Comparison of ephedrine and pseudoephedrine contents in 34 Kampo extracts containing Ephedrae Herba used clinically in Japan.

Ephedrae Herba is among the important crude drugs prescribed in Kampo medicine for the treatment of cold, flue, rhinitis, nasal congestion, cough, and asthma. The active ingredients of Ephedrae Herba, ephedrine (E) and pseudoephedrine (PE), are potent sympathomimetic compounds that stimulate α-, ß1-, and ß2-adrenoceptors resulting in dilatation and alleviation of nasal mucosal hyperemia. Hypertension, palpitations, insomnia, and dysuria are the main adverse effects of E and PE, which can be avoided by determining the actual contents of these alkaloids in Kampo extracts containing Ephedrae Herba. However, the extraction efficiencies of E and PE from Ephedrae Herba contained in Kampo formulas in combination with other crude drugs remain unknown. Therefore, we comprehensively determined the E and PE contents of 34 Kampo extracts containing Ephedrae Herba used clinically in Japan. The E and PE contents per daily dosage in Kampo extracts were generally proportional to the compounding amount of Ephedrae Herba. In contrast, the extraction efficiencies of E or PE were not constant and not influenced by the pH of the extracts. We assume that the extraction efficiencies of E and PE may be independently affected by other constituent crude drugs. Thus, it is necessary to investigate the cause and mechanism in the future. In conclusion, these results show that the E and PE content of each Kampo formulation can be estimated from the compounding amount of Ephedrae Herba. Therefore, the amount of Ephedrae Herba should be carefully considered to ensure the safe use of Kampo formulations containing Ephedrae Herba.

Hochuekkito exerts the anti-allergic effects via activating regulatory T cells in a murine model of contact hypersensitivity.

Hochuekkito (HET) is a Kampo prescription, used for the clinical treatment of skin diseases such as atopic dermatitis (AD), in Japan. Oral administration of HET exerts anti-allergic effects in an experimental dermatitis mice model and in patients with atopic dermatitis; however, the mechanism underlying the anti-allergic effects of HET is still unclear. Therefore, we investigated the immunopharmacological properties of the anti-allergic actions of HET using a 2,4,6-trinitrochlorobenzene (TNCB)-induced murine contact hypersensitivity (CHS) model and adoptive cell transfer experiments. Oral administration of HET (1.4 g/kg) exhibited anti-allergic effects in a TNCB-induced CHS model via activation of Tregs; this activation was observed even without antigen sensitization in donor mice. Activation was dependent on the duration of HET administration and required at least 4 days of dosing. In addition, the anti-allergic effects of HET through the activation of Tregs were not antigen specific. Flow cytometry results indicated that the proportion of CD4+CD25+Foxp3+ cells in the splenic lymphocytes increased after oral administration of HET. Therefore, oral administration of HET induced both inducible regulatory T cells (iTregs) and thymus-derived naturally occurring regulatory T cells (nTregs). Ginseng radix and Bupleuri radix were involved in the anti-allergic actions of HET through the induction and/or activation of Tregs; Bupleuri radix participated in the activation of nTregs. In conclusion, our findings suggest that HET exerts the anti-allergic effects through the induction and/or activation of Tregs. These findings elucidate the usefulness of HET as an immunomodulator.

Orengedokuto exerts anti-allergic effects via inhibition of effector T cell activation in a murine model of contact hypersensitivity.

Orengedokuto (OGT) is a Kampo prescription that has been used for the treatment of inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. It is also used for the treatment of skin diseases such as urticaria and atopic dermatitis. We previously studied its anti-allergic effects of OGT on the murine model of 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity (CHS) and demonstrated that it significantly suppresses ear swelling in a dose-dependent manner. However, the mechanism underlying this activity remained unknown. Here, we sought to identify the mechanism involved. Using a murine model of TNCB-induced CHS, together with adoptive cell transfer experiments, we found that the anti-allergic effects of OGT may be due to the inhibition of effector T cell activation and not the induction and/or activation of regulatory T cells. Flow cytometry analysis revealed that oral administration of OGT suppressed the increase in CD8+CD44highCD62L+ cell number in draining lymph nodes (dLNs) of mice sensitized with 5% TNCB. Additionally, ex vivo experiments confirmed the suppressive effect of OGT on the activation of effector T cells, as interferon-γ (IFN-γ) production by cultured lymphocytes obtained from 5% TNCB-sensitized mice and stimulated with anti-CD3ε and anti-CD28 monoclonal antibodies was reduced by OGT administration. In conclusion, our finding suggests that OGT exerts anti-allergic effects by regulating the activation of effector T cells involved in inflammatory skin diseases such as atopic dermatitis.

