Takuhiro Uto

Eriobotryae Folium Extract Suppresses LPS-Induced iNOS and COX-2 Expression by Inhibition of NF-κB and MAPK Activation in Murine Macrophages

Eriobotryae folium (EF), the dried leaves of Eriobotrya japonica (Thunb.) Lindl. has been traditionally used to treat various diseases such as chronic bronchitis, cough, inflammation, skin diseases, and diabetes. In this study, we examined the effects of Eriobotryae folium extract (EFE) on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2(PGE2) in RAW264 murine macrophage cells. EFE suppressed LPS-induced NO and PGE2 production in a dose-dependent manner. Consistent with these observations, EFE reduced the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both protein and mRNA levels. Furthermore, EFE significantly inhibited LPS-induced NF-kappaB binding activity, which was associated with the inhibition of IkappaB-alpha degradation. EFE also attenuated LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK). These results suggest that the anti-inflammatory properties of EF might result from inhibition of iNOS and COX-2 expression through the downregulation of NF-kappaB activation and MAPK phosphorylation in LPS-stimulated RAW264 cells.

Analysis of the synergistic effect of glycyrrhizin and other constituents in licorice extract on lipopolysaccharide-induced nitric oxide production using knock-out extract.

The pharmacological evidence for synergism between natural compounds is not fully elucidated. In this study, we investigated the synergistic function of one target compound in medicinal plant extract by using knock-out (KO) extract, which is one target compound-eliminated extract from whole crude extract. Licorice is the most important ingredient used in the traditional Chinese medicine (TCM) and the Japanese Kampo medicine, and one of the major active components of licorice is glycyrrhizin (GC). To identify the potential role of GC, we prepared GC-removed extract (GC-KO extract) from licorice extract (LE) using immunoaffinity column conjugated with anti-GC monoclonal antibody (MAb), which could eliminate 99.5% of GC from LE. LE inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264 murine macrophage cells. However, treatment of GC alone could not show the suppression of NO production and iNOS expression. Interestingly, the inhibitory effect of GC-KO extract was significantly attenuated compared with LE. Furthermore, the combined treatment with GC-KO extract and GC could improve the attenuated inhibition. Taken together, our results indicate that GC may exert synergistic suppression of iNOS expression when coexisting with the other constituents contained in LE, and KO extract is a useful approach for determination of real pharmacological functions of natural compound in the phytochemical mixture.

Very long-chain fatty acid accumulation causes lipotoxic response via 5-lipoxygenase in cerebral adrenoleukodystrophy

Abstract Childhood adrenoleukodystrophy (cALD) is a metabolic disorder in which very long-chain fatty acids (VLCFA) accumulate due to ALD protein gene defects, ultimately leading to lipotoxicity-induced neuroinflammatory demyelinating disease. Therefore, we examined VLCFA-mediated alterations in the metabolism of lipoxidative enzymes and inflammatory mediators in the cALD brain. 5-Lipoxygenase (5-LOX)-derived leukotrienes were significantly elevated in all the areas of white matter in the cALD brain. Unlike cyclooxygenase-2 expression, which was moderately high only in the plaque area, expression of 5-LOX and cytosolic phospholipase A2 was prominent in all the areas. This lipoxidative burden in the cALD brain was further shown by reduced levels of glutathione and enhanced expression of heat shock protein-70/manganese superoxide dismutase. These pathological observations were confirmed through in vitro mechanistic investigation. After increasing VLCFA through silencing Abcd1+Abcd2 in mouse primary astrocytes, enhanced expression of 5-LOX was observed, and this increased expression was blocked by treatment with monoenoic fatty acids. These results link the previously observed accumulation of VLCFA in cALD to the 5-LOX enzyme pathway. A similar increase in 5-LOX expression in astrocytes was also detected following treatment with exogenous VLCFA (C26:0). In sum, through 5-LOX activation, VLCFA accumulation causes a lipotoxic response consistent with cALD brain pathology.

Anti-Proliferative Activities and Apoptosis Induction by Triterpenes Derived from Eriobotrya japonica in Human Leukemia Cell Lines.

