Yuk-Gunja-Tang attenuates neuronal death and memory impairment via ERK/CREB/BDNF signaling in the hippocampi of experimental Alzheimer's disease model.

Yuk-Gunja-Tang (YG) is the Korean traditional medicine in East Asia for gastrointestinal disorders. In the present study, we determined the protective effects of YG on glutamate-induced cytotoxicity in HT22 hippocampal neuronal cells and mice with scopolamine-induced memory impairment. In vitro assessments were performed using a cell viability assay, flow cytometry, and Western blotting, while in vivo assessments were performed in C57BL/6 mice administered with YG for 7 days and injected with scopolamine (1 mg/kg) for 7 days. We assessed the memory function using the Y-maze, novel object recognition, and passive avoidance tests. Protein expression analyses and histological analyses were performed using hippocampal tissues. YG treatment significantly restored cell viability against glutamate-induced apoptosis. It significantly suppressed glutamate-induced reactive oxygen species accumulation and mitochondrial dysfunction. It also increased Bcl-2 protein expression and decreased HO-1 protein expression. It activated the extracellular signal-regulated kinase/cAMP response element binding protein (ERK/CREB) signaling pathway and increased the expression of brain-derived neurotrophic factor (BDNF) under excitotoxic conditions. In the scopolamine-injected mice, YG ameliorated memory impairment in the Y-maze, novel object recognition, and passive avoidance tests; restored dysfunction in the acetylcholine, acetylcholinesterase expression levels; reduced neuronal damage in Nissl staining; and increased BDNF and phosphorylated ERK and CREB levels in Western blotting and immunofluorescence staining. Thus, YG exerted neuroprotective effects by activating ERK/CREB/BDNF signaling in the hippocampus, indicating its potential cognition-enhancing effects, especially in Alzheimer's disease.

Menthae Herba Attenuates Neuroinflammation by Regulating CREB/Nrf2/HO-1 Pathway in BV2 Microglial Cells.

Chronic inflammation and oxidative stress cause microglia to be abnormally activated in the brain, resulting in neurodegenerative diseases such as Alzheimer's disease (AD). Menthae Herba (MH) has been widely used as a medicinal plant with antimicrobial, anti-inflammatory, and antioxidant properties. In this study, we sought to evaluate the effects of MH on the inflammatory response and possible molecular mechanisms in microglia stimulated with lipopolysaccharide (LPS). Transcriptional and translational expression levels of the proinflammatory factors were measured using ELISA, RT-qPCR, and Western blot analysis. MH extract inhibited the production of proinflammatory enzymes and mediators nitric oxide (NO), NO synthase, cyclooxygenase-2, tumor necrosis factor-α, and interleukin-6 in LPS-stimulated cells. Our molecular mechanism study showed that MH inhibited the production of reactive oxygen species (ROS) and the phosphorylation of mitogen-activated protein kinase and nuclear factor (NF)-κB. In contrast, MH activated HO-1 and its transcriptional factors, cAMP response element-binding protein (CREB), and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways. Thus, MH reduces ROS and NF-κB-mediated inflammatory signaling and induces CREB/Nrf2/HO-1-related antioxidant signaling in microglia. Together, these results may provide specific prospects for the therapeutic use of MH in the context of neuroinflammatory diseases, including AD.

Samhwangsasim-tang attenuates neuronal apoptosis and cognitive decline through BDNF-mediated activation of tyrosin kinase B and p75-neurotrophin receptors.

