Yeokwisan, a Standardized Herbal Formula, Enhances Gastric Emptying via Modulation of the Ghrelin Pathway in a Loperamide-induced Functional Dyspepsia Mouse Model.

Background: Yeokwisan, a standardized herbal formula, has exhibited clinical benefit for patients suffering from refractory functional dyspepsia (FD) in Korea since 2016. However, data about the mechanism of action of this formula are yet not available. Aim of the study: To evaluate and explore the effects of Yeokwisan on gastric emptying, a major symptom of functional dyspepsia, and its underlying mechanisms of action using a mouse model. Materials and methods: BALB/C mice were pretreated with Yeokwisan (100, 200, and 400 mg/kg, po) or mosapride (3 mg/kg, po) for 5 days and then treated with loperamide (10 mg/kg, ip) after 20 h of fasting. A solution of 0.05% phenol red (500 µL) or diet of 5% charcoal (200 µL) was orally administered, followed by assessment of gastric emptying or intestinal transit. Plasma acyl-ghrelin (ELISA), C-kit (immunofluorescence and western blotting), nNOS (western blotting) and gastric contraction- and ghrelin-related gene/protein expression levels were examined in stomach and small intestine tissues. Results: Loperamide injection substantially delayed gastric emptying, while Yeokwisan pretreatment (especially 200 and 400 mg/kg Yeokwisan) significantly attenuated this peristaltic dysfunction, as evidenced by the quantity of phenol red retained in the stomach (p < 0.05 or 0.01) and stomach weight (p < 0.05 or 0.01). The levels of plasma acyl-ghrelin and expression of gastric ghrelin-related genes, such as growth hormone secretagogue receptor (GHSR), ghrelin-O-acyltransferase (GOAT), adrenergic receptor ß1 (ADRB1) and somatostatin receptor (SSTR), were significantly normalized (p < 0.05 or 0.01) by Yeokwisan (400 mg/kg). Yeokwisan (400 mg/kg) significantly tempered the loperamide-induced alterations in the c-kit and nNOS levels (p < 0.01) as well as the expression of contraction- and ghrelin-related genes, such as 5-HT4 receptor (5-HT4R), anoctamin-1 (ANO1), ryanodine receptor 3 (RYR3) and smooth muscle myosin light chain kinase (smMLCK), in the stomach, but not in the small intestine. Conclusion: The present results showed the clinical relevance of Yeokwisan, in treating FD, especially in promoting gastric emptying but not small intestinal transit. The main mechanisms corresponding to these effects may involve the modulation of the ghrelin pathway and activation of interstitial cells of Cajal in stomach tissue.

Genotoxicity of Water Extract from Bark-Removed Rhus verniciflua Stokes.

Rhus verniciflua Stokes (RVS) has been traditionally used as an herbal remedy to support the digestive functions in traditional Korean medicine. Additionally, the pharmacological effects of RVS, including antioxidative, antimicrobial and anticancer activities, have been well-reported. The genotoxicity of RVS, however, is elusive; thus, we evaluated the genotoxicity of RVS without bark (RVX) for safe application as a resource of functional food or a medical drug. To evaluate the genotoxicity of RVX, we used a bacterial reverse mutation test, chromosomal aberration test and comet assay, according to the "Organization for Economic Co-operation and Development" (OECD) guidelines. Briefly, for the reverse mutation test, samples (5000, 1667, 556, 185, 62 and 0 µg/plate of RVX or the positive control) were treated with a precultured strain (TA98, TA100, TA1535, TA1537 or WP2µvrA) with or without the S9 mix, in which RVX partially induced a reverse mutation in four bacterial strains. From the chromosomal aberration test and comet assay, the RVX samples (556, 185, 62, 20 and 0 µg/mL of RVX or the positive control) were treated in a Chinese hamster ovary cell line (CHO-K1 cells) in the conditions of the S9 mix absent or S9 mix present and in Chang liver cells and C2C12 myoblasts, respectively. No chromosomal aberrations in CHO-K1 or DNA damage in Chang liver cells and C2C12 myoblasts was observed. In conclusion, our results suggest the non-genotoxicity of RVX, which would be helpful as a reference for the safe application of bark-removed Rhus verniciflua Stokes as functional raw materials in the food, cosmetics or pharmaceutical fields.

