EXTRACT OF Morinda lucida LEAF RESCUES HYPOGLYCAEMIC AND DYSLIPIDAEMIC CONDITIONS IN Plasmodium berghei-INFECTED MICE

Increasing drug resistance is a great threat to malaria control. Therefore, a continuous investigation into alternative therapy to mitigate malaria-associated damages is important. In this study, we investigated the anti-hypoglycaemic and anti-hyperlipidaemic effects of aqueous extract of Morinda lucida leaf in Plasmodium berghei-infected mice. Twenty-five mice were randomly grouped into five: Uninfected, infected-untreated, chloroquine (20 mg/kg, per oral), and extract-treated (400 mg/kg and 800 mg/kg, respectively, per oral) groups. Fasting blood glucose was measured before parasite inoculation and after the last treatment. Blood was collected for lipid profile assay at the end of the 4-day treatment. Our results revealed that both chloroquine and the extract lowered parasite growth (p <0.05), while chloroquine and 400 mg/kg of the extract improved blood glucose in Plasmodium berghei- infection. More so, all the treated groups showed attenuated Plasmodium berghei- induced dyslipidaemia, with 400 mg/kg of the extract exhibiting better efficacy. Therefore, this study suggests that Morinda lucida leaf extract can be harnessed as a therapeutic regimen for improved malaria treatments and associated complications. Further study is recommended to elucidate the mechanism of anti-hypoglycaemic and anti-hyperlipidaemic activities of the extract and the possible bioactive compound(s) involved.


INTRODUCTION
Despite  a remarkable reduction in global incidence and mortality rate in the previous decade, malaria remains one of the life-threatening diseases, especially in sub-Saharan Africa and Southeast Asia (WHO, 2020). As of 2018, these regions accounted for approximately 85% of global malaria deaths, most of which occur in children under five. Meanwhile, Nigeria with 23%, leads the six most affected countries, followed by the Democratic Republic of the Congo (11%). Others include the United Republic of Tanzania (5%), Burkina Faso, Mozambique, and Niger (4% each) (WHO, 2019). The majority of malariarelated death in sub-Saharan Africa, including Nigeria, is attributed to Plasmodium falciparum (Satish & Ranjana, 2013;Oladeji et al., 2020a) with complex pathological progression as * Corresponding author: abdulkareem.ao@unilorin.edu.ng previously reported (Babamale et al., 2017;Geleta & Ketema, 2016;Plewes et al., 2018).
During adaptation to the intracellular compartment of the host's erythrocyte, Plasmodium spp. require a large amount of glucose. Therefore, they increase their hexose permeability through hexose transporter of the erythrocyte for improved glucose uptake that then results in reduced host blood glucose level -a condition called hypoglycemia, accompanied by suppression of erythropoiesis (Zijlmans et al., 2014;Pathak & Gosh, 2016). This is the hallmark of several pathological conditions such as anaemia, thrombocytopenia, and nutrient deficiency that are associated with severe malaria, as reported (Slavic et al., 2010;Babamale et al., 2017;Plewes et al., 2018).
Lipid derangement is another diagnostic and prognostic indicator of parasitic infection, including malaria (Mohapatra et al. 2014, Kullu et al., 2018. Lipids play crucial roles in Plasmodium spp. metabolism, facilitating their proliferation and transmission (Gulati et al., 2015;Kilian et al., 2018). Their quest for host lipids during the pre-erythrocytic stage of development leads to a lipid homeostatic imbalance and hepatocellular damage (Warjri et al., 2016).
Multiple drug resistance has been a major setback in combating malaria and its complicated outcome until the discovery of Artemisinin-based Combination Therapy (ACT) in the 20 th century. The efficacy of ACT was again short-lived by resistance (WHO, 2015), thus necessitating the continuous search for alternative antimalarial drugs that are not only safe but are also accessible and affordable. In many African countries, the use of medicinal plants and their products has gained tremendous recognition as an alternative treatment strategy for many infectious diseases, including malaria (Idowu et al., 2010;Oladeji et al., 2020b). One of such medically-important plants is Morinda lucida-a tropical rainforest plant (family Rubiaceae) which occurs throughout the year in the South-Western part of Nigeria (Adeneye, 2013;Adeleye et al., 2018;Oladeji et al., 2020a).
Despite the wide usage of crude extracts of the plant for local malaria treatment in Nigeria (Adebayo et al., 2010, Idih et al., 2017Afolabi & Abejide, 2020), information regarding the efficiency of M. lucida on malaria-induced hypoglycaemia and dyslipidaemia is scarce. Therefore, this study investigated the effect of aqueous leaf extract of M. lucida on blood glucose and lipid profile in P. berghei-infected mice. We reported that the extract has the potential to ameliorate pathological alterations in the glucose and lipid profiles of the infected mice.