BACKGROUND: Samhwangsasim-tang (SST) is a traditional medicine used to treat hypertension and arteriosclerosis. Additionally, due to the effects of its constituent herbs, SST is considered effective for memory-related disorders. PURPOSE: We investigated the effects of SST on neuronal survival and memory in glutamate-induced hippocampal cells and in a mouse model of scopolamine-induced memory impairment. METHODS: SST components were identified using 3D-ultra performance liquid chromatography (3D-UPLC). In vitro, we induced glutamate-induced excitotoxicity in HT22 cells after SST pretreatment. We used a cell counting kit-8 and cell cytotoxicity assay, flow cytometry, and western blotting to test the protective effects of SST on neuronal death. In vivo, C57BL/6J mice were administered with 150 and 300 mg/kg SST once daily for 7 days and then intraperitoneally injected with 1 mg/kg scopolamine for 7 days to induce cognitive impairment. We then measured cognitive behavior using a novel object recognition test (NORT) and passive avoidance test (PAT) and analyzed the histological and protein changes. RESULTS: Our results showed that treatment with 50 and 100 µg/ml SST provided significant protection against glutamate-induced cell death. Flow cytometry and western blotting results suggested that 100 µg/ml SST treatment reduced oxidative stress and mitochondrial dysfunction. SST treatment also increased brain-derived neurotrophic factor (BDNF), its receptor, TrkB receptor, and cAMP-response element binding protein (CREB) activation while reducing the P75NTR and JNK signaling activation. Our in vivo results showed that SST administration improved cognitive impairment, similar to donepezil treatment (as a positive control), in NORT and PAT. SST and donepezil decreased neuronal cell death and apoptosis, and acetylcholine levels were increased in the scopolamine-treated hippocampus. Additionally, SST promoted CREB phosphorylation and BDNF maturation while reducing JNK and P75NTR activation; in contrast, donepezil did not alter levels of these proteins in the scopolamine-treated mouse hippocampus. CONCLUSION: Our results suggest that SST has neuroprotective effects to attenuate neuronal cell death and oxidative stress through CREB/JNK signaling via BDNF activation. SST may regulate endogenous survival factors in the hippocampus, which may be a safe and potential clinical treatment for cognitive impairment in AD.

Phenolic Constituents from Platycodon grandiflorum Root and Their Anti-Inflammatory Activity.

Six lignols (1-6), including two new compounds (+)-(7R,8R)-palmitoyl alatusol D (1) and (+)-(7R,8R)-linoleyl alatusol D (2), along with four phenolics (7-10), a neolignan (11), three alkyl aryl ether-type lignans (12-14), two furofuran-type lignans (15-16), three benzofuran-type lignans (17-19), a tetrahydrofuran-type lignan (20), and a dibenzylbutane-type lignan (21) were isolated from the ethyl acetate-soluble fraction of the methanol extract of Platycodon grandiflorum (Jacq.) A. DC. root. The chemical structures of the obtained compounds were elucidated via high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy analyses. The obtained spectroscopic data agreed well with literature. Among the isolated compounds, eighteen (1-7 and 11-21) were isolated from P. grandiflorum and the Campanulaceae family for the first time. This is the first report on lignol and lignan components of P. grandiflorum. The anti-inflammatory effects of the isolated compounds were examined in terms of their ability to inhibit the production of pro-inflammatory cytokines IL-6, IL-12 p40, and TNF-α in lipopolysaccharide-stimulated murine RAW264.7 macrophage cells. Nine compounds (4-6, 12, and 15-19) exhibited inhibitory effects on IL-12 p40 production, eleven compounds (1-6, 12, 15-17, and 19) exhibited inhibitory activity on IL-6 production, and eleven compounds (1-6 and 15-19) exhibited inhibitory effects against TNF-α. These results warrant further investigation into the potential anti-inflammatory activity and general benefits of the phenolic constituents of P. grandiflorum root.

Isolation and Characterization of Compounds from Glycyrrhiza uralensis as Therapeutic Agents for the Muscle Disorders.

Skeletal muscle is the most abundant tissue and constitutes about 40% of total body mass. Herein, we report that crude water extract (CWE) of G. uralensis enhanced myoblast proliferation and differentiation. Pretreatment of mice with the CWE of G. uralensis prior to cardiotoxin-induced muscle injury was found to enhance muscle regeneration by inducing myogenic gene expression and downregulating myostatin expression. Furthermore, this extract reduced nitrotyrosine protein levels and atrophy-related gene expression. Of the five different fractions of the CWE of G. uralensis obtained, the ethyl acetate (EtOAc) fraction more significantly enhanced myoblast proliferation and differentiation than the other fractions. Ten bioactive compounds were isolated from the EtOAc fraction and characterized by GC-MS and NMR. Of these compounds (4-hydroxybenzoic acid, liquiritigenin, (R)-(-)-vestitol, isoliquiritigenin, medicarpin, tetrahydroxymethoxychalcone, licochalcone B, liquiritin, liquiritinapioside, and ononin), liquiritigenin, tetrahydroxymethoxychalcone, and licochalcone B were found to enhance myoblast proliferation and differentiation, and myofiber diameters in injured muscles were wider with the liquiritigenin than the non-treated one. Computational analysis showed these compounds are non-toxic and possess good drug-likeness properties. These findings suggest that G. uralensis-extracted components might be useful therapeutic agents for the management of muscle-associated diseases.

Effect of Rhus verniciflua Extract on IgE-Antigen-Mediated Allergic Reaction in Rat Basophilic Leukemic RBL-2H3 Mast Cells and Passive Cutaneous Anaphylaxis in Mice.

