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BackgroundRecent 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.MethodsUsing 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.ResultsECZ 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.ConclusionThese 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.

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 β-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.

Because tyrosinase catalyzes essential steps in melanin synthesis, tyrosinase inhibitors are widely used for skin–lightening. To reduce the side effects of synthetic compounds, safer tyrosinase inhibitors isorated from natural products have gained attention. Here, we examined the anti-melanogenic effect of the seed of Ziziphus jujuba and identified eight major compounds, of which spinosin exhibited the strongest tyrosinase inhibitory activity (IC50 = 47 µM). When further examined, spinosin suppressed αMSH- or UVB-induced melanogenesis in B16F10 cells without cytotoxicity. The anti-melanogenic effect was also shown in a human skin model. As an underlying mechanism, in silico analysis showed that spinosin may bind to and suppress tyrosinase activity by forming multiple hydrogen bonds and hydrophobic interactions with the binding pocket of tyrosinase. Lineweaver-Burk and Dixon plots further revealed that spinosin acts as a competitive inhibitor of tyrosinase (Vmax, 11.61 × 103, 11.63 × 103, and 11.62 × 103, Km, 0.139, 0.185, and 0.239 for the con, 10 µM, 50 µM of spinosin, respectively). In conclusion, spinosin, a major compound in the seed of Ziziphus jujuba, could be a novel additive for treating pigmentation disorders or skin-lightening cosmetics.

Viscum coloratum has been used as a component for traditional medicine for therapy of inflammatory diseases. Nonetheless, effect of Viscum coloratum on inflammatory bowel disease is unknown. Therefore, we investigated whether the ethanol extract of Viscum coloratum (VCE) could suppress inflammatory responses in dextran sodium sulfate (DSS)-treated mice and mast cell-derived inflammatory mediator (MDIM)-activated Caco-2 cells. VCE significantly attenuated body weight loss, shortened colon length, enteric epithelium disruption, enterorrhagia and colonic edema in DSS-treated mice. Additionally, VCE decreased the levels of immunoglobulin E, interleukin-6 and tumor necrosis factor-[Formula: see text] in serum and the activity of myeloperoxidase in colonic tissue. Moreover, VCE inhibited the infiltration of immune cells as well as the activity and expression of both matrix metalloprotease-2 and matrix metalloprotease-9. Furthermore, VCE restored zonula occludens-1 expression. Consistent with in vivo studies, VCE suppressed the activity and expression of matrix metalloprotease-2 and matrix metalloprotease-9 in MDIM-activated Caco-2 cells. In addition, VCE reinstated the expression of zonula occludens-1 through inhibiting activation of janus kinase 2/signal transducer and activator of transcription 3 in the cells. In conclusion, VCE exerts anticolitic action through inhibiting the activation of mast cells. Therefore, VCE may be useful as a phytomedicine or functional food for inflammatory bowel disease.

Aloe vera ethanol extract (AVE) reportedly has significant anti-influenza virus activity, but its underlying mechanisms of action and constituents have not yet been completely elucidated. Previously, we have confirmed that AVE treatment significantly reduces the viral replication of green fluorescent protein-labeled influenza A virus in Madin-Darby canine kidney (MDCK) cells. In addition, post-treatment with AVE inhibited viral matrix protein 1 (M1), matrix protein 2 (M2), and hemagglutinin (HA) mRNA synthesis and viral protein (M1, M2, and HA) expressions. In this study, we demonstrated that AVE inhibited autophagy induced by influenza A virus in MDCK cells and also identified quercetin, catechin hydrate, and kaempferol as the active antiviral components of AVE. We also found that post-treatment with quercetin, catechin hydrate, and kaempferol markedly inhibited M2 viral mRNA synthesis and M2 protein expression. A docking simulation suggested that the binding affinity of quercetin, catechin hydrate, and kaempferol for the M2 protein may be higher than that of known M2 protein inhibitors. Thus, the inhibition of autophagy induced by influenza virus may explain the antiviral activity of AVE against H1N1 or H3N2. Aloe vera extract and its constituents may, therefore, be potentially useful for the development of anti-influenza agents.

The biological effect of lappaol A, a lignan, is derived from Arctium lappa, and is relatively unknown; however, lignans have various beneficial properties. Therefore, we investigated the effects of lappaol A on the immunoglobulin E/antigen (IgE/Ag)-mediated allergic response in mast cells and mice. Lappaol A significantly reduced the release of β-hexosaminidase, IL-4, TNF-α, PGD2 and LTC4 with IC50 values of 131.3 μM, 11.58 μM, 59.50 μM, 17.85 μM and 10.99 μM, respectively, in IgE/Ag-activated RBL-2H3 cells. Moreover, lappaol A suppressed the activation of the arachidonate and FceRI signaling cascades in the cells. Furthermore, lappaol A suppressed the IgE/Ag-mediated passive cutaneous anaphylaxis reaction, ear edema and mast cell infiltration in the ear tissues of IgE/Ag-exposed mice. In conclusion, these findings suggest that lappaol A exhibits anti-allergic activity by inhibiting the activation of the arachidonate and FceRI signaling cascades in IgE/Ag-activated mast cells. Therefore, this information may help to elucidate the anti-allergic action of lappaol A and Arctium lappa L.

Background: The leaf of Pinus densiflora known as pine needles has been used to treat vascular disease, gastrointestinal diseases, and urinary diseases in traditional medicine. We evaluated anti-osteoporotic effect of water extract of Pinus densiflora (WEPN) on acute bone loss and osteoclastogenesis induced by receptor activator for nuclear factor-κB ligand (RANKL). Methods: After oral administration of WEPN (0.25 g/kg) for 5 days, femora were collected, and bone parameter [trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), trabecular number (Tb. N), and bone mineral density (BMD)] were analyzed by micro-CT analysis. Anti-osteoclastic effect of WEPN was examined using tartrate-resistant acid phosphatase activity and activation of RANKL signaling pathway. Results: We found that WEPN significantly attenuated RANKL-induced decrease of BV/TV, Tb.Th., Tb.N, and BMD but increase of Tb. Sp in femora. WEPN dose-dependently decreased osteoclastogenesis accompanied by inhibiting the activation of RANKL signaling components (JNK, p38, and p65) and mRNA expression level of osteoclast specific genes (NFATc1, c-Fos, TRAP, cathepsin K, DC-STAMP, and carbonic anhydrate). Conclusion: WEPN inhibition on osteoclastogenesis could contribute to attenuate RANKL-induced trabecular bone loss in vivo. Therefore, it might suggest that WEPN could be prescribed in traditional medicine or used in health functional food to prevent or treat osteoporotic bone diseases. Keywords: Pinus densiflora, Osteoclast, Receptor activator for nuclear factor-κB ligand, Nuclear factor of activated T cells cytoplasmic 1

Six diarylheptanoids (1–6) and two flavonoids (7 and 8) derived from Alpinia officinarum were evaluated for their ability to inhibit acetylcholinesterase. Compound 1 showed the highest degree of inhibition, with an IC50 of approximately 2 μM, followed by moderate degrees of inhibition by 2, 4 and 7, with IC50 values ranging from 20 to 40 μM. The remaining isolated compounds 3, 5, 6 and 8 had IC50 values greater than 50 μM. Enzyme kinetic studies showed that the compounds with high or moderate activity were competitive inhibitors, anchored to the active site of acetylcholinesterase. In particular, compounds 1 and 2 were docked at slightly different positions from those occupied by 4 and 7. Furthermore, molecular dynamics studies showed that compound 1 maintained its interactions with residues Thr74 and Phe295 throughout the simulation trajectory. Our findings suggest that compound 1 is a potential therapeutically relevant inhibitor of acetylcholinesterase.

