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British Journal of Healthcare and Medical Research - Vol. 10, No. 6

Publication Date: December 25, 2023

DOI:10.14738/bjhmr.106.14541.

Chimbevo, L. M., Munyekenye, G. O., Ngeny, C., Juma, A. M., & Gicharu, G. K. (2023). Antioxidant Activity of Annona Squamosa (L.)

and Annona Muricata (L.) Fruit Pulp Extracts Against Leishmania major Induced Oxidative Stress in BALB/c Mice Model. British

Journal of Healthcare and Medical Research, Vol - 10(6). 235-252.

Services for Science and Education – United Kingdom

Antioxidant Activity of Annona Squamosa (L.) and Annona

Muricata (L.) Fruit Pulp Extracts against Leishmania major

Induced Oxidative Stress in BALB/c Mice Model

Lenny Mwagandi Chimbevo

Department of Biochemistry and Biotechnology,

School of Pure and Applied Science, Pwani University, Kilifi, Kenya

Godwil Otsyula Munyekenye

School of Health Sciences, Kirinyaga University, Kerugoya, Kenya

Chris Ngeny

Department of Biochemistry and Biotechnology,

School of Pure and Applied Science, Pwani University, Kilifi, Kenya

Amana Mzee Juma

Department of Biochemistry and Biotechnology,

School of Pure and Applied Science, Pwani University, Kilifi, Kenya

Gibson Kamau Gicharu

Department of pure and Applied Sciences, School of Applied and

Health Sciences, Technical University of Mombasa (TUM), Mombasa, Kenya

ABSTRACT

Animals remain the best models for the characterization of any disease and its

impact on the host. The BALB/c mice model provides a unique opportunity to study

Cutaneous Leishmaniasis (CL) in human in its active form due to its susceptibility to

L. major infection, developing clinical and pathological features of CL similar to

those found in human. During leishmaniasis progression, there is damage of

internal organs such as the liver and spleen due to high parasite burden or

metabolic processes produced by the host. Reactive oxygen species (ROS) and

reactive nitrogen species (RNS) are generated upon macrophages exposure to

Leishmania leading to the regulation of the inflammatory response controlled by

the cellular antioxidant defense system. The ripe fresh fruits of A. muricata and A.

squamosa were collected. The pulp, peel and seeds from the fruits were dried and

grinded into fine powder. Aqueous and organic extraction of phytochemicals was

undertaken and the extracts used to treat the BALB/c mice. The A. muricata and A.

squamosa fruits aqueous and methanol extracts showed marked antioxidative

activities against CL accompanied with DNA protective effects against H2O2-induced

toxicity both in vitro and in vivo Methanol and aqueous extracts of the fruits were

evaluated for in vivo antioxidant activities using both non-enzymatic and enzymatic

antioxidants and organ protection ability using serum. The findings of this study

showed that these extracts possess antioxidant potential in BALB/c mice. Extracts

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236

British Journal of Healthcare and Medical Research (BJHMR) Vol 10, Issue 6, December- 2023

Services for Science and Education – United Kingdom

strengthened both enzymatic and non-enzymatic antioxidants to prevent the extent

of lipid peroxidation and normalized level of both cardiac and hepatic markers in

serum.

Keywords: Antioxidants, Induced Oxidative stress, Leishmaniasis, Reactive Oxygen

Species

INTRODUCTION

Animals remain the best model for the characterization of any disease and its impact on the

host. Hamster and mouse are the two well-studied and suitable models for studying the

infection and chemotherapy while monkey model is used for vaccine trials (Awasthi et al.,

2004). The BALB/c mice model provides a unique opportunity to study Cutaneous

Leishmaniasis (CL) in human in its active form due to its susceptibility to L. major infection,

developing clinical and pathological features of CL similar to those found in human (Awasthi et

al., 2004). However, different strains of mice show variability in their susceptibility to

Leishmania parasite (Bradley & Kirkley, 1977). BALB/c mice have been shown to be highly

susceptible to L. major, showing signs of slow breeding, are small and delicate to breed (Bradley

& Kirkley, 1977). The Swiss Albino mice on the other hand are easy to breed. Both have the same

appearance and are quite resilient but are known to show resistance to L. major (Santos et al.,

2003; Santos et al., 2008). Although infection in BALB/c mice is a well-studied susceptible host

model, it is not suitable for trial of chemotherapeutic purpose due to much higher effective dose

required to cure Leishmania infection compared to humans (Croft & Yardley, 2002; Awasthi et

al., 2004). However, it can form the basis of selecting higher animal models. The matching

physiology with human and availability, BALB/c mice had been chosen as a model in this study.

