Antiallergic activities of Japanese leaf burdock extract in a rat basophilic leukemia cell line.

Although primarily cultivated for its edible root, the leaves and stems of "Japanese leaf burdock (Arctium lappa L)" are also edible. This study determined the antiallergic activity of root, leaf, and stem extracts of Japanese leaf burdock using the rat basophilic leukemia cell line. We demonstrate that the hot water extract of Japanese leaf burdock stems has a strong inhibitory effect on antigen-stimulated degranulation. This extract was further fractionated into five fractions using a glass column. The fraction obtained with 99% ethanol extracts (99% EtEx) elicits a concentration-dependent increase in degranulation inhibition. One of antiallergic compounds from the separation of components of the 99% EtEx fraction using high-performance liquid chromatography and mass spectrometry analyses had a value of m/z 393.16. From these results and those of previous studies, one of the antiallergic components was hypothesized to be onopordopicrin. Our findings demonstrate that the extract of Japanese leaf burdock exerts a strong antiallergic activity. PRACTICAL APPLICATIONS: "Japanese leaf burdock (Arctium lappa L)" is a traditional Japanese vegetable with edible leaves and stems. The antiallergic activity of the hot water extracts has been shown in vitro. One of the antiallergic components was hypothesized to be onopordopicrin. Therefore, "Japanese leaf burdock" could serve as a functional food for patients with allergic disorders, such as allergic rhinitis.

A novel challenge of nondestructive analysis on OGATA Koan's sealed medicine by muonic X-ray analysis.

OGATA Koan (1810-63) was a physician and the director of Tekijuku, and he contributed to Western medicine in the late Edo period. Osaka University preserves two of his medicine chests. One of the chests, which was used in his last years (the second chest) contained 22 glass bottles and 6 wooden cylinders. These bottles and cylinders contained formulated medicines; however, about half cannot be opened because of the long-term storage. It is necessary to comprehend the physical property of both the containers and their contents for investigation of this adequate preservation method; however, destructive analysis is not allowed. To analyze the medicines sealed in the glass bottles, we focused on muonic X-ray analysis, which has high transmittance. First, we certified the analytical methods using a historical medicinal specimen preserved in Osaka University. Thereafter, we applied the method on the bottles stored in the second chest. X-ray fluorescence identified the glass of those bottles to be lead potash glass. Among these bottles, we chose the bottle with the label "," which contains white powdered medication, for muonic X-ray analysis. We identified the contents of the medication in the glass to be Hg2Cl2. Through this study, we first applied muonic X-ray analysis on the medical inheritances and succeeded to detect the elements contained both in the container and in the contents of the sealed bottle. This would be a new method for nondestructive analysis of such cultural properties.

Retinoic Acid Ameliorates the Severity of Food Allergy under Allergen Exposure in a Mouse Model with Food Allergy.

Effectiveness of retinoic acid (RA) in treating food allergy is not yet clear. Using an allergic mouse model, we examined the amelioration of the severity of food allergy by daily RA intake with allergen or without. Female Balb/c mice were systemically sensitized to egg white (EW) and alum by intraperitoneal injection. Sensitized mice were provided diets supplemented with 0% (non-treated group), 0.1% EW (allergen group), 0.0017% RA (RA group), or 0.1% EW plus 0.0017% RA (RA+allergen group) with 20% casein for 4 wk. Oral food challenge (OFC) and allergic biomarkers were quantified. The decrease in rectal temperature post-OFC was significantly suppressed in the RA and RA+allergen groups compared to those in the non-treated and allergen groups, respectivety. The plasma levels of ovalbumin-specific IgE, IgA and IgG1 at the study endpoint were higher in the allergen and RA+allergen groups than those in the non-treated and RA+allergen groups, respectivety. Plasma ovalbumin-specific IgG2a levels at the study endpoint were significantly higher in the RA+allergen group than those in the RA groups. The supernatant concentrations of interleukin-10 and interferon-γ in the cultured spleen lymphocytes were highest in the RA+allergen group compared to those in the other groups. Thus, continuous intake of RA under allergen exposure ameliorated the severity of food allergy in a mouse model with food allergy.

