Evodiae Fructus extract suppresses inflammatory response in HaCaT cells and improves house dust mite-induced atopic dermatitis in NC/Nga mice.

This study was conducted to assess the effect of Evodiae Fructus 70% ethanol extract (EFE) on the pathology of atopic dermatitis using in vitro and in vivo models. The major compounds in EFE were identified by ultra-performance liquid chromatography with tandem mass spectrometry as rutaecarpine, evodiamine, evodol, dehydroevodiamine, limonin, synephrine, evocarpine, dihydroevocarpine, and hydroxyevodiamine. EFE significantly decreased chemokine levels in tumor necrosis factor-α/interferon-γ-stimulated HaCaT cells. In house dust mite-treated NC/Nga mice, topical application of EFE significantly decreased the dermatitis score, epidermal hyperplasia and thickening, mast cell infiltration, and plasma levels of histamine and corticosterone. Thymic stromal lymphopoietin, CD4+ T cells, interleukin-4, and intercellular adhesion molecule-1 expression in the lesioned skin was reduced in the treated mice. The mechanism of EFE was elucidated using transcriptome analysis, followed by experimental validation using Western blotting in HaCaT cells. EFE down-regulated the activation of Janus kinase (JAK)-signal transducers and activators of transcription (STAT) and mitogen-activated protein kinases (MAPK) signaling pathways in HaCaT cells. EFE improves atopic dermatitis-like symptoms by suppressing inflammatory mediators, cytokines, and chemokines by regulating the JAK-STAT and MAPK signaling pathways, suggesting its use as a potential agent for the treatment of atopic dermatitis.

Anti-inflammatory and anti-allergic effects of Cheonwangbosim-dan water extract: An in vitro and in vivo study.

Ethnopharmacological relevance: Cheonwangbosim-dan is a traditional herbal prescription that is widely used to improve or treat physical and mental illnesses in East Asian countries.Aim of the study: The aim of the present study was to investigate the preventive and protective effects of a Cheonwangbosim-dan water extract (CBDW) against allergic inflammation using in vitro and in vivo models. Materials and methods: BEAS-2B and MC/9 cells were treated with various concentrations of CBDW and stimulated with different inducers of inflammatory mediators. The production of various inflammatory mediators was subsequently evaluated. BALB/c mice were sensitized and challenged by repeated application of ovalbumin (OVA). CBDW was administered by oral gavage once daily for 10 consecutive days. We assessed the number of inflammatory cells and production of Th2 cytokines in bronchoalveolar lavage fluid (BALF), the plasma levels of total and OVA-specific immunoglobulin E (IgE), and histological changes in lung tissue. Results: Our findings showed that CBDW significantly decreased the levels of various inflammatory mediators (eotaxin-1, eotaxin-3, RANTES, LTC4, TNF-α, MMP-9, 5-LO, ICAM-1, and VCAM-1) in vitro, significantly reduced the accumulation of total inflammatory cells, the production of Th2 cytokines (IL-5 and IL-13), the levels of IgE (total and OVA-specific) in vivo, and remarkably inhibited histological changes (infiltration of inflammatory cells and goblet cell hyperplasia) in vivo. Conclusions: These results suggest that CBDW possesses anti-inflammatory and anti-allergic properties by lowering allergic inflammation.

In Vitro and In Vivo Genotoxicity Assessments and Phytochemical Analysis of the Traditional Herbal Prescription Siryung-Tang.