During leishmaniasis progression, there is damage of internal organs such as the liver and

spleen due to high parasite burden (Makwali et al., 2012; Jarallah, 2015; Jarallah, 2016) or

increased secondary metabolic processes produced by the host (Oliveira & Cecchini, 2000;

Oliveira et al., 2011b). Damage of DNA and nitric oxide production has been reported in mice

following infection with L. chagas (Oliveira et al., 2011b; Inacio et al., 2014). Besides, adverse

effects of leishmania chemotherapy such as Amphotericin B (Am B) interacting with both

parasite and host cell membrane induce lipid peroxidation (LPO) of the plasma membrane (Roy

et al., 2012; Fernandes et al., 2013; Alkathiri et al., 2017) complicating further the management

of the diease. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated

upon macrophages exposure to Leishmania leading to the regulation of the inflammatory

response controlled by the cellular antioxidant defense system (Inacio et al., 2014; Paiva &

Bozza, 2014; Alkathiri et al., 2017).

Internal and external pathological factors such as viral, bacterial, and parasitic infections

disrupt the body’s oxidant/antioxidant balance initiating oxidative stress mechanisms such as

oxidation of lipids, proteins, and nucleic acids (Eissa et al., 2012; Jafari et al., 2014; Aguiar et al.,

2010). Accumulation of ROS and RNS in cells, damage membrane lipids if not prevented by an

appropriate antioxidant scavenging system (Alkathiri et al., 2017). However, good antioxidant

defense system is closely associated with good Protein Energy (PE) or Protein calories (PC)

nutrition. Although endogenous antioxidants are available to reduce ROS and RNS

accumulation, exogenous antioxidants obtained from appropriate diet can also play a crucial

role (Dkhil et al., 2016). The exogenous and endogenous antioxidant defense systems act in

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Chimbevo, L. M., Munyekenye, G. O., Ngeny, C., Juma, A. M., & Gicharu, G. K. (2023). Antioxidant Activity of Annona Squamosa (L.) and Annona

Muricata (L.) Fruit Pulp Extracts Against Leishmania major Induced Oxidative Stress in BALB/c Mice Model. British Journal of Healthcare and Medical

Research, Vol - 10(6). 235-252.

URL: http://dx.doi.org/10.14738/bjhmr.106.14541.

coordination, with their levels being regulated by each other, to avoid oxidative stress (Dkhil et

al., 2013; Dkhil et al., 2016). Based on these facts, it is important to carry out studies to

investigate the potential antioxidant property of A. Squamosa and A. Muricata fruit pulp against

L. major induced oxidative stress in BALB/c mice model to provide insights on the pathogenesis

of the disease.

MATERIALS AND METHODS

Collection of Plant Materials

Ripe fresh fruits of A. muricata and A. squamosa were collected between March and September

2014 from farms in Kilifi and Kwale Counties at the coastal Kenya. The National Museum of

Kenya, Nairobi, identified the species where voucher numbers for A. muricata and A. squamosa

were deposited. The harvested fruits were washed with chlorinated water to retard aging and

remove fungi and bacteria. The pulp, peel and seeds from the fruits were separated and then

dried at 40oC and 95% relative humidity using a constant temperature and humidity chamber

(Tokyo Thermo Tech Co. Ltd, Japan). The dry pulp, peel and seeds were then grinded separately

into fine powder using a grinding machine (Mitamura Riken, Kogyo Inc. Tokyo, Japan). The

ground fruit parts in form of powder were weighed using a top-loading balance, transferred

into polythene bags, sealed, and stored at 4oC.

Extraction of Phytochemicals

Aqueous extraction was done by macerating 50g of A. muricata and A. squamosa fruits powder

in 100 mL sterile distilled water in a Warring blender for 10 minutes. The macerate was then

filtered through double-layered muslin cloth and centrifuged at 4000g for 30 minutes then the

supernatant was filtered through Whatman No.1 filter paper. The extracts were then preserved

aseptically in sterile airtight bottle at 4oC for later use (Biba et al., 2013). The organic solvent

extraction was carried out using 50g of powdered pulp from A. muricata and A. squamosa fruits

sequentially using 100 mL of solvents of increasing polarity starting with n-hexane followed by

ethyl acetate and finally Methanol (MeOH) for 48 hours each with occasional swirling to ensure

thorough extraction. The extracts were decanted and filtered through Whatman filter paper

and the macerate steeped in solvents (n-hexane, ethyl acetate and MeOH) again for 48 hrs.

Extraction process was repeated twice and the filtrates combined and concentrated on a rotary

vacuum evaporator (Bibby Sterilin Ltd, RE 100B, UK) under reduced pressure at a temperature

of 50°C, packed and stored in an airtight bottle at 4oC for later use (Biba et al., 2013) as

described and abbreviated in Table 1.

Table 1: Abbreviations of different crude extracts of A. muricata and A. squamosa pulp,

peel and seeds

Extract Abbreviation Full name of the extract

ASPUAE Annona squamosa pulp aqueous extract

ASPUME Annona squamosa pulp methanol extract

AMPUAE Annona muricata pulp aqueous extract

AMPUME Annona muricata pulp methanol extract

Animals

BALB/c mice (3 - 4 weeks old) obtained from Animal House Unit, Kenya Medical Research

Institute (KEMRI), Nairobi, Kenya were used. BALB/c mice were maintained with standard rat

pellets (Rate pellets®, Unga Feeds Ltd, Kenya) and water ad libitum. They were then kept under