Transition in Longgu Surface Associated with Decoction Process of Keishikaryukotsuboreito.

We aim to attain the sustainable use of longgu and have investigated the significance of longgu in Keishikaryukotsuboreito (KRB) decoction. We have already reported that longgu alters compound profiles in KRB decoction and hypothesized that it does so by adsorbing foreign organic compounds into its superficial pores. In the present study, we focused on the adsorbability of organic materials onto longgu surface as the cause of component profile alteration. We analyzed the physical changes in longgu through the decoction process by measuring the adsorbed water on longgu surface. 1H magic angle spinning NMR (1H-MASNMR) spectroscopic analysis revealed that raw longgu (R-raw) as well as decocted longgu [whether single (R-r) or KRB (R-krb) decoction] adsorbed water. However, the amount of adsorbed water in R-krb was smaller than that in R-raw and R-r. The nitrogen adsorption isotherms of longgu samples indicated that longgu was macroporous. The Brunauer-Emmett-Teller (BET) surface area of R-krb was smaller than that of R-raw and R-r. Further, thermogravimetric analysis of longgu samples showed that R-krb adsorbed matter that R-raw and R-r did not adsorb. The above findings and the 1H-MASNMR analysis of heated longgu samples suggested that longgu adsorbed organic compounds into the pores. We considered that longgu adsorbed organic compounds during KRB decoction into its superficial pores through the decoction process.

Diets Supplemented with 1% Egg White Induce Oral Desensitization and Immune Tolerance in an Egg White-Specific Allergic Mouse Model.

BACKGROUND: The objective of this study was to determine the required concentration of egg white (EW) in the diet to induce oral desensitization and/or immune tolerance within 4 weeks of oral immunotherapy (OIT) in an EW allergic mouse model. METHODS: Female BALB/c mice were systemically sensitized to EW by intraperitoneal injections and subsequently subjected to oral allergen gavage. Sensitized mice were provided 4 weeks of OIT by supplementing with 0 (non-OIT), 0.01, 0.1, or 1% EW in a 20% casein diet. Nonsensitized mice served as the nonallergy group. We performed oral and intraperitoneal EW challenges, assessed vascular permeability in the dorsal skin, and measured allergic biomarkers. RESULTS: The change in rectal temperature after oral challenge was not significantly different between the nonallergy and 1% EW groups, and the frequency of diarrhea in the 1% EW group was lower than that in the non-OIT group. The levels of plasma ovomucoid-specific IgE, IgA, and IgG2a in the 1% EW group at the study endpoint were significantly lower than those in the non-OIT group. IFN-γ and IL-10 secretions of spleen lymphocytes in the 1% EW group were significantly higher than those in the non-OIT group, and the percentage of CD4+Foxp3+ cells in the 1% EW group was higher than that in the non-OIT group. CONCLUSION: These results suggested that diet supplemented with 1% EW can induce oral desensitization and immune tolerance in the EW allergic mouse model.

Quality Characterization of Japanese Medicinal Paeoniae Radix by Metallomic Analysis.