Eriobotrya japonica leaf is a traditional herbal medicine that contains numerous triterpenes, which have various pharmacological properties. In this study, we investigated the anti-proliferative activity of four triterpenes derived from E. japonica, including corosolic acid (CA), ursolic acid (UA), maslinic acid (MA) and oleanolic acid (OA), in human leukemia cell lines. CA showed the strongest anti-proliferative activity in all of the leukemia cell lines tested, but not in normal human skin fibroblast cell lines. To determine the mechanism underlying the anti-proliferative effect of CA, we examined the effect of CA on molecular events known as apoptosis induction. CA induced chromatin condensation, DNA fragmentation, sub-G1 phase DNA, activation of caspase-3, -8 and -9 and the cleavage of PARP in HL-60. CA also activated Bid and Bax, leading to the loss of mitochondrial membrane potential (Δψm) and cytochrome c release into the cytosol, whereas Bcl-2 and Bcl-xL were unaffected by CA. These results suggest that CA has an anti-proliferative effect on leukemia cells via the induction of apoptosis mediated by mitochondrial dysfunction and caspase activation. CA may be a potential chemotherapeutic agent for the treatment of human leukemia.

Pharmacological Effects of Ginseng on Liver Functions and Diseases: A Minireview

Ginseng, an ancient and famous medicinal herb in the Orient, has been used as a valuable tonic and for the treatment of various diseases including hepatic disorders. Ginseng saponins, commonly known as ginsenosides, are principal constituents and have believed to be responsible for multiple ginseng health benefits. There are more 40 ginsenosides isolated from ginseng. To date, treatment options for common liver diseases such as cirrhosis, fatty liver, and chronic hepatitis remain problematic. In this regard, ginseng extracts and individual ginsenosides have shown a wide array of beneficial role in the regulation of regular liver functions and the treatment of liver disorders of acute/chronic hepatotoxicity, hepatitis, hepatic fibrosis/cirrhosis, hepatocellular carcinoma, and so on in various pathways and mechanisms. In this paper, we first outline the pharmacological effects of ginseng and ginsenosides on the liver functions.

17-Hydroxy-jolkinolide B, a diterpenoid from Euphorbia fischeriana, inhibits inflammatory mediators but activates heme oxygenase-1 expression in lipopolysaccharide-stimulated murine macrophages

Jolkinolides are the main abietane-type diterpenoids isolated from the root of Euphorbia fischeriana Steud. In the present study, we investigated in vitro anti-inflammatory activity of four structural analogs of jolkinolide in lipopolysaccharide (LPS)-stimulated RAW264 macrophages. Among these jolkinolides, 17-hydroxy-jolkinolide B (HJB) exhibited the most potent inhibition of LPS-induced production of inflammatory mediators such as prostaglandin E(2) (PGE(2)), nitric oxide (NO), and pro-inflammatory cytokines [interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)]. HJB could decrease LPS-induced protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and the mRNA expressions of COX-2, iNOS, IL-6, and TNF-α in a concentration-dependent manner. These inhibitory effects were caused by suppression of MAPK phosphorylation and NF-κB activation. Furthermore, we demonstrated that HJB strongly induced heme oxygenase-1 (HO-1) protein and mRNA expressions. These findings suggest that HJB possesses anti-inflammatory actions in macrophages and may provide a potential therapeutic approach for inflammatory disorders.

Development of a monoclonal antibody-based immunochemical assay for liquiritin and its application to the quality control of licorice products.

Liquiritin was reacted with a keyhole limpet hemocyanin (KLH) to synthesize a liquiritin-KLH conjugate as an immunogen for mice. A hybridoma cell line named 2F8 secreted a monoclonal antibody (mAb) against liquiritin, which was applied to an enzyme-linked immunosorbent assay (ELISA) for liquiritin. ELISA showed a good linear range from 0.39 to 25 μg/mL of liquiritin. The maximum relative standard deviation (RSD) values for the intra-assay and interassay were approximately 5%. The recovery rates of liquiritin were in the range of 100.9-103.7%, and the concentrations of liquiritin in various licorice roots, as determined by ELISA, showed a good correlation with those analyzed by high-performance liquid chromatography (HPLC; R² = 0.948). These results suggested that ELISA with anti-liquiritin mAb could be a simple, rapid, convenient, and accurate method for the high-throughput analysis of liquiritin in various licorice products including liqueurs, sweets, and food supplements.

