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Ethnopharmacological relevance Gleditsia sinensis Lam. (G. sinensis) has been used in Oriental medicine for tumor, thrombosis, inflammation-related disease, and obesity. Aim of the study The pharmacological inhibitory effects of fruits of G. sinensis (GFE) on hyperlipidemia have been reported, but its inhibitory effects on adipogenesis and underlying mechanisms have not been elucidated. Herein we evaluated the anti-adipogenic effects of GFE and described the underlying mechanisms. Materials and methods The effects of ethanol extracts of GFE on adipocyte differentiation were examined in 3T3-L1 cells using biochemical and molecular analyses. Results During the differentiation of 3T3-L1 cells, GFE significantly reduced lipid accumulation and downregulated master adipogenic transcription factors, including CCAAT/enhancer-binding protein-α and peroxisome proliferator-activated receptor-γ, at mRNA and protein levels. These changes led to the suppression of several adipogenic-specific genes and proteins, including fatty acid synthase, sterol regulatory element-binding protein 1, stearoyl-CoA desaturase-1, and acetyl CoA carboxylase. However, the inhibitory effects of GFE on lipogenesis were only shown when GFE is treated in the early stage of adipogenesis within the first two days of differentiation. As a potential mechanism, during the early stages of differentiation, GFE inhibited cell proliferation by a decrease in the expression of DNA synthesis-related proteins and increased p27 expression and suppressed signal transducer and activator of transcription 3 (STAT3) activation induced in a differentiation medium. Conclusions GFE inhibits lipogenesis by negative regulation of adipogenic transcription factors, which is associated with GFE-mediated cell cycle arrest and STAT3 inhibition.

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.

Ethnopharmacological relevance Perillae Herba is a perennial plant that is widely distributed throughout Asia. The leaves of Perillae Herba have been widely used to treat various diseases, such as cold due to wind-cold, headache, cough, abdominal fullness, distention, and fish and crab poisoning. Materials and methods To assess the anti-inflammatory activity of Perillae Herba leaf ethanolic extract (PHE) in human keratinocytes, we measured the tumor necrosis factor (TNF)-α/interferon (IFN)-γ-induced mRNA expression and production of proinflammatory chemokines such as thymus and activation-regulated chemokines; regulated on activation, normal T cell expressed and secreted; interleukin (IL)-6; and IL-8 in HaCaT cells. We evaluated the ability of PHE to decrease the expression of proinflammatory marker proteins, such as mitogen-activated protein kinase (MAPK), STAT-1, and NK-κB, using western blot analysis and immunocytochemistry. Results PHE inhibited activation of p38, ERK, and JNK and suppressed the phosphorylation of STAT-1 and NK-κB in TNF-α/IFN-γ-stimulated HaCaT cells. PHE also suppressed chemokine mRNA and protein levels in TNF-α/IFN-γ-stimulated HaCaT cells. PHE appears to regulate chemokine formation by inhibiting activation of MAPK, as well as the STAT-1 and NK-κB pathways. Conclusions PHE suppresses the expression and production of TNF-α/IFN-γ-stimulated proinflammatory chemokines by blocking NF-κB, STAT-1, and MAPK activation.

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.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.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 (P0.05). 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.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.

Ethnopharmacological relevance In Eastern Asia, E. koreanum Nakai (EKN) has traditionally been used as an aphrodisiac herbal medicine. However, there was no available information for its genotoxicity. This study was conducted to evaluate the genotoxic potentials of EKN. Materials and methods The phytochemicals of EKN were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Three standard battery of genotoxicity assay for bacterial reverse mutation, mammalian chromosomal aberration and in vivo micronuclei formation was employed. Results The LC/MS/MS analysis revealed four hydroxybenzoic acids, three lignans, and ten flavonoid glycosides in EKN. The bacterial reverse mutation assay revealed no mutagenic effects of EKN. Moreover, EKN did not show any clastogenic effects in the in vivo and in vitro assays. Conclusion EKN water extract was shown to be a non-genotoxic herbal medicine under the conditions tested in this study.