Rhus verniciflua is widely known for its antioxidant, antibacterial, anticancer, and antiaging efficacy and α-glucosidase inhibition. This study was designed whether Rhus verniciflua extracts inhibit the IgE-antigen-mediated allergic reaction in RBL-2H3 mast cells, and it further investigated the FcεRI- and arachidonate-signaling by which Rhus verniciflua extracts exert its antiallergic effects. IgE-antigen-sensitized RBL-2H3 mast cells were investigated for the cytotoxicity of Rhus verniciflua extracts and ß-hexosaminidase release, and inflammatory mediators (e.g., TNF-α, IL-4, IL-6, histamine, and PGD2) were then assessed. Additionally, we examined expressions of genes involved in arachidonate- and FcεRI-signaling pathway in RBL-2H3. Rhus verniciflua extracts inhibited ß-hexosaminidase release and production of the inflammatory mediators in RBL-2H3. Rhus verniciflua extracts reduced amounts of histamine and expressions of FcεRI signaling-related genes such as Lyn and Syk and phosphorylation of extracellular signal-regulated kinase in mast cells. Finally, in late allergic responses, Rhus verniciflua extracts reduced PGD2 release and COX-2 and cPLA2 phosphorylation expressions from IgE-antigen-mediated mast cells. Lastly, 250-500 mg/kg RVE significantly attenuated the Ag/IgE-induced passive cutaneous anaphylaxis (PCA) reaction in mice. These findings provide novel information on the molecular mechanisms underlying the antiallergy properties of Rhus verniciflua extracts in FcɛRI-mediated allergic reaction.

Ethanol extract of Chrysanthemum zawadskii Herbich induces autophagy and apoptosis in mouse colon cancer cells through the regulation of reactive oxygen species.

BACKGROUND: Recent research has suggested that autophagy can provide a better mechanism for inducing cell death than current therapeutic strategies. This study investigated the effects of using an ethanol extract of Chrysanthemum zawadskii Herbich (ECZ) to induce apoptosis and autophagy associated with reliable signal pathways in mouse colon cancer CT-26 cells. METHODS: Using ECZ on mouse colon cancer CT-26 cells, cell viability, annexin V/propidium iodide staining, acridine orange staining, reactive oxygen species (ROS) and western blotting were assayed. RESULTS: ECZ exhibited cytotoxicity in CT-26 cells in a dose-dependent manner. ECZ induced apoptosis was confirmed by caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and increased production of reactive oxygen species (ROS). Furthermore, it was shown that ECZ induced autophagy via the increased conversion of microtubule-associated protein 1 light chain 3II, the degradation of p62, and the formation of acidic vesicular organelles. The inhibition of ROS production by N-Acetyl-L-cysteine resulted in reduced ECZ-induced apoptosis and autophagy. Furthermore, the inhibition of autophagy by 3-methyladenine resulted in enhanced ECZ-induced apoptosis via increased ROS generation. CONCLUSION: These findings confirmed that ECZ induced ROS-mediated autophagy and apoptosis in colon cancer cells. Therefore, ECZ may serve as a novel potential chemotherapeutic candidate for colon cancer.

Lycopus lucidus Turcz. ex Benth. Attenuates free fatty acid-induced steatosis in HepG2 cells and non-alcoholic fatty liver disease in high-fat diet-induced obese mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is closely related to metabolic diseases such as obesity and insulin resistance. PURPOSE: We studied whether an ethanol extract of Lycopus lucidus Turcz. ex Benth (LLE) exhibited effects on lipid metabolism in NAFLD. STUDY DESIGN: An in vitro modelwas established by treatment of HepG2 cells with a 1 mM free fatty acid (FFA) mixture (oleic acid/palmitic acid, 2:1). C57BL/6 mice were fed a high-fat diet (HFD; 60 kcal% fat) for 14 weeks to induce obesity and were treated with or without LLE (100 or 200  mg/kg daily by oral gavage). METHODS: HepG2 cells were exposed to 1 mM FFA, with or without LLE (250 - 1000  mg/ml). Intracellular lipid contents were measured by Oil Red O staining and a Nile Red assay. The body weight, relative liver weight, hepatic lipids, triglycerides (TGs), and total cholesterol (TC) were measured in the mice. Serum alanine aminotransferase (ALT), TG, TC, glucose, insulin, leptin, and tumor necrosis factor-alpha (TNF-α) levels were determined by biochemical or enzyme-linked immunosorbent assays. Histologic analysis was performed in the liver. Western blotting and quantitative real-time polymerase chain reaction were used to analyze the expression of key enzymes of hepatic lipid metabolism. RESULTS: LLE significantly decreased the intracellular lipid accumulation in FFA-treated HepG2 cells. LLE not only remarkably decreased the expression of lipogenesis genes but also increased ß-oxidation in FFA-induced HepG2 cells. In the in vivo study, LLE treatment significantly decreased the body weight, relative liver weight, serum ALT, TC, and low-density lipoprotein cholesterol, as well as the serum glucose, insulin, leptin, and TNF-α levels in HFD-fed mice. The hepatic TG and TC contents were significantly reduced in the LLE-treated groups. Western blot analysis showed that the expression of sterol-regulatory element-binding protein 1 decreased, while that of phosphorylated AMP-activated protein kinase and peroxisome proliferator-activated receptor α increased in the LLE-treated mice. CONCLUSION: These results suggest that LLE may exert protective effects against NAFLD-related obesity and metabolic disease.

