Determination of Short-Chain Fatty Acids in Mouse Feces by High-Performance Liquid Chromatography Using 2-Nitrophenylhydrazine as a Labeling Reagent.

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.

Daiokanzoto (Da-Huang-Gan-Cao-Tang) is an effective laxative in gut microbiota associated with constipation.

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-ß-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.