Paeoniae Radix is one of the crude drugs frequently used in traditional Japanese medicine (Kampo medicine). It takes abundant labor and time to cultivate Paeonia lactiflora for medicinal use; high production cost is one of the main reasons why the domestic production of Paeoniae Radix is decreasing in Japan. To promote the production of Paeoniae Radix, we focused on Paeonia cultivars that produce commercially valuable flowers and investigated their possibility for medicinal use. We prepared 28 batches of peony roots derived from P. lactiflora, which were cultivated in Japan; 4 batches were crude drug samples, and 24 batches were cultivar roots. The elements contained in these samples were measured using inductively coupled plasma (ICP)-MS. The obtained data were then analyzed by principal component analysis (PCA) and back propagation artificial neural network (BPANN) analysis. No significant differences were found between the profiles of elements contained in crude drugs and cultivar roots. However, PCA results indicated a high similarity of the multielement fingerprints of crude drugs. Using the PCA results, we also assessed visible cluster trends and found that 5 batches of cultivars also showed fingerprints related to those of crude drugs. We certified this classification by BPANN. From the perspective of metallomics, our findings suggest that these 5 batches of Paeonia cultivars could be alternatives to crude drugs.

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor.

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.

Taxonomic examination of longgu (Fossilia Ossis Mastodi, "dragon bone") and a related crude drug, longchi (Dens Draconis, "dragon tooth"), from Japanese and Chinese crude drug markets.

Longgu ("dragon bone," Ryu-kotsu, Fossilia Ossis Mastodi, or Os Draconis) is the only fossil crude drug listed in the Japanese Pharmacopoeia. All longgu in the current Japanese market is imported from China, where its resources are being depleted. Therefore, effective countermeasures are urgently needed to prevent resource depletion. One possible solution is the development of a substitute made from bones of contemporary animals that are closely related to the original animal source of the current longgu. However, no research has been conducted on the original animal source of longgu, except for a report on the longgu specimens present in the Shosoin Repository. Taxonomic examination was performed on the fossil specimens related to longgu which are owned by the Museum of Osaka University, Japan. In total, 20,939 fossil fragments were examined, of which 20,886 were mammalian fossils, and 246 of these fossils were classified into nine families. The longgu specimens from the Japanese market belonged to a relatively smaller variety of taxa than those from the Chinese market. Despite the variety of taxa in longgu, medical doctors using Kampo preparations with longgu have not reported any problems due to the presence of impurities in the original animal source. These results suggest that the effect of longgu is independent of its origin as long as it is closely related to the origin of the current longgu. Thus, despite the considerable effects of fossilization, our results could help in developing an optimal substitute for longgu.

Longgu (Fossilia Ossis Mastodi) alters the profiles of organic and inorganic components in Keishikaryukotsuboreito.

Longgu (Fossilia Ossis Mastodi) is a non-botanical crude drug, defined as "the ossified bone of large mammal" in the Japanese Pharmacopoeia sixteenth edition (JP16). It is a non-reproducible drug and is now facing the threat of exhaustion. To solve this problem, we aimed to clarify the role of longgu in Kampo prescriptions, which has not yet been scientifically ascertained. In this study, we focused on decoction of Keishikaryukotsuboreito (KRB). The profile of inorganic and organic components in the extract was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and gas chromatography flame ionization detection (GC/FID), respectively. Twenty-five elements were detected by ICP-MS in KRB and longgu-free KRB (KB) decoctions. However, 23 elements were detected in unadultrated longgu (R) decoctions, and their total amount was 30 times lower than those of the KRB and KB decoctions were. No organic compounds were detected in R decoctions by GC/FID, though many were detected in KRB and KB decoctions. KRB decoctions were distinguished from KB decoctions by multivariate analysis. The only difference in the crude drugs was the presence of longgu, and therefore the difference in the profiles was attributed to the effect of longgu. Longgu was submitted to terahertz (THz) wave spectrometry after the decocting process. The THz spectra indicated that longgu adsorbed compounds during the KRB decoction. These results suggested that longgu not only releases its components, but also adsorbs ingredients from other crude drugs during decoction.

[Visualization for Traditional Quality Management Techniques--Characterization Method for Spikenard of INUBUSHI SEIYAKU Established in the Edo Period].