Molecular Mechanisms Underlying Anti-Inflammatory Actions of 6-(Methylsulfinyl)hexyl Isothiocyanate Derived from Wasabi (Wasabia japonica)

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a major bioactive compound in wasabi (Wasabia japonica), which is a typical Japanese pungent spice. Recently, in vivo and in vitro studies demonstrated that 6-MSITC has several biological properties, including anti-inflammatory, antimicrobial, antiplatelet, and anticancer effects. We previously reported that 6-MSITC strongly suppresses cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokines, which are important factors that mediate inflammatory processes. Moreover, molecular analysis demonstrated that 6-MSITC blocks the expressions of these factors by suppressing multiple signal transduction pathways to attenuate the activation of transcriptional factors. Structure-activity relationships of 6-MSITC and its analogues containing an isothiocyanate group revealed that methylsulfinyl group and the length of alkyl chain of 6-MSITC might be related to high inhibitory potency. In this paper, we review the anti-inflammatory properties of 6-MSITC and discuss potential molecular mechanisms focusing on inflammatory responses by macrophages.

Development of an Eastern Blotting Technique for the Visual Detection of Aristolochic Acids in Aristolochia and Asarum Species by Using a Monoclonal Antibody Against Aristolochic Acids I and II

INTRODUCTION Aristolochic acids (AAs) are naturally occurring nephrotoxicants and human carcinogens. Aristolochic acid I (AA-I) and aristolochic acid II (AA-II) are two important AAs with clear toxicity. OBJECTIVE To obtain a monoclonal antibody (MAb) recognising AA-I and AA-II and develop an Eastern blotting technique for the specific visualisation and easy determination of AA-I and AA-II in plant extracts or tissues of Aristolochia and Asarum species. METHODS A hybridoma secreting MAb against AAs was prepared by cell fusion with splenocytes derived from a mouse immunised with AA-I-keyhole limpet haemocyanin (KLH) conjugate and the myeloma cell line SP2/0-Ag14. AA-I and AA-II were separated by thin-layer chromatography (TLC) and then blotted onto a positively charged polyethersulphone (PES) membrane using a modified carbodiimide method. The resulting membrane-bound AA-protein conjugates were linked to the newly prepared MAb and then to the secondary antibody labelled with peroxidase. 4-Chloro-1-naphthol was then added as the peroxidase substrate for staining. RESULTS MAb 2A10-10B showed a high specificity for AA-I (100%) and AA-II (69.3%) and low cross reactivity (≤ 2.2%) toward analogues that may disrupt detection of AA-I and AA-II in plants. An established Eastern blotting method was applied to the immunohistolocalisation of AA-I and AA-II in dry plant tissues, and this analysis showed that the phelloderm, cortex and phloem of Aristolochia manshuriensis stem may contain higher amounts of total AA-I and AA-II as compared with the pith and xylem. CONCLUSION This method was extremely useful for the visual screening of AA-I and AA-II among easily mistaken herbal medicines.

Preparation of knockout extract for determination of really active compound using MAb.

The crude-rhizome extract of P. japonicus was loaded on the immunoaffinity column conjugated with anti- ginsenoside-Rb1 monoclonal antibody (MAb) and washed with the washing solvent, followed by elution solvent, to give fraction 2 containing higher concentration of compound 1. Compound 1 clearly indicated a dammarane saponin having protopanaxadiol as a framework and three sugars in a molecule suggesting that compound 1 is chikusetsusaponin III. Compound 2 was also determined as chikusetsusaponin VI compared to the staining color, its Rf value and the comparison with ginsenoside Rb1. We succeeded in one step purification of ginsenoside-Rb1 by immunoaffinity column conjugated with anti- ginsenoside-Rb1 MAb leading to the knock-out extract which will be useful for pharmacological investigation. The antibody was stable when exposed to the eluent, and the immunoaffinity column showed almost no decrease in capacity after repeated use more than 10 times under the same conditions. From the crude extract of licorice we isolated glycyrrhizin by one-step purification by the immunoaffinity column using anti-glycyrrhizin MAb. Washing fraction contained all components except for only glycyrrhizin and was named as the knockout extract. We confirmed the synergic effect of glycyrrhizin with some other components for the inhibition of nitric oxide (NO) production by blocking inducible nitric oxide synthase (iNOS) expression by using its knockout extract.