Maydis Stigma Elicits Analgesia and Blocks Edema in Mice and Inhibits Inflammation in Macrophages.

Maydis Stigma (MS) is an herb traditionally used in many parts of the world. Previous studies have reported that MS plays a role in several biological activities, including antidiabetic and anticancer activities. However, the effects of a MS ethanolic extract (MSE) on the anti-inflammatory cellular mechanism remain unclear. Here, we investigated the anti-inflammatory properties of MSE and its molecular mechanism both in vitro and in vivo. The effects of MSE on the production of inflammatory mediators, cytokines, and related proteins and the identification of target genes were determined using LPS-stimulated macrophages. We also determined the analgesic and anti-inflammatory effects of MSE by examining acetic acid-induced writhing responses and xylene-induced ear edema in mice. Our results indicated that MSE markedly decreased iNOS and COX-2 levels without causing cytotoxicity and suppressed the secretion of NO in LPS-stimulated macrophages. MSE also inhibited the production of proinflammatory cytokines, such as TNF-[Formula: see text], IL-6, and IL-1[Formula: see text], and induced the expression of HO-1. Moreover, MSE treatment significantly reduced the LPS-stimulated activation of MAPK, NF-[Formula: see text]B, and AP-1. Furthermore, MSE exerted an analgesic effect on the acetic acid-induced abdominal writhing response test and an anti-inflammatory effect on xylene-induced ear edema in ICR mice. Finally, we investigated the components of MSE using UPLC-ESI-MS and found that it contains the maysin as a marker component. Overall, these observations demonstrate that MSE has anti-inflammatory and antinociceptive effects both in vitro and in vivo, which may provide new scientific evidence for its use as a potential therapeutic agent for the treatment of inflammation.

Anti-inflammatory effects of Forsythia suspensa in dextran sulfate sodium-induced colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Forsythia suspensa Fructus (FS) is used to treat various inflammatory disorders in traditional Oriental medicine, including gastrointestinal diseases, but its therapeutic potential in ulcerative colitis is unclear. Thus, we investigated any potential therapeutic effects of FS against intestinal inflammation and the bioactive constituents in FS. MATERIALS AND METHODS: After the induction of colitis using 3% dextran sulfate sodium, FS (100mg/kg/day) was administered orally during the experimental period. We evaluated body weight, bloody diarrhea, colon length, and pro-inflammatory cytokine levels. Subsequently, the bioactive constituents of FS were identified using UPLC/MS/MS. RESULTS: FS significantly decreased the body weight loss, colon length shortening, and tumor necrosis factor-α and interleukin-6 elevations induced by colitis compared with the negative control (P < 0.05). Moreover, FS improved the colitis-induced histopathological damage to the colon, including epithelial necrosis, infiltration of inflammatory cells, ulceration, and submucosal edema. In phytochemical analyses, 7 flavonoids, 9 lignans, 13 phenolics, and 2 triterpenes were identified by comparison with the retention times and mass fragmentations of authentic standards. CONCLUSIONS: We demonstrated beneficial effects of FS and its constituents, suggesting their potential for treatment of intestinal inflammation. These data could provide useful information for managing ulcerative colitis.

Simultaneous Determination of Three Furanocoumarins by UPLC/MS/MS: Application to Pharmacokinetic Study of Angelica dahurica Radix after Oral Administration to Normal and Experimental Colitis-Induced Rats.