A standardized herbal combination of Astragalus membranaceus and Paeonia japonica, protects against muscle atrophy in a C26 colon cancer cachexia mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus membranaceus (Fisch.) and Bunge and Paeonia japonica (Makino)Miyabe & H.Takeda have been traditionally used to improve the poor quality of life such as weakness, lack of appetite, fatigue, and malaise which is considered with cachexia condition. AIM OF THE STUDY: We investigated anti-cachectic effects of a herbal formula composed of Astragalus membranaceus and Paeonia japonica (APX) and the molecular mechanisms of APX in C26 cancer-induced cachexia mice and TNF-a-treated C2C12 myotubes. Additionally synergistic anti-cachectic effects of APX were compared to those of individual herbal extracts and megestrol acetate. METHODS AND MATERIALS: The forty-two BALB/c mice were randomly divided into 6 groups: normal (nontreatment), control (C26 injection), AM (C26 injection with Astragalus membranaceus), PJ (C26 injection with Paeonia japonica), APX (C26 injection with combination of Astragalus membranaceus and Paeonia japonica and MA (C26 injection with megestrol acetate). All mice were orally administered DW (normal and control groups) or 100 mg/kg AM, PJ, APX or MA for 10 days. In the animal model, several tissues were weighed, and muscle tissue and blood were used to measure pro-inflammatory cytokines. C2C12 myotubes were exposed to 100 ng/mL TNF- α with or without 10 µg/mL of AM, PJ, APX or MA for 48 h. The cells were used to immunofluorescence staining and western blot analyses. RESULTS: C26 injection induced notable body and muscle weight loss while APX administration significantly attenuated these alterations and the decrease of muscle weights and strength. APX also significantly attenuated the abnormal elevations in the concentration of three muscle atrophy-inducible cytokines; serum and muscle TNF-α,muscle TWEAK and IL-6 in C26 tumor-bearing mice. In the TNF-α-treated C2C12 myotube model, TNF-α treatment notably decreased MyH but activated atrophic proteins (MuRF and Fbx32) along with p38 and NFκB while these molecular alterations were significantly ameliorated by APX treatment. These pharmacological actions of APX were supported by the results of immunofluorescence staining to MyH expression and the translocation of NFκB into the nucleus in C2C12 myotubes. CONCLUSIONS: Our data indicate the potential of an herbal formula, APX as an anti-cachexia agent; the effect of APX was superior to that of megestrol acetate overall especially for muscle atrophy. The underlying mechanisms of this herbal formula may involve the modulation of muscle atrophy-promoting molecules including p38, NFκB, TNF-α and TWEAK.

Synergistic hepatoprotective effects of CGplus on CCl4-induced acute injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese/Korean medicine suggests "blood stasis ()", "stagnation of vital energy ()" and "dampness and Phlegm ()" as the main etiologies of liver disorders, and multiherbal formulas are generally believed to exert synergistic action. AIM OF THE STUDY: The present study aimed to investigate the synergistic hepatoprotective effects of CGplus (a mixture of Salviae miltiorrhiza, Artemisia iwayomogi and Ammomum xanthioides) compared to those of the individual herbs. METHODS AND MATERIALS: A total of fifty-six male Balb/C mice were randomly divided into eight groups and were administered water (normal and CCl4 groups), 100 mg/kg S. miltiorrhiza, A. iwayomogi, or A. xanthioides, 50 or 100 mg/kg CGPlus or dimethyl dimethoxybiphenyl dicarboxylate (DDB) as a positive control for 4 consecutive days. After a single CCl4 injection (i.p., 10 mL/kg of 0.2% CCl4 in olive oil), blood and liver tissues were collected after 18 h of fasting for serum biochemistry, histopathological examination and molecular analyses. RESULTS: CCl4 injection induced drastic hepatic injury characterized by a more than 30-fold increase in the release of AST and ALT into the serum. These alterations were significantly attenuated by pretreatment with each of the three herbs, while the effects of the individual herbs were synergistically augmented by CGPlus pretreatment. The synergistic hepatoprotective actions of CGPlus were demonstrated consistently by analyses of oxidative stress (oxidative stressors, oxidation products and antioxidant enzymes), pro-/anti-inflammatory cytokines (TNF-ɑ, IL-1ß, IL-6, IL-10), and apoptosis (caspase-3, p53 and BAX) and histopathology. CONCLUSIONS: These data suggest that CGPlus exerts its hepatoprotective effects in a synergistic manner, and further studies are required for clinical application using other chronic models.