Siryung-tang (SRT) is a traditional herbal prescription containing Oryeong-san and Soshiho-tang that is used to treat digestive system diseases. We performed safety evaluations of SRT based on genotoxicity and developed an assay for quality control using high-performance liquid chromatography with a photodiode array detector. Genotoxicity was evaluated based on bacterial reverse mutation (Salmonella typhimurium TA1535, TA98, TA100, and TA1537, and Escherichia coli WP2 uvrA), chromosomal aberration (Chinese hamster lung cells), and micronucleus (mouse) tests. Quality control analysis was conducted using a SunFire C18 column and gradient elution with a distilled water-acetonitrile mobile phase system containing 0.1% (v/v) formic acid for 12 markers (5-(hydroxy-methyl)furfural, 3,4-dihydroxybenzaldehyde, liquiritin apioside, liquiritin, coumarin, baicalin, wogonoside, cinnamaldehyde, baicalein, glycyrrhizin, wogonin, and atractylenolide III). SRT showed no genotoxicity in three tests. Ames tests showed that SRT at 313-5000 µg/plate did not significantly increase the number of revertant colonies with or without metabolic activation among five bacterial strains. Moreover, in vivo micronucleus testing showed that SRT did not increase the frequency of bone marrow micronuclei. The number of chromosomal aberrations associated with SRT was similar to that observed in the negative controls. The 12 markers were detected at 0.04-16.86 mg/g in a freeze-dried SRT sample and completely eluted within 45 min. The extraction recovery was 95.39-104.319% and the relative standard deviation value of the precision was ≤2.09%. Our study will be used as basic data for the safety and standardization of SRT.

Ojeok-san ameliorates visceral and somatic nociception in a mouse model of colitis induced colorectal cancer.

Cancer patients can develop visceral, somatic, and neuropathic pain, largely due to the malignancy itself and its treatments. Often cancer patients and survivors turn to the use of complementary and alternative medicine (CAM) to alleviate pain and fatigue. Thus, it is necessary to investigate how CAM therapies work as novel analgesics to treat cancer pain. Ojeok-san (OJS) is an herbal formula consisting of seventeen herbs. This herbal formula has been shown to possess anti-inflammatory, immunoregulatory, and analgesic properties. In this study, we examined the potential beneficial effects and mechanism of action of OJS in a preclinical model of colitis-associated colorectal cancer. Male and female C57BL/6J mice were exposed to the carcinogen, azoxymethane (AOM, 10 mg/kg) and a chemical inflammatory driver, dextran sulfate sodium (DSS1-2%), to promote tumorigenesis in the colorectum. OJS was given orally (500, 1000, and 2000 mg/kg) to determine its influence on disease activity, tumor burden, nociception, sedation, Erk signaling, and behavioral and metabolic outcomes. In addition, in vitro studies were performed to assess CT-26 cell viability, dorsal root ganglia (DRG) activation, and bone-marrow-derived macrophage (BMDM) inflammatory response to lipopolysaccharide stimulation after OJS treatment. We found that administration of 2000 mg/kg of OJS was able to mitigate mechanical somatic and visceral nociception via Erk signaling without affecting symptom score and polyp number. Moreover, we discovered that OJS has sedative properties and elicits prolonged total sleeping time in AOM/DSS mice. Our in vitro experiments showed that OJS has the capacity to reduce TNFα gene expression in LPS-stimulated BMDM, but no changes were observed in DRG spike number and CT-26 cell proliferation. Taken together, these data suggest that OJS ameliorates nociception in mice and warrants further examination as a potential CAM therapy to promote analgesia.

Amelioration of Hypertension by Oryeongsan through Improvements of Body Fluid and Sodium Balance: Roles of the Renin-Angiotensin System and Atrial Natriuretic Peptide System.

Oryeongsan (Wulingsan in China and Goreisan in Japan), a formula composed of five herbal medicines, has long been used for the treatment of imbalance of the body fluid homeostasis in Asian countries. However, the mechanism by which Oryeongsan (ORS) improves the impaired body fluid and salt metabolism is not clearly defined. The present study was performed to define the role of the cardiorenal humoral system in the ORS-induced changes in blood pressure and renal function in hypertension. Experiments were performed in normotensive and two-kidney, one-clip hypertensive rats. Changes in the fluid and salt balance were measured in rats individually housed in metabolic cages. Changes in the systemic and local renin-angiotensin system (RAS) and cardiac natriuretic peptide hormone system (NPS) were evaluated. ORS water extract was administered by oral gavage (100 mg/kg daily) for 3 weeks. ORS induced diuresis and natriuresis along with an increase in glomerular filtration rate and downregulation of the Na+/H+ exchanger 3 (NHE3) and aquaporin 2 expression in the renal cortex and medulla, respectively. Furthermore, treatment with ORS significantly decreased systolic blood pressure with contraction of body sodium and water accumulation in hypertensive rats. ORS-induced changes were accompanied by modulation of the RAS and NPS, downregulation of the systemic RAS and cardiorenal expression of angiotensin-converting enzyme (ACE) and angiotensin II subtype 1 (AT1) receptor, and upregulation of the plasma ANP concentration and cardiorenal expression of ANP, ACE2, Mas receptor, and AT2 receptor. These findings indicate that ORS induces beneficial effects on the high blood pressure through modulation of the RAS and NPS of the cardiorenal system, suppression of the prohypertensive ACE-AT1 receptor pathway and NHE3, accentuation of the antihypertensive ACE2-Mas axis/AT2 receptor pathway in the kidney, suppression of the systemic RAS, and elevation of the plasma ANP levels and its synthesis in the heart. The present study provides a biological basis for the use of ORS in the treatment of impaired volume and pressure homeostasis.