In traditional oriental medicine, Angelica dahurica Radix (ADR) is used in the treatment of gastrointestinal, respiratory, neuromuscular, and dermal disorders. We evaluated the pharmacokinetic profiles of oxypeucedanin, imperatorin, and isoimperatorin, major active ingredients of ADR, in normal and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rats. A rapid, sensitive, and validated UPLC/MS/MS method was established for evaluating the pharmacokinetics of three furanocoumarins. After oral administration of ADR (0.5 and 1.0 g/kg), blood samples were collected periodically from the tail vein. In colitis rats, the time to reach the peak concentration (Tmax) of imperatorin and isoimperatorin was significantly delayed (p < 0.05). Lower peak plasma concentrations (Cmax) and longer mean residence times for all furanocoumarins were also observed (p < 0.05) compared with normal rats. There was no significant difference in the area under the plasma concentration-time curve or elimination half-lives. Thus, the delayed Tmax and decreased Cmax, with no influence on the elimination half-life, could be colitis-related changes in the drug-absorption phase. Therefore, the prescription and use of ADR in colitis patients should receive more attention.

Factors associated with medication adherence in patients with human immunodeficiency virus in South Korea.

This study aimed to identify the factors associated with medication adherence in human immunodeficiency virus (HIV) patients in South Korea. A cross-sectional study was conducted from six hospitals participating in the Nationwide Specialized Counseling Program for HIV infected patients from 22 February to 10 May 2010. A total of 300 HIV patients have completed a self-administered questionnaire. Among 300 patients, 230 patients had above 95% medication adherence. Binary logistic regression analysis revealed that having medical insurance (p = .003) and a good relationship with the medical team (p = .046) were the main factors affecting medication adherence in HIV patients. In conclusion, medical insurance through the National Health Insurance Service and a good relationship between HIV infected patients and physicians are the main influencing factors that impact medication adherence in countries with low economic barriers to treatment.

Inhibitory Effects of Multiple-Dose Treatment with Baicalein on the Pharmacokinetics of Ciprofloxacin in Rats.

Ciprofloxacin is used as a treatment for urinary and respiratory tract infections in clinical practice. Baicalein, a major flavonoid present in Scutellaria baicalensis, is a well-known and potent antibacterial compound used in complementary and alternative medicine practices. The present study aimed to clarify the effects of multiple-dose treatment with baicalein on the pharmacokinetics of ciprofloxacin in rats. Following the oral administration of baicalein (20, 40, or 80 mg/kg) for five consecutive days, the rats received an oral administration of ciprofloxacin (20 mg/kg). Blood samples were collected at specific time points, and the plasma concentrations of ciprofloxacin were determined by using high-performance liquid chromatography. To evaluate the mechanisms underlying the interaction between baicalein and ciprofloxacin, a rhodamine 123 accumulation assay was performed in LS-180 cells. A pharmacokinetic study revealed that multiple-dose treatment with baicalein significantly decreased the peak serum concentration (Cmax ), area under the curve (AUC0 → 480 min ), and relative bioavailability (Frel ) of ciprofloxacin (p < 0.05). The rhodamine 123 accumulation assay revealed that treatment with baicalein for 48 h markedly reduced the intracellular accumulation of rhodamine 123. Taken together, these findings suggest that baicalein may result in the therapeutic failure of ciprofloxacin or other quinolone-based antibiotics used for chemotherapy in clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibitory Effects of Viscum coloratum Extract on IgE/Antigen-Activated Mast Cells and Mast Cell-Derived Inflammatory Mediator-Activated Chondrocytes.

The accumulation and infiltration of mast cells are found in osteoarthritic lesions in humans and rodents. Nonetheless, the roles of mast cells in osteoarthritis are almost unknown. Although Viscum coloratum has various beneficial actions, its effect on allergic and osteoarthritic responses is unknown. In this study, we established an in vitro model of mast cell-mediated osteoarthritis and investigated the effect of the ethanol extract of Viscum coloratum (VEE) on IgE/antigen (IgE/Ag)-activated mast cells and mast cell-derived inflammatory mediator (MDIM)-stimulated chondrocytes. The anti-allergic effect of VEE was evaluated by degranulation, inflammatory mediators, and the FcεRI signaling cascade in IgE/Ag-activated RBL-2H3 cells. The anti-osteoarthritic action of VEE was evaluated by cell migration, and the expression, secretion, and activity of MMPs in MDIM-stimulated SW1353 cells. VEE significantly inhibited degranulation (IC50: 93.04 µg/mL), the production of IL-4 (IC50: 73.28 µg/mL), TNF-α (IC50: 50.59 µg/mL), PGD2 and LTC4, and activation of the FcεRI signaling cascade in IgE/Ag-activated RBL-2H3 cells. Moreover, VEE not only reduced cell migration but also inhibited the expression, secretion, and/or activity of MMP-1, MMP-3, or MMP-13 in MDIM-stimulated SW1353 cells. In conclusion, VEE possesses both anti-allergic and anti-osteoarthritic properties. Therefore, VEE could possibly be considered a new herbal drug for anti-allergic and anti-osteoarthritic therapy. Moreover, the in vitro model may be useful for the development of anti-osteoarthritic drugs.