Juzentaihoto Exerts Anti-Allergic Effects by Inhibiting Effector T-Cell Activation and Inducing and/or Activating Regulatory T Cells in a Murine Model of Contact Hypersensitivity.

BACKGROUND: Juzentaihoto (JTT) is a Kampo prescription that has been used clinically for treating skin diseases such as atopic dermatitis in Japan. We have previously studied the anti-allergic effects of JTT on 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity (CHS) in mice and demonstrated that it significantly suppresses ear swelling in a dose-dependent manner. However, the mechanism underlying the anti-allergic actions of JTT is obscure. METHODS: We investigated the mechanism underlying the anti-allergic effects of JTT using a TNCB-induced murine CHS model and adoptive cell transfer experiments. RESULTS: We showed that the anti-allergic effects of JTT are due to inhibition of effector T-cell activation and induction and/or activation of regulatory T cells. Furthermore, ex vivo experiments confirmed the effect of JTT on the activation of effector T cells and regulatory T cells, as interferon-γ production decreased, whereas interleukin (IL)-10 production increased, in the cultured lymphocytes obtained from 5% TNCB-sensitized mice treated with anti-CD3ε and anti-CD28 monoclonal antibodies. Flow cytometry showed that the CD4+CD25+Foxp3+, CD4+CD25+Foxp3-, and CD8+CD122+ cell population increased after oral administration of JTT. Finally, the anti-allergic effect of JTT by inducing and/or activating regulatory T cells (Tregs) was confirmed to be mediated by IL-10 through in vivo neutralization experiments with anti-IL-10 monoclonal antibodies. CONCLUSION: We suggested that JTT exerts anti-allergic effects by regulating the activation of effector T cells and Tregs involved in murine CHS model.

Effect of glucoglycyrrhizin on IgE-mediated immediate hypersensitivity in mice.

To evaluate the pharmacological property of glucoglycyrrhizin (GGL), a unique glycoside of glycyrrhetinic acid (GA), we investigated the anti-allergic effect of GGL on IgE-mediated immediate hypersensitivity in mice. GGL exhibited the antiallergic effect against IgE-mediated immediate hypersensitivity. At a dose of 100 mg/kg, GGL exhibited antiallergic activity equivalent to that of glycyrrhizin (GL). Furthermore, the pretreatment with anti-GA monoclonal antibody eliminated the antiallergic action of GGL. These results indicated that GGL may act in the same way as GL in the human body. Its safety should be verified for its use as a drug similar to GL.

Effect of hot water extract of a glycyrrhizin-deficient strain of Glycyrrhiza uralensis on contact hypersensitivity in mice.

To evaluate the medicinal properties of a glycyrrhizin (GL)-deficient strain of Glycyrrhiza uralensis, we investigated the anti-allergic effect of the hot water extract obtained from its roots on contact hypersensitivity in mice, and compared it with that of the hot water extract of a commercial crude drug, Glycyrrhiza Radix. The hot water root extract of the GL-deficient strain contained glucoglycyrrhizin (GGL) and rhaoglucoglycyrrhizin (RGL) instead of GL, and it showed anti-allergic activity against contact hypersensitivity in a fashion similar to that of the crude drug extract. We further confirmed the presence of glycyrrhetinic acid (GA), a major metabolite of GL, in mice serum after oral administration of the hot water root extract of a GL-deficient strain. We demonstrated that GGL underwent hydrolysis by intestinal microflora of mice to form GA. These results suggest that a GL-deficient strain of G. uralensis is a useful medicinal resource since the glycosides of GA work in a fashion similar to that of GL when orally administered.