Simultaneous Analysis of 19 Marker Components for Quality Control of Oncheong-Eum Using HPLC-DAD.

Oncheong-eum (OCE) is a traditional herbal prescription made by combining Samul-tang and Hwangryunhaedok-tang. It is primarily used to treat gynecological disorders such as metrorrhagia and metrostaxis. In the present study, we focused on developing and validating a simultaneous assay for the quality control of OCE using 19 marker components (gallic acid, 5-(hydroxymethyl)furfural, chlorogenic acid, geniposide, coptisine chloride, jatrorrhizine chloride, paeoniflorin, berberine chloride, palmatine chloride, ferulic acid, nodakenin, benzoic acid, baicalin, benzoylpaeoniflorin, wogonoside, baicalein, wogonin, decursin, and decursinol angelate). This analysis was performed using high-performance liquid chromatography coupled with a diode array detector, and chromatographic separation of the 19 markers was carried out using a SunFireTM C18 reversed-phase column and gradient elution conditions with two mobile phases (0.1% aqueous formic acid-0.1% formic acid in acetonitrile). The developed analytical method was validated through linearity, limits of detection and quantification, recovery, and precision. Under this assay, 19 markers in OCE samples were detected at not detected-9.62 mg/g. The analytical methods developed and validated in our research will have value as basic data for the quality control of related traditional herbal prescriptions as well as OCE.

Combination therapy with tamsulosin and traditional herbal medicine for lower urinary tract symptoms due to benign prostatic hyperplasia: A double-blinded, randomized, pilot clinical trial.

OBJECTIVES: To evaluate the efficacy and safety of tamsulosin and Hachimijiogan or Ryutanshakanto in patients with lower urinary tract symptoms due to benign prostatic hyperplasia. METHODS: A prospective, randomized, double-blind method was used to determine the efficacy and safety of the combination or placebo at baseline and 4, 8, and 12 weeks of study. The International Prostate Symptom Score, quality of life index, complete voiding diary, and National Institutes of Health-Chronic Prostatitis Symptom Index were studied. Uroflowmetery and postvoid residual urine volume were measured and compared. Laboratory tests including prostate-specific antigen were performed. RESULTS: In all groups, International Prostate Symptom Score and quality of life showed improvement, but no significant differences were shown among the groups. Prostate volume increased after treatment, and uroflowmetric parameters showed improvements after treatment without significance among the three groups. The total score of the National Institutes of Health-Chronic Prostatitis Symptom Index showed a significant improvement in all groups, without significant differences among the groups. Only the pain sub-score of the National Institutes of Health-Chronic Prostatitis Symptom Index showed a significant decrease in the tamsulosin with Ryutanshakanto group compared to the control group. A total of 11 adverse reactions occurred, but they were mild and not related to the study drugs. CONCLUSION: Ryutanshakanto can provide pain relief in patients with chronic prostatitis and chronic pelvic pain syndrome. If more research is conducted, Hachimijiogan and Ryutanshakanto may be applied as add-on treatments in patients with storage symptoms with alpha-blocker monotherapy.

Simultaneous Analysis for Quality Control of Traditional Herbal Medicine, Gungha-Tang, Using Liquid Chromatography-Tandem Mass Spectrometry.