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy. Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effects against respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration of SFE significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation. Moreover, oral inoculation of SFE significantly improved RSV clearance from the lungs of BALB/c mice. Interestingly, the phenolic compounds eicosane, docosane, and tetracosane were identified as active components of SFE. Treatment with a non-cytotoxic concentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection.

Ulcerative colitis is one inflammatory bowel disease (IBD) and is caused by diverse factors, including the extent and duration of intestinal inflammation. We investigated the effect of Acer palmatum thumb. ethanol extract (KIOM-2015E) on the expression of tight junction proteins and the levels of inflammation in the cell model induced with interleukin-6- (IL-6-) and mouse model of dextran sodium sulfate (DSS) induced with acute colitis. KIOM-2015E (100 mg/kg) was orally administered once per day to BALB/C mice with colitis induced by administration of 5% DSS in drinking water. KIOM-2015E did not affect viability in Caco-2 cells. Also, KIOM-2015E repaired the IL-6-induced intestinal barrier dysfunction in Caco-2 cells. Furthermore, KIOM-2015E recovered the loss of body weight and the abnormally short colon lengths in the DSS-induced model of acute colitis. Moreover, KIOM-2015E significantly inhibited the decrease of zonula occluden-1 and occludin in colonic tissue relative to the DSS-treated control group. KIOM-2015E also significantly inhibited the expression of IL-6 and tumor necrosis factor-α in the level of serum relative to the control group. Collectively, these data suggest that KIOM-2015E protects colitis principally by improving intestinal barrier function and promoting anti-inflammatory responses. In turn, these effects inhibit macrophage infiltration into the colon and thus may be a candidate treatment for IBD.

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.

Lycopus lucidusTurcz. ex Benth (LT) has been broadly used as a traditional medicinal herb in Asia including Korea, China, and Japan due to its noted ability to promote blood circulation and remove blood stasis. However, its anticancer mechanism is not understood. This study aims to elucidate the effects of ethanol extracts of LT (ELT) relative to the role of Runt-related transcription factor- (Runx-) 2 in the invasive and metastatic potentials of mouse colon cancer to determine the underlying mechanisms involved. ELT was evaluated for the antimetastasis activity using CT-26 colon cancer using wound healing, transwell matrigel, and western blot analysis. We used Runx-2-specific siRNA to further determine the relationship between Runx-2 and matrix metalloprotease- (MMP-) 9 in the migration and invasion of CT-26 cells. Runx-2 was first demonstrated to be a transcription factor that plays a remarkable role in diverse biological processes of chondrocytes and osteoblasts, but recently, Runx-2 has been reported to be associated with the progression of certain human cancers. ELT was not altered in its effects on growth inhibition. However, ELT significantly inhibited wound closure and cell invasion in a dose-dependent manner. ELT decreased the metastasis by regulating the activity of MMP-9 and Runx-2 at the translational levels. Our results demonstrate that ELT decreases metastasis by inhibiting the Runx-2–MMP-9 axis. We suggest that it can be used as a novel agent in therapeutic strategies for combating colon cancer.

Rhaponticin (RA; 3'5-dihydroxy-4'-methoxystilbene 3-O-β-D‑glucopyranoside) is a component isolated from various medicinal herbs including Rheum undulatum L. RA has been reported to be an effective treatment for allergy, diabetes, thrombosis, liver steatosis, lung fibrosis and colitis. In addition, RA effectively inhibits tumor growth and induces apoptosis; however, the effects of RA, at non-cytotoxic doses, on the metastasis and angiogenesis of malignant cancer cells have, to be the best of our knowledge, not been identified. In the present study, it was identified that RA suppressed the metastatic potential of MDA‑MB231 breast cancer cells, including colony formation, migration and invasion. Human umbilical vein endothelial cells (HUVECs) treated with RA exhibited a decreased ability to form tube-like networks and to migrate across a Transwell membrane, when compared with RA‑untreated HUVECs. Using the chick chorioallantoic membrane assay, RA treatment significantly suppressed spontaneous and vascular endothelial growth factor (VEGF)-induced angiogenesis. Furthermore, RA inhibited the production of pro-angiogenic factors, including matrix metalloproteinase (MMP)-9, pentraxin‑3, interleukin-8, VEGF and placental growth factor under normoxic and hypoxic conditions, and suppressed the phorbol 12-myristate 13-acetate-induced increase in the gelatinolytic MMP‑9 activity and MMP‑9 expression in HT1080 cells. RA also significantly inhibited the hypoxia-inducible factor (HIF)-1α pathway, leading to decreased HIF‑1α accumulation and HIF‑1α nuclear expression under hypoxia. These results indicated that RA exhibits potent anti‑metastatic and anti‑angiogenic activities with no cytotoxicity via suppression of the HIF‑1α signaling pathway. Thus, RA may control malignant cancer cells by inhibiting the spread from primary tumors and expansion to distant organs.

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.

// Aeyung Kim 1 , Minju Im 1 and Jin Yeul Ma 1 1 Korean Medicine (KM) Application Center, Korea Institute of Oriental Medicine (KIOM), Dong-gu, Daegu 701-300, Republic of Korea Correspondence to: Jin Yeul Ma, email: jyma@kiom.re.kr Keywords: colon cancer; cachexia; weight loss; muscle wasting; herbal medicine Received: August 24, 2017 Accepted: February 23, 2018 Published: March 27, 2018 ABSTRACT Cachexia in cancer patients, characterized by marked involuntary weight loss and impaired physical function, is associated with a poor prognosis in response to conventional treatment and with an increase in cancer-related mortality. Prevention of skeletal muscle loss under cancer-induced cachexia via inhibition of pro-cachectic factors, as well as a reduction in tumor mass, has been considered reasonable pharmacological and nutritional interventions to treat cancer patients. In this study, we constructed a novel herbal formula, SGE, which contains Ginseng Radix alba, Atractylodis Rhizoma alba , and Hoelen , examined its anti-cancer and anti-cachexia efficacies. In in vitro experiments, SGE induced death of CT-26 murine colon carcinoma cells via endoplasmic reticulum stress, and suppressed the production of inflammatory cytokines in Raw 264.7 murine macrophage-like cells. In addition, SGE treatment attenuated CT-26-induced C2C12 skeletal muscle cell atrophy as well as CT-26-induced reduction in lipid accumulation in 3T3-L1 adipocyte. In CT-26 tumor-bearing mice, daily oral administration of 10 and 50 mg/kg SGE remarkably attenuated the cachexia-related symptoms, including body weight and muscle loss, compared with saline treatment, while food intake was not affected. These data collectively suggest that SGE is beneficial as an anti-cancer adjuvant to treat cancer patients with severe weight loss.