Plant collection, identification and extraction
The fresh leaves of M. lucida were obtained from Rot-Tund farms Nigeria investment limited, Ila-Orangun, Osun State, Nigeria, and identified at Forest Research Institute of Nigeria, Ibadan, Nigeria. The modified method of Ojewunmi et al. (2013) was used for the extraction. Briefly, the plant was thoroughly washed, air-dried at room temperature, and macerated. Macerated leaf (100 g) was soaked in 1000 mL of distilled water and stirred intermittently for 24 hours. Thereafter, the mixture was filtered with muslin cloth and then with filter paper. The filtrate was evaporated to dryness using a rotary evaporator at 55 0 C. The extract was refrigerated at 4 0 C before use.

Experimental animals
Twenty-five male Swiss albino mice with an average weight of 20g were obtained from the central animal house, Department of Anatomy, University of Ibadan, Ibadan Oyo state. They were subsequently transferred to the animal house, Zoology department, University of Ilorin, for acclimation and experimental set-up under standard conditions including free access to food and clean water. The rats were handled in accordance with the rules and regulations of the Animals Care and Use Committee (ACUC) and the Institutional Ethical Review Board of the University of Ilorin throughout the study.

Parasite inoculation
Plasmodium berghei (ANKA strain)-bearing mice were obtained from the laboratory, Biochemistry Department, University of Ilorin, Ilorin, Nigeria. 1ml of blood was taken from donor mice and diluted with normal saline; such that 0.2 ml of the infected blood contained inoculums of 1 × 10 7 infected red blood cells. Twenty out of the total used parasite-free mice were inoculated intraperitoneally with 0.2 ml of infected blood and parasites were allowed to incubate before treatment commenced.

Experimental design and animal grouping
After parasite incubation, the mice were randomly grouped as follows: Groups 1 (uninfected) and 2 (infected-untreated): received distilled water. Group 3 (chloroquine): infected and treated with 20 mg/kg body weight (b.w) chloroquine. Groups 4 and 5 (extract): infected and treated with 400 mg/kg and 800 mg/kg b.w aqueous extract of M. lucida leaf, respectively. Both the vehicle (distilled water) and the extract were orally administered for 4 days.

Estimation of parasitaemia and percentage parasite inhibition
Blood was obtained from the caudal tip of the animals on the first treatment day (Day 0), smeared on a clean glass slide, and stained with Giemsa for parasite count. The procedure was repeated on Days 2 and 4. The parasitaemia and percentage (%) parasite inhibition were then determined as previously described (Adetutu et al., 2016).

Blood glucose determination
The blood glucose was measured by obtaining a drop of blood from the tail after a 24hr fast using a Glucometer (Elased et al., 1996). Fasting blood glucose was measured before parasite inoculation and after the last treatment.

Lipid profile assay
Estimations of total cholesterol (TC) and triglyceride (TG) were carried out by colorimetric methods, using assay kit obtained from Fortress Diagnostics Ltd. (Antrim, United Kingdom).

Aqueous extract of Morinda lucida inhibits plasmodia growth in P. berghei mice
The efficacy of malaria therapy is a measurement of its ability to reduce the burden of the infection associated with BIOLOGY parasite proliferation. Our data in Table 1 show that P. berghei infection significantly increased (p <0.05) parasitaemia after a 4day curative test. However, treatments with chloroquine (CQ) and aqueous leaf extract of M. lucida lowered (p <0.05) the parasitaemia and significantly inhibited (p <0.05) parasite proliferation (Fig. 1). Percentage parasite inhibition recorded in the CQ-treated group (75.88 %) was higher than the values observed in groups treated with 400 mg/kg extract (52.79 %) and 800 mg/kg extract (51.34 %).  There was progressive parasite growth in the infected-untreated group, whereas, CQ and Ext significantly inhibited parasite growth, on Day 4 post-treatment. Values were expressed as mean ± S.E.M of 3 mice per group (+++ p < 0.001 vs infecteduntreated).

Aqueous extract of Morinda lucida improves fasting blood glucose and lipid profile
Hypoglycaemia and lipid derangements are important prognostic indicators of malaria complications; hence, amelioration of hypoglycaemia and dyslipidaemia offers a better treatment option in malarial infection. Our results reveal that, Plasmodium berghei infection significantly lowered fasting blood glucose level by Day 4 post-treatment (p <0.05) compared with the uninfected group (Fig 2). In contrast, both chloroquine and 400 mg/kg M. lucida aqueous leaf extract improved the fasting blood glucose. On the other hand, total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-c) levels were higher (p <0.05) in the infected-untreated group compared with the uninfected group (Table 2). Treatments with chloroquine and aqueous leaf extract of M. lucida significantly reduced (p <0.05) both TC and LDL-c, with the best effect in 400 mg/kg extract. No significant differences were observed in triglycerides and high-density lipoprotein-cholesterol (HDL-c) levels, though; there was a slight decrease in HLD-c level in the infecteduntreated level when compared with the uninfected group which was relatively improved in all the treated groups.