Evaluation of the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems III: anti-allergic effects of hot water extracts on IgE-mediated immediate hypersensitivity in mice.

To evaluate the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems, we investigated anti-allergic action in mice using IgE-mediated immediate hypersensitivity. Hot water extracts obtained from the roots of Glycyrrhiza uralensis cultivated using two systems were orally administered at a dose of 100 mg/kg as glycyrrhizin (GL) and compared with the commercial crude drug, Glycyrrhizae Radix. Both the artificial hydroponic and artificial hydroponic-field hybrid cultivated root extracts showed anti-allergic effects on IgE-mediated immediate hypersensitivity in mice, as did the commercial crude drugs. These results highlight the potential for artificially cultivated roots of Glycyrrhiza uralensis to be used as an alternative medicinal source.

Effect of Schisandrae Fructus on glycyrrhizin content in Kampo extracts containing Glycyrrhizae Radix used clinically in Japan.

Glycyrrhizae Radix is an important crude drug in Japan and is the most frequently prescribed drug in Kampo medicines for the treatment of a wide range of diseases. Glycyrrhizin (GL), the major active ingredient of Glycyrrhizae Radix, has various pharmacological actions but causes adverse effects such as pseudoaldosteronism. In a previous study, the GL content of shoseiryuto was found to be unexpectedly low, and Schisandrae Fructus in shoseiryuto reduced the pH value of the decoction and drastically decreased the extraction efficiency of GL from Glycyrrhizae Radix. In the present study, we investigated the extraction efficiency of GL from Glycyrrhizae Radix in decoctions comprising Glycyrrhizae Radix and five different fruit-derived crude drugs. Among the five fruit-derived crude drugs tested, Schisandrae Fructus markedly decreased both the pH value of the decoction and the extraction efficiency of GL. A comparison of the pH value of the decoction and the GL content of 12 Kampo prescriptions (containing at least Glycyrrhizae Radix and Schisandrae Fructus) showed that the GL content per daily dose was proportional to the compounding amount of Glycyrrhizae Radix, and that the extraction efficiency of GL from Glycyrrhizae Radix was strongly correlated with the pH value of the decoction. In addition, the pH value of the decoction was similar to the pH value documented in interview forms provided by pharmaceutical companies. These results suggested that the GL content in Glycyrrhizae Radix-containing Kampo products can be estimated from both the compounding amounts of Glycyrrhizae Radix and the pH value documented in their interview forms. Knowledge of GL content will help avoid adverse reactions due to Glycyrrhizae Radix.

Evaluation of the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic-field hybrid cultivation systems II: comparison of serum concentration of glycyrrhetinic acid serum concentration in mice.

To evaluate the safety and efficacy of Glycyrrhiza uralensis root produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems, we investigated the pharmacokinetics of a major metabolite of glycyrrhizin (GL), glycyrrhetinic acid (GA). Hot water extracts obtained from the roots of the artificial hydroponic-field hybrid cultivated Glycyrrhiza uralensis were orally administered at a dose of 100 mg/kg as GL in mice and, compared with a commercial crude drug, Glycyrrhizae Radix. The temporal changes in serum GA concentration was found to depend on the GL concentration of the hot-water extracts. When hot-water extracts containing relatively high GL were administered, bimodal peaks appeared. In contrast, a broad single peak was detected when a hot-water extract containing relatively low GL content was administered. These tendencies in the serum GA concentration time course were observed for all samples, regardless of their derivation. Moreover, we compared the pharmacokinetic parameters and found that the Cmax and AUC0-48 values after oral administration of the extracts from Glycyrrhiza uralensis roots produced by the artificial cultivation system are within the range of variation for the commercial crude drugs. These results suggest the possibility that roots of Glycyrrhiza uralensis cultivated by the artificial hydroponic-field hybrid cultivation system can be used in addition to currently available commercial crude drugs produced from wild plant resources.

Mutagenetic and anti-allergic studies for evaluation of extracts of Coptis Rhizome produced by an artificial hydroponic system.