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.

// Kwang-Il Park 1 , Kwang-Youn Kim 1 , Tae Woo Oh 1 , Du-Seock Kang 2 , Eung-Kyun Kim 3 , Yong Ryoul Yang 2 , Young-Kyo Seo 2 , Jin-Yeul Ma 1 and Pann-Ghill Suh 2 1 Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM), Cheomdan-ro 70, Dong-gu, Daegu 41062, Republic of Korea 2 School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan 44919, Republic of Korea 3 Gene and Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan 50612, Republic of Korea Correspondence to: Pann-Ghill Suh, email: pgsuh@unist.ac.kr Keywords: phospholipase C γ1; inflammation; colorectal cancer; intestinal barrier dysfunction; inflammatory bowel disease Received: June 26, 2017 Accepted: November 29, 2017 Published: December 19, 2017 ABSTRACT Colorectal cancer (CRC) is the third diagnosed cancer and the second leading cause of cancer-related deaths in the United States. Colorectal cancer is linked to inflammation and phospholipase Cγ1 (PLCγ1) is associated with tumorigenesis and the development of colorectal cancer; however, evidence of mechanisms connecting them remains unclear. The tight junctions (TJ), as intercellular junctional complexes, have an important role for integrity of the epithelial barrier to regulate the cellular permeability. Here we found that PLCγ1 regulated colitis and tumorigenesis in intestinal epithelial cells (IEC). To induce the colitis-associated cancer (CAC), we used the AOM/DSS model. Mice were sacrificed at 100 days (DSS three cycles) and 120 days (DSS one cycle). In a CAC model, we showed that the deletion of PLCγ1 in IEC decreased the incidence of tumors by enhancing apoptosis and inhibiting proliferation during tumor development. Accordingly, the deletion of PLCγ1 in IEC reduced colitis-induced epithelial inflammation via inhibition of pro-inflammatory cytokines and mediators. The PLCγ1 pathway in IEC accelerated colitis-induced epithelial damage via regulation of TJ proteins. Conclusions: Our findings suggest that PLCγ1 is a critical regulator of colitis and colorectal cancer and could further help in the development of therapy for colitis-associated cancer.

Background: The hooks and stems of Uncaria sinensis have been used to mitigate cardiovascular and central nervous system disorders in Asia traditional medicine. Regulation of osteoclast differentiation and activity is a major target for preventing and treating pathological bone diseases. Methods: Tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-stained multinucleated cells were used to examine receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. The activation of RANKL-induced signaling pathways and the expression of transcription factors were investigated by western blot analysis and quantitative real-time polymerase chain reaction. The bone resorption activity of osteoclast was studied using a plate coated with hydroxyl-apatite. Trabecular bone destruction was investigated using a RANKL-induced trabecular bone loss mouse model. Results: We found that water extract of the hooks and stems of U. sinensis (WEUS) inhibits RANKL-induced differentiation of murine bone marrow macrophages and RAW264.7 cells into osteoclasts. WEUS inhibited the activation of NF-κB and the expression of nuclear factor of activated T-cells, cytoplasmic 1. In addition, WEUS suppressed the bone resorbing activity of mature osteoclasts without affecting their survival. Furthermore, oral administration of WEUS suppressed RANKL-induced bone loss with a significant amelioration of trabecular bone micro-structures. WEUS also reduced RANKL-induced increase in serum TRAP5b activity and C-terminal cross-linked telopeptide of type I collagen levels. Conclusion: The present study demonstrates that WEUS has a pharmacological activity that inhibits osteoclast-mediated bone destruction by suppressing osteoclast differentiation and function. These results suggest that U. sinensis could be a promising herbal candidate for preventing and treating bone diseases such as osteoporosis. Keywords: Uncaria sinensis, Osteoclasts, Receptor activator for nuclear factor-κB ligand, Nuclear factor of activated T-cells, Cytoplasmic 1

// Kyoung Mi Moon 1, 5, * , Bonggi Lee 1, 5, * , Ji Won Jeong 1 , Dae Hyun Kim 1 , Yun Jung Park 2 , Hye Rim Kim 2 , Ji Young Park 2 , Min Jo Kim 1 , Hye Jin An 1 , Eun Kyeong Lee 1 , Young Mi Ha 3 , Eunok Im 1 , Pusoon Chun 4 , Jin Yeul Ma 5 , Won-Kyung Cho 5 , Hyung Ryong Moon 2 and Hae Young Chung 1 1 Molecular Inflammation Research Center for Aging Intervention, College of Pharmacy, Pusan National University, Busan, Korea 2 Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan, Republic of Korea 3 Department of Chemistry, Dong-A University, Busan, Republic of Korea 4 College of Pharmacy, Inje University, Inje-ro, Gyeongnam, Korea 5 Korean Medicine Application Center, Korea Institute of Oriental Medicine, Daegu, Republic of Korea * These authors have contributed equally to this work Correspondence to: Hae Young Chung, email: hyjung@pusan.ac.kr Hyung Ryong Moon, email: mhr108@pusan.ac.kr Keywords: oxidative stress, antioxidant, compound 2d, compound 2l, inflammation Received: May 24, 2017 Accepted: July 12, 2017 Published: October 10, 2017 ABSTRACT Liver inflammation is closely associated with metabolic syndrome. Oxidative stress plays a synergistic role in inflammation by activating nuclear factor kappa B (NF-κB) signaling in the liver. Therefore, substantial efforts have been made to develop compounds that inhibit the generation of oxidative stress and activation of NF-κB. We synthesized twenty-six novel 5-(substituted benzyl)-2-oxo- and 5-(substituted benzyl)-2-thioxo-dihydropyrimidine-4,6(1 H ,5 H )-dione derivatives for the development of potential antioxidants and examined their biological activities in vitro and in vivo . Thio-barbiturate-derived compounds 5-[4-hydroxy-3-methoxybenzy]-2-thioxodihydropyrimidine-4,6[1 H ,5 H ]-dione (2d) and 5-[4-hydroxy-3,5-methoxybenzy]-2-thioxodihydropyrimidine-4,6[1 H ,5 H ]-dione (2l) had the strongest inhibitory effect on reactive oxygen species and peroxynitrite generation in vitro . Furthermore, oral administration of compounds 2d and 2l in mice notably suppressed lipopolysaccharide (LPS)-induced oxidative stress and NF-κB activation in the liver. Because macrophages play an essential role in liver inflammation, we investigated the effects of these compounds on inflammatory signaling in LPS-induced RAW264.7 macrophages. LPS-induced NF-κB activation and protein expression of cyclooxygenase 2 and inducible nitric oxide synthase were inhibited by pretreatment of these compounds in macrophages. In parallel with this finding, the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and AKT signalings, which are upstream activators of p65, were decreased by these compounds in macrophages. Our study suggests that compounds 2d and 2l inhibit oxidative stress and NF-кB-mediated inflammation, at least partially, through suppressing PTEN/AKT signaling. Therefore, these compounds may be useful as therapeutic agents for the amelioration of inflammatory diseases.