Gungha-tang (GHT), a traditional herbal medicine, consists of nine medicinal herbs (Cnidii Rhizoma, Pinelliae Tuber, Poria Sclerotium, Citri Unshius Pericarpium, Citri Unshius Pericarpium Immaturus, Aurantii Fructus Immaturus, Atracylodis Rhizoma Alba, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens). It has been used for various diseases caused by phlegm. This study aimed to develop and verify the simultaneous liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis method, using nine marker components (liquiritin apioside, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, and 6-shogaol) for quality control of GHT. LC-MS/MS analysis was conducted using a Waters TQ-XS system. All marker analytes were separated on a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) using gradient elution with a distilled water solution (containing 5 mM ammonium formate and 0.1% [v/v] formic acid)-acetonitrile mobile phase. LC-MS/MS multiple reaction monitoring (MRM) analysis was carried out in negative and positive ion modes of an electrospray ionization source. The developed LC-MS/MS MRM method was validated by examining the linearity, limits of detection and quantification, recovery, and precision. LOD and LOQ values of nine markers were calculated as 0.02-8.33 ng/mL and 0.05-25.00 ng/mL. The recovery was determined to be 89.00-118.08% and precision was assessed with a coefficient of variation value of 1.74-8.64%. In the established LC-MS/MS MRM method, all markers in GHT samples were detected at 0.003-16.157 mg/g. Information gathered during the development and verification of the LC-MS/MS method will be useful for the quality assessment of GHT and other herbal medicines.

Genotoxicity of Asiasari Radix et Rhizoma (Aristolochiaceae) ethanolic extract in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional herbal medicines have diverse efficacy and are increasingly used worldwide. However, some of these herbal medicines have toxicities or side effects, but the scientific understanding of traditional herbal medicine toxicity has not yet been established. Asiasari Radix et Rhizoma (ARE) is known as a herbal medicine used to relieve pain, and recent studies have shown that ARE has anticancer and antimelanogenesis efficacy. AIM OF THE STUDY: Current study was conducted to assess the potential genotoxicity of an ethanolic extract of ARE. MATERIALS AND METHODS: The genotoxixity of ARE was confirmed by the bacterial reverse mutation assay (Ames test), a mammalian chromosomal aberration test, and a micronucleus test in vivo using ICR mice and comet assay using Sprague-Dawley rats. RESULTS: ARE showed no genotoxicity in a micronucleus test up to 2000 mg/kg body weight in vivo. By contrast, the chromosomal aberration test showed that ARE induced an increase in the number of chromosomal aberrations after treatment for 6 h with a metabolic activation system and for 6 and 22 h without the metabolic activation system when compared with vehicle control. In the Ames test, all strains except TA1535, with or without a metabolic activation system, showed an increase in the number of revertant mutant colonies in the ARE-treated group. In comet assay, DNA damage was observed in the stomach when ARE was administered. CONCLUSION: ARE potentially shows genotoxicity by inducing DNA damage.

Quality assessment of traditional herbal formula, Hyeonggaeyeongyo-tang through simultaneous determination of twenty marker components by HPLC-PDA and LC-MS/MS.

Simultaneous analysis of 20 marker components (gallic acid, cimifugin, geniposide, paeoniflorin, ferulic acid, nodakenin, narirutin, naringin, neohesperidin, arctiin, baicalin, oxypeucedanin hydrate, wogonoside, baicalein, arctigenin, glycyrrhizin, wogonin, pulegone, decursin, and decursinol angelate) for quality assessment of the traditional herbal formula, Hyeonggaeyeongyo-tang (HYT) was carried out by using high-performance liquid chromatography (HPLC) with photodiode array detection (PDA) and liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS/MS). The coefficient of determination showed excellent linearity of more than 0.9999 for all analytes. The recovery of 20 marker components was 93.92 to 102.66% with relative standard deviation (RSD) < 3.00% and RSD value of precision was ≤ 3.44%. The amounts of 20 marker components using HPLC-PDA and LC-MS/MS were determined to be 0.18-14.60 and 0.01-1.76 mg/freeze-dried g, respectively.