As a part of the investigation of the safety and efficacy of the cultivated Coptis japonica rhizome extracts using an artificial hydroponic cultivation system, the mutagenetic and anti-allergic activities were evaluated. Some extracts of commercial crude drugs of Coptis sp. were also evaluated for the comparison. None of the extracts showed a significant mutagenicity in Salmonella typhimurium TA102 by the Ames tests, but all the extracts showed in S. typhimurium TA98. The extracts of the hydroponically cultivated rhizomes showed anti-allergic activities against contact hypersensitivity as well as those of commercial crude drugs of Coptis sp. These results suggested the potential of the hydroponically cultivated rhizomes as one of the alternative sources for the medicinal usage.

Correction to: Comparison of glycyrrhizin content in 25 major kinds of Kampo extracts containing Glycyrrhizae Radix used clinically in Japan.

The article Comparison of glycyrrhizin content in 25 major kinds of Kampo extracts containing Glycyrrhizae Radix used clinically in Japan, written by Mitsuhiko Nose, Momoka Tada, Rika Kojima, Kumiko Nagata, Shinsuke Hisaka, Sayaka Masada, Masato Homma and Takashi Hakamatsuka, was originally published Online First without open access. After publication in volume 71, issue 4, page 711-722 the author decided to opt for Open Choice and to make the article an open access publication. Therefore, the copyright of the article has been changed to

Comparison of glycyrrhizin content in 25 major kinds of Kampo extracts containing Glycyrrhizae Radix used clinically in Japan.

Glycyrrhizae Radix is the most frequently used crude drug in Japan and is prescribed in Kampo medicine for the treatment of a wide range of diseases. The major active ingredient of Glycyrrhizae Radix, glycyrrhizin (GL), has been shown to possess various pharmacological actions, but is also known to cause adverse effects such as pseudoaldosteronism. To avoid the adverse effects of GL, precautions have been indicated on the package inserts of Glycyrrhizae Radix-containing formulas depending on the amount of Glycyrrhizae Radix they contain. However, it remains unknown whether the extraction efficiency of GL from Glycyrrhizae Radix is constant throughout the different combinations of crude drugs in Glycyrrhizae Radix-containing formulas. To confirm the basis of the safety regulation, in this study we comprehensively determined the GL content of 25 major kinds of Kampo extracts compounding Glycyrrhizae Radix. We found that the GL content per daily dosage in all Kampo extracts are generally proportional to the compounding amount of Glycyrrhizae Radix, except in the case of shoseiryuto (Sho-seiryu-To). We also found that Schisandrae Fructus in Sho-seiryu-To decoction caused a lowered pH condition and drastically decreased the extraction efficacy of GL from Glycyrrhizae Radix. Moreover, we were able to confirm that the extraction efficiency of GL from Glycyrrhizae Radix is dependent on the pH value of the extraction solvent. The extraction efficiency of GL in the 25 kinds of Kampo extracts was not constant but it correlates significantly with the pH value of the decoction. Furthermore, the GL contents are well correlated with pseudoaldosteronism incidence data obtained from the Japanese Adverse Drug Event Report (JADER) database on the 25 kinds of Kampo extracts. This suggests that the GL content is a better index to consider to avoid the adverse effects of Glycyrrhizae Radix-containing Kampo formulas.

Effect of the hot water extract of Artocarpus camansi leaves on 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity in mice.

Medicinal plants with reported anti-inflammatory activity could have the potential use as anti-allergens and inhibitors of allergic contact dermatitis reactions produced by allergens and chemicals. Some species from the genus Artocarpus were reported to have anti-inflammatory activity. In the Philippines one local source is Artocarpus camansi BLANCO (Moraceae), which is utilized as an ingredient of their cuisine, and decoction of leaves is used for diabetes and baths of people with rheumatism. The objective of this study was to evaluate the effect of the hot water extract of A. camansi leaves on contact hypersensitivity (CHS) in mice. Contact hypersensitivity was induced using 2,4,6-trinitrochlorobenzene (TNCB). The results showed that the A. camansi hot water extract exhibited significant activity against the swelling produced during 24 h and 48 h post-challenge. The same responses were observed from the mice that received the kamansi ethanol-precipitate (KEP) and kamansi ethanol precipitate water-soluble (KEPWS) fractions. Since the high molecular mass fraction showed the significant activity, we therefore speculate that the compound responsible might be a polysaccharide and/or glycoprotein. In conclusion, our results suggest that the hot water extract of A. camansi leaves might be an effective natural product to treat allergic contact dermatitis. However, further investigations are required to understand the mechanisms involved.