Banha-sasim-tang improves gastrointestinal function in loperamide-induced functional dyspepsia mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Banha-sasim-tang (BST; Hange-shashin-to in Kampo medicine; Banxia xiexin tang in traditional Chinese medicine) is a traditional Chinese harbal medicine that has been commonly used for gastrointestinal disorders. AIM OF THE STUDY: To investigate the pharmacological effects of BST, a standardized herbal drug, on main symptoms of functional dyspepsia including delayed gastric emptying, and underlying mechanisms of action in mouse model. METHODS AND MATERIALS: Balb/C mice were pretreated with BST (25, 50, 100 mg/kg, po) or mosapride (3 mg/kg, po) for 3 days, and then treated with loperamide (10 mg/kg, ip) after 19 h fasting. A solution of 0.05% phenol red (500 µL) or 5% charcoal diet (200 µL) was orally administered, followed by scarifying and assessment of gastric emptying or gastro-intestinal motility. C-kit (immunofluorescence), nNOS (western blot) and gastric contraction-related gene expression were examined in stomach tissue. RESULTS: The loperamide injection substantially delayed gastric emptying, while the BST pretreatment significantly attenuated this peristaltic dysfunction, as evidenced by the quantity of stomach-retained phenol red (p < 0.05 or 0.01) and stomach weight (p < 0.05 or 0.01). The BST pretreatment significantly tempered the loperamide-induced inactivation of c-kit and nNOS (p < 0.05 or 0.01) as well as the contraction-related gene expression, such as the 5HT4 receptor (5HT4R), anoctamin-1 (ANO1), ryanodine receptor 3 (RYR3) and smooth muscle myosin light chain kinase (smMLCK). The BST pretreatment also significantly attenuated the alterations in gastro-intestinal motility (p < 0.01). CONCLUSION: Our results are the first evidence of the prokinetic agent effects of Banha-sasim-tang in a loperamide-induced FD animal model. The underlying mechanisms of action may involve the modulation of peristalsis via activation of the interstitial cells of Cajal and the smooth muscle cells in the stomach.

Standardization of the Manufacturing Process of Bee Venom Pharmacopuncture Containing Melittin as the Active Ingredient.

BACKGROUND: Pharmacopuncture is a unique treatment in oriental medicine that combines chemical stimulation with conventional acupuncture. However, there are no standardized methods for preparing the herbal medicines used in pharmacopuncture, and it is not clear whether the active ingredients are safe and stable. Several studies have investigated nonstandardized preparation processes, but few investigations have addressed safety and preparation methods. Pharmacopuncture may provide an alternative treatment for incurable diseases. However, it must be as valid and safe as standardized medicine. In this way, the present project may contribute to the industrialization of medicine in Korea. It may also expand health insurance coverage by promoting evidence-based medical insurance benefits. Thus, the present study attempted to standardize and improve the raw materials, preparation, and efficacy of bee venom pharmacopuncture (BVP), which is a highly effective technique in oriental medicine. METHOD: To purify the crude bee venom, the extract was subjected to a stepped-gradient open column (ODS-A; 120 Å, 150 mesh). Using this method, the yield of melittin was significantly increased and the allergen proteins were effectively removed. The melittin content of the purified bee venom was determined using HPLC, and the product was then diluted to 0.1 mg/mL using injection water in preparation for BVP. RESULTS: In the present study, we standardized the purification process to provide safe and stable BVP by increasing the main effective components and eliminating allergens. This study will be seminal in the industrialization and regulation of BVP. CONCLUSION: We developed an effective strategy for melittin purification and allergen removal from bee venom to create safe BVP.

Anti-inflammatory Effects of KOTMIN13: A Mixed Herbal Medicine in LPS-stimulated RAW 264.7 Cells and Mouse Edema Models.