DISCUSSION
The pathological impact of aqueous leaf extract of M. lucida was investigated in this study. The results showed that 400 mg/kg and 800 mg/kg aqueous extract of M. lucida leaf significantly reduced P. berghei proliferation in mice. We also showed that the extract (400 mg/kg) improved fasting blood glucose and dyslipidaemia in P. berghei infection. Comparatively, our findings emphasized that 400 mg/kg of aqueous leaf extract of M. lucida efficiently ameliorated P. berghei-associated dyslipidaemia better than 800 mg/kg of the extract and chloroquine.
Our findings of a higher curative effect in rats treated with 400 mg/kg extract, compared to those treated with 800 mg/kg, agree with the previous study of Afolabi and Abejide (2020) where the lowest of the used doses of M. lucida plant extract showed a highest curative effect in P. berghei-infected mice. However, 52.79 % parasite inhibition observed in the 400 mg/kg extract-treated group of this study is lower than the 83 % curative effect reported in their study. Similarly, the antiplasmodial activity of M. lucida leaf extract reported in our study is lower than that earlier reported by Idowu et al. (2014). The difference in these findings may be due to the different extracting solvents used, period of extraction, and/or varied sites of leaf collection. Extracting solvent and the geographical location of a plant are key important factors that affect the quantitative yield of a plant's bioactive components, thereby affecting its presumed medicinal activity (Sultana et al., 2009;Gupta et al., 2011).
Hypoglycaemia is a common complication of malaria in pregnant women and children, which increases the risk of mortality in them (Ounjaijean et al., 2019). In this study, P. berghei infection resulted in decreased fasting blood glucose. This concurs with previous findings (Sihabud et al., 2015;Ounjaijeana et al., 2018). Although the underlying mechanism of hypoglycaemia in malaria is not well understood, increasing evidence suggests that suppression of gluconeogenesis by the parasites, and the host's inability to compensate for the parasite glucose uptake play major roles (Mehta et al., 2005;Zijlmans et al., 2014). Thus, the hypoglycaemia observed in this study following P. berghei infection may be attributed to increased glucose uptake by the parasite without compensatory production in the hosts. Administration of 400 mg/kg aqueous extract of M. lucida leaf improved blood glucose level, which compares favourably with chloroquine's effect. The observed improvement in blood glucose in the 400 mg/kg administered group may imply that the lower dose of M. lucida leaf extract enhanced gluconeogenesis in the hosts or it inhibited hexose transporter of the infected erythrocytes, thereby decreasing glucose uptake by the parasite (Ruan et al., 2012;Ahmad et al., 2016). Lipid metabolism is compromised during the exoerythrocytic stage of Plasmodium in parenchyma and Kupffer cells, leading to alterations in lipid profile and subsequently dyslipidaemia (Oluba et al., 2012). The present study shows evidence of dyslipidaemia in P. berghei infection. This is in conformity with the previous studies that demonstrated increased TC and LDL-c in human and rodent malaria (Ngou-Milama et al., 1995;Krishna et al., 2009;Jacob, 2014) and rodents (Joshua et al., 2020). Treatments with both chloroquine and the extract lowered TC and LDL-c in P. berghei-infected mice, suggesting the efficiency of the extract in mitigating malaria-induced dyslipidaemia. However, a better ameliorative effect was observed in the 400 mg/kg extract-treated group. This may imply that aqueous extract of M. lucida leaf at 400 mg/kg possesses a better therapeutic potential to suppress dyslipidaemia in malaria than chloroquine.
Aqueous leaf extract of M. lucida has been previously shown to possess flavonoids, among other bioactive components (Unekwuojo et al., 2011). Flavonoids are known for their antioxidant and anti-inflammatory properties. They can also inhibit low-density lipoprotein (LDL) oxidation and regulate serum lipid metabolism (Kerry & Abbey, 1997;Chen et al., 2011). Therefore, the anti-hyperlipidaemic effect of M. lucida leaf extract observed in the present study may be due to the bioactive components of the extract, especially flavonoids, which then aid in bringing lipid metabolism back to normal.

CONCLUSION
This study justifies the common practice of using M. lucida crude extracts for malaria treatment in the South-Western part of Nigeria. It also demonstrates that the aqueous extract of M. lucida leaf improved blood glucose and ameliorated lipid profile derangement in P. berghei-infected mice. We, therefore, suggest that M. lucida leaf extract can be harnessed as a therapeutic regimen for better treatment of malaria and associated complications. Further study is recommended to investigate the mechanism of antihypoglycaemic and anti-hyperlipidaemic activities of the extract and the possible bioactive compound(s) involved.