Background Black ginseng has a more potent biological activity than non-steamed ginseng. We investigated the effects of long-term intake of dietary black ginseng extract (BG) on antioxidant activity in aged mice. We also compared the effects of BG on cognitive deficits with those of white ginseng extract (WG) and red ginseng extract (RG). Methods Ten-month-old mice were fed an AIN-93G-based diet containing 10 g/kg (low dose, L) or 30 g/kg (high dose, H) WG powder, RG powder, or BG powder for 24 wk. We measured serum lipids, the activities of antioxidant enzymes, and malondialdehyde levels. Additionally, the protein expression levels of choline acetyltransferase and vesicular acetylcholine transporter, which are presynaptic cholinergic markers in the cortex and hippocampus of the brain, were measured by western blotting. Results Triglyceride levels were reduced in all the extract-treated mice, except those in the LBG group. High-density lipoprotein cholesterol levels in the HBG group were higher than those in the control group. Total cholesterol levels were reduced in the LBG group. Additionally, glucose levels in the HBG group were significantly reduced by 41.2%. There were lower levels of malondialdehyde in the LBG group than in the control group. Furthermore, glutathione reductase activity increased in the HWG group and the HRG group. The protein expression levels of choline acetyltransferase and vesicular acetylcholine transporter significantly increased in all the ginseng-treated groups. Conclusion The results suggest that supplementation with the tested ginseng extracts may suppress the cognitive decline associated with aging, via regulation of the cholinergic and antioxidant defense systems.

// Bonggi Lee 1, 5, * , Kyoung Mi Moon 2, 5, * , Jong Seung Lim 2 , Yeojin Park 2 , Do Hyun Kim 1, 2 , Sujin Son 1, 2 , Hyoung Oh Jeong 2 , Dae Hyun Kim 1, 2 , Eun Kyeong Lee 2 , Ki Wung Chung 2 , Hye Jin An 2 , Pusoon Chun 3 , Arnold Y. Seo 4 , Ju-Hye Yang 5 , Bong-Seon Lee 5 , Jin Yeul Ma 5 , Won-Kyung Cho 5 , Hyung Ryong Moon 1, 2 and Hae Young Chung 1, 2 1 Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National University, Busan, Republic of Korea 2 College of Pharmacy, Pusan National University, Busan, Republic of Korea 3 College of Pharmacy, Inje University, Gimhae, Gyeongnam, Republic of Korea 4 Janelia Research Campus, Howard Huge Medical Institute, Ashburn, VA, USA 5 Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM), Dong-gu, Daegu, Korea * These authors have contributed equally to this work Correspondence to: Hae Young Chung, email: hyjung@pusan.ac.kr Hyung Ryong Moon, email: mhr108@pusan.ac.kr Keywords: skin, melanogenesis, tyrosinase inhibitor, pigmentation, photoaging Received: May 27, 2017 Accepted: July 13, 2017 Published: September 06, 2017 ABSTRACT Tyrosinase is a key player in ultraviolet-induced melanogenesis. Because excessive melanin accumulation in the skin can induce hyperpigmentation, the development of tyrosinase inhibitors has attracted attention in cosmetic-related fields. However, side effects including toxicity and low selectivity have limited the use of many tyrosinase inhibitors in cosmetics. We synthesized 12 novel 2-(substituted benzylidene)malononitrile derivatives and investigated their anti-melanogenic activities. Of these 12 compounds, 2-(3, 4-dihydroxy benzylidene)malononitrile (BMN11) exhibited the strongest inhibitory activity against tyrosinase (IC 50 = 17.05 μM). In parallel with this, BMN11 treatment notably decreased alpha-melanocyte-stimulating hormone-induced melanin accumulation in B16F10, cells without toxicity and also decreased melanin accumulation in a human skin model. As a mechanism underlying the BMN11-mediated anti-melanogenic effect, docking simulation showed that BMN11 can directly bind to tyrosinase by forming two hydrogen bonds with GLY281 and ASN260 residues, and via three hydrophobic interactions with VAL283, PHE264, and ALA286 residues in the tyrosinase binding pocket, and this likely contributes to its inhibitory effect on tyrosinase. Consistently, Lineweaver-Burk and Cornish-Bowden plots showed that BMN11 is a competitive inhibitor of tyrosinase. We concluded that BMN11 may be a novel tyrosinase inhibitor that could be used in cosmetics.

// Bonggi Lee 1, * , Kyoung Mi Moon 1, * , Bong-Seon Lee 1 , Ju-Hye Yang 1 , Kwang Il Park 1 , Won-Kyung Cho 1 and Jin Yeul Ma 1 1 Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM), Daegu 41062, Republic of Korea * These authors have contributed equally to this work Correspondence to: Jin Yeul Ma, email: jyma@kiom.re.kr Won-Kyung Cho, email: wkcho@kiom.re.kr Keywords: swertiajaponin; melanogenesis; pigmentation; human skin; tyrosinase Received: July 27, 2017 Accepted: August 10, 2017 Published: September 15, 2017 ABSTRACT Many skin-whitening compounds target tyrosinase because it catalyzes two rate-limiting steps in melanin synthesis. Although many tyrosinase inhibitors are currently available for a skin–whitening purpose, undesirable adverse effects are also reported. Thus, numerous efforts have been made to develop safer tyrosinase inhibitors from natural products. In line with this, we tested fifty flavonoids, a group of naturally occurring antioxidants and metal chelators, and screened swertiajaponin as the strongest tyrosinase inhibitor in cell-free experiments. Swertiajaponin did not show cytotoxicity in B16F10, HaCat, and Hs27 cells and exhibited strong anti oxidative activity in experiments using the cell-free system and B16F10 cells. It markedly inhibited αMSH- or UVB-induced melanin accumulation in B16F10 cells and suppressed skin pigmentation in a human skin model. As underlying mechanisms, in silico and Lineweaver-Burk plot analyses exhibited that swertiajaponin may directly bind to and inhibit tyrosinase activity by forming multiple hydrogen bonds and hydrophobic interactions with the binding pocket of tyrosinase. In addition, western blotting results indicated that swertiajaponin inhibited oxidative stress-mediated MAPK/MITF signaling, leading to decrease in tyrosinase protein level. Together, swertiajaponin suppresses melanin accumulation by inhibiting both activity and protein expression levels of tyrosinase. Thus, it would be a novel additive for whitening cosmetics.

A new 8-prenyl-flavonoid, epimedokoreanoside III, and 11 other flavonoids, were isolated from the aerial parts of Epimedium koreanum Nakai. Their chemical structures were elucidated by nuclear magnetic resonance and mass analyzes. All isolates were evaluated for their soluble epoxide hydrolase and tyrosinase inhibitory activities. Among the isolates, compounds 1, 5, 9, 10, and 12 exerted soluble epoxide hydrolase inhibitory activity in dose-dependent manner, with IC50 values ranging from 26.0 ± 1.2 to 37.6 ± 1.0 µM, whereas compounds 4 and 9 showed potent inhibition of tyrosinase in a dose-dependent manner, having IC50 values of 16.8 ± 2.7 and 8.7 ± 2.7 µM, respectively.