BACKGROUND: A Korean herbal medicine, KOTMIN13, composed of Inula japonica Thunberg, Trichosanthes kirilowii Maximowicz var. japonica kitamura, Peucedanum praeruptorum Dunn, and Allium macrostemon Bge, has been used for anti-allergic and anti-asthmatic treatment in oriental clinics, but its activity has not been investigated. MATERIALS AND METHODS: To evaluate the anti-inflammatory activity of KOTMIN13 for in vitro study, LPS-stimulated RAW 264.7 cells were used to induce the production and expression of inflammatory mediators and its mechanisms. 12-O-Tetradecanoylphorobol-13 aceate (TPA)-induced ear edema and carrageenan-induced paw edema models were also used to evaluate the effect of KOTMIN13 on acute inflammation in vivo. RESULTS: KOTMIN13 reduced the release of inflammatory mediators [nitric oxide, prostaglandin E2, interleukin (IL)-1ß, and IL-6] and the protein expression of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-stimulated RAW 264.7 cells. Mechanism studies showed the attenuation of LPS-induced NF-κB activation by KOTMIN13 via IκBα degradation abrogation and a subsequent decrease in nuclear p65 levels. Activation of mitogen-activated protein kinases (ERK, JNK, and p38) was also suppressed. Furthermore, KOTMIN13 ameliorated the development of TPA-induced ear edema and carrageenan-induced paw edema in acute inflammatory edema mouse models. CONCLUSION: Our study demonstrates that KOTMIN13 inhibits inflammatory mediators through the inhibitions of NF-κB and MAPK activities in LPS-induced RAW 264.7 cells, as well as acute inflammation in edema models, indicating that KOTMIN13 is an effective suppressor for anti-inflammatory activities. SUMMARY: KOTMIN13 decrease the production of No, PGE2, and proinflammatory cytokine (TNF-∝, IL-1ß,IL-6).KOTMIN13 Suppressed the degradation of NF-kß and IKßα and the phosorylation of MAP Kinases.Topical application of KOTMIN13 reduced mouse ear edema.Oral administration of KOTMIN13 decreased carrageenan-induced paw edema. Abbreviations used: NO: nitric oxide; PGE2: prostaglandin E2; iNOS: inducible NO synthase; COX-2: cyclooxygenase-2; TNF-α: tumor necrosis factor-α; IL: interleukin; NF-κB: nuclear factor kappaB; MAPK: mitogen-activated protein kinases; ERK: extracellular signal regulated kinase; JNK: c-jun N terminal kinase; TPA: 12-O-tetradecanoylphorbol-13-acetate.

KOTMIN13, a Korean herbal medicine alleviates allergic inflammation in vivo and in vitro.

BACKGROUND: The ethanol extract of KOTMIN13, composed of Inula japonica Flowers, Trichosanthes kirilowii Semen, Peucedanum praeruptorum Radix, and Allium macrostemon Bulbs, was investigated for its anti-asthmatic and anti-allergic activities. METHODS: The anti-asthmatic effects of KOTMIN13 were evaluated on ovalbumin (OVA)-induced murine asthma model. Anti-allergic properties of KOTMIN13 in bone-marrow derived mast cells (BMMC) and passive cutaneous anaphylaxis (PCA) in vivo were also examined. RESULTS: In asthma model, KOTMIN13 effectively suppressed airway hyperresponsiveness induced by aerosolized methacholine when compared to the levels of OVA-induced mice. KOTMIN13 treatment reduced the total leukocytes, eosinophil percentage, and Th2 cytokines in the bronchoalveolar lavage fluids in OVA-induced mice. The increased levels of eotaxin and Th2 cytokines in the lung as well as serum IgE were decreased by KOTMIN13. The histological analysis shows that the increased inflammatory cell infiltration and mucus secretion were also reduced. In addition, the degranulation and leukotriene C4 production were inhibited in BMMC with IC50 values of 3.9 µg/ml and 1.7 µg/ml, respectively. Furthermore, KOTMIN13 treatment attenuated mast-mediated PCA reaction. CONCLUSIONS: These results demonstrate that KOTMIN13 has anti-asthmatic and anti-allergic effects in vivo and in vitro models.

Anti-allergic effect of a Korean traditional medicine, Biyeom-Tang on mast cells and allergic rhinitis.

BACKGROUND: Biyeom-Tang, a medicine prescribed by oriental clinics, has been used for the treatment of the allergic rhinitis (AR). In the present study, an ethanol extract of Biyeom-Tang (EBT) was investigated for anti-allergic properties on bone-marrow derived mast cells (BMMC) and in vivo models. METHODS: The anti-allergic properties of EBT were evaluated by measuring ß-Hex release and the production of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) on BMMC in vitro and PCA and OVA-induced AR models in vivo. RESULTS: EBT strongly inhibited a degranulation reaction in a dose dependent manner with an IC50 value of 35.6 µg/ml. In addition, the generation of PGD2 and LTC4 was inhibited in BMMC in a concentration-dependent manner with IC50 values of 7.0 µg/ml and 10.9 µg/ml, respectively. When administrated orally, EBT ameliorated the mast cell-mediated PCA reaction. In the OVA-induced AR model, the increased levels of IgE were reduced by EBT. The levels of cytokines, such as IL-4, IL-5, IL-10, and IL-13 decreased in the splenocytes of EBT-treated mice. The histological analysis shows that the infiltration of inflammatory cells increased by OVA-sensitization was also reduced. CONCLUSIONS: Taken together, these results suggested that EBT has anti-allergic and anti-inflammatory effects in vitro and in vivo models.