Intervention in gut microbiota increases intestinal γ-aminobutyric acid and alleviates anxiety behavior: a possible mechanism via the action on intestinal epithelial cells.

The role of the gut microbiota in the gut-brain axis has attracted attention in recent years. Some gut microbiota produces γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in mammals, in vitro, but the correlation between gut microbiota composition and intestinal GABA concentration, as well as the action of intestinal GABA in vivo, are poorly understood. Herein, we found that the intestinal GABA concentration was increased in mice by the intervention of the gut microbiota with neomycin or Bifidobacterium bifidum TMC3115 (TMC3115). Administration of TMC3115 reduced anxiety without affecting serum levels of serotonin, corticosterone, or GABA. We further found that intestinal epithelial cells expressed GABA receptor subunits and mediated mitogen-activated protein kinase signaling upon GABA stimulation. In addition, administration of TMC3115 induced mitogen-activated protein kinase signaling in colonic epithelial cells but not in small intestinal epithelial cells in mice. These results indicate that GABA produced by the gut microbiota, mainly in the colon, may affect host behavioral characteristics via GABA receptors expressed in intestinal epithelial cells without being transferred to the blood. This study suggests a novel mechanism by which intestinal GABA exerts physiological effects, even in the presence of the blood-brain barrier.

Molecular Identification of UDP-Sugar-Dependent Glycosyltransferase and Acyltransferase Involved in the Phenylethanoid Glycoside Biosynthesis Induced by Methyl Jasmonate in Sesamum indicum L.

Sesame (Sesamum indicum L.) plants contain large amounts of acteoside, a typical phenylethanoid glycoside (PhG) that exhibits various pharmacological activities. Although there is increasing interest in the biosynthesis of PhGs for improved production, the pathway remains to be clarified. In this study, we established sesame-cultured cells and performed transcriptome analysis of methyl jasmonate (MeJA)-treated cultured cells to identify enzyme genes responsible for glucosylation and acylation in acteoside biosynthesis. Among the genes annotated as UDP-sugar-dependent glycosyltransferase (UGT) and acyltransferase (AT), 34 genes and one gene, respectively, were upregulated by MeJA in accordance with acteoside accumulation. Based on a phylogenetic analysis, five UGT genes (SiUGT1-5) and one AT gene (SiAT1) were selected as candidate genes involved in acteoside biosynthesis. Additionally, two AT genes (SiAT2-3) were selected based on sequence identity. Enzyme assays using recombinant SiUGT proteins revealed that SiUGT1, namely, UGT85AF10, had the highest glucosyltransferase activity among the five candidates against hydroxytyrosol to produce hydroxytyrosol 1-O-glucoside. SiUGT1 also exhibited glucosyltransferase activity against tyrosol to produce salidroside (tyrosol 1-O-glucoside). SiUGT2, namely, UGT85AF11, had similar activity against hydroxytyrosol and tyrosol. Enzyme assay using the recombinant SiATs indicated that SiAT1 and SiAT2 had activity transferring the caffeoyl group to hydroxytyrosol 1-O-glucoside and salidroside (tyrosol 1-O-glucoside) but not to decaffeoyl-acteoside. The caffeoyl group was attached mainly at the 4-position of glucose of hydroxytyrosol 1-O-glucoside, followed by attachment at the 6-position and the 3-position of glucose. Based on our results, we propose an acteoside biosynthetic pathway induced by MeJA treatment in sesame.

Differential regulation of fluorescent alkaloid metabolism between idioblast and lacticifer cells during leaf development in Catharanthus roseus seedlings.

Bioactive specialized (secondary) metabolites are indispensable for plant development or adjustment to their surrounding environment. In many plants, these specialized metabolites are accumulated in specifically differentiated cells. Catharanthus roseus is a well-known medicinal plant known for producing many kinds of monoterpenoid indole alkaloids (MIAs). C. roseus has two types of specifically differentiated cells accumulating MIAs, so-called idioblast cells and laticifer cells. In this study, we compared each of the cells as they changed during seedling growth, and found that the fluorescent metabolites accumulated in these cells were differentially regulated. Analysis of fluorescent compounds revealed that the fluorescence observed in these cells was emitted from the compound serpentine. Further, we found that the serpentine content of leaves increased as leaves grew. Our findings suggest that idioblast cells and laticifer cells have different biological roles in MIA biosynthesis and its regulation.

Development of an HPLC method with relative molar sensitivity based on 1H-qNMR to determine acteoside and pedaliin in dried sesame leaf powders and processed foods.

A high-performance liquid chromatography (HPLC) method with relative molar sensitivity (RMS) based on 1H quantitative NMR spectroscopy (1H-qNMR) has been developed for food ingredients such as acteoside (verbascoside) and pedaliin (pedalitin-6-O-glucoside) without requiring authentic and identical standards as the reliable analytical methods. This method is used methyl 4-hydroxybenzoate (MHB) as an alternative reference standard. Each RMS is also calculated from the ratio of each analyte's molar absorption coefficient to that of MHB after correcting the purities of the analytes and reference standard by 1H-qNMR. Therefore, this method can quantify several analytes with metrological traceability to the International System of Units (SI) using the RMS and one alternative reference standard. In this study, the content of acteoside and pedaliin in several samples, such as dried sesame leaf powders and commercially processed foods, can be determined by the proposed RMS method and demonstrated in good agreement that obtained by a conventional method. Moreover, the proposed method yields analytical data with SI-traceability without the need for an authentic and identical analyte standard. Thus, the proposed RMS method is a useful and practical tool for determining acteoside and pedaliin in terms of the accuracy of quantitative values, the routine analysis, and the cost of reagents.

Chemical characterization and biological activity in young sesame leaves (Sesamum indicum L.) and changes in iridoid and polyphenol content at different growth stages.

Three iridoids (lamalbid (I1), sesamoside (I2) and shanzhiside methyl ester (I3)) and seven polyphenols (cistanoside F (P1), chlorogenic acid (P2), pedalitin-6-O-laminaribioside (P3), pedaliin (P4), isoacteoside (P6), pedalitin (P7) and martynoside (P8)) were identified in young sesame leaves (Sesamum indicum L.) other than the acteoside (P5) reported previously. P3 was a new compound, and I1, I3, P2 and P8 were found in a species of Sesamum for the first time. HPLC analyses revealed that the compounds I1 (0.29-1.75% of dry leaves), I2 (0.38-0.87%), I3 (0.04-1.07%), P4 (0.01-2.05%) and P5 (0.13-4.86%) were present primarily in young sesame leaves and were found in plants cultivated on different farms (plant height, 30-70 cm). Of the identified compounds, P5 and P6 showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, oxygen radical absorbance capacity (ORAC), and in vitro antiglycation activities. Given its content, P5 makes a major contribution to the biological activities of young sesame leaves. The compounds were examined at six different growth stages of plants cultured in a greenhouse to determine the optimum harvest stage and for end-use assessment. P5 accumulated in the leaves during growth, and the content reached a maximum of 12.9% of dry leaves in the 4th stage (plant height, 74.5±9.7 cm), which is one of the highest percentages reported in plants from nature.