MOLECULAR INVESTIGATION ON THE TOXICOLOGICAL EFFECTS OF Anonna muricata LEAF EXTRACT IN MALE AND FEMALE WISTAR RATS

Abstract

Natural products, especially phytochemicals, have been used to help mankind sustain its health since the dawn of medicine. Anonna muricata, a lowland tropical, fruit-bearing tree of the family Annonaceae have been used in traditional medicine for diverse biological activities including anti-hyperglycemia activities, anti-inflammatory activities, and protective effect against liver problem, arthritis, hypertension and cancer treatments due to its richness in phytochemicals. However, adverse effects associated with herbal medicines makes it pertinent that pre-clinical toxicological studies be carried out on these natural products. This study seeks to carry out molecular investigations on the toxicological effects of methanol extract of A. muricata leaf in Wistar rats. A. muricata leaves were air-dried at room temperature and pulverized into fine particles. The powdered leaves were soaked in 80% methanol for 72 hours, filtered, lyophilized and kept at 4°C for further analysis. Animals were divided into four groups of 12 animals (6 male and 6 female) per group. Group 1 served as control and received the vehicle (distilled water) only, Group 2 received 50 mg/kg of the extract, Group 3 received 100 mg/kg of the extract daily and Group 4 received 200 mg/kg of the extract daily. All administrations were done orally. The treatments were carried out for 90 days after which the animals in each group were examined for signs of weakness and death and then sacrificed. Blood and organs such as the Liver, heart, kidney, spleen, pancreas, testes/ovary and brain were collected, processed and used for haematological, histological, biochemical and gene expression analyses. Haematological evaluations carried out include total white blood cell (WBC) count, packed cell volume (PCV), haemoglobin concentration (HB) and red blood cell (RBC) count while the biochemical assays are reduced glutathione (GSH), superoxide dismutase (SOD), xanthine oxidase (XO), nitric oxide (NO) and lactate

dehydrogenase (LDH) levels/activities were accessed. The histological examination of kidney, liver, heart and spleen was done using H&E staining. Finally, gene expression analysis by RT-PCR was also carried out on interleukin1 (IL-1β), tumor necrotic factor (TNF-α), cytochrome P450 (CYP3A4 and CYP2D6), glucose kinase, pyruvate kinase and endothelial nitric oxide synthase (eNOS). The results revealed that chronic administration of A. muricata methanol leaf extract is safe for consumption with records of improved haematological parameters as evident by significant (**p<0.01, ***p<0.001, ****p<0.0001) increase in PCV, eosinophil, monocytes levels and platelet count amongst others while also revealing increasing antioxidant potentials which is evident by increased GSH content, increased activities of SOD, and reductions in the level of NO and activities of XO and LDH. Molecular studies revealed that the extract favourably modulates the expression of glucose/drug metabolising and inflammatory genes glucose kinase, pyruvate kinase, cytochrome P450 (CYP3A4 and CYP2D6), interleukin1β (IL-1β), tumor necrotic factor (TNF-α), endothelial nitric oxide synthase (eNOS) respectively in both sexes of the animals. Given its rich prowess in combating oxidative stress and preventing inflammation with no recorded toxicity, it can thus be considered a safe alternative for the management of certain diseases characterized by oxidative stress and inflammation.