Ethnopharmacological relevance The root of Platycodon grandiflorum (PG), commonly known as Kilkyong in Korea, Jiegeng in China, and Kikyo in Japan, has been extensively used as a traditional anti-inflammatory medicine in Asia for the treatment of respiratory conditions, such as bronchitis, asthma, and tonsillitis. Platycosides isolated from PG are especially well-known for their anti-cancer effects. Aim of the study We investigated the involvement of autophagic cell death and other potential molecular mechanisms induced by the platycoside-containing butanol fraction of PG (PGB) in human lung carcinoma cells. Materials and methods PGB-induced growth inhibition and cell death were measured using a 5-diphenyl-tetrazolium bromide (MTT) assay. The effects of PGB on autophagy were determined by observing microtubule-associated protein 1 light chain 3 (LC3) redistribution with confocal microscopy. The PGB-mediated regulation of autophagy-associated proteins was investigated using Western blotting analysis. Furthermore, the anti-cancer mechanism of PGB was confirmed using chemical inhibitors. A high-performance liquid chromatography (HPLC)-DAD system was used to analyze the platycosides in PGB. Results In A549 cells, PGB induced significant autophagic cell death. Specifically, PGB upregulated LC3-II in a time- and dose-dependent manner, and it redistributed LC3 via autophagosome formation in the cytoplasm. PGB treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and subsequently suppressed the AKT/mammalian target of the rapamycin (mTOR) pathway. Furthermore, PGB inhibited cell proliferation by regulating the mitogen-activated protein kinase (MAPK) pathways. In this study, six types of platycosides were identified in the PGB using HPLC. Conclusions PGB efficiently induced cancer cell death via autophagy and the modulation of the AMPK/mTOR/AKT and MAPK signaling pathways in A549 cells. Therefore, PGB may be an efficacious herbal anti-cancer therapy.

Ethnopharmacological relevance The peel of Citrus unshiu Markovich fruits (CUP), called “Jinpi” in Korea, and “Chenpi” in China, has been used for the treatment of respiratory and blood circulation disorders in traditional oriental medicine (TOM). Despite its widespread uses in TOM, no information on the safety of CUP has been reported. Thus, genotoxicity and systemic toxicity of CUP were evaluated in the current studies. Materials and methods We conducted a toxicological evaluation of CUP water extracts using acute and subchronic (13-week repeated-dose) toxicity tests and three genotoxicity assays (bacterial reverse mutation, mammalian chromosomal aberration, and micronuclei formation). Results In acute and subchronic toxicity tests, both the median lethal dose (LD50) and no-observed-adverse-effect level (NOAEL) were more than 4000 mg/kg/day in rats. None of the genotoxicity assays revealed any mutagenicity or clastogenicity in in vitro and in vivo systems. Conclusion CUP water extracts were found to be nongenotoxic under our testing conditions and had low acute and subchronic toxicity.

Ethnopharmacological relevance Dianthi Herba is a traditional herbal medicine used to treat inflammatory-related diseases including acute pyelonephritis, cystitis, laryngopharyngitis, and urethritis. Aim of the study We investigated the effects of Dianthi Herba ethanolic extract (DH) on lipopolysaccharide (LPS)-mediated inflammatory responses in murine macrophages including RAW 264.7 cell line and mouse peritoneal macrophages as well as nociceptive and edema mouse models. Materials and methods The biological effects of DH on inflammatory cytokine, mediator, and related protein production were assessed using enzyme-linked immunosorbent assay (ELISA), Western blotting, and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). Additionally, Western blotting was performed to investigate intracellular signaling pathways, and the anti-nociceptive activity of three doses of DH (100, 200, and 300 mg/kg) against acetic acid-induced writhing responses and its inhibitory effects on xylene-induced ear edema were researched in mice through oral administration. Results DH treatment significantly inhibited nitric oxide (NO) secretion and inflammatory cytokine production in RAW 264.7 cells and mouse peritoneal macrophages and induced heme oxygenase (HO)−1 expression. DH strongly inhibited the transcriptional activity of nuclear factor (NF)-κB and phosphorylation of mitogen-activated protein kinases (MAPK) in LPS-stimulated macrophages. Meanwhile, DH exerted anti-nociceptive effects on writhing responses and anti-edema effects in mice. Conclusion We confirmed the anti-inflammatory activities and inhibitory mechanism of DH in macrophages and clarified its inhibitory effects in vivo. These findings illustrate the therapeutic potential of DH as a natural anti-inflammatory agent.