Simultaneous Determination of Eight Bioactive Compounds in Dianthus superbus by High-performance Liquid Chromatography.

BACKGROUND: Dianthus superbus, one of traditional herbal medicine, is widely used to treat urethritis, carbuncles and carcinoma. OBJECTIVE: A simultaneous determination method was established for controlling the quality of D. superbus using the eight compounds, (E)-methyl-4-hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl) (1), diosmetin-7-O(2'',6''-di-O-α-L-rhamnopyranosyl)-ß-D-glucopyranoside (2), vanillic acid (3), 4-hydroxyphenyl acetic acid (4), 4-methoxyphenyl acetic acid (5), (E)-4-methoxycinnamic acid (6), 3-methoxy-4-hydroxyphenylethanol (7), and methyl hydroferulate (8) isolated from D. superbus. MATERIALS AND METHODS: This analysis method was developed using high performance liquid chromatography coupled with diode array detector with a Shishedo C18 column at a column temperature of 3°C. The mobile phase was composed of 0.1% trifluoroacetic acid in water and acetonitrile. The flow rate was 1 ml/min and detection wavelength was set at 205 nm and 280 nm. Validation was performed in order to demonstrate selectivity, accuracy and precision of the method. RESULTS: The calibration curves showed good linearity (R (2) > 0.99). The limits of detection and limits of quantification were within the ranges 0.0159-0.6205 µg/ml and 0.3210-1.8802 µg/ml, respectively. Moreover, the relative standard deviations of intra- and inter-day precision were both <2.98%. The overall recoveries were in the range of 96.23-109.87%. Quantitative analysis of eight compounds in 12 D. superbus samples (D-1-D-12) from various regions were analyzed and compared by developed method. CONCLUSION: As a result, this established method was accurate and sensitive for the quality evaluation of eight compounds isolated from D. superbus and may provide a new basis for quality control of D. superbus. SUMMARY: A simultaneous determination method of eight compounds in Dianthus superbus was established by high performance liquid chromatography-diode array detectorDeveloped analysis method is validated with linearity, precious and accuracyThe newly established method was successfully evaluated contents of eight compounds in 12 D. superbus samples (D.1.D.12) from various regions and compared. Abbreviations used: HPLC: High performance liquid chromatography, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation.

Neuroprotective Properties of Compounds Extracted from Dianthus superbus L. against Glutamate-induced Cell Death in HT22 Cells.