Suppressive effect of the hot-water extract of Ficus pseudopalma Blanco leaves on the postprandial increase in blood glucose level in mice.

The use of medicinal plants with anti-diabetic properties continues because of the high cost of diabetes mellitus treatment. In the Bicol region of the Philippines, one local source is the leaves of Ficus pseudopalma Blanco (Philippine fig), which is utilized as an ingredient of their cuisine, and the decoction of its leaves is believed to have a blood-glucose lowering effect. The aim of this study was to evaluate the blood-glucose lowering effect of F. pseudoplama using sugar/carbohydrate-loaded and normoglycemic mice. The results showed that the hot-water extract of the leaves significantly suppressed the increase of blood glucose levels after glucose, maltose and starch loading. On the other hand, the extract did not show any hypoglycemic activity in either fasted or non-fasted mice as compared to the positive control drugs. These results suggest that F. pseudopalma is potentially useful for the management of blood glucose levels in the postprandial condition, as believed in the Bicol region of the Philippines.

The herbal medicine compound falcarindiol from Notopterygii Rhizoma suppresses dendritic cell maturation.

Dendritic cells (DCs) are important for regulating the immune response. We report an herbal medicine compound called falcarindiol that affects DC function. Ethanol extracts of 99 crude drugs that are the main components of 210 traditional Japanese medicines (Kampo medicine) approved by the Ministry of Health, Labor and Welfare in Japan were prepared and screened using the murine epidermal-derived Langerhans cell line XS106. Notopterygii Rhizoma strongly suppressed major histocompatibility complex (MHC) class II expression in XS106 cells. Activity-guided fractionation led to the isolation and identification of falcarindiol as a principal active compound in Notopterygii Rhizoma. Falcarindiol (1-5 microM) dose-dependently suppressed MHC II expression in XS106 cells. Fresh-isolated bone marrow-derived DCs were examined for the production of MHC II, CD80, CD86, interleukin (IL)-12p70, and IL-10. Treatment of bone marrow-derived DCs with 5 muM falcarindiol significantly inhibited lipopolysaccharide-induced phenotype activation and cytokine secretion and inhibited MHC II expression by CD40 ligation, but not phorbol 12-myristate 13-acetate + ionomycin or IL-12. Falcarindiol inhibited DC maturation by blocking the canonical pathway of nuclear factor-kappaB and phosphorylated p38. Topical application of 0.002 and 0.01% falcarindiol before sensitization dose-dependently suppressed delayed-type hypersensitivity to ovalbumin (p < 0.01). Falcarindiol induces immunosuppressive effects in vitro and in vivo and might be a novel therapy for autoimmune or allergic diseases.

Novel functions of herbal medicines in dendritic cells: role of Amomi Semen in tumor immunity.

Dendritic cells (DCs) have a major role in regulating immune responses, including tumor immunity and peripheral tolerance. In the present study, we identified novel functions of herbal medicines in DCs by screening 99 herbal medicines, most of which are among the 210 Chinese medicines approved by the Ministry of Health, Labour, and Welfare, Japan. Ethanol extracts were prepared, and a murine epidermal-derived Langerhans cell line, XS106, was used to screen the 99 extracts by analyzing major histocompatibility complex (MHC) class II expression. Amomi Semen (amomum seed), Polyporus (polyporus sclerotium), and Plantaginis Semen (plantago seed) potently activated XS106 and were selected for further analysis. The effects of these extracts on bone marrow-derived DCs (BM-DCs) generated in vitro were then analyzed using surface phenotype (MHC class II, CD80, and CD86) and interleukin (IL)-12p70 production as indicators. BM-DCs treated with Amomi Semen extract exhibited activated phenotypes and secreted IL-12p70. The activation level was similar to that induced by lipopolysaccharides. Finally, an E.G7-OVA tumor model (E.L4-OVA transfectant) was used to examine the anti-tumor effects of Amomi Semen extract. Vaccination of mice with a subcutaneous injection of BM-DCs treated with Amomi Semen extract and OVA peptide significantly inhibited the growth of tumor cells and prolonged survival time compared to controls. Furthermore, therapeutic effects were observed on established tumors. The inhibition rates for both the prophylactic and therapeutic protocols were comparable to those of lipopolysaccharides. These results indicate that Amomi Semen extract potently activate DCs and is potentially useful for DC vaccination.