Background: Traditional medicine and herbal prescriptions are becoming more popular, and they account for a large share of the worldâs healthcare research studies, developments, and market demands. Increasing scientific evidence of the substantive efficacies such as preventive health keeping pharmaceutical materials and dietary supplements can be found elsewhere. Above all, safety should be the critical premise for considering developmental materials such as pharmaceuticals without side effects and toxicity. Methods: The authors formulated KIOM2012H (K2H) using four herbs that were reported to have medicinal effectsâincluding anticancer, antiaging, antimicrobial, inflammation, and neuroprotective properties. In order to examine the toxicity, single and repeated dose toxicity, and genotoxicities of bacterial mutation, micronucleus, and chromosomal aberration assays were conducted. Results: All experimental observations and results showed normal findings. Toxicities or abnormal signs were not observed in all experimental assays, including oral administration, animal behavior, clinical findings, and changes in body weight in vivo. In vitro bacterial cultures produced no revertant colonies, and no increased numbers of structural or numerical aberrant metaphases were found in the metaphase chromosomes examined. Moreover, no significant increased frequency of micronucleus was observed in any of the doses used. Overall, no acute toxicity or genotoxicity was found in all analysis parameters in all the assays conducted. Conclusion: Reviewing the results as a whole, K2H extract was regarded as a safe material with no toxicity, and can be applied for the research and development of complementary and alternative medicines with improved efficacy in current therapeutic healthcare, based on traditional medicine and herb resources. Keywords: genotoxicity, herb, toxicity, traditional medicine

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.

Methylglyoxal (MG) is a reactive dicarbonyl intermediate and a precursor of advanced glycation end products (AGEs). The authors investigated the role played by AGEs in muscle myopathy and the amelioration of its effects by curcumin and gingerol. In addition to producing phenotypical changes, MG increased oxidative stress and reduced myotube formation in C2C12 cells. RAGE (receptor for AGEs) expression was up-regulated and MYOD and myogenin (MYOG) expressions were concomitantly down-regulated in MG-treated cells. Interestingly, AGE levels were higher in plasma (~32 fold) and muscle (~26 fold) of diabetic mice than in control mice. RAGE knock-down (RAGEkd) reduced the expressions of MYOD and MYOG and myotube formation in C2C12 cells. In silico studies of interactions between curcumin or gingerol and myostatin (MSTN; an inhibitor of myogenesis) and their observed affinities for activin receptor type IIB (ACVRIIB) suggested curcumin and gingerol reduce the interaction between MSTN and ACVRIIB. The findings of this study suggest enhanced AGE production and subsequent RAGE-AGE interaction obstruct the muscle development program, and that curcumin and gingerol attenuate the effect of AGEs on myoblasts.

Background: The traditional medicine oyaksungi-san (OY) has been prescribed in East Asia for hundreds of years for the treatment of stroke, paralysis, and ataxia. OY also has therapeutic effects on arthralgia, myalgia, and rheumatoid arthritis, and recent studies have shown its protective effects against apoptosis of hippocampal cells and its anti-inflammatory effects on the peripheral blood cells of patient with cerebral infarction. Many studies have explored the use of traditional medicine and herb materials in the development of safe, novel, and effective pharmaceuticals with fewer side effects. These efforts commonly adopt a bioconversion tool for fermentation with beneficial microbes. However, only pharmaceuticals with high levels of safety and low levels of toxicity can be used in healthcare system. Methods: OY water extract was fermented with Lactobacillus and assayed for acute toxicity and genotoxicity. Single dose acute toxicity, bacterial reverse mutation, chromosome aberrations, and micronucleus were observed and assayed in rats, histidine/tryptophan auxotrophic bacteria, Chinese hamster ovary fibroblast cells, and mice bone marrow cells, respectively. Results: All the experimental animals showed no abnormal behavior, clinical signs, body weight increases, or mortality. In the bacterial cultures, no revertant colonies were observed. Morphological and numerical chromosomal aberrations were not found in all metaphases examined. Frequency of induced micronuclei was not significantly increased in all doses applied. Conclusion: As a whole, no acute toxicity or genotoxicity were observed in all the assays examined. Therefore, fermented OY is considered to be a safe material that can be used for development of complementary and alternative medicine using bioconversion. Keywords: fermentation, genotoxicity, oyaksungi-san, traditional medicine, toxicity

Agastache rugosa (Fisch. & C.A. Mey.) Kuntze (A. rugosa), commonly known as Korean mint, has traditionally been used for both food and medicine, depending on which part of the plant is used. We investigated the anti-proliferative activities of A. rugosa extract in vascular smooth muscle cells (VSMCs) and analysed several molecular pathways to determine the active component of the extract. A. rugosa extract inhibited PDGF-BB-stimulated VSMC proliferation and DNA synthesis. A. rugosa extract caused arrest of the cell cycle at the G0/G1 transition by up-regulating the levels of p21WAF1/CIP1 and p27KIP1. Among the components of the A. rugosa extract reported in a previous study, rosmarinic acid (RA) only inhibited DNA synthesis and dose-dependently suppressed PCNA expression. These results indicate that A. rugosa extract, which includes the active component RA, demonstrates anti-proliferative activity by arresting the cell cycle at the G0/G1 phase through the upregulation of p21WAF1/CIP1 and p27Kip1 expression.

Lonicera japonica Thunb. (L. japonica T.) has historically been used in Korean herbal medicine due to its anticancer and protective effects on the respiratory system. In the present study, the polyphenolic compounds in L. japonica T. were investigated using high-performance liquid chromatography coupled with tandem mass spectrometry, and its anticancer effects on A549 non-small-cell lung cancer cells were studied. Polyphenolic compounds potentially inhibit A549 cells in a dose-dependent manner. Flow cytometry and western blot analysis demonstrated that polyphenolic compounds induce apoptosis by regulating the protein expression levels of caspases, poly-(ADP-ribose) polymerase and the B-cell lymphoma-2-associated X-protein/B-cell lymphoma-extra large ratio. Furthermore, polyphenolic compounds inhibited mitochondrial membrane potential activity. Caspase-3 activity was increased in a dose-dependent manner and polyphenolic compounds inhibited the activation of protein kinase B by dephosphorylation. These results suggest that polyphenolic compounds in A549 cells indicate the anticancer activity through the induction of apoptosis.

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.

Background Galla Rhois has been considered to have medicinal properties against diarrhea, excessive sweating, bleeding, and chronic cough in Asian countries. Gallotannins, which are Galla Rhois-derived tannins, have been reported to possess biological and pharmacological activities, especially anticancer activity. In this study, we evaluated the effect of steaming at a temperature over 120 °C on the chemical constituents and biological activities of the water extract of Galla Rhois (GRE). Methods GRE was steamed at a temperature over 120 °C (AGRE), and its specific constituents were analyzed; the results were validated using a high-performance liquid chromatography–diode array detector system. To evaluate the anticancer effect of GRE and AGRE, cell viability assay, cell cycle analysis, and Western blot analysis were performed in HCT116 human colon cancer cells. Results Steaming markedly increased the contents of gallic acid and ellagic acid in GRE, and GRE or AGRE treatment reduced the viability of HCT116 cells. Notably, the steaming process enhanced the growth inhibitory effect of GRE in cancer cells. AGRE induced apoptosis through the activation of caspase-3, caspase-8, and caspase-9. Additionally, AGRE regulated the activation of mitogen-activated protein kinases including extracellular signal-regulated kinase, p38, and c-Jun NH2-terminal kinase, whereas GRE did not. However, both GRE and AGRE inhibited the activation of AKT. Conclusion Compared with GRE, AGRE is more potent in its ability to induce apoptosis in HCT116 cells; therefore, we suggest that the steaming process may be useful as a feasible method for improving the anticancer effect of GRE.

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.

Toosendanin (TSN) is a major bioactive component of Melia Fructus (MF) with anti-inflammatory, anti-botulinum, anti-microbial, and analgesic efficacy. Our previous study demonstrated that MF has anti-influenza A virus activity; however, the contribution of TSN is still unclear. In this study, we found that TSN suppressed influenza A virus infection when administered before or concurrent with the virus, but not after infection. TSN pretreatment inhibited viral hemagglutinin (HA), nucleoprotein (NP), polymerase acidic (PA) protein, and matrix protein 2 (M2) mRNA synthesis as well as NP, PA, M2, and nonstructural protein 1 (NS1) expression but had no effect on HA or neuraminidase (NA) activity. In addition, TSN induced cytoplasmic location of PA protein disrupting nuclear translocation. Docking simulation suggested that the binding affinity of TSN to PA protein may be stronger than that of a known PA protein inhibitor. Pretreatment with TSN also suppressed the infection-induced phospho-AKT expression but not the host immune response. Oral pretreatment with TSN enhanced the survival of infected mice. These results suggest that TSN inhibits influenza A virus infection at an early stage by altering PA protein nuclear localization. Thus, TSN may be a promising candidate for anti-influenza agent targeting the PA protein of the influenza A virus RNA polymerase complex.

The herbs Plantago asiatica and Clerodendrum trichotomum have been commonly used for centuries in indigenous and folk medicine in tropical and subtropical regions of the world. In this study, we show that extracts from these herbs have antiviral effects against the respiratory syncytial virus (RSV) in vitro cell cultures and an in vivo mouse model. Treatment of HEp2 cells and A549 cells with a non-cytotoxic concentration of Plantago asiatica or Clerodendrum trichotomum extract significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and also blocked syncytia formation. Interestingly, oral inoculation with each herb extract significantly improved viral clearance in the lungs of BALB/c mice. Based on reported information and a high-performance liquid chromatography (HPLC) analysis, the phenolic glycoside acteoside was identified as an active chemical component of both herb extracts. An effective dose of acteoside exhibited similar antiviral effects as each herb extract against RSV in vitro and in vivo. Collectively, these results suggest that extracts of Plantago asiatica and Clerodendrum trichotomum could provide a potent natural source of an antiviral drug candidate against RSV infection.

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

Several components isolated from Glycyrrhizae radix rhizome (GR), including glycyrrhizin, liquiritin, and liquiritigenin, have been shown to induce cancer cell death and inhibit cancer metastasis. Isoliquiritin apioside (ISLA), a component isolated from GR, has been effective for treating tetanic contraction and genotoxicity. However, the effects of ISLA on the metastasis and angiogenesis of malignant cancer cells and endothelial cells (ECs) have not been reported. In this study, we found that up to 100 μM ISLA did not affect cell proliferation but efficiently suppressed the metastatic ability of HT1080 cells, as assessed by scratch-wound migration, Transwell® migration, scratch-wound invasion, Transwell® invasion, and three-dimensional spheroid invasion. ISLA significantly decreased phorbol 12-myristate 13-acetate (PMA)-induced increases in matrix metalloproteinase (MMP) activities and suppressed PMA-induced activation of mitogen-activated protein kinase as well as NF-κB, which are involved in cancer metastasis. In addition, ILSA treatment reduced the production of pro-angiogenic factors in HT1080 cells, including MMP-9, placental growth factor, and vascular endothelial growth factor under normoxia as well as hypoxia conditions, by impairing the hypoxia-inducible factor-1α pathway. We also found that the abilities of human umbilical vein ECs to migrate across the Transwell® and to form tube-like structures were significantly reduced by ISLA treatment. Moreover, using the chorioallantoic membrane assay, vessel formation with or without vascular endothelial growth factor was significantly suppressed by ISLA. These results suggested that ISLA possesses anti-metastatic and anti-angiogenic abilities in malignant cancer cells and ECs, with no cytotoxicity. ISLA may therefore be a safe and effective lead compound to develop anti-cancer drug for limiting the spread of primary tumors to distant organs to form secondary tumors.

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

Background: Yijin-tang (YJ) has been used traditionally for the treatment of cardiovascular conditions, nausea, vomiting, gastroduodenal ulcers, and chronic gastritis. In this study, a simple and sensitive high-performance liquid chromatography (HPLC) method was developed for the quantitation of nine bioactive compounds in YJ: homogentisic acid, liquiritin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, 6-gingerol, and pachymic acid. Methods: Chromatographic separation of the analytes was achieved on an RS Tech C18 column (4.6 mm × 250 mm, 5 μm) using a mobile phase composed of water containing 0.1% (v/v) trifluoroacetic acid (TFA) and acetonitrile with a gradient elution at a flow rate of 1.0 mL/min. Results: Calibration curves for all analytes showed good linearity (R2 ≥ 0.9995). Lower limits of detection and lower limits of quantification were in the ranges of 0.03–0.17 μg/mL and 0.09–0.43 μg/mL, respectively. Relative standard deviations (RSDs; %) for intra- and interday assays were < 3%. The recovery of components ranged from 98.09% to 103.78%, with RSDs (%) values ranging from 0.10% to 2.59%. Conclusion: This validated HPLC method was applied to qualitative and quantitative analyses of nine bioactive compounds in YJ and fermented YJ, and may be a useful tool for the quality control of YJ.

Aloe has long been used in food products, beverages and cosmetics, and as a traditional medicine to treat various diseases in many countries. In the present study, a new chromone, aloe resin E ( 1 ), and a new pyrone, aloenin C ( 11 ), together with thirteen known compounds were isolated from aqueous dissolved Aloe exudates and their structures were identified by spectroscopic analysis. Nuclear factor kappa B (NF-κB) inhibitory activity of the isolated compounds was evaluated using an NF-κB luciferase assay in HepG2 cells. Among them, 7-hydroxy-5-(hydroxymethyl)-2-methylchromone ( 4 ), 5-(( S )-2′-oxo-4′-hydroxypentyl)-2-hydroxymethylchromone ( 9 ), and aloenin aglycone ( 13 ) showed significant inhibitory effects against TNFα-induced NF-κB transcriptional activity in a dose-dependent manner with IC 50 values ranging from 14.92 to 18.70 µM. Furthermore, the transcriptional inhibition of compounds 4 , 9 , and 13 was confirmed by a decrease in the expression of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) genes in HepG2 cells.