INUBUSHI SEIYAKU, a Japanese pharmaceutical company established in 1807, manufactures KEISHIN-TAN. This is an original drug developed by the company, and consists of 14 exotic natural medicines, spikenard, oriental bezoar, musk, agarwood, etc. It has been used for adjusting the autonomic nervous system and physical conditions. We studied the original methods of the traditional quality management techniques handed down within INUBUSHI SEIYAKU in selecting the appropriate spikenard (Nardostachys chinensis) for medicinal use. Currently, spikenards are mainly used as incense rather than medicine. KEISHIN-TAN is a rare case in that the bulk powder of the spikenards is used for pharmaceutical products in Japan. We examined the morphological characteristics and made an analysis of the component of spikenards selected by traditional methods. The raw material of the spikenards was purchased from the Japanese market, and was classified into two categories-superior, fit for medicinal use and defective, to be discarded-by traditional methods of INUBUSHI SEIYAKU. The methods of the characterization of the spikenard by INUBUSHI SEIYAKU were investigated. As a result, only thick spikenard roots over 2.0 cm in length and approximately 0.5 cm in diameter were found to be used, and the total weight of the superior was only 15% of the raw material. By comparing the weights of hexane extracts and GC-MS analyses, the content of calarene--main sedative compound in spikenards--in the superior material was 2.8 times higher than the raw material and 4.3 times higher than the defective material. The ways to devise how to enhance the pharmacological effects of spikenards may be contained in this method. These results revealed the traditional spikenard selection criteria, and may show the indications of using spikenard or its compounds for medicinal purposes.

Establishment of a methodology for identifying Paeoniae Radix based on metallomic analysis.

We aimed to establish a methodology for identifying Paeonia samples based on metallomic analysis. We prepared 66 batches of samples (16 batches of crude drugs and 50 batches of cultivars, comprising 64 batches of Paeonia lactiflora and 2 batches of P. veitchii) collected from Japan and China (Inner Mongolia and elsewhere) between 1996 and 2008. P. lactiflora samples were genetically classified into white peony root (WPR) type and red peony root (RPR) type. Up to 47 elements were measured by inductively coupled plasma-mass spectrometry, and RPR type crude drug samples contained up to five times as much calcium as the others. Principal component analysis (PCA) of the multi-element fingerprints obtained suggested that P. veitchii, which grows wild, were distinguishable from the other cultivated P. lactiflora samples. This was confirmed perfectly by soft independent modeling of class analogy (SIMCA). The PCA of the fingerprints of P. lactiflora crude drug samples also suggested that it was possible to classify them by production area (Japan, Inner Mongolia, and China excluding Inner Mongolia) and genetic type (RPR and WPR types). They were also classified 100 % to the predicted class by SIMCA in both cases. These analyses were successful among the samples whose collection dates varied. This simple metallomic method is an efficient approach for verifying the complex origin of Paeoniae Radix.

Andrographolide inhibits the expression and metabolic activity of cytochrome P450 3A4 in the modified Caco-2 cells.

AIM OF THE STUDY: The aim of this study is to examine the effects of andrographolide on intestinal enzyme cytochrome P450 3A4 (CYP3A4) and predict whether oral administration of andrographolide-containing remedy leads to herb-drug interaction. MATERIALS AND METHODS: Caco-2 cells are treated with 1α, 25-dihydroxyvitamin D3 for 3 wks to induce the expression of CYP3A4, and then andrographolide (1, 10, 100 µM) is added and treated for 72 h. Upon the further 4-h testosterone (250 µM) or nifedipine (200 µM) treatment, the basolateral medium samples and the Caco-2 monolayers are collected for analyses. RESULTS: Andrographolide (1, 10, 100 µM) significantly down-regulates the mRNA level and protein level of CYP3A4, and inhibits nifedipine oxidation and testosterone 6ß-hydroxylation. CONCLUSION: Oral administration of andrographolide likely leads to reduction of the metabolic activity of intestinal CYP3A4, therefore herb preparations containing andrographolide may result to herb-drug interactions in combination therapy.

