In vitro and in vivo screening of herbal extracts against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Abstract

This study investigated 354 herbal extracts prepared from 115 herbs via 4 extraction methods. The extracts' antibacterial activities against Streptococcus agalactiae were sequentially evaluated in vitro by two methods: (1) microplate assay and (2) agar well diffusion. Then the herbal extracts with high antibacterial activity in vitro were mixed into feed at 100 mg/g (dry herb/dry feed, w: w) and fed to fishes with S. agalactiae infection for in vivo evaluation of their therapeutic efficacy. Subsequently, the EC50 values (the concentrations result in the relative percent survival (RPS) of 50%) of the three herbal extracts with the highest therapeutic efficacies were calculated according to fit equations. Finally, phytochemical analyses of the herbs with the highest therapeutic efficacies were conducted using ultra performance liquid chromatography-Q Exactive Orbitrap-Mass spectrometry (UPLC-QE-MS). The results showed the following: in detections from the microplate assay method, 62 of the 354 candidate herbal extracts had antibacterial activity with inhibition ratios above 5%; in detections from the agar well diffusion method, 29 of the 62 the candidate herbal extracts showed clear inhibition zones and 33 showed semi-clear inhibition zones. Aristolochia debilis (fruit), Lonicera japonica (flower), Spatholobus suberectus (stem), and Arecae Semen (fruit) extracts prepared by 3 h of maceration at 50 °C had RPSs of 100.0%, 55.6%, 75.0%, and 33.3%, respectively; Panax ginseng (leaf), Rheum palmatum L. (root), and Semen Alpiniae (seed) extracts prepared by 72 h of maceration at room temperature had RPSs of 77.8%, 25.0%, and 12.5%, respectively; and Radix Isatidis (root) extract prepared by 30 min of ultrasonic-extraction at room temperature had an RPS of 62.5%. A. debilis, P. ginseng, and S. suberectus extracts had EC50s of 44.03, 41.19, 39.54 mg/g, respectively. Phytochemical analyses of A. debilis, P. ginseng, and S. suberectus extracts using UPLC-QE-MS produced the following results: the negative model detected 270, 623, and 326 peaks and identified 54, 70, and 52 compounds, respectively; the positive model detected 446, 1401, and 400 peaks and identified 61, 107, and 44 compounds, respectively. Taken together, these results indicate that the extracts of A. debilis, P. ginseng, and S. suberectus could prove useful for the treatment of S. agalactiae infection in Nile tilapia and suggest that appropriate bioactive compounds from these three herbal extracts may be developed as alternates to antibiotics.