The mistletoeLoranthus parasiticushas been used as a compound for traditional medicine in Northeast Asia for a long time and is known to possess neuroprotective action. Nonetheless, the effect ofLoranthus parasiticuson allergic responses remains unknown. In the present study, we evaluated whether the water extract ofLoranthus parasiticus(LPE) could inhibit IgE-mediated allergic responses in RBL-2H3 cells. LPE inhibited the release ofβ-hexosaminidase (IC50, 184.5 μg/mL) and the formation of tumor necrosis factor-α(IC50, 84.27 μg/mL), interleukin-4 (IC50, 93.43 μg/mL), prostaglandin E2(IC50, 84.10 μg/mL), prostaglandin D2, and leukotriene C4(IC50, 43.27 μg/mL) in a concentration-dependent manner. Moreover, LPE inhibited phosphorylation of Syk, PLCγ1/2, PKCδ, ERK, JNK, p38, and Akt. In the late phase, LPE decreased 5-lipoxygenase phosphorylation and COX-2 expression but not cPLA2phosphorylation. Additionally, LPE included total phenolic compounds (10.72 mg/g dry weight) and total flavonoids (56.20 mg/g dry weight). These results suggest that the phenolic compounds or flavonoids contained in LPE may be associated with antiallergic activity. The phenolic compounds and flavonoids in LPE are antiallergic phytochemicals capable of inhibiting the activation of the FcεRI signaling cascade in mast cells. Such effects may provide further information for the development of a phytomedicine for allergic diseases.

Artemisia capillaris has been used to treat jaundice and relieve high liver-heat in traditional medicine. In this study, we found that the administration of a water extract from A. capillaris (WEAC) to the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced bone loss model significantly prevents osteoporotic bone loss, increasing bone volume/trabecular volume by 22% and trabecular number by 24%, and decreasing trabecular separation by 29%. WEAC stimulated in vitro osteoblast mineralization from primary osteoblasts in association with increasing expression of osterix, nuclear factor of activated T cells cytoplasmic 1, and activator protein-1, as well as phosphorylation of extracellular signal-regulated kinase. In contrast to the anabolic effect of WEAC, WEAC significantly suppressed in vitro osteoclast formation from bone marrow macrophages by inhibiting the RANKL signaling pathways and bone resorption by downregulating the expression of resorption markers. Therefore, this study demonstrated that WEAC has a beneficial effect on bone loss through the regulation of osteoblast mineralization, as well as osteoclast formation and bone resorption. These results suggest that A. capillaris may be a promising herbal candidate for therapeutic agents to treat or prevent osteoporotic bone diseases.

Rhapontici Radix (RR) has been used in traditional medicine in East Asia and has been shown to have various beneficial effects. However, its biological properties or mechanism on inflammation-related diseases is unknown. The goal of this study was to determine the anti-inflammatory activity and underlying molecular mechanisms of Rhapontici Radix ethanol extract (RRE). The inhibitory effect of RRE on the production of NO, cytokines, inflammatory-related proteins, and mRNAs in LPS-stimulated macrophages was determined by the Griess assay, ELISA, Western blot analysis, and real-time RT-PCR, respectively. Our results indicate that treatment with RRE significantly inhibited the secretion of NO and inflammatory cytokines in RAW 264.7 cells and mouse peritoneal macrophages without cytotoxicity. We also found that RRE strongly suppressed the expression of iNOS and COX-2 and induced HO-1 expression. It also prevented nuclear translocation of NF-κB by inhibiting the phosphorylation and degradation of IκBα. Furthermore, the phosphorylation of MAPKs in LPS-stimulated RAW 264.7 cells was significantly inhibited by RRE. These findings suggest that RRE may operate as an effective anti-inflammatory agent by inhibiting the activation of NF-κB and MAPK signaling pathways and inducing HO-1 expression in macrophages. Our results suggest that RRE has potential value as candidate to inflammatory therapeutic phytomedicine.

Alpinia officinarum rhizome has been used as a traditional herbal remedy to treat inflammatory and internal diseases. Based on the previously observed inhibitory effect of A. officinarum rhizome in an arthritis model, we evaluated whether a water extract of A. officinarum rhizome (WEAO) would enhance in vitro osteoblast mineralization using calvarial osteoblast precursor cells or would inhibit in vitro osteoclast differentiation and bone resorption using bone marrow derived macrophages. In osteoblasts, WEAO enhanced the mRNA levels of transcription factor (runt-related transcription factor 2, smad1, smad5, and junB) and marker (bone morphogenetic protein-2, collagen type 1alpha1, and osteocalcin) genes related to osteoblast mineralization, consistent with increased alizarin red S staining intensity. WEAO markedly inhibited osteoclast differentiation by suppressing the receptor activator for nuclear factor-[Formula: see text]B ligand-induced downregulation of inhibitor of DNA binding 2 and V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and the phosphorylation of c-Jun N-terminal kinase, p38, nuclear factor-[Formula: see text]B, c-Src, and Bruton’s tyrosine kinase to induce nuclear factor of activated T cells cytoplasmic 1 expression. WEAO also suppressed the resorbing activity of mature osteoclasts by altering actin ring formation. Therefore, the results of this study demonstrate that WEAO stimulates osteoblast mineralization and inhibits osteoclast differentiation. Thus, WEAO may be a promising herbal candidate to treat or prevent pathological bone diseases by regulating the balance between osteoclast and osteoblast activity.

Angelica tenuissima Nakai is a widely used commodity in traditional medicine. Nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of Angelica tenuissima Nakai. In the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of Angelica tenuissima Nakai both in vitro and in vivo. In vitro, an effective dose of Angelica tenuissima Nakai markedly inhibited the replication of Influenza A virus (PR8), Vesicular stomatitis virus (VSV), Herpes simplex virus (HSV), Coxsackie virus, and Enterovirus (EV-71) on epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. Such inhibition can be described by the induction of the antiviral state in cells by antiviral, IFNrelated gene induction and secretion of IFNs and pro-inflammatory cytokines. In vivo, Angelica tenuissima Nakai treated BALB/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3, and H9N2). We also found that Angelica tenuissima Nakai can induce the secretion of IL-6, IFN-λ, and local IgA in bronchoalveolar lavage fluid (BALF) of Angelica tenuissima Nakai treated mice, which correlating with the observed prophylactic effects. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. Therefore, an extract of Angelica tenuissima Nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents.