Curdione Plays an Important Role in the Inhibitory Effect of Curcuma aromatica on CYP3A4 in Caco-2 Cells.

Curcuma aromatica is a plant belonging to genus Curcuma of family Zingiberaceae and is widely used as supplements in Japan. Rhizomes of C. aromatica have curcumin as a major yellow pigment and curdione as a main ingredient of essential oils. In this study, we investigated the affect of C. aromatica on CYP3A4 using 1α,25-(OH)(2)-D(3)-treated Caco-2 clone cells. Caco-2 cells were treated with methanol extract (0.1 mg ml(-1)), its hexane soluble fraction (0.1 mg ml(-1)), curcumin (4 µM) and curdione (20 µM) for 72 hours. Nifedipine was used as a substrate of CYP3A4. Methanol extract, hexane fraction and curdione inhibited the formation of oxidized nifedipine by 50-70%, and curcumin showed no effect. The IC50s of methanol extract, hexane fraction and curdione to oxidized nifedipine formation were 21, 14 and 3.9 µg ml(-1) (16.9 µM), respectively. The content of curdione in methanol extract was 11.4%. Moreover, all of methanol extract, hexane fraction and curdione decreased CYP3A4 protein expression but had no affect on CYP3A4 mRNA expression. Our results showed that these drugs further decreased the CYP3A4 protein expression level after the protein synthesis was inhibited by cychroheximide. These findings suggest that curdione plays an important role in the CYP3A4 inhibitory activity of C. aromatica and curdione might inhibit the activity by accelerating the degradation of CYP3A4.

Experimental Adjustment on Drug Interactions through Intestinal CYP3A Activity in Rat: Impacts of Kampo Medicines Repeat Administered.

TO PROVIDE THE INFORMATION THAT IS NECESSARY FOR MAKING THE PROPER USE OF KAMPO MEDICINES, WE HAVE PROPOSED THE ADEQUATE METHODOLOGY FOCUSED ON THE FOLLOWING ISSUES: (i) kampo medicines emphasize the effects produced by the combination of herbal drugs rather than the individual effect of any single herb and (ii) Intestinal CYP3A has become a key factor for the bioavailability of orally administrated drugs. In the present study, we investigated both the in vivo and in vitro effects of Saireito and Hochuekkito (kampo formulas) on CYP3A activities. From our study, oral pre-treatment with Saireito or Hochuekkito did not affect the pharmacokinetics of nifedipine after intravenous administration to rats. When nifedipine was administered to rat intrajejunum, a significant decrease of AUC was showed by pre-treatment with both kampo formulas. Saireito pre-treatment led to 80% decrease in C(max) of nifedipine. Saireito caused significant increases in both protein expression and metabolic activity of CYP3A in intestinal microsome, whereas it had no effect on CYP3A in hepatic microsome. Our result also showed that this affect of Saireito can be gone by wash-out with 1 week. These findings demonstrated that Saireito may induce CYP3A activity of intestine but not of liver in rats. When resources for research are limited, well-designed scientific studies except clinical trials also have many advantages.

Quality and safety issues related to traditional animal medicine: role of taurine.

