Yukihiro Shoyama

Dietary Crocin Inhibits Colitis and Colitis-Associated Colorectal Carcinogenesis in Male ICR Mice

A natural carotenoid crocin is contained in saffron and gardenia flowers (crocuses and gardenias) and is used as a food colorant. This study reports the potential inhibitory effects of crocin against inflammation-associated mouse colon carcinogenesis and chemically induced colitis in male ICR mice. In the first experiment, dietary crocin significantly inhibited the development of colonic adenocarcinomas induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) in mice by week 18. Crocin feeding also suppressed the proliferation and immunohistochemical expression of nuclear factor- (NF-) κB but increased the NF-E2-related factor 2 (Nrf2) expression, in adenocarcinoma cells. In the second experiment, dietary feeding with crocin for 4 weeks was able to inhibit DSS-induced colitis and decrease the mRNA expression of tumor necrosis factor α, interleukin- (IL-) 1β, IL-6, interferon γ, NF-κB, cyclooxygenase-2, and inducible nitric oxide synthase in the colorectal mucosa and increased the Nrf2 mRNA expression. Our results suggest that dietary crocin suppresses chemically induced colitis and colitis-related colon carcinogenesis in mice, at least partly by inhibiting inflammation and the mRNA expression of certain proinflammatory cytokines and inducible inflammatory enzymes. Therefore, crocin is a candidate for the prevention of colitis and inflammation-associated colon carcinogenesis.

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.

Crocin promotes non-rapid eye movement sleep in mice.

Crocus sativus L. (saffron) has been traditionally used for the treatment of insomnia and other diseases of the nervous systems. Two carotenoid pigments, crocin and crocetin, are the major components responsible for the various pharmacological activities of C. sativus L. In this study, we examined the sleep-promoting activity of crocin and crocetin by monitoring the locomotor activity and electroencephalogram after administration of these components to mice. Crocin (30 and 100 mg/kg) increased the total time of non-rapid eye movement (non-REM) sleep by 60 and 170%, respectively, during a 4-h period from 20:00 to 24:00 after its intraperitoneal administration at a lights-off time of 20:00. Crocetin (100 mg/kg) also increased the total time of non-REM sleep by 50% after the administration. These compounds did not change the amount of REM sleep or show any adverse effects, such as rebound insomnia, after the induction of sleep.

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.

Immunofluorescence and Immunoelectron Microscopic Localization of Medicinal Substance, Rb1, in Several Plant Parts of Panax ginseng

It is important to know the localization of medicinal substance, Rb1, of Ginseng, Panax ginseng, in this plant in order to achieve efficient extraction of Rb1 or to culture producing cells. In this report, we describe the localization of Rb1 in various parts of the plant as determined by immunofluorescence (IF) and immunoelectron microscopies (IEM). Using IF, we show that Rb1 is localized to chloroplasts, peroxisomes and cytoplasm but not to vacuoles of leaf parenchymal cells. In the leaf stem, Rb1 is localized to the vascular bundles as well as vacuoles. In the root, vacuoles of parenchymal cells are stained at various intensities. Using IEM, gold particles showing Rb1 antigenic sites are present in the compartments stained by IF technique. In addition, Rb1 is localized in the sieve elements of the phloem and degrading primary cell wall of xylem, and in the root parenchymal cells Rb1 is associated with electron dense polymorphic materials but not in starch granules. Translocation and storage of Rb1 and effective utilization of leaves are discussed.

Naphthoquinone Components from Alkanna tinctoria (L.) Tausch Show Significant Antiproliferative Effects on Human Colorectal Cancer Cells

Our research to seek active compounds against human colorectal cancer from the root of Alkanna tinctoria (L.) Tausch led to the isolation of two naphthoquinones, alkannin (1) and angelylalkannin (2). The antiproliferative effects of the two compounds on human colon cancer cells HCT‐116 and SW‐480 were determined by the 3,4‐(5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium salt (MTS) method. Cell cycle profile and cell apoptosis were determined using flow cytometry. Both of the two compounds showed significant inhibitory effects on the cancer cells. For alkannin (1) and angelylalkannin (2), the median inhibitory concentration (IC50) values were 2.38 and 4.76 µ m for HCT‐116 cells, while for SW‐480 cells they were 4.53 and 7.03 µ m, respectively. The potential antiproliferative mechanisms were also explored. At concentrations between 1–10 µ m, both compounds arrested the cell cycle at the G1 phase and induced cell apoptosis. Copyright

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.

Antiproliferative and apoptotic effects of compounds from the flower of Mammea siamensis (Miq.) T. Anders. on human cancer cell lines.

On the search for anti-cancer compounds from Thai traditional herb medicines, a bioassay-guided fractionation and chemical investigation of the methanol extract of Mammea siamensis flower resulted in the isolation and identification of eight compounds (1-8) including a novel geranylated coumarin, namely mammeanoyl (2), and seven known compounds (1 and 3-8). The structure of new compound 2 was elucidated based on the extensive spectroscopic and chemical methods. Among the isolated compounds, three structurally related coumarins 3, 4, and 5 showed significant antiproliferative activities against human leukemia and stomach cancer cell lines. However, these compounds did not affect the cell viabilities of colon cancer, hepatoma, and normal skin fibroblast cell lines. Further analysis demonstrated that the morphological features of apoptosis including DNA fragmentation and chromatin condensation were observed in human leukemia HL-60 cells treated with compounds 3, 4, and 5. In addition, compound 3 led to caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP), and compound 3-induced DNA fragmentation was inhibited by caspase-specific inhibitors. These results suggest that compound 3, 4, and 5 exert antiproliferative actions through apoptotic cell death in leukemia cells and these compounds may have the potential to be developed into new anti-cancer drug candidates.

Quality control of traditional chinese medicine by monoclonal antibody method.

In a previous study, we reported the preparation, characterization, variation, specificity, and sensitivity of an anti-aristolochic acid-II (AA-II) monoclonal antibody. The preparation procedure was as follows. AA-II conjugated with bovine serum albumin was used as an antigen for immunizing BALB/c mice. Splenocytes isolated from the immunized mice were fused with an aminopterin-sensitive mouse myeloma cell line to produce hybridoma cells secreting a mono-clonal antibody (MAb) against AA-II. The selected MAb was subsequently cloned. Hapten number, isotype, and an esti-mated dissociation constant (KD) of the secreted MAb were determined. This MAb was used to establish an ELISA method. The linear range was 0.19-13 µg/ml. Anti-AA-II MAb showed extremely high specificity for AA-II, low cross-reactivity (CR) against other AAs or aristololactam-I, and negligible CR (<0.5%) toward other natural compounds with different chemical structures. This study describes the successful application of the ELISA method using anti-AA-II MAb to determine AA-II concentration in several crude drugs derived from Aristolochia species. The highest AA-II concentration (2.82 µg/mg) was observed in the stem of A. manshuriensis, followed by that in the fruit of A. contorta (0.81 µg/mg). In case of A. indica, AA-II concentration in the root was higher than that in the aerial parts. These data indicated that the established ELISA method can be used for the quality control of crude drugs derived from Aristolochia plants.

Preparation of a Single-Chain Variable Fragment and a Recombinant Antigen-Binding Fragment against the Anti-Malarial Drugs, Artemisinin and Artesunate, and Their Application in an ELISA

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.