In vivo toxicity study in Sprague–Dawley rats receiving different doses of Moringa oleifera extract

Background: Protein deficiency poses a significant challenge during the growth and development of children. Moringa oleifera Lam (MO) leaves, renowned for their high protein content, present a potential solution to address amino acid imbalances in protein-deficient conditions. Aim: This study aimed to evaluate the toxicity of MO leaf extract in Sprague-Dawley Rats. Methods: Protein extraction from Moringa oleifera (MO) leaves was performed using ultrasonic-assisted extraction (UAE) with ethanol. The ethanol leaf extract of MO (EEMO) was then characterized for protein content, amino acids, minerals, phytic acid, and phytochemicals. Antioxidant activity was assessed using the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) test, while cytotoxicity was evaluated using the MTT (3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide) assay on HepG2 and Madin-Darby canine kidney (MDCK) cells. EEMO was tested for acute toxicity in 60 healthy male and female Sprague–Dawley rats. The rats were divided into five groups of five rats each, receiving single oral doses of 5, 50, 300, 2,000, and 5,000 mg/kg body weight (BW) of EEMO. Observations were conducted daily till day 14 before the necropsy of rats. Liver and kidney tissues were harvested and preserved in 10% formalin for histopathological analysis. Results: The extraction process revealed a protein content of 45.5%, with phenylalanine being the predominant essential amino acid at 22.25 mg/g, and glutamic acid as the dominant non-essential amino acid at 60.03 mg/g. Potassium (1174.23 mg/100 g) and selenium (149 mg/100 g) were identified as the primary macro and micro minerals, respectively. The IC₅₀ and CC₅₀ values for antioxidant and cytotoxic activities in HepG2 and MDCK were found to be 41.04,182.66, and 121.04 ppm, respectively. Toxicity testing on experimental animals resulted in an LD₅₀ value of 5,000 mg/kg BW for EEMO, indicating its relative safety upon oral administration. Conclusion: MO extract produced by the UAE extraction method, containing high-quality food-grade protein, showed no cytotoxic effects on HepG2 and MDCK cells and exhibited no acute toxicity in rats.