BACKGROUND: Dianthus superbus L. has been used in Chinese herbal medicine as a diuretic and anti-inflammatory agent. OBJECTIVE: In this study, we isolated ten bioactive compounds from D. superbus and evaluated their neuroprotective activity against glutamate-induced cell death in the hippocampal neuronal HT22 cells. MATERIALS AND METHODS: New compound, (E)-methyl-4-hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl) (1) and, nine known compounds, diosmetin-7-O (2'',6''-di-O-α-L-rhamnopyranosyl)-ß-D-glucopyranoside (2), 4-hydroxy-3-methoxy-pentyl ester benzenepropanoic acid (3), vanillic acid (4), 4-hydroxy-benzeneacetic acid (5), 4-methoxybenzeneacetic acid (6), (E)-4-methoxycinnamic acid (7), 3-methoxy-4-hydroxyphenylethanol (8), hydroferulic acid (9), and methyl hydroferulate (10), were isolated by bioactivity-guided separation. Structures of the isolated compounds were identified on the basis of (1)H nuclear magnetic resonance (NMR), (13)C NMR, and two-dimensional NMR spectra, while their neuroprotective properties were evaluated by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: D. superbus extract had a neuroprotective effect and isolated 10 compounds. Among the compounds, compounds 5 and 6 effectively protected HT22 cells against glutamate toxicity. CONCLUSION: In conclusion, the extract of D. superbus and compounds isolated from it exhibited neuroprotective properties, suggesting therapeutic potential for applications in neurotoxic diseases. SUMMARY: D. superbus extract significantly protected on glutamate-induced cell death in HT22 cellsNew compound, (E)-methyl-4-hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl) (1) and, nine known compounds, diosmetin-7-O(2'',6''-di-O-α-L-rhamnopyranosyl)-ß-D-glucopyranoside (2), 4-hydroxy-3-methoxy-pentyl ester benzenepropanoic acid (3), vanillic acid (4), 4-hydroxy-benzeneacetic acid (5), 4-methoxybenzeneacetic acid (6), (E)-4-methoxycinnamic acid (7), 3-methoxy-4-hydroxyphenylethanol (8), hydroferulic acid (9), and methyl hydroferulate (10) were isolated from D. superbus extract4-hydroxy-benzeneacetic acid and 4-methoxybenzeneacetic acid showed significant protective activity against glutamate-induced toxicity in HT22 cells. Abbreviations used: CNS: Central nervous system, ROS: Reactive oxygen species, CHCl3: Chloroform, EtOAc: Ethyl acetate, BuOH: Butanol, HPLC: High performance liquid chromatography, TLC: Thin layer chromatography, MPLC: Middle performance liquid chromatography, MeOH: Methanol, OD: Optical density, COSY: Correlation spectroscopy, HMQC: Heteronuclear multiple-quantum correlation, HMBC: Heteronuclear multiple-bond correlation, HR-MS: High-resolution molecular spectroscopy, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Inhibitory effect of fermented Arctium lappa fruit extract on the IgE-mediated allergic response in RBL­2H3 cells.

Arctium lappa fruit has been used in traditional medicine, and it is known to exert beneficial effects, such as antioxidant, anti-inflammatory and anticancer effects. However, the effects of the Arctium lappa fruit on the allergic response remain unknown. In this study, we evaluated the anti-allergic effects of Arctium lappa fruit extract (AFE) and its fermented form (F-AFE) using immunoglobulin E (IgE)-activated RBL­2H3 cells. To investigate the anti-allergic effects of AFE or F-AFE, we examined the release of ß-hexosaminidase, a key biomarker of degranulation during an allergic reaction, and the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) in the cells treated with or without the above-mentioned extracts. AFE weakly inhibited the release of ß-hexosaminidase, whereas F-AFE significantly suppressed the release of ß-hexosaminidase in a dose-dependent manner. Consistently, F-AFE suppressed the production of TNF-α and PGE2 in a dose-dependent manner. F-AFE exerted an inhibitory effect on the production of ß-hexosaminidase, TNF-α and PGE2 with an IC50 value of 30.73, 46.96 and 36.27 µg/ml, respectively. Furthermore, F-AFE inhibited the phosphorylation of Lyn, Fyn and Syk, which are involved in the FcεRI signaling pathway, that of phosphoinositide phospholipase C (PLC)γ1/2 and protein kinase C (PKC)δ, which are associated with the degranulation process, as well as that of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK), p38 and Akt, which are associated with cytokine expression. In the late phase, F-AFE partially suppressed the phosphorylation of cytosolic phospholipase A2 (cPLA2), but not the expression of cyclooxygenase (COX)-2. To compare and identify the major components of the two extracts, we used high-performance liquid chromatography. The levels of arctigenin, one of the major compounds, were elevated 6-fold in F-AFE compared with AFE, whereas the levels of arctiin, an arctigenin glycoside, were decreased in F-AFE by approximately 57.40%. These results suggest that arctigenin plays an important role in the anti-allergic effects of F-AFE. Taken together, F-AFE containing anti-allergic phytochemicals, including arctigenin, inhibited the activation of the FcεRI receptor induced by the antigen­IgE complex. Such effects may provide further information for the development of a phytomedicine for allergic diseases.

The novel herbal cocktail MA128 suppresses tumor growth and the metastatic potential of highly malignant tumor cells.