Ethnopharmacological relevance Dryopteris crassirhizoma has been traditionally used for the treatment of tapeworm infestation, the common cold and cancer in Korea, China and Japan. Despite various pharmacological properties of Dryopteris crassirhizoma , there is no available information about the safety of Dryopteris crassirhizoma . Aim of this study To ensure more information about the safety of Dryopteris crassirhizoma , we performed the acute oral toxicity and genotoxicity tests of Dryopteris crassirhizoma . Materials and methods The acute oral toxicity test of Dryopteris crassirhizoma was performed in rats. Genotoxicity of Dryopteris crassirhizoma was evaluated by bacterial reverse mutation, chromosomal aberration and bone marrow micronucleus tests. Results In acute toxicity test, Dryopteris crassirhizoma exhibited no mortality, body weight and behavioral changes and adverse effects in male and female rats. Dryopteris crassirhizoma did not significantly increase the number of the bacterial revertant and chromosomal aberration in both in vitro assays. Moreover, the Dryopteris crassirhizoma -related increases of micronucleated polychromatic erythrocytes (MNPCE) in mouse bone marrow were not observed. Conclusion Therefore, Dryopteris crassirhizoma is non-genotoxic in a three standard battery of tests and the oral LD 50 of Dryopteris crassirhizoma is >2000 mg/kg.

Ethnopharmacological relevance Samul-tang (Si-Wu-tang in Chinese, Shimotsu-to in Japanese), widely used in eastern Asia, is composed of Angelica gigas (Angelicae Gigantis Radix), Cnidium officinale (Cnidii Rhizoma), Paeonia lactiflora (Paeonia Radix) and Rehmannia glutinosa (Rehmanniae Radix Preparata). Paeoniflorin, one of active components in Samul-tang has anti-platelet, anti-inflammation, anti-cancer and neuroprotective properties. However, there is no information about the effects of gender and food intake on the pharmacokinetics of paeoniflorin till now. Aim of the study This study was conducted to investigate whether food and gender could influence pharmacokinetic profiles of paeoniflorin after oral administration of Samul-tang. Materials and methods Male and female rats were administered with a single oral dose of Samul-tang equivalent to 80 mg/kg of paeoniflorin. Plasma concentrations of paeoniflorin were measured by high-performance liquid chromatography. The statistical differences of each group were evaluated using the analysis of variance (ANOVA) or Student t-test. Results The pharmacokinetic parameters of paeoniflorin were not significant different by gender difference. However, the maximum plasma concentration (Cmax, 0.47±0.29 μg/mL versus 1.10±0.35 μg/mL), area under the concentration-time curve (AUC0→∞, 1.41±0.89 h·μg/mL versus 3.12±1.61 h·μg/mL) and relative bioavailability (Frel=2.21) of fed rats were significantly increased in comparison with those of fasted rats (P Conclusion Taken together, food intake can affect both the rate and extent of absorption of paeoniflorin when Samul-tang was administered orally. Furthermore, this study demonstrates a readily preparative HPLC method in the research of traditional herbal medicine.

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.

Shibimijihwang-tang (SJT) has been used traditionally to improve systemic blood circulation and biological energy production in patients with circulatory and neuronal diseases. The object of this study is to determine the effect of SJT extract on the intracellular adenosine triphosphate (ATP) and choline content in the cerebral cortex of ovariectomized (OVX) rats. Bilateral ovaries of 8-week-old rats were removed. Rats were maintained for 12 weeks to deplete ovarian steroid hormones, followed by treatment with SJT at 500 mg/kg body weight per day for 12 weeks. High rate of body weight increase in the OVX rats was markedly reduced by treatment with SJT, but the change in body weight of normal rats was not affected by it. SJT also significantly reduced the decline of cerebral weight in the OVX rats (P

