Indole is produced from dietary tryptophan by tryptophanase in intestinal bacteria, such as Escherichia coli. In the liver, indole is converted into indoxyl sulfate, a uremic toxin and risk factor for chronic kidney disease (CKD). Probiotics and prebiotics are currently used for suppressing CKD, but there are no drugs that directly suppress indole production. In this study, we developed an optimized HPLC method for analyzing indole production and evaluated the effect of diets and rhubarb on indole production via the changes of gut microbiota. In high-carbohydrate and high-fat diet-fed mice, the indole production was significantly higher than in high-fiber diet-fed mice. We further used the high-carbohydrate diet-fed mice as a model for examining the effect of rhubarb on indole production. The 20% methanol-eluted fraction of aqueous rhubarb extract significantly suppressed indole production, and the eluate constituent rhein 8-O-β-D-glucopyranoside (RG) contributed to this effect in a concentration-dependent manner. The effect of RG depended on the anthraquinone core substructure, i.e., the aglycone moiety (rhein) of RG, which appeared to inhibit the tryptophanase function in gut microbiota. Thus, in addition to earlier reports that rhubarb is an effective CKD treatment, our study demonstrated that the anthraquinone moiety in rhubarb prevents uremic toxin production via functional changes in gut microbiota, which suppresses CKD progression.

Interindividual differences affect the purgative activities of sennoside A (SA) and Daiokanzoto (Da-Huang-Gan-Cao-Tang, DKT). In this study, we manipulated gut microbiota in mice to establish laxative responders and non-responders by feeding them a high-carbohydrate, a high-fat or a high-fibre diet. To assess the relationship between laxatives and gut microbiota, we monitored the gut microbiota before and after administering laxatives. Twenty mice per diet were divided into four groups of five mice to evaluate purgative activities of four laxative preparations, DKT, SA, SA plus rhein 8- O - β -D-glucopyranoside (SA + RG), and SA plus liquiritin (SA + LQ). Gut microbiota changes were monitored by next-generation sequencing of 16 S rRNA gene amplicons. In high-carbohydrate and high-fat diet-fed mice, DKT exerted a significantly higher purgative activity than SA alone, and RG contributed to this activity. DKT and SA + RG administration increased the Enterobacteriaceae content of gut microbiota, which was associated with an increased purgative activity. In contrast, DKT activity was significantly suppressed by high-fibre diet. Hence, diet-induced differences in gut microbiota determined the effect of DKT, which is interesting, considering that Oriental medicines are formulated for a specific functional state or “pattern”. These results demonstrated that the purgative activity of anthranoid laxatives is susceptible to diet-induced alterations in gut microbiota.

It has been suggested that imbalances in gut microbiota are related to diseases associated with metabolism, the central nervous system, etc. Therefore, analysis of short-chain fatty acids (SCFAs) produced by gut microbiota is very important as an indicator of causation, demonstrating the effects on the host due to changes in the gut microbiota. We developed a HPLC method for the determination of SCFAs in mouse feces. After homogenization, the SCFAs in mouse feces and 2-ethylbutyric acid (internal standard) were derivatized with 2-nitrophenylhydrazine (2-NPH) in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. The 2-NPH derivatives of SCFAs and the internal standard were separated on a reversed-phase column (octadecyl silyl column) by gradient elution using phosphoric acid (pH 2.5)-acetonitrile at 50°C and detected by absorbance measurement at 400 nm. The recovery of the method was 90-115%, with a precision (relative standard deviation) of 1.3-7.7%. The determination of SCFAs by the present method can provide useful information for biological and clinical research.

Sennoside A (SA), the main purgative constituent of Daiokanzoto (da-huang-gan-cao-tang; DKT), is generally regarded as a prodrug that is transformed into an active metabolite by β-glucosidase derived from Bifidobacterium spp. It has been suggested that antibiotics would promote dysbiosis, and thereby inhibit the purgative activity of DKT. In this study, ampicillin was administered to mice for 8 d, and the changes in the SA metabolism of SA alone and of DKT were investigated. The results showed that the SA metabolism of SA singly continued to be inhibited by ampicillin, but that of DKT was activated from day 3 under the same conditions. In order to investigate the mechanism of SA metabolism activated by DKT in the mice administered ampicillin, changes in the SA metabolism were observed in the presence of rhein 8-O-β-D-glucopyranoside (RG) in rhubarb and liquiritin in glycyrrhiza, both of which accelerated the SA metabolism. In fact, RG achieved an activation of SA metabolism similar to that by DKT. The purgative action of DKT, which was continued treatment of the ampicillin, was significantly greater than that by SA alone, and it was shown that RG was involved in this effect. We also analyzed changes in the intestinal microbiota before and after administration of ampicillin. No Bifidobacteria were detected throughout the treatment, but the population of Bacteroides was significantly increased after 3 d under the same conditions. Taken together, these results strongly suggested that the RG in DKT changed the function of Bacteroides and thereby allowed DKT to metabolize SA.

