Efficacy and Safety, Nutritional andAntioxidant Activityof Kenyan Annona muricata (L.) and Annona squamosa (L.) Fruits Extracts in BALB/c Mice Model of L. major Leshmaniasis

Abstract

Leishmaniasis is classified by the WHO as an emergent category of one of the uncontrolled and NTDsco-exist with malnutrition and HIV/AIDS. Due to lack of vaccine, control relies on ineffective, toxic and expensive chemotherapy and vector control, which are not sustainable. The study was aimed at determination of efficacy and safety, nutritional and antioxidant activity of Kenyan A. muricata (L.) and A. squamosa (L.) fruits extracts in BALB/c mice model of L. majorleishmaniasis to develope supportive therapy and efficacious and safe antilrishmanicidal agents. Ripe fresh fruitswere collected from farms in Kilifi and Kwale Counties, Coast regionof Kenya. Dried fruits were powdered, subjected to extraction with solvents of increasing polarity (Hexane, Ethyl Acetate, Methanol and Water) for 48 hours. Proximate and nutritional composition of various partsof the plants, phytochemicals screening, carotenoidsanalysis, in vitroand in vivo cytotoxicity, antioxidant, DNAbinding and DNA Topo I inhibitory activities of the extracts wereassesed. The efficacies of thedifferent extracts against L. major parasites (IDUB/KE/83=NLB-144 strain) wasassessed in vitro in inbred infected BALB/c mice maintained on AFPP and RP and in vitro using promastigotes. The results were expressed as mean analyzed using mean separation done through Fischer least significance difference by GenStat program. Comparisons between two treatments were done by Student’s t-test and significance established by ANOVA. Differences of P<0.05 were considered statistically significant.Majority of the phytochemicalswere detected inthe extracts.The fruitdemonstrated high dry matter, moisture content, crude fat, crude protein, crude fibre, total carbohydrates, oil content, reducing sugar, TSS, ascorbic acid, tocopherol, titratable acidity and ash content. Fatty acid profile showed presence of SFA, MUFAand PUFA. Different parts revealed varying amounts of P, Na, Ca, Mg and P whereas Cu, Fe, Zn and Se were detected in trace amounts. The HPLC profilingof carotinoids revealed presence of neoxanthin, violaxanthin and zeaxanthin, α-carotene, β-carotene, γ-caroteneand chlorophyll. The extracts showed in vitro and in vivoantioxidant activitiesagainst non-enzymatic and enzymatic antioxidants. The BALB/c mice fed with AFPP showed improve growth, metabolic efficiency and feed utilization.The extracts showed a dose dependent in vitro antileishmanial activity with MIC values ranged between 12.50±1.03 μg/mL to 55.0±2.97 μg/mL. The extracts showed antileishmanial activity in vivo through reduction ofLHFD swelling and lesion sizes. The extracts ranged from highly toxic (IC50 2.54±0.44-7.55±1.19µg/mL), toxic (IC50 12.85±2.80-94.07±5.81µg/mL) and moderately toxic (IC50 104.81±1.16-292.94±10.10µg/mL). No mortality was observed even at highest dose of 2500 mg/Kg suggesting LD50>2500mg/Kg. The extractsshowed DNA binding interaction via displacement of methyl green in DNA methyl green test and DNA Topo I inhibitory activity by inhibiting relaxation of supercoiled DNA pBR322 at 5, 25 and 100 µM. It can be concluded that the nutritional components of the fruits were responsible for the improve growth, metabolic efficiency and feed utilization of BALB/c mice fed with AFFP. Thephytochemicals detected were responsible for the in vitro and in vivo antioxidant and antileishmanial activities besides DNA binding interaction and DNA Topo I invibitory activity. Low IC50 and lack of mortality in extracts treated mice suggests safety. The study recommends further investigation using purified fractionsof these extracts on higher animal model of the lishmaniasis.