Shao-Yin-Ren Shi-Quang-Da-Bu-Tang (SDT) has been used traditionally to improve the systemic blood circulation and biological energy production in the body. The object of this study is to determine the effect of SDT extract on the decline of cerebral adenosine triphosphate (ATP) and choline content associated with learning and memory impairments in senescence-accelerated mice prone 8 (SAM P8). Twenty-four-week old mice were orally treated with SDT at 400 mg/kg body weight per day, and continued for 12 consecutive weeks. At the termination of the treatment, the body weight of SAM P8 was markedly lower than that of the equal aged senescence-resistant prone 1 (SAM R1), but this was conspicuously recovered to the level of SAM R1 by SDT treatment. SDT also significantly reduced the decline of cerebral weight (P < 0.05). By comparison with normal mice, a spontaneous decrease of cerebral ATP was observed in the SAM P8. Two- and 6-fold increases of cerebral ATP content were found in SAM R1 and SAM P8 by SDT administration, respectively. The cerebral choline content was significantly different between SAM R1 and SAM P8 aged 36-week old (P < 0.01). SDT remarkably restored the decrease of cerebral choline content in SAM P8 (P < 0.01). Taken together, these results demonstrate that SDT can reduce the decrease of cerebral weight, and restore the decline of cerebral ATP and choline content associated with an alteration of neuronal metabolism in SAM P8 brain. This suggests that pharmacological properties of SDT may participate in improvement of declined cerebral energy production and cholinergic neurotransmitter synthesis in senile dementia.

Ethnopharmacological relevance Acer tegmentosum , which contains salidroside and tyrosol, has been used for the treatment of hepatic disorders in eastern Asia. However, little is known about its safety. Aim of the study To determine the safety of Acer tegmentosum , we evaluated its acute oral toxicity and genotoxicity profiles. Materials and methods Salidroside and tyrosol present in Acer tegmentosum were quantified using high-performance liquid chromatography. Acute oral toxicity testing of Acer tegmentosum was performed in rats. Genotoxicity of Acer tegmentosum was assessed by bacterial reverse mutation, chromosomal aberration, and bone marrow micronucleus tests. All the tests were conducted in accordance with the good laboratory practices. Results The amounts of salidroside and tyrosol in Acer tegmentosum were found to be 85.01±1.21 mg/g and 3.12±0.04 mg/g, respectively. In the bacterial reverse mutation test, Acer tegmentosum increased the number of revertant Salmonella typhimurium TA98 colonies, regardless of metabolic activation by S9 mixture. In contrast, Acer tegmentosum application did not significantly increase the number of chromosomal aberrations in Chinese hamster ovary (CHO)-K1 cells and micronucleated polychromatic erythrocytes in mice. In the acute oral toxicity test, the median lethal dose (LD 50 ) of Acer tegmentosum was found to be >2000 mg/kg in rats. Conclusion Take together, Acer tegmentosum exhibits mutagenicity, which was evident from the bacterial reverse mutation test. Further studies are needed to identify the components responsible for such an effect and the underlying mechanisms.

Ethnopharmacological relevance Yijung-tang (YJ), a traditional Asian medicine, is used to treat various diseases. However, its anti-osteoporotic effect and mechanism of action remain unclear. The aim of the present study was to evaluate the anti-osteoporotic effect of YJ in ovariectomized (OVX) rats. Materials and methods Sprague-Dawley rats were divided into five groups as follows: sham-operated, ovariectomized (OVX), OVX rats treated with 100 g/kg/day 17-estradiol, and OVX rats treated with 0.3 and 1.0 g/kg/day YJ for 12 weeks. Trabecular bone mineral density (BMD) and bone microarchitecture were evaluated by microcomputed tomography. The effects of YJ on osteoblast and osteoclast formation were also investigated in an in vitro model using primary murine bone marrow-derived macrophages and murine calvarial preosteoblasts. mRNA expression of osteoclast differentiation-related genes was measured by real-time quantitative reverse transcription-polymerase chain reaction. Activation of the mitogen-activated protein kinases and nuclear factor-κB (NF-κB) were determined by Western blot. Results The decrease of BMD and destruction of bone microarchitecture were significantly reduced in the OVX-induced osteoporosis rat model after 12 weeks of YJ treatment. The anti-osteoporotic effect of YJ on bone loss was due to inhibition of osteoclast differentiation through down-regulation of the NF-κB pathway. In addition, YJ suppressed the induction of nuclear factor of activated T-cells, cytoplasmic 1 and c-Fos following receptor activator of nuclear factor kappa-B ligand stimulation. Conclusions These results suggest that YJ possess potent anti-osteoporotic activity in OVX rats and may be a useful remedy for the treatment of postmenopausal osteoporosis.