BACKGROUND: Calculus Bovis (:C.Bovis) is one of the most precious and commonly-used medicinal materials in Japan and China. As the natural occurrence is very rare, a source of supply for C. Bovis is far behind the actual need and great efforts have been taken for some substitutes of natural C. Bovis. Unfortunately, very little information is available on the quality and/or clinical efficacy of medication based on C. Bovis. To ensure sustainable use of traditional therapeutic agents derived from C. Bovis, we felt that several issues needed to be addressed: 1) the source of the C. Bovis materials and quality control; 2) the role of taurine in the efficacy of C. Bovis. METHODS: Nine samples of natural C. Bovis and its substitutes were collected. ICP-MS was used for elemental analysis and the characterization was performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) as multivariate approaches. The efficacy of C. Bovis was evaluated for morphology, viability and beating pattern on cultured cardiac myocytes and/or fibroblasts. RESULTS: PCA and multi-elemental focus was effective in discriminating C. Bovis samples derived from different habitats. A satisfactory classification using SIMCA was obtained among Australia C. Bovis, other habitats and the substitutes. Australian samples had better batch uniformity than other habitats and were composed of fewer elements. We have used Australian C. Bovis for assessment on its bioactive compounds. Rat cardiac cells incubated with C. Bovis extract (0.01-0.1 mg/ml) maintained normal morphology, viability and beating pattern. Cardiac myocytes and fibroblasts treated for 48 h with CA (0.5 mM) or DCA (0.1 mM) caused cell injury, as reflected by changes in appearance and a reduction of viability detected by the MTS assay. In cardiomyocytes, 0.5 h exposure of CA (0.5 mM) markedly decreased the velocity ratio of beating, whereas the simultaneous addition of 1 mM taurine largely prevented the decrease. CONCLUSIONS: The multi-elemental focus provided some references for the quality control and the efficacy of C. Bovis. Taurine partly attenuated the harmful actions of bile acids. It is plausible that the relationship between taurine and the bile acids contributes to therapeutic effect of C. Bovis.

The relation between clinical effects of Tokishakuyakusan and the identity of Paeonia lactiflora materials.

AIM OF THE STUDY: To investigate the relation between the clinical effects and the quality of crude drugs, we focused on Tokishakuyakusan (TS), consisted of 6 crude drugs. MATERIALS AND METHODS: We prepared two kinds of TS containing either medicinal cultivar of Paeonia lactiflora (MTS) or ornamental one (OTS). Other components were the same. First, we assessed the clinical effects of two TS formulations by cross-over study among the anemia patients. Second, we investigated the chemical differences between them by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Mössbauer analysis. RESULTS: The clinical effects of these formulations (3 g/day for 8 weeks) were tested in the cross-over study consisted of 12 women patients who were diagnosed as having anemia (Hb ≤ 11 g/dl) and consented to participate to this study. Both TS formulations were effective for anemia symptoms as shown by the improvement of several hematological parameters, whereas their comprehensive effects were distinguishable by Genetic Algorithm Partial Least Squares (GA-PLS) analysis. There were no significant differences in organic ingredients and Fe content measured by ultra performance liquid chromatography (UPLC) and ICP-MS, respectively. Interestingly, Mössbauer spectra of Fe ion were remarkably different between two formulations. Fe ion in MTS was only one form, but that in OTS was at least two forms. CONCLUSIONS: This study suggested that clinical effects of TS formulation reflect the quality of Paeoniae Radix.

Tool from traditional medicines is useful for health-medication: Bezoar Bovis and taurine.

Bezoar Bovis (BB:dried cattle gallbladder stones) has been used empirically in Asia for over 3000 years to treat heart and liver disorders. Yet its therapeutic potential remains unexplored by Western researchers. The aim of this study has been to clarify the actions of BB on cultured cardiomyocytes and to identify its active component(s). BB is a component of 98.7% of the Japanese over the counter (OTC) cardioactive drugs. The water-extract of BB exhibits protection action against arrhythmias produced by low Ca2+ and high Ca2+ in the medium. On the other hand, the Ca(2+)-antagonist, verapamil, did not suppress arrhythmias that developed in cell culture. Rather, it aggravated the beating status of the cardiomyocytes. The major constituents of the BB extract are bile salts (cholate, deoxycholate, taurocholate) and amino acids (taurine, cysteine, leucine, isoleucine). Most cells incubated with bile salts developed morphological damage. However, one of the major constituents of the BB extract, taurine, was effective in protecting against the abnormal beating pattern induced by high Ca2+. Since beta-alanine, an inhibitor of taurine transport, antagonized the protective effects of both BB and taurine, it is likely that the effect of BB is partly mediated by taurine.