Antiplasmodial activities of Morinda lucida (Benth) and Alstonia boonei (De wild) in mice infected with Plasmodium berghei

This study focused on the antiplasmodial activities of two botanicals: Morinda lucida and Alstonia boonei used in malaria treatment. The in vivo activity of the plants against established Plasmodium berghei NK65 infection was evaluated in 75 experimental mice randomly distributed into 15 groups and treated with extracts of M. lucida and A. boonei, combined recipe of the two plants at graded doses of 400, 600, and 800 mg/kg and chloroquine at 10 mg/kg. The results revealed that the percentage parasitemia was higher in the infected untreated mice (18.40%) than the treated mice. Among the treated mice, the highest percentage parasitemia (6.0%) was obtained in mice treated with 800 mg/kg of A. boonei while the lowest percentage parasitemia (0.0%) was obtained in mice treated with 10 mg/kg of chloroquine. Chloroquine eliminated all the parasites (100% clearance) when compared to the percentage clearance by the plant extracts. For the plant extracts, antiplasmodial activity was highest with the combined recipe of M. lucida and A. boonei at 800 mg/kg (92%) and lowest at 800 mg/kg (45%) of M. lucida. Meanwhile, the optimal antiplasmodial activity (83%) of M. lucida was obtained at 400 mg/kg and that of A. boonei was obtained at 600 mg/kg (85%). This study revealed that the two plant extracts under the study have antiplasmodial activities which are promising in reducing the morbidity and mortality of drug-resistant malaria in endemic communities.


Introduction
Malaria is an infectious disease with significant health challenges in Nigeria and other countries where the disease is endemic. Over the last 10 years, malaria prevalence has been on the increase, especially in third world countries. It was reported that over three billion people live in risk areas of the disease in 109 countries and territories around the world (Umar et al. 2012). The disease is predominant in subtropical and tropical regions such as the Americas, Asia, and Africa (Rowe 2006). Each year, there are approximately three hundred million people who suffered from the disease, the majority of whom are children below 5 years domiciled in sub-Saharan Africa (Snow et al. 2005). The disease is a major killer disease responsible for the death of millions of children, pregnant women, and adults (Satish and Ranjana 2013). About 80% of malaria mortality is concentrated in 13 countries in Africa, and over half of the percentage mortality was recorded in Nigeria, Congo, Ethiopia, Tanzania, and Kenya (World Health Organization 2008a;World Health Organization 2014). The disease transmission occurs all year round in the southern part of the country while in the north it is more seasonal (Adebayo and Krettli 2011).
Malaria is treated with chloroquine, quinine, derivatives of artemisinin, and other herbal antimalarials by suppressing the virulence of the parasites or kills them (Martey 2013). The well-known use of chloroquine (CQ) and antifolates (sulfadoxine-pyrimethamine) for malaria treatment is no longer effective in most endemic areas including Nigeria. The synthetic drugs are expensive with a lot of side effects, and parasites have also developed resistance to them as a result of a long-term usage abuse (Martey et al. 2013).
To overcome all these limitations on the drugs, combination therapy has currently be introduced and has shown to be the best practical solution in overcoming the resistant strains of the parasites (World Health Organization 2008b). However, clinical resistance to these drug combinations has been reported in Cambodia (Noedl et al. 2008). P. falