Ameliorative effect of Alnus japonica ethanol extract on colitis through the inhibition of inflammatory responses and attenuation of intestinal barrier disruption in vivo and in vitro.

Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract caused by high levels of pro-inflammatory cytokines and epithelial barrier dysfunction. Alnus japonica Steud. (Betulaceae) has been used in traditional Asian medicine. However, the potential of A. japonica for the treatment of intestinal inflammation has not been investigated. This study investigated the effects of ethanol extract from A. japonica bark (AJE) on colonic mucosa injury in mice with dextran sodium sulfate (DSS)-induced colitis. Treatment with AJE ameliorated pathological damage and the histopathologic features of DSS-induced colitis. The administration of AJE also inhibits DSS-induced pro-inflammatory cytokines expression, including interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2. Notably, AJE administration attenuated the reduction of tight junction proteins, zonula occludens (ZO)-1 and occludin, in DSS-induced colitis. In addition, AJE increased heme oxygenase (HO)-1 expression and prevented DSS-induced apoptosis in colonic epithelial cells. Furthermore, in vitro studies demonstrated that AJE inhibits TNF-α-induced IL-8, IL-1ß, and COX-2 expression in human intestinal epithelial HT-29 cells and tert-butyl hydroperoxide-induced reduction of ZO-1 and occludin expression in human intestinal epithelial Caco-2 cells. AJE-induced HO-1 protein expression was also found in both HT-29 and Caco-2 cells. Taken together, our findings demonstrated that AJE inhibits intestinal inflammation and protects against intestinal barrier disruption in mice with DSS-induced colitis in vivo and human intestinal epithelial cells in vitro. These results suggest that AJE might have beneficial effects for the treatment of IBD.

PF2405, standardized fraction of Scutellaria baicalensis, ameliorates colitis in vitro and in vivo.

Standardized extraction procedures for herb are as important as their authentication to maintain their quality and ensure their safe use. We had prepared a standardized and purified Scutellaria baicalensis Georgi extract, PF2405, which was enriched with three major components, baicalein, oroxylin A and wogonin. In the present study, we investigated the potential anti-inflammatory effects of PF2405 in vitro and in two different experimental animal models of inflammatory bowel disease. Effect of PF2405 studied in tumor necrosis factor (TNF)-α-induced HT-29 cells in vitro. In vivo experimental colitis models were induced by administration of trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS). PF2405 (50 µg/ml) decreased TNF-α-induced cyclooxygenase (COX)-2 expressions through inhibition of phosphorylation of c-Jun N-terminal kinases and p38 mitogen-activated protein kinase in HT-29 cells. Combination of baicalein (20 µg/ml), oroxylin A (8 µg/ml), and wogonin (2 µg/ml) markedly inhibits TNF-α-induced COX-2 expression when compared with individual components. PF2405 (25 mg/kg b.w.) treatment significantly reduced histopathological severity; suppressed expression of COX-2, TNF-α, and interleukin-1ß in TNBS-induced mice. Moreover, PF2405 (25 mg/kg b.w.) has both potent preventive and therapeutic activities in DSS-induced colitis. Collectively, PF2405 shows prominent anti-inflammatory effect that can be used as a new therapeutic approach for intestinal inflammatory disorders.

Anti-fibrotic effect of PF2401-SF, a standardized fraction of Salvia miltiorrhiza, in thioacetamide-induced experimental rats liver fibrosis.

We previously reported the in vitro and in vivo hepatoprotective and anti-fibrotic effects of PF2401-SF, a standardized fraction of Salvia miltiorrhiza, against acute and subacute liver injury. The aim of this study was to investigate the effect of PF2401-SF on liver fibrosis induced by thioacetamide (TAA), a chronic liver injury model (12 weeks) that closely resembles fibrosis and cirrhosis in humans. Hepatoprotective activity was indicated by low serum levels of the markers aspartate amino transferase and alanine amino transferase .In addition, compared to the TAA-group livers, the PF2401-SF-treated liver tissues showed no fibrous tissue deposition in the portal areas, hepatocyte morphology more closely resembling normal tissue morphology, and significantly reduced collagen deposition. Furthermore, downregulation of collagen 1(α) and tissue inhibitor of metalloproteinase (TIMP)1 protein and mRNA expression also supports PF2401-SF's anti-fibrotic effect. We also observed reduced expression of α-smooth muscle actin (α-SMA), an important marker of hepatic stellate cells (HSCs) activation. From these results, we conclude that PF2401-SF's anti-fibrotic mechanism in the TAA model involves reduced HSC activation, and may be mediated by downregulation of central markers of fibrosis, including collagen 1(α), TIMP1, and α-SMA.

PF2401-SF, standardized fraction of Salvia miltiorrhiza shows anti-inflammatory activity in macrophages and acute arthritis in vivo.

Standardization of processing methods for herbs is as important as authentication to maintain their quality and ensure their safe use. We had previously prepared a standardized and purified Salvia miltiorrhiza Bunge extract, PF2401-SF, and showed that it protects against liver injury in vivo, at a greater potency than an ethanol extract. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). In this study, we investigated potential anti-inflammatory effects of PF2401-SF in vitro and in vivo. We demonstrated that PF2401-SF shows anti-inflammatory potency on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. A mechanistic study indicated that PF2401-SF induced heme oxygenase (HO)-1 expression through extracellular signal-regulated kinases (ERK1/2) phosphorylation. Moreover, we also evaluated that PF2401-SF significantly reduced inflammation on carrageenan- or dextran-induced acute arthritis in rats. Our results suggest that PF2401-SF may be a potential candidate for the treatment of various inflammatory diseases.

PF2401-SF, standardized fraction of Salvia miltiorrhiza, induces apoptosis of activated hepatic stellate cells in vitro and in vivo.

During the course of our attempts to develop a potential herbal medicine, we had previously prepared PF2401-SF, a standardized fraction of S. miltiorrhiza, and reported its hepatoprotective activity in vitro as well as in vivo. Since apoptosis of activated hepatic stellate cells (HSCs) is a well-accepted anti-fibrotic strategy, in this study, we investigated the direct effect of PF2401-SF on t-HSC/Cl-6 cells in vitro and on CCl4-induced liver injury in vivo. We evaluated the activation and cleavage of hallmarkers of apoptosis, namely, caspase 3, 8, 9 and PARP. Upregulation of the pro-apoptotic Bax protein and downregulation of the anti-apoptotic Bcl2 protein were also analyzed. Furthermore, in the PF2401-SF treated rats, apoptosis induction of activated HSCs was demonstrated by reduced distribution of α-SMA-positive cells and the presence of high number of TUNEL-positive cells in vivo. Our data suggest that PF2401-SF can mediate HSCs apoptosis induction, and may be a potential herbal medicine for the treatment of liver fibrosis.

Involvement of heme oxygenase-1 induction in inhibitory effect of ethyl gallate isolated from Galla Rhois on nitric oxide production in RAW 264.7 macrophages.

In the present study, we investigated an anti-inflammatory effect of ethyl gallate (EG) isolated from Galla Rhois as evaluated by inhibition of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and a potential role of heme oxygenase-1 (HO-1) in the inhibition of NO production elicited by EG. Treatment of RAW264.7 macrophages with EG significantly inhibited the production of NO and iNOS expression stimulated by lipopolysaccharide (LPS). We also demonstrated that EG treatment increased HO-1 mRNA and protein expression, as assessed by quantitative RT-PCR and Western blot analysis. EG treatment also increased the levels of nuclear factor-erythroid 2-related factor 2, which is critical for transcriptional induction of HO-1. In addition, treatment with SnPP (tin protoporphyrin IX), a selective HO-1 inhibitor, counteracted the inhibitory effect of EG on nitrite production, suggesting that HO-1 is, at least in part, implicated in the inhibition of NO production induced by EG treatment. Taken together, these results indicate that EG isolated from Galla Rhois suppresses NO production in LPS-stimulated RAW 264.7 macrophages via HO-1 induction.

In Vitro and In Vivo Antibacterial Activity of Punica granatum Peel Ethanol Extract against Salmonella.

Punica granatum is commonly used in Korea as a traditional medicine for the treatment of pathogenic bacteria. In this study, we investigated the in vitro and in vivo antimicrobial activity of P. granatum peel EtOH extract (PGPE) against 16 strains of Salmonella. The minimal inhibitory concentrations of PGPE were in the range of 62.5-1000 x03BCg mL(-1). In addition, the in vivo antibacterial activity of the PGPE extract was examined in a S. typhimurium infection mouse model. Mice were initially infected with S. typhimurium and then with PGPE. The extract was found to have significant effects on mortality and the numbers of viable S. typhimurium recovered from feces. Although clinical signs and histological damage were rarely observed in the treated mice, the untreated controls showed signs of lethargy and histological damage in the liver and spleen. Taken together, the results of this study indicate that PGPE has the potential to provide an effective treatment for salmonellosis.

A new diarylheptanoid from the bark of Alnus japonica.

A new diarylheptanoid, epihirsutanonol (1), was isolated from the bark of Alnus japonica, along with two known ones (2 and 3). Their structures were elucidated on the basis of extensive spectroscopic evidence. The new compound 1 showed significant hepatoprotective activity on the basis of t-butylhydroperoxide-induced hepatocyte injury in vitro assay.

Tanshinone II A induces apoptosis and S phase cell cycle arrest in activated rat hepatic stellate cells.

Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, improves blood circulation and treats chronic hepatitis and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is the predominant event in liver fibrosis. The therapeutic goal in liver fibrosis is the reversal of fibrosis and selective clearance of activated HSCs. We used rat HSCs transformed by Simian virus 40 (t-HSC/Cl-6) to overcome the limitations inherent in studying subcultures of HSCs. Treatment of t-HSC/Cl-6 cells with tanshinone IIA inhibited cell viability in a dose- and time-dependent manner. Tanshinone IIA induced apoptosis as demonstrated by DNA fragmentation, poly(ADP-ribose) polymerase and caspase-3 cleavage, increased Bax/Bcl-2 protein ratio, and depolarization of mitochondrial membranes to facilitate cytochrome c release into the cytosol. Furthermore, this compound markedly induced S phase cell cycle arrest, and down-regulated cyclins A and E, and cdk2. Thus, tanshinone IIA induces apoptosis and S phase cell cycle arrest in rat HSCs in vitro.

Antioxidative and hepatoprotective diarylheptanoids from the bark of Alnus japonica.

The present study to evaluate the potential of constituents of the bark of Alnus japonica as a functional food with medicinal properties led to the identification of one new diarylheptanoid, named alusenone (1A), and 11 known ones (1B and 2-11). Their antioxidative and hepatoprotective activities were accessed by, respectively, a TOSC assay and a TBH-induced hepatotoxicity rat model. Mixtures 1, 2-6, 10, and 11 showed good antioxidative and hepatoprotective effects as compared with the positive controls.

Chromone glycosides and hepatoprotective constituents of Hypericum erectum.

Two chromone glycosides, hyperimone A [7-(beta-D-glucopyranosyloxy)-5-hydroxy-2-(1-methylethyl)-4H-1-benzopyran-4-one (1)] and hyperimone B [7-(beta-D-glucopyranosyloxy)-5-hydroxy-3-methyl-4H-1-benzopyran-4-one (2)], together with six known compounds were isolated from the methanolic extract of the whole plant of Hypericum erectum. 1,3,5,6-Tetrahydroxyxanthone (5) and I3, II8-biapigenin (6) showed moderate hepatoprotective activity with EC(50) values of 160.2 +/- 0.6 microM and 217.7 +/- 1.3 microM, respectively, against tacrine-induced cytotoxicity in HepG2 cells.

Preventive effects of a purified extract isolated from Salvia miltiorrhiza enriched with tanshinone I, tanshinone IIA and cryptotanshinone on hepatocyte injury in vitro and in vivo.

Salvia miltiorrhiza is traditionally used to treat liver disease in Asia. In this study, we tested the ability of a purified extract of S. miltiorrhiza (PF2401-SF) and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, to protect against acute and subacute liver damage induced by carbon tetrachloride by measuring serum transaminase levels, the reduced form of glutathione (GSH), antioxidant enzyme activities, and lipid peroxidation levels in the liver. We also evaluated their ability to protect primary cultured rat hepatocytes from tertiary-butylhydroperoxide (tBH) or d-galactosamine (GalN). PF2401-SF was protective at 50-200mg/kg per day in acute liver injury and 25-100mg/kg per day in subacute liver injury. Tanshinone I, tanshinone IIA, and cryptotanshinon (40 microM), inhibited lactate dehydrogenase leakage, GSH depletion, lipid peroxidation and free radical generation in vitro. PF2401-SF and its major constituents, tanshinone I, tanshinone IIA and cryptotanshinone, can protect against liver toxicity in vivo and in vitro due to its antioxidant effects.

Dibenzocyclooctadiene lignans and lanostane derivatives from the roots of Kadsura coccinea and their protective effects on primary rat hepatocyte injury induced by t-butyl hydroperoxide.

A new dibenzocyclooctadiene lignan, acetylepigomisin R ( 1), and a new 3,4-seco-lanostane-type triterpene, seco-coccinic acid F ( 2), along with three known dibenzocyclooctadiene lignans, isovaleroylbinankadsurin A ( 3), kadsuralignan J ( 4), and binankadsurin A ( 5), and one lanostane-type triterpene, 20( R),24( E)-3-oxo-9 beta-lanosta-7,24-dien-26-oic acid ( 6), were isolated from the methanol extract of the Kadsura coccinea roots. Their structures were elucidated on the basis of spectroscopic evidence including ESI-MS, HR-EI-MS, 1D and 2D NMR. The protective effects of these compounds were evaluated in primary cultured rat hepatocytes intoxicated with 1.2 mM T-butyl hydroperoxide. Compounds 1, 3, and 5 showed protective effects with ED (50) values of 135.7, 26.1, and 79.3 microM, respectively.

Salvia miltiorrhiza Bunge and its active component cryptotanshinone protects primary cultured rat hepatocytes from acute ethanol-induced cytotoxicity and fatty infiltration.

Alcoholic liver disease involves hepatocellular injury induced by the acute or chronic consumption of ethanol. Fatty infiltration is usually followed by inflammation and focal necrosis, which can lead to cirrhosis if not treated properly in the initial stage. There have been many attempts to develop effective therapies for the disease, using natural products derived from medicinal plants. In this study, we report that the standardized fraction of Salvia miltiorrhiza Bunge (Sm-SF) and its active component, cryptotanshinone, were able to protect hepatocytes from lipopolysaccharide- and ethanol-induced cell death. They also suppressed ethanol-induced lipid accumulation as evidenced by the Nile red binding assay. The ethanol-induced activation and nuclear translocation of sterol regulatory element-binding protein-1 and the consequent transactivation of the target genes involved in fatty acid biosynthesis were inhibited by Sm-SF and cryptotanshinone in a dose-dependent manner. Cryptotanshinone, an active component of S. miltiorrhiza, has the potential to ameliorate alcoholic liver disease by blocking hepatic cell death and fatty acid synthesis.

In vitro activity of methyl gallate isolated from galla rhois alone and in combination with ciprofloxacin against clinical isolates of salmonella.

Salmonella remains a primary cause of food poisoning worldwide, and massive outbreaks have been witnessed in recent years. Therefore, this study investigated the antimicrobial activity of methyl gallate (MG), which exhibited good antibacterial activity (MIC=3.9-125 mg/ml) against all the bacterial strains tested. In a checkerboard dilution test, MG markedly lowered the MICs of ciprofloxacin (CPFX) against Salmonella. The combined activity of CPFX and MG against Salmonella resulted in fractional inhibitory concentrations (FICs) ranging from 0.0037 to 0.015 and from 0.24 to 7.8 mg/ml, respectively. Meanwhile, the FIC index ranged from 0.31-0.37, indicating a marked synergistic relationship between CPFX and MG against Salmonella. Time-kill assays also showed a decrease in the CFU/ml between the combination and the more active compound. Therefore, this study demonstrated that MG and CPFX can act synergistically in inhibiting Salmonella in vitro.

Anti-nociceptive and anti-inflammatory effects of Angelicae dahuricae radix through inhibition of the expression of inducible nitric oxide synthase and NO production.

The extract of Angelicae dahuricae radix has traditionally been used as an anti-noceptive remedy in China. In this study, the methanol extract of Angelicae dahuricae radix (MEAD) was evaluated to determine if it has anti-noceptive and anti-inflammatory action. The anti-nociceptive activities of MEAD were evaluated by determining the writhing response and sleeping time, as well as by a formalin test. In addition, the anti-inflammatory activities of MEAD were evaluated by a vascular permeability test as well as by measuring the carrageenan-induced paw edema and conducting a myeloperoxidase (MPO) assay. MEAD (600 and 1200 mg/kg) exhibited anti-inflammatory effects on acetic acid-induced vascular permeability, carrageenan-induced paw edema, and MPO activity. Moreover, the results of the formalin test, the acetic acid-induced writhing response and the pentobarbital-induced sleeping time indicated that MEAD had anti-nociceptive effects that occurred in a concentration-dependent manner. To determine the mechanism by which MEAD exerted its effects on the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) by treated murine macrophage RAW 264.7 cells was evaluated. Similar to the in vivo activities, both the iNOS expression and NO production were significantly suppressed by MEAD in a dose-dependent manner. Furthermore, MEAD inhibited the activating phosphorylation of ERK1/2. These results provide a scientific basis that explains the mechanism by which Angelicae dahuricae radix relieves inflammatory pain.

1,2,3,4,6-penta-O-galloyl-beta-D-glucose from Galla Rhois protects primary rat hepatocytes from necrosis and apoptosis.

In this study, we investigated the hepatoprotective effects of four compounds from Galla Rhois [gallic acid methyl ester, gallic acid, an equilibrium mixture of 3-galloyl-gallic acid and 4-galloyl-gallic acid isomers, and 1,2,3,4,6-penta- O-galloyl- beta- D-glucose (PGG)] in primary rat hepatocytes undergoing necrosis or apoptosis. Treatment with gallic acid methyl ester (12.5 and 50 microM) or PGG (3.125, 12.5 and 50 microM) reduced hepatocyte necrosis induced by tert-butyl hydroperoxide. PGG treatment (4 and 20 microM) also altered hepatocyte apoptosis induced by glycochenodeoxycholic acid. Based on these results, we propose that PGG warrants further evaluation as a hepatoprotective agent, because it protected primary rat hepatocytes from both necrosis and apoptosis.

Synergistic effects of the combination of galangin with gentamicin against methicillin-resistant Staphylococcus aureus.

The antimicrobial killing activity toward methicillin-resistant Staphylococcus aureus (MRSA) has been a serious emerging global issue. New effective antimicrobials and/or new approaches to settle this issue are urgently needed. The oriental herb, Alpinia officinarum, has been used in Korea for several hundreds of years to treat various infectious diseases. As it is well known, one of the active constituents of Alpinia officinarum is galangin. Against the 17 strains, the minimum inhibitory concentrations (MICs) of galangin (GAL) were in the range of 62.5 ~ 125 microg/ml, and the MICs of gentamicin (GEN) ranged from 1.9 microg/ml to 2,000 microg/ml. The fractional inhibitory concentrations (FICs) of GAL, in combination with GEN, against 3 test strains were 0.4, 3.9, and 250 microg/ml, and were all 15.62 microg/ml in GEN. The FIC index showed marked synergism in the value range of 0.19 to 0.25. By determining time-kill curves, also confirmed the low synergism of the GAL and GEN combination against 4 h, 8 h, 12 h, and 24 h cultured MRSA. The time-kill study results indicated a low synergistic effect against 3 test strains. Thus, the mixture of GAL and GEN could lead to the development of new combination antibiotics against MRSA infection.

Isoliquiritigenin inhibits cell proliferation by a heme oxygenase-dependent pathway in rat hepatic stellate cells.

We evaluated whether the antiproliferative effects of isoliquiritigenin (ISL) on rat hepatic stellate cells (HSCs) are related to the induction of heme oxygenase 1 (HO-1) expression. ISL significantly inhibited serum- or growth factor-induced HSC proliferation. The inhibition of platelet-derived growth factor (PDGF)-induced proliferation by ISL was associated with the mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-p70 (S6K) pathways. ISL induced the expression of HO-1 in HSCs. Using the chemical inhibitor tin protoporphyrin, we also found that the inhibitory action of ISL on PDGF-induced proliferation is mediated by HO-1. These data suggest that HO-1 expression is responsible for the antiproliferative effect of ISL on HSCs.

Fatty acids ameliorate doxorubicin-induced intracellular ca2+ increase and apoptosis in rat cardiomyocytes.

Doxorubicin (Dox) is a highly effective anticancer drug but exhibits cumulative dose-dependent cardiomyopathy. In this study, we investigated effects of Magnolia seed extract (MagS) on the Dox-induced cardiotoxicity. The results showed that MagS significantly reduces doxorubicin (Dox)-induced increase in intracellular Ca2+ concentration ([Ca2+]i), generation of reactive oxygen species (ROS), and apoptosis in rat cardiomyocytes. Analyses of the bioactive compounds in MagS by thin layer chromatography and gas chromatography/mass spectroscopy revealed that bioactive compounds in MagS are linoleic acid, oleic acid, and palmitic acid. All three fatty acids were able to inhibit the Dox-induced increase in [Ca2+]i, ROS generation, and apoptosis with a similar potency. Efficacy of MagS was examined in in vivo using a murine Dox-induced cardiomyopathy model. Dox (12 mg/kg, intravenously) was administered to mice and treated with the MagS (2 mg/kg/d, intraperitoneally) or saline for three weeks. Dox-treated mice showed structural disarray in heart tissue, including lymphocyte infiltration and loss of body weight. In contrast, treatment of the MagS substantially attenuated the Dox-induced cardiac damages including the loss of body weight. These results indicate that fatty acids in MagS and other seeds may ameliorate cardiotoxicity of the anticancer drug.