RESUMEN
Yerba-mate (Ilex paraguariensis) is recognized for its biocompounds and bioactive properties. This study aimed to assess the potential of yerba-mate extract to modulate the intestinal microbiota in rats. After the ethical committee approval (CEUA - UPF, number 025/2018), the Wistar rats were given a daily dose of 3.29 mg of phenolic compounds per animal for 45 days. The antioxidant activity of the extract was assessed by ABTS and FRAP assays and the total phenolic compounds was measured at different pH levels. Identification and quantification of chlorogenic acid isomers were carried out using high-performance liquid chromatography (HPLC). Intestinal microbiota modulation was evaluated by administering the yerba-mate extract or water (control) to Wistar rats via intragastric gavage and its efficiency was measured through PCR. The antioxidant capacity of the yerba-mate extract was 64.53 ± 0.26 µmol Trolox/mL (ABTS) and 52.96 ± 0.86 µmol Trolox/mL (FRAP). The total phenolic compounds showed higher levels at pH 7.5 compared to pH 2.0. Chlorogenic acid isomers were found in greater abundance, with a concentration of 14.22 g/100 g. The administration of the extract resulted in positive modulation of the intestinal microbiota, specifically for the genera Lactobacillus sp. and Prevotella sp. The increase of these genera is related to the promotion of homeostasis of the gut microbiota. Therefore, these findings indicate that yerba-mate extract possesses significant antioxidant activity and can effectively modulate the intestinal microbiota in rats. These results support the potential use of yerba-mate as an alternative for controlling and preventing diseases associated with intestinal dysbiosis.
Asunto(s)
Microbioma Gastrointestinal , Ilex paraguariensis , Ratas , Animales , Ilex paraguariensis/química , Ratas Wistar , Antioxidantes/farmacología , Extractos Vegetales/farmacología , Extractos Vegetales/química , Ácido Clorogénico/farmacologíaRESUMEN
BACKGROUND: The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis. METHODS: Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200 ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). RESULTS: The expected cDNA fragment of approximately 1240 bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot. CONCLUSIONS: The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.