was thought to be caused by the conversion of SGT phytochemicals by fermentation. SGT166 shows potential for treating neurological damage-related diseases.

Jageum-Jung improves 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice and suppresses pro-inflammatory chemokine production by inhibiting TNF-α/IFN-γ-induced STAT-1 and NFκB signaling in HaCaT cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Jageum-Jung (JGJ) is an oriental herbal formula comprising five herbs (Melaphis chinensis Bell, Cremastra variabilis Nakai, Knoxia valerianoides Thorel, Euphorbia lathyris L., and Moschus moschiferus L.). It has been used for detoxification and treating cancer and inflammatory diseases in China, Japan, and Korea. However, the mechanism of action of JGJ on keratinocyte inflammatory response is poorly understood. AIM OF THE STUDY: In the present study, we investigated the anti-inflammatory mechanism of JGJ and studied the effects of JGJ on atopic dermatitis-like skin lesions in mice. MATERIALS AND METHODS: We elucidated the anti-inflammatory and anti-inflammatory effects of JGJ on tumor necrosis factor-α/interferon-γ (TNF-α/IFN-γ)-treated human keratinocyte cells, IgE-sensitized RBL-2H3 cells, and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)-like mice, respectively. RESULTS: The results showed that JGJ suppressed the production and mRNA revels of IL-8, IL-6 and, conspicuously, both TARC and RANTES. JGJ inhibited nuclear translocation of the inflammatory transcription factors NFκB and STAT-1. Moreover, JGJ improved AD-like skin lesions in DNCB-treated mice and inhibited degranulation of mast cell. CONCLUSIONS: The results of this study suggest that JGJ can be considered as a candidate agent for AD treatment.

Anti-Inflammatory and Anti-Apoptotic Effects of Acer Palmatum Thumb. Extract, KIOM-2015EW, in a Hyperosmolar-Stress-Induced In Vitro Dry Eye Model.

The aim of this study was to assess the anti-inflammatory and anti-apoptotic effects of KIOM-2015EW, the hot-water extract of maple leaves in hyperosmolar stress (HOS)-induced human corneal epithelial cells (HCECs). HCECs were exposed to hyperosmolar medium and exposed to KIOM-2015EW with or without the hyperosmolar media. Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 production and apoptosis were observed, and the activation of mitogen-activated protein kinases (MAPKs) including extracellular signal regulated kinase (ERK), p38 and c-JUN N-terminal kinase (JNK) signaling and nuclear factor (NF)-κB was confirmed. Compared to isomolar medium, the induction of cell cytotoxicity significantly increased in HCECs exposed to hyperosmolar medium in a time-dependent manner. KIOM-2015EW-treatment significantly reduced the mRNA and protein expression of pro-inflammatory mediators and apoptosis. KIOM-2015EW-treatment inhibited HOS-induced MAPK signaling activation. Additionally, the HOS-induced increase in NF-κB phosphorylation was attenuated by KIOM-2015EW. The results demonstrated that KIOM-2015EW protects the ocular surface by suppressing inflammation in dry eye disease, and suggest that KIOM-2015EW may be used to treat several ocular surface diseases where inflammation plays a key role.

Protective Effects of Spatholobi Caulis Extract on Neuronal Damage and Focal Ischemic Stroke/Reperfusion Injury.

Neuronal apoptotic cell death plays an important role in many neurological disorders, including Alzheimer's disease, Parkinson's disease, and ischemic stroke. Spatholobi Caulis (SC) has been widely used in traditional herbal medicine for the treatment of cancer, inflammation, viral infection, and anemia. However, the protective effects of SC extract (SCE) against apoptotic cell death in the brain have not been reported. We investigated the protective effects of SCE against neuronal injury etoposide-induced neurotoxicity and in rats subjected to focal transient ischemic stroke middle cerebral artery occlusion (MCAO) for 45 min, followed by 7 days of reperfusion. The in vitro study demonstrated that SCE protected cells against etoposide-induced cell viability loss in SH-SY5Y cells. Apoptotic phenotypes, such as cleaved PARP and caspase-3, and oxidative stress in etoposide-treated cells were ameliorated by SCE treatment. In MCAO-reperfusion injury, SCE promoted neuronal survival and level of brain-derived neurotrophic factor (BDNF) by reducing glial activation, oxidative stress, and apoptosis in the ipsilateral cortex. These results indicated that SCE exerted protective effects under etoposide treatment and in a MCAO-reperfusion model by reducing JNK and p38 MAPK activation. This study presents the first evidence that SCE has therapeutic potential for the treatment of ischemic stroke or neurological disorder-related cell death.

Protective Effect of Panax notoginseng Root Water Extract against Influenza A Virus Infection by Enhancing Antiviral Interferon-Mediated Immune Responses and Natural Killer Cell Activity.

Influenza is an acute respiratory illness caused by the influenza A virus, which causes economic losses and social disruption mainly by increasing hospitalization and mortality rates among the elderly and people with chronic diseases. Influenza vaccines are the most effective means of preventing seasonal influenza, but can be completely ineffective if there is an antigenic mismatch between the seasonal vaccine virus and the virus circulating in the community. In addition, influenza viruses resistant to antiviral drugs are emerging worldwide. Thus, there is an urgent need to develop new vaccines and antiviral drugs against these viruses. In this study, we conducted in vitro and in vivo analyses of the antiviral effect of Panax notoginseng root (PNR), which is used as an herbal medicine and nutritional supplement in Korea and China. We confirmed that PNR significantly prevented influenza virus infection in a concentration-dependent manner in mouse macrophages. In addition, PNR pretreatment inhibited viral protein (PB1, PB2, HA, NA, M1, PA, M2, and NP) and viral mRNA (NS1, HA, PB2, PA, NP, M1, and M2) expression. PNR pretreatment also increased the secretion of pro-inflammatory cytokines [tumor necrosis factor alpha and interleukin 6] and interferon (IFN)-beta and the phosphorylation of type-I IFN-related proteins (TANK-binding kinase 1, STAT1, and IRF3) in vitro. In mice exposed to the influenza A H1N1 virus, PNR treatment decreased mortality by 90% and prevented weight loss (by approximately 10%) compared with the findings in untreated animals. In addition, splenocytes from PNR-administered mice displayed significantly enhanced natural killer (NK) cell activity against YAC-1 cells. Taking these findings together, PNR stimulates an antiviral response in murine macrophages and mice that protects against viral infection, which may be attributable to its ability to stimulate NK cell activity. Further investigations are needed to reveal the molecular mechanisms underlying the protective effects of PNR and its components against influenza virus A infection.