MA128, a novel herbal medicine, was previously identified and its effectiveness in the treatment of asthma and atopic dermatitis (AD) was demonstrated. In particular, post-inflammatory hyperpigmentation (PIH) in AD mice was improved by treatment with MA128. In addition, MA128 exhibited anti-melanogenic activity by inhibiting tyrosinase activity via the p38 MAPK and protein kinase A signaling pathways in B16F10 cells. In the present study, we examined whether oral administration of MA128 suppressed the in vivo tumor growth of HT1080 cells in athymic nude mice. The results showed that the daily oral administration of 75 and 150 mg/kg MA128 suppressed the tumorigenic growth of HT1080 cells efficiently. Since metastasis is a major cause of cancer-associated mortality and the greatest challenge during cancer treatment, we investigated the effect of non-toxic concentrations of MA128 on the metastatic potential of HT1080 cells. MA128 inhibited anchorage-independent colony formation, migration and invasion. Matrix metalloproteinase-9 (MMP-9) activity under resting and PMA-stimulated conditions was decreased in a dose-dependent manner by MA128 in HT1080 cells. In addition, the daily oral administration of MA128 at doses of 75 and 150 mg/kg efficiently blocked the lung metastasis of B16F10 cells that had been injected into the tail veins of C57BL/6 mice. In particular, none of the mice treated with MA128 exhibited systemic toxicity, such as body weight loss or liver and kidney dysfunction. MA128 also inhibited tumor­induced angiogenesis. Taken together, the results suggest that MA128 is a potential therapeutic agent and a safe herbal medicine for controlling malignant and metastatic cancer.

Jaeumganghwa-Tang Induces Apoptosis via the Mitochondrial Pathway and Lactobacillus Fermentation Enhances Its Anti-Cancer Activity in HT1080 Human Fibrosarcoma Cells.

Jaeumganghwa-tang (JGT, Zi-yin-jiang-huo-tang in Chinese and Jiin-koka-to in Japanese) is an oriental herbal formula that has long been used as a traditional medicine to treat respiratory and kidney diseases. Recent studies revealed that JGT exhibited potent inhibitory effects on allergies, inflammation, pain, convulsions, and prostate hyperplasia. Several constituent herbs in JGT induce apoptotic cancer cell death. However, the anti-cancer activity of JGT has not been examined. In this study, we investigated the anti-cancer effects of JGT using highly tumorigenic HT1080 human fibrosarcoma cells and elucidated the underlying mechanisms. In addition, we examined whether the Lactobacillus fermentation of JGT enhanced its anti-cancer activity using an in vivo xenograft model because fermentation of herbal extracts is thought to strengthen their therapeutic effects. Data revealed that JGT suppressed the growth of cancer cells efficiently by stimulating G1 cell cycle arrest and then inducing apoptotic cell death by causing mitochondrial damage and activating caspases. The phosphorylation of p38 and ERK also played a role in JGT-induced cell death. In vitro experiments demonstrated that JGT fermented with Lactobacillus acidophilus, designated fJGT162, elicited similar patterns of cell death as did non-fermented JGT. Meanwhile, the daily oral administration of 120 mg/kg fJGT162 to HT1080-bearing BALB/c nude mice suppressed tumor growth dramatically (up to 90%) compared with saline treatment, whereas the administration of non-fermented JGT suppressed tumor growth by ~70%. Collectively, these results suggest that JGT and fJGT162 are safe and useful complementary and alternative anti-cancer herbal therapies, and that Lactobacillus fermentation improves the in vivo anti-cancer efficacy of JGT significantly.

Acute toxicity and genotoxicity study of fermented traditional herb formula Guibi-tang.

ETHNOPHARMACOLOGY RELEVANCE: Guibi-tang (Guipi-tang in Chinese and Kihi-to in Japanese) is a multi-herb traditional medicine commonly prescribed to treat psychoneurosis in East Asia. Although this medicine has been widely used, there is little available information on the safety and toxicity of Guibi-tang, especially on the fermented one. MATERIALS AND METHODS: Guibi-tang, composed of 12 herbs, was fermented with bacteria and lyophilized. Single dose acute toxicity in rats was observed for 14 days after administration. Genetic toxicity of fermented Guibi-tang was evaluated on bacterial reverse mutation in Salmonella and Escherichia spp., chromosome aberrations in Chinese hamster ovary cells, and micronucleus formation in mice. Ingredients in FGBT were identified and quantified by high performance liquid chromatography-mass spectrometry. RESULTS: In acute oral toxicity study, behavior, clinical signs and body weight changes were normal observing in all experimental animals. No revertant colonies were found in any bacterial cultures examined. Morphological or numerical anomalies and significant increased number of aberrant metaphases were not observed. Micronucleus assay showed no significant increases in the frequency of inducing micronuclei in any dose examined. Decursinol, decursin, glycyrrhizin, and 6-gingerol in fermented Guibi-tang were identified and quantitated. As a whole, no acute and genotoxic effects were found in all the assays and parameters analyzed. CONCLUSION: Fermented Guibi-tang was recognized as safe and non-toxic, and therefore can be used for applications of traditional medicine in modern complementary and alternative therapeutics and health care.