In Silico and In Vivo Potential of Fraction Red Betel Leaf as an Immunostimulant Agent in White-leg Shrimp

Abstract

Production of white-leg shrimp (Litopenaeus vannamei) in aquaculture with advanced technology ultimately results in high mortality rates in cultivation. Infectious diseases, including Vibrio spp., can cause mortality with necrosis symptoms in the white-leg shrimp hepatopancreas. Disease prevention through enhancing immunity is highly effective in controlling diseases in shrimps. The current study aimed to obtain information on the compounds present in the fraction of Piper (P.) crocatum as an immunostimulant agent used in silico. The current study investigated the absorption, distribution, metabolism, excretion, and toxicity (ADME/T), and determined the optimal fraction dosage of P. crocatum when injected as an immunostimulant substance. In this study, in silico analysis was conducted by accessing several servers, while the shrimp’s immune response was evaluated using a completely randomized design experiment with four treatments (10 individuals/container) and three replications, including 0 (control), 0.5 µg/g, 1 µg/g, and 1.5 µg/g. The shrimp’s immunity was examined by injecting the P. crocatum fraction initially, followed by a second injection 24 hours later. Shrimp hemolymph was collected before the injection of the P. crocatum fraction and 24 hours after the injection. Hemolymph was collected at both time intervals to assess total hemocyte count (THC), differential hemocyte count (DHC), and phenoloxidase (PO) as the immune response of shrimp before and after administration of P. crocatum fraction. Two compounds were confirmed immunostimulant agents in a fraction of P. crocatum, 2-Amino-1,3,4-octadecanetriol, and erucamide. The immune response values for THC (14.17 ± 2.45 × 106 cells mL-1), DHC hyaline (53 ± 4.5%), semi-granular cells (52 ± 4.0%), and granular cells (43 ± 40%), and PO (0.112 ± 0.016 units/λ=490) at a concentration of 1.5 µg/g showed a significant increase in number and percentage compared to the control. These results indicate the presence of two compounds in fraction one P. crocatum, as candidates for immunostimulant agents. Administration of 1.5 µg/g of a fraction of P. crocatum is the appropriate dose as an immunostimulant agent when administered via injection method for white-leg shrimp.