Subchronic toxicological evaluation of Bojungikki-tang water extract: 13-Week oral repeated-dose toxicity study in Crl:CD (SD) rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Bojungikki-tang is a traditional herbal medicine used to boost immunity and reduce fatigue. However, there is not enough scientific evidence about its toxicological safety profile to support its continued clinical application. AIM OF THE STUDY: The objective of this study was to investigate the subchronic toxicity profile of Bojungikki-tang water extract (BITW) in Sprague Dawley rats who were exposed to it in multiple doses and various concentrations. MATERIALS AND METHODS: BITW was administered to rats orally, once daily at doses of 0, 500, 1000, or 2000 mg/kg/day for 13 weeks. We checked toxicological parameters including general observations, organ/body weights, food consumption, ophthalmological signs, hematological and serum biochemical values, urinalysis values and histopathological findings. RESULTS: The 13 week repeated oral administration of BITW to rats at doses at doses levels of less than or equal to 2000 mg/kg/day caused no significant toxicological changes and only minor nonsignificant changes. CONCLUSIONS: Our findings indicate that administration of BITW for up to 13 weeks may be safe and nontoxic, with a no-observed-adverse-effect-level of >2000 mg/kg/day for both male and female rats.

Effect of Samryungbaekchul-san Combined with Otilonium Bromide on Diarrhea-Predominant Irritable Bowel Syndrome: A Pilot Randomized Controlled Trial.

Conventional and herbal drugs are frequently used together to treat many disorders. Samryungbaekchul-san (SRS, a herbal formula) and otilonium bromide (OB, an antispasmodic agent) are widely used to treat diarrhea-predominant irritable bowel syndrome (D-IBS) in Eastern Asian countries. However, there have been no studies on the co-administration of SRS and OB. Therefore, we aimed to preliminarily assess the feasibility of SRS combined with OB for D-IBS treatment in a pilot double-blind, four-arm, parallel-group, randomized controlled trial (RCT), including 80 patients diagnosed with D-IBS according to the Rome III criteria. The patients were randomly assigned to four treatment groups and were administered drugs for eight weeks after a two-week preparatory period. Follow-up was conducted four weeks after the administration period. The primary outcome was evaluated by using a global D-IBS symptom improvement score; no statistically significant difference was observed between the groups. However, multiple logistic regression analysis of primary outcome scores shows that SRS significantly improved D-IBS symptoms (p < 0.05). For secondary outcomes, better results were observed in the SRS + OB group, in terms of symptoms, including abdominal pain, discomfort, frequency of abdominal pain, and stool form than in OB alone or placebo groups (p < 0.05). In conclusion, the co-administration of SRS and OB might be an effective and safe strategy for the treatment of D-IBS. Large-scale RCTs are warranted to further confirm and clarify these findings.

A 4-Week Repeated Oral Dose Toxicity Study of Ssanghwa-Tang in Crl:CD Sprague Dawley Rats.

Ssanghwa-tang (SHT), a traditional herbal formula, has been widely used to recover fatigue or consumptive disease after an illness. Along with much attention to herbal formula, the concerns about the safety and toxicity have arisen. To establish the safety information, SHT was administrated in Crl:CD Sprague Dawley rats at a daily dose of 0, 1000, 2000, and 5000 mg/kg for 4 weeks. During the test periods, we examined the mortality, clinical observation, body weight change, food consumption, organ weights, hematology, serum biochemistry, and urinalysis parameters. No changes of mortality and necropsy findings occurred in any of the groups during the experimental period. In either sex of rats treated with SHT at 5000 mg/kg/day, changes were observed in food intake, reticulocyte, total bilirubin, some urinalysis parameters, and relative organ weights. The results indicated that SHT did not induce toxic effects at a dose level up to 2000 mg/kg in rats. This dosage was considered no observed adverse effect level (NOAEL) and was appropriate for a 13-week subchronic toxicity study.

