Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Evaluation of the acute toxicity of Thymus linearis ethanol extract and its effect on the hemato-biochemical and behavioural response of the Golden mahseer, Tor putitora (Hamilton, 1923).

The present investigation was conducted to estimate the acute toxicity of Thymus linearis plant extract, its effect on hemato-biochemical parameters and behavioural response in the golden mahseer (Tor putitora). The phytochemical composition present in T. linearis plant extrat were Alkaloids, Flavonoids, Phenols and Tannin. The fishes were subjected to eight different concentrations of T. linearis leaves extract (8.25, 8.50, 8.75, 9.00, 9.25, 9.50 and 9.75 mg/kg) and the control group without plant extract for 96-h LD50 study. The mortality was recorded every 24 h post-treatment. Minimum mortality was recorded in the 8.25 mg/kg, whereas 100% mortality was recorded in the 9.75 mg/kg T. linearis extract after 96-h periods. The LD50 was estimated by probit analysis, and the value of T. linearis at 96 h was found to be 8.71 mg/kg for golden mahseer. A non-lethal dose of 1/10th of 96-h LD50 value (0.87 mg/kg) was taken for the sublethal study. After 96 h, the red blood cell (RBC), white blood cell (WBC), haemoglobin (Hb), packed cell volume (PCV) and blood glucose were measured. RBC (×106/mm3), Hb (%) and PCV (%) significantly decreased at 8.25, 8.50, 8.75, 9.00 mg/kg, but WBC and blood glucose significantly increase at 8.25, 8.50, 8.75, 9.00 mg/kg of T. linearis plant extract. The observations on behaviour response of golden mahseer were also recorded. In the present study, the acute toxicity of wild ajwain was more significant than short-term toxicity. The mortality rate was very high during the study period of T. linearis exposure.