Development of nanostructured lipid carrier clove oil as anesthetic agent in white shrimp (Litopenaeus vannamei)

Abstract

Clove oil is an essential oil extracted from the dried buds and leaves of the clove plant (Syzygium aromaticum). Clove oil contains eugenol as the main active ingredient. Clove oil used as an anesthesia functions as calming effect, loss of equilibrium, non-reactivity and consciousness in lead to reduce activity, physiological stress, mortality rate, and preventing cannibalism method during handling and transportation. However, the component of clove oil is insoluble in water and must be dissolved in ethanol. So, clove oil ethanolic solution leading to toxic and irritation in animal and user. In this study, to avoid this side effect, the anesthetic preparation of clove oil without ethanol is an attempt to be developed. We have prepared a new generation of clove oil in the form of NLCs to enhance water miscibility, high efficacy, better physical stability, and low toxicity compared with STD clove oil. The clove oil NLCs obtained were characterized in terms of physicochemical, efficacy, toxicity and biodistribution of clove oil NLCs. The results showed that clove oil NLCs had particle size of 175.07 ± 0.72 nm, zeta potential of -48.37 ± .38, PDI of 0.115 ± 0.230. Clove oil NLCs have spherical particle shape, %encapsulation efficiency of clove oil NPs was 88.55% and demonstrated sustained release of active ingredients. The anesthetic effects of clove oil were studied in white shrimp (average 3 g). Clove oil concentration at 10, 20, 30, 40, 50, 60, 70, 100, 120, 140, 160, 180 and 200 ppm of clove oil NLCs and STD clove oil were used in study. The results showed no effect of clove oil to white shrimp at 10 ppm of both groups. After exposure to clove oil NLCs at 30 ppm, showed the shrimp can be started to stage 2 (Complete loss of equilibrium) while shrimp of STD clove oil group be started to stage 2 within 5 minutes at concentrations of 50 ppm. The optimum concentration of anesthetic was 30 ppm in shrimp which induced shrimp to stage 2 within 5 minutes after exposure to Clove oil NLCs. The toxicity of clove oil to different concentration of clove oil was studied and median lethal concentration (LC50) at 48 h were report in toxicity study. The result show that the STD clove oil concentration of 140 ppm resulted in median lethal concentration (LC50 48h), whereas the concentration of 140 ppm of clove NLCs led to a 0% mortality over a 48-h period. And the result of biodistribution study of clove, it was found that shrimp in clove oil NLCs group was able to excrete clove oil from the body within 30 minutes but shrimp in STD clove oil group was still detected clove oil in shrimp. In conclusion, the clove oil NLCs has been successfully developed with enhanced water miscibility, increased high anesthetic efficacy with a sustained release, reduction of clove oil toxicity and clove oil NLCs do not accumulate on the shrimp body.