Safety assessment of Gyejibokryeong-hwan water extract: Study of acute and subacute toxicity, and influence on drug metabolizing enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: Gyejibokryeong-hwan is a traditional herbal medicine and is reported to have various pharmacological actions. Despite many reports of previous studies, there is limited scientific evidence concerning its safety and few drug-metabolism profiles to support the continued therapeutic application of Gyejibokryeong-hwan. AIM OF THE STUDY: The purpose of the present study was to investigate the acute and subacute toxicity profile of a Gyejibokryeong-hwan water extract (GBHW) in vivo, and its effects on the activities of drug-metabolizing enzymes in vitro. MATERIALS AND METHODS: Acute and subacute toxicity was evaluated by giving GBHW to rats. In a study of acute toxicity, the rats were given GBHW by single oral gavage administration at 0 and 5000 mg/kg. In a study of subacute toxicity, rats were given GBHW by oral gavage at 0, 1000, 2000, and 5000 mg/kg/day daily for 28 days. The activities of the major human microsomal cytochrome P450 (CYP450) and UDP-glucuronosyltransferase (UGT) isozymes were investigated using fluorescence- and luminescence-based enzyme assays in vitro, respectively. RESULTS: GBHW did not cause any mortality in the study of acute toxicity. In the study of subacute toxicity, GBHW at more than 2000 mg/kg/day was observed with minor changes in the absolute and relative organ weight, hematology, serum biochemistry and urinalysis parameters in rats of either sex. However, these changes were not considered to be important toxicologically. GBHW moderately inhibited the activities of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A4, and UGT1A1. CONCLUSIONS: Our present data suggest that GBHW does not cause toxicologically important adverse events at doses up to 2000 mg/kg/day in the 4-week repeated dose toxicity study and provide valuable information concerning its potential to interact with conventional medicine.

Evaluation of the subacute toxicity of Yongdamsagan-tang, a traditional herbal formula, in Crl:CD Sprague Dawley rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Yongdamsagan-tang, a traditional herbal formula, is used widely for the treatment of inflammatory and viral diseases. However, the safety of Yongdamsagan-tang has not been established. AIM OF THE STUDY: To evaluate the subacute toxicity of Yongdamsagan-tang water extract (YSTE) in Crl:CD Sprague Dawley rats. MATERIALS AND METHODS: We evaluated the subacute toxicity of YSTE in male and female Crl:CD Sprague Dawley rats (n = 5 per group). Rats were treated with YSTE at doses of 0, 1000, 2000 and 5000 mg/kg administered once a day by oral gavage for 4 weeks. RESULTS: There were no significant changes in mortality, body weight, food intake, serum biochemistry, or results of hematology and urinalysis after YSTE administration. However, all rats treated with 5000 mg/kg/day YSTE exhibited excessive salivation and discolored urine. Necropsy findings showed discoloration in the liver of both male (n = 1) and female (n = 3) rats treated with 5000 mg/kg/day YSTE, and an increase in the relative weights of kidney and liver was also found in male rats treated with 5000 mg/kg/day. In addition, decreases in serum creatinine, total bilirubin, alanine transaminase, and alkaline phosphatase were observed in male rats treated with 2000 or 5000 mg/kg/day YSTE. CONCLUSIONS: Abnormalities in some rats are considered to be independent of YSTE toxicity. Therefore, the results suggest that oral administration of YSTE in rats for 4 weeks is safe at doses of up to 5000 mg/kg/day.

Effects of Herbal Formulas Bojungikgi-tang and Palmijihwang-hwan on Inflammation in RAW 264.7 Cells and the Activities of Drug-Metabolizing Enzymes in Human Hepatic Microsomes.