Pharmacological characterization of a chronic pruritus model induced by multiple application of 2,4,6-trinitrochlorobenzene in NC mice.

Female NC/Jic mice were sensitized and challenged repeatedly at 48 h intervals for 10 and 30 days by painting 1% 2,4,6-trinitrochlorobenzene (TNCB) on both ears. Mice challenged with TNCB for 30 days developed an inflammatory dermatitis with high immunoglobulin E (IgE) titer. Histological analysis with acidic Toluidine Blue staining revealed that dermal mast cells markedly differentiated and intensely degranulated, consistent with a dramatic increase in scratching behavior. A significant increase in total scratching events could be observed in mice treated with TNCB for a short period of 10 days. Extending the term of TNCB application to 30 days, the IgE titer and number of mast cells elevated significantly, and thus various drugs were evaluated pharmacologically by using the mice treated with TNCB for 30 days. Terfenadine and cyproheptadine attenuated the chronic scratching behavior. Tacrolimus and dexamethasone were less effective and cromolyn showed no effect. In addition, terfenadine and tacrolimus suppressed the degranulation of mast cells. The present chronic scratching model could be suitable to evaluate drugs effective for suppression of mast cell differentiation and degranulation by irritation, and may represent a promising tool to develop new drugs for inflammatory pruritus associated with, for example, atopic dermatitis.

Sho-saiko-to and Saiko-keisi-to, the traditional Chinese and Japanese herbal medicines, altered hepatic drug-metabolizing enzymes in mice and rats when administered orally for a long time.

As the consumption of herbal remedies has increased, the opportunity that such herbal medicines are co-administered with other drugs has also risen gradually and we are, therefore, very much concerned about herb-drug interactions. We examined the effects of pre-administration of Kampo medicines (Sho-saiko-to, Saiko-keishi-to, Shigyaku-san and Dai-saiko-to) on the pentobarbital-induced sleeping time in mice and rats, to clarify the possibility that they could affect the drug-metabolizing enzymes. The administration of Sho-saiko-to and Saiko-keishi-to for 4 weeks significantly shortened the pentobarbital-induced sleeping time in mice and the administration of Sho-saiko-to for 2 weeks significantly reduced the sleeping time in rats. Furthermore, we tried to identify the molecular species of rat cytochrome P450s (CYPs) affected by Sho-saiko-to and Saiko-keishi-to by competitive RT-PCR. The oral administration of Sho-saiko-to for 2 weeks upregulated the mRNA expression of CYP2B, CYP3A1, CYP2E1 and CYP4A1 in rats. The treatment with Saiko-keishi-to for 2 weeks also up-regulated the mRNA expression of CYP2B, CYP3A1 and CYP4A1. Sho-saiko-to and Saiko-keishi-to may potentially influence the drug-metabolizing enzymes in man, and would thus require much attention when used in the clinical situation.

Comparative study of the high molecular mass fraction and low molecular mass fraction of Sho-saiko-to in a murine immunologically induced liver injury model.

We compared the pharmacological actions of the high and low molecular mass fractions of Sho-saiko-to using a murine immunologically induced liver injury model to estimate the roles of these fractions in the expression of the pharmacological action. In a Bacillus Calmette-Guerin (BCG)/lipopolysaccharide (LPS)-induced liver injury model, Sho-saiko-to and both of its fractions significantly reduced the increases in the aminotranseferase levels in serum. They also reduced the increase in the nitric oxide (NOx) level in serum. On the other hand, Sho-saiko-to and its high molecular mass fraction suppressed the increase in plasma NOx level in an LPS-induced endotoxin shock model but its low molecular mass fraction did not. These results suggest the possibility that both fractions act hepatoprotectively in a different manner. We believe that these results can help to elucidate the mechanism of action of ingredients in Sho-saiko-to.