Inflammatory bowel disease (IBD) comprises Crohn’s disease (CD) and ulcerative colitis (UC). IBD increases the risk of colorectal cancer (CRC), depending on the extent and duration of intestinal inflammation. Increased IL-6 expression has been reported in IBD patients, which may be associated with intestinal barrier function through discontinuous tight junction (TJ). KIOM-MA is a specific agent for allergic diseases and cancer, and it is composed of several plants; these herbs have been used in traditional oriental medicine. We fermented KIOM-MA, the product of KIOM-MA128, using probiotics to improve the therapeutic efficacy via the absorption and bioavailability of the active ingredients. In this study, we demonstrated that KIOM-MA/MA128 exhibited anticolitis effects via the modulation of TJ protein. Interleukin-6 resulted in a dose-dependent decrease in the TER and an increase in the FITC-dextran permeability; however, pretreatment with 400 µg/ml KIOM-MA/MA128 resulted in a significant increase in the TER and a decrease in the FITC-dextran permeability via IL-6 induction. Furthermore, protein and mRNA TJ levels remained stable after pretreatment with 400 µg/ml KIOM-MA/MA128. Moreover, KIOM-MA/MA128 suppressed the expression of PLCγ1 and PKC. Taken together, these findings suggest novel information and clue of the anticolitis effects of KIOM-MA128 via regulation of tight junction.

Background Several components isolated from rhubarb, the root of Rheum undulatum L., including emodin, rhein, rhaponticin, and piceatannol, have been reported to induce cell death and inhibit metastasis in various types of cancer. Recently, piceatannol-3-O-β-D-glucopyranoside (PG) isolated from rhubarb was demonstrated to improve vascular dysfunction by inhibiting arginase activity. Purpose In this study, we examined the anti-cancer activities of PG, including effects on the proliferation, metastasis, and angiogenesis of endothelial and malignant cancer cells. Results We found that PG did not affect the proliferation of human fibrosarcoma (HT1080) and human umbilical vein endothelial cells (HUVECs) at treatments up to 100 μM. However, PG efficiently suppressed the metastatic ability of HT1080 cells, as determined by scratch wound migration, transwell migration/invasion assay, and three-dimensional (3D) spheroid invasion assay. PG significantly suppressed the phorbol 12-myristate 13-acetate (PMA)-induced increase of matrix metalloproteinase (MMP)-9 expression as well as gelatinolytic MMP-9 activity, which are essential for cancer metastasis. In addition, PG treatment reduced the production of proangiogenic factors in HT1080 cells under normoxic and hypoxic conditions and suppressed hypoxia-induced activation of the hypoxia-inducible factor (HIF)-1α pathway. We also found that HUVEC angiogenic activity, including migration and tubular structure formation, were significantly reduced by PG treatment. Moreover, in an in ovo chick chorioallantoic membrane assay, spontaneous and vascular endothelial growth factor (VEGF)-induced vessel formation were significantly inhibited by PG treatment. Conclusion These results collectively indicate that PG has potent anti-metastatic and anti-angiogenic activities with no cytotoxicity. Thus, PG may be useful to limit the hyperplasia of malignant tumors and the spread of cancer to distant secondary organs.

Salinomycin, a monocarboxylic ionophore in Streptomyces albus, has been studied as an anti-cancer agent. However, we wondered whether salinomycin has another effect such as an anti-oxidant and hepatic protectant, because some chemical drugs treating human diseases were sometimes related with their toxic effects. Therefore, this study was conducted to examine the effects of salinomycin against oxidative stress and mitochondrial impairment in vivo and in vitro as well as the cellular mechanisms of action. In hepatocyte, salinomycin inhibited arachidonic acid (AA) + iron-induced apoptosis, mitochondrial dysfunction and ROS production. As a molecular mechanism, salinomycin induced autophagy through AMP-activated protein kinase (AMPK) activation, as assessed by the accumulation of acidic vesicle organelles, p62 and LC3-II. Moreover, these protective effects were blocked by AMPK inhibition, which indicates the importance of AMPK in the process of salinomycin's effects. In mice, oral administration of salinomycin protected against carbon tetrachloride (CCl4)-induced oxidative stress and liver injury, and also activated AMPK as well as autophagy-related proteins in the liver. Collectively, salinomycin had the ability to protect hepatocytes against AA+iron-induced reactive oxygen species production and mitochondrial dysfunction, as well as CCl4-induced liver injury. Although this beneficial effect was demonstrated under severe oxidative stress, this study showed that salinomycin protected the liver against the oxidative stress and liver damage through AMPK and autophagy, and suggest that salinomycin has a possibility to treat a broad range of diseases.

Artemisia apiacea Hance is a traditional herbal medicine used for treating eczema and jaundice in Eastern Asia including China, Korea, and Japan. However, the biological and pharmacological actions of Artemisia apiacea Hance in atopic dermatitis (AD) are not fully understood. An ethanolic extract of Artemisia apiacea Hance (EAH) was tested in vitro and in vivo to investigate its anti-inflammatory activity and anti-atopic dermatitis effects. The results showed that EAH dose-dependence inhibited production of regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin (IL)-6, IL-8, and thymus and activation-regulated chemokine (TARC). EAH inhibited the activation of p38, extracellular signal-regulated kinases (ERK), and STAT-1 and suppressed the degradation of inhibited both nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (I&kappa, B-&alpha, ) in TNF-&alpha, /IFN-&gamma, &ndash, stimulated HaCaT cells. EAH also suppressed the translocation of inflammation transcription factors such as NF-&kappa, B p65 in TNF-&alpha, stimulated HaCaT cells. In addition, EAH reduced 2,4-dinitrochlorobenzene (DNCB)-induced ear thickness and dorsal skin thickness in a dose-dependent manner. EAH appeared to regulate chemokine formation by inhibiting activation of and ERK as well as the NK-&kappa, B pathways. Furthermore, EAH significantly improved the skin p38 conditions in a DNCB-induced AD-like mouse model.

Although traditionally used to improve indigestion, diarrhea, dysentery, and constipation, the therapeutic effects of Citrus aurantium on intestinal inflammation remain unclear. The aim of this study was to evaluate the beneficial effects and to identify the active components of a hydroalcoholic extract of C. aurantium (HECA) on ulcerative colitis. HECA was prepared with 70% ethanol solution in water and extracted at 37 °C for 12 h in triplicate, filtered through a sieve, and lyophilized. Phytochemical identification of HECA was performed by ultra-performance liquid chromatography-diode array detector-tandem mass spectrometry (UPLC-DAD-MS/MS). Animals were randomly assigned to one of four groups based on the treatment conditions. Ulcerative colitis was induced by administration of 2.5% dextran sodium sulfate (DSS) in drinking water for 5 d. Body weight, clinical signs, colon length, pro-inflammatory cytokine expression levels, and histopathological findings were evaluated. In UPLC-DAD-MS/MS analysis, the identified phytochemical components of HECA included four alkaloids, seven coumarins, 18 flavonoids, two lignans, two phenolics, and 10 terpenoids. HECA markedly protected against body weight loss and colon shortening. In pathological examination, HECA alleviated DSS-related mucosal inflammatory lesions in the colon. Moreover, HECA markedly reduced the expression levels of interleukin-6, interferon-γ, tumor necrosis factor-α, and monocyte chemotactic protein-1 in colonic inflammation. Taken together, HECA has potential to relieve mucosal inflammation in the colon, suggesting that the putative active ingredients are responsible for the anti-ulcerative effects.