In the present study, Bojungikgi-tang (BJIKT: Buzhongyiqi-tang, Hochuekki-to) and Palmijihwang-hwan (PMJHH: Baweidìhuang-wan, Hachimijio-gan), traditional herbal formulas, investigated anti-inflammatory efficacies in murine macrophage cell line and the influence on the activities of drug-metabolizing enzymes (DMEs). The anti-inflammatory potentials of the herbal formulas were evaluated to inhibit the production of the inflammatory mediators and cytokines and the protein expression of inducible nitric oxide and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The activities of the major human DMEs, cytochrome P450 isozymes (CYP450s) and UDP-glucuronosyltransferase isozymes (UGTs), were measured by in vitro enzyme assay systems. BJIKT and PMJHH significantly suppressed the prostaglandin E2 (PGE2) production (IC50 = 317.3 and 282.2 µg/mL, respectively) and the protein expression of COX-2 in LPS-treated RAW264.7 cells. On the human microsomal DMEs, BJIKT inhibited the activities of CYP1A2 (IC50 = 535.05 µg/mL), CYP2B6 (IC50 > 1000 µg/mL), CYP2C9 (IC50 = 800.78 µg/mL), CYP2C19 (IC50 = 563.11 µg/mL), CYP2D6 (IC50 > 1000 µg/mL), CYP2E1 (IC50 > 1000 µg/mL), CYP3A4 (IC50 = 879.60 µg/mL), UGT1A1 (IC50 > 1000 µg/mL), and UGT1A4 (IC50 > 1000 µg/mL), but it showed no inhibition of the UGT2B7 activity at doses less than 1000 µg/mL. PMJHH inhibited the CYP2D6 activity (IC50 = 280.89 µg/mL), but IC50 values of PMJHH exceeded 1000 µg/mL on the activities of CYP1A2, CYP2C19, CYP2E1, and CYP3A4. At concentrations less than 1000 µg/mL, PMJHH did not affect the activities of CYP2B6, CYP2C9, UGT1A1, UGT1A4, and UGT2B7. The results indicate that both BJIKT and PMJHH may be potential candidates to prevent and treat PGE2- and COX-2-mediated inflammatory diseases. In addition, this study will expand current knowledge about herb-drug interactions by BJIKT and PMJHH.

Single Oral Acute Toxicity of Banhasasim-Tang and Its Antiobesity Effect on Diet-Induced Obese Mice and 3T3-L1 Adipocytes.

We had tested antiobesity effect of 52 traditional herbal formulas in 3T3-L1 adipocyte, and Banhasasim-tang (BHSST) was chosen as one of the effective medications to inhibit triglyceride accumulation. We investigated the antiobesity effect of BHSST on 3T3-L1 adipocytes and high-fat diet- (HFD-) induced obese mice. In addition, we evaluated the acute toxicity of BHSST in Sprague Dawley (SD) rats. Differentiated 3T3-L1 cells were treated with various concentrations of BHSST for 8 days. Accumulated triglyceride level and the expressions of adipogenesis-related genes and proteins were subsequently investigated. To evaluate the single oral toxicity of BHSST, the SD rats of each sex were administered a single dose (5000 mg/kg) of BHSST via oral gavage; the control group received vehicle only. After a single administration, the mortality, clinical signs, gross findings, and body weight were monitored for 15 days. Male C57BL/6J mice were fed HFD for 4 weeks to induce obesity and randomly received 50 mg/kg of Orlistat (n=12, OR), 200 mg/kg of BHSST (n=12, B200), and 1000 mg/kg of BHSST (n=12, B1000) for another 8 weeks. BHSST suppressed the triglyceride contents and lipid accumulation in a dose-dependent manner in 3T3-L1 adipocytes. BHSST also downregulated the adipogenesis-related gene levels and protein expression compared with those in undifferentiated adipocytes. In a single oral dose toxicity study, there was no adverse effect on mortality, clinical signs, body weight changes, and gross findings in the treatment group. HFD-fed mice treated with BHSST showed significantly reduced body weight gain, food efficiency ratio, and white adipose tissue weight. The medial lethal dose (LD50) of BHSST was 5000 mg/kg/day body weight for each sex in the rats. BHSST decreased the body weight gain in HFD-fed obese mice and inhibited triglyceride accumulation via a cascade of multiple factors at the mRNA and protein levels in 3T3-L1 adipocytes.

The Inhibitory Effect of Ojeoksan on Early and Advanced Atherosclerosis.

Atherosclerosis is closely related to vascular dysfunction and hypertension. Ojeoksan (OJS), originally recorded in an ancient Korean medicinal book named "Donguibogam", is a well-known, blended herbal formula. This study was carried out to investigate the beneficial effects of OJS on atherosclerosis in vitro and in vivo. Western-diet-fed apolipoprotein-E gene-deficient mice (ApoE -/-) were used for this study for 16 weeks, and their vascular dysfunction and inflammation were analyzed. OJS-treated ApoE -/- mice showed lowered blood pressure and glucose levels. The levels of metabolic parameters with hyperlipidemia attenuated following OJS administration. Hematoxylin and eosin (H&E) staining revealed that treatment with OJS reduced atherosclerotic lesions. OJS also suppressed the expression of adhesion molecules and matrix metalloproteinases (MMPs) compared to Western-diet-fed ApoE -/- mice and tumor necrosis factor-alpha (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs). Expression levels of MicroRNAs (miRNA)-10a, -126 3p were increased in OJS-fed ApoE -/- mice. OJS significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) and protein kinase B (Akt), which are involved in nitric oxide (NO) production. OJS also regulated eNOS coupling by increasing the expression of endothelial GTP Cyclohydrolase-1 (GTPCH). Taken together, OJS has a protective effect on vascular inflammation via eNOS coupling-mediated NO production and might be a potential therapeutic agent for both early and advanced atherosclerosis.