BACKGROUND:There are problems in diagnosis of allergy to amide-type local anesthetic agents (ALAs), because definitive diagnosis is not obtained by in vivo tests, which are used for the diagnosis. Consequently, patients may be exposed to risk. There are few diagnoses based on in vitro tests, and there are almost no relevant studies.METHODS:Authors examined involvement of allergic reaction using the leukocyte migration test (LMT) through multiple standpoints in 43 patients who underwent suspected diagnosis of allergy to ALAs.RESULTS:Rate of LMT-positives was 54%, and especially the positive rate of lidocaine hydrochloride preparations was significantly high. In 15 positives to lidocaine hydrochloride preparations, all cases were indicated as positive in a test with drugs containing antiseptic agent, but only 3 cases were indicated as positive in a test with lidocaine hydrochloride alone. In addition, test with paraben was conducted in 4 cases; 2 cases were confirmed as positive. In relevance of histories of drug or food allergies, development rates of ALAs-allergies were the highest in both allergies, and were 35% and 13%, respectively.CONCLUSIONS:There is a high possibility that these adverse reactions were caused by pseudoallergy to drug. Even by allergic reactions, it was assumed that 80% of them might be caused by antiseptic agents such as paraben. In addition, it was suggested that ALAs, especially lidocaine hydrochloride preparations have high antigenicity (sensitizing property). Furthermore, it was considered that patients with past history of drug or food allergies have a high potential for manifestation of the reactions.

In vivo tests may be used for the diagnosis of allergy to iodinated contrast media (ICM); however, the tests do not provide definitive diagnosis and are associated with risks for patients. Diagnoses based on in vitro tests are limited, and there are almost no relevant studies. Herein, the authors examined involvement of allergic reaction from a multilateral standpoint in 39 patients suspected of having ICM allergies using leukocyte migration test (LMT). The positive rate of LMT was 44%. A comparison with the positive rate of LMT in drugs other than ICM (74%) indicated 30% difference, which was significantly low value, suggesting that there is poor involvement of these drugs in the allergic reaction. In LMT positives, 76% of hypersensitivity reactions were skin rash mainly erythema, and 18% was anaphylactic reactions. Cases considered as non-immediate hypersensitivity accounted for about 4 times as many as immediate-type hypersensitivity. In examination of relevancy between a history of drugs or food allergies, the incidence of ICM allergies was 35%. There is a high possibility that these adverse reactions were caused by pseudoallergy to drug. It was suggested that most hypersensitivity reactions were skin rash related to non-immediate hypersensitivity, and approximately 20% of the reaction was immediate anaphylactic reaction. Therefore attention should be paid not only to immediate-type hypersensitivity but also delayed reactions. Moreover, it was considered that patients with past history of drug or food allergies have a high potential for manifestation of the reactions.

Daiokanzoto (DKT), a Kampo medicine that includes the combination of two crude drugs (rhubarb and glycyrrhiza), is clinically effective for constipation. Sennoside A, the main purgative constituent of rhubarb, is a prodrug that is transformed into an active metabolite, rheinanthrone, by -glucosidase derived from biÞ dobacteria. Thus far, we have presented evidence that anthraquinones in rhubarb, rhein 8-O-D-glucopyranoside (RG) and rhein, contribute to the purgative action of sennoside A by accelerating its metabolic activity. The aim of this study was to clarify the mechanism of action by which anthraquinones accelerate sennoside A metabolism. In pre-incubated fecal suspensions, mixed with anthraquinones, there was significant acceleration of the metabolic activity of sennoside A compared with that in the control. This finding suggested that anthraquinones induced metabolism by bacteria or their enzyme of sennoside A. The mechanism of action by RG was thus further investigated with puromycin, which is a protein synthesis inhibitor, in terms of quantitative or qualitative changes of sennoside A-metabolic enzyme. The results suggested that RG increased the synthesis of sennoside A-metabolic enzyme by bifidobacteria. Therefore, it is assumed that the anthraquinones included in DKT intensiÞ ed the activation of sennoside A by increasing the synthesis of sennoside A-metabolic enzyme derived from biÞ dobacteria, which is the main metabolic bacteria. rhein 8-O-D-glucopyranoside RG LQ A SA SA RG RG Rhein LQ SA RG Rhein SA puromycin RG SA RG SA SA RG RG SA puromycin

Borrelia duttonii, a causative agent of relapsing fever, is transmitted between the distinct microenvironments of the vector tick, Ornithodoros moubata, and a mammalian host. We identified the total and membrane fraction proteins of B. duttonii strain Ly isolated from a patient in Tanzania by using two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The analyses of the total and membrane fractions from bacterial cultures incubated at 37°C identified 68 and 15 proteins, respectively. Since spirochaete clearance in mice is associated with an immunoglobulin M (IgM) and immunoglobulin G3 (IgG3)-mediated response, immunoblot analyses were used to identify the proteins reactive with IgM and IgG3 of gerbil serum against B. duttonii strain Ly. The outcome showed that six proteins (antigen p83/100, membrane-associated protein P66 (P66), flagellar filament outer layer protein, hypothetical protein BDU_412, vlp protein gamma subfamily (γ-Vlp) and flagellin (FlaB)) were identified against IgM, and four (antigen p83/100, P66, γ-Vlp and FlaB) of the six proteins also reacted with IgG3. It is believed that these proteins are immunodominant antigens for the host immune response. Some of these immunogenic proteins might be used as molecular diagnostic tools in the study of relapsing fever in Tanzania.

In recent years, it is necessary to acquire knowledge not only about medicine but also over-the-counter (OTC) drugs and health food for children, because lowering trend in the age of the health hazard by improper use of health food is reported. Therefore, in order to estimate the extent of use of OTC drugs and health food, the school pharmacists administered a questionnaire to students in grade-school (n=123), junior high school (n=303), and high school (n=115) in Fukuyama city. As a result of the questionnaire survey, surprisingly, the usage ratio of OTC drugs and health food showed the most increase in grade-schooler. The trigger of use of health food is "parents' recommendations" in the lower grades, otherwise the ratio of "use by themselves" was increased in the higher grades. Moreover, a remarkable difference was observed by the kinds of use in students with or without exercise. Interestingly, exercise group expected "physical strength" effects than no exercise group. In addition, the ratio of consultation to the pharmacist at the time of purchase of OTC drugs and health food was low in all grade students. In particular, the ratio of consultation to the pharmacist at the time of purchase of health food was very low in high school students. Therefore, to provide accurate information of medicine and health food for students, the school pharmacist should engage not only in routine work but also in positive guidance about OTC drugs and health food in the future.

The effect of high molecular weight chitosan supplement (HMCS), a natural polymer derived from chitin, on indices of oxidative stress was investigated in normal volunteers. The use of HMCS for 8 weeks resulted in a significant decrease in total cholesterol levels and atherogenic index, and increased levels of high density lipoprotein (HDL) cholesterol. HMCS treatment also resulted in a lowered ratio of oxidized to reduced albumin and an increase in total plasma antioxidant activity. A good correlation between the atherogenic index and oxidized albumin ratio was found. The results suggest that the ratio of oxidized to reduced albumin ratio represents a potentially useful marker of the metabolic syndrome. In in vitro studies, HMCS slightly reduced the levels of two stable radicals in a dose- and time-dependent manner. The strong binding capacity of indoxyl sulfate and low density lipoprotein (LDL) cholesterol was also observed with HMCS. These results suggest that HMCS reduces significant levels of pro-oxidants such as cholesterol and uremic toxins in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation in humans.

ライム病(Lyme disease)は,野ネズミやシカ,野鳥などを保菌動物とし,マダニによって媒介されるスピロヘータの一種,ボレリアBorrelia感染によって引き起こされる人獣共通感染症のひとつである。一方,回帰熱(relapsing fever)は,齧歯類や鳥類等を保菌動物とし,ヒメダニ科のダニやシラミによって媒介されるボレリア感染症で,アメリカ大陸,アフリカ,中東や欧州で患者の発生が報告されている。近年のボレリア研究の進展はめざましく,特にライム病ボレリアでは遺伝子組換え用ベクターが開発されたことから遺伝子欠損株が作成されて,ボレリアライフサイクル中における特異的発現タンパク質の機能解析が可能となってきた。現在ではボレリアの宿主への感染成立のためのボレリアタンパク質―宿主側結合因子や媒介ベクターへのボレリア定着因子など,ボレリアと宿主,ボレリアと媒介ダニのインターフェイスにおける発現タンパク質の同定に注目が集まっている。

We examined the vector competence of the tick, Ornithodoros moubata, using laboratory-reared gerbils as hosts. Transmission of the relapsing fever agent Borrelia duttonii occurred efficiently from infected ticks to uninfected gerbils and from infected gerbils to uninfected ticks. Spirochetes were maintained stably in the ticks for at least 3 months, but they disappeared from the bloodstream of infected gerbils after three episodes of spirochetemia. We also examined transovarial transmission of B. duttonii during the gonotrophic cycle and filial generation. No spirochetes could be detected from the offspring generation of the ticks by culture and polymerase chain reaction (PCR) methods, although spirochetes were still found in the female ticks. The results indicate that, because of the rarity of transovarial infection, the role of transovarial passage of B. duttonii to eggs and larval O. moubata ticks is limited in maintaining B. duttonii. Our findings strongly suggest that B. duttonii is maintained through the O. moubata tick-human transmission cycle in tick-borne relapsing fever endemic areas.