Safety assessment of Oryeong-san, a traditional herbal formula: Study of subacute toxicity and influence of cytochrome P450s and UDP-glucuronosyltransferases.

Oryeong-san is a traditional herbal formula that is used for the treatment of common genitourinary diseases in Korea and other Asian countries. However, little is known about its safety and influence on drug metabolism. In the present study, we investigated the subacute toxicity of an Oryeong-san water extract (OSWE) in rats and its effects on activities of drug-metabolizing enzymes. Subacute toxicity was modeled in animals exposed to treatment with the extract at multiple doses. Rats were given OSWE by oral gavage at 0, 1000, 2000 and 5000 mg/kg/day for 4 weeks. We checked general observations and investigated any changes of body/organ weight, food consumption, hematology, serum biochemistry, and urinalysis in vivo; and the activities of human microsomal cytochrome P450s (CYP450s) and UDP-glucuronosyltransferase (UGT) isozymes in vitro. We found that OSWE caused no significant toxicological changes at the doses tested. Therefore, the no observed adverse effect level of OSWE was more than 5000 mg/kg/day for male and female rats. OSWE inhibited the activities of CYP2C19 (IC50: 737.69 µg/mL) and CYP2E1 (IC50: 177.77 µg/mL). These results indicate that OSWE may be safe with no drug-related toxicity for up to 4 weeks and provide useful information concerning its potential to interact with conventional drugs or other herbal medicines.

Sub-chronic toxicity of Gyejibokryeong-hwan in Sprague-Dawley rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional herbal formula Gyejibokryeong-hwan (GJBRH; Guizhifuling-wan, Keishibukuryo-gan) consisting five medicinal herbs has been used to treat uterine disorders, gynecological diseases and blood stasis syndrome in Asia. AIM OF THE STUDY: We evaluated the safety of GJBRH in Crl:CD Sprague-Dawley (SD) rats over a period of 13 weeks. MATERIALS AND METHODS: To confirm the stability of the components of GJBRH, we analyzed the component contents in GJBRH at different storage periods, using high-performance liquid chromatography. Male and female SD rats were orally administered with GJBRH at doses of 0, 1000, 2000 and 5000 mg/kg/day for 13 weeks and assessed after a 4-week recovery period. Mortality, changes in body weight and food consumption, organ weights, hematology and serum biochemistry were monitored during the experimental period, along with clinical observations, ophthalmological examinations, urinalysis and histopathology. RESULTS: There were no significant differences among the eight marker compounds in GJBRH according to storage period. No significant GJBRH-treatment-related toxicological changes were observed in mortality or ophthalmological examinations in either sex. However, soft feces were observed in the male 5000 mg/kg/day group. In addition, there were significant changes in body weight and food consumption in both male and female rats treated with GJBRH at a dose of 5000 mg/kg/day. In the hematological examinations, we found a significant increase in white blood cells, neutrophils and fibrinogen in the 5000 mg/kg/day groups. In the urinalysis, a decrease in the total protein and albumin and an increase in the ovalbumin/globulin ratio were observed in both male and female rats treated with GJBRH at a dose of 5000 mg/kg/day. Histopathological examinations revealed erosion/ulcers and dilated glands in the stomachs of males from the 5000 mg/kg/day group, and squamous cell hyperplasia and epithelial atrophy was observed in the stomachs of both male and female rats treated with GJBRH at a dose of 5000 mg/kg/day. CONCLUSION: The no-observed-adverse-effect level (NOAEL) was 2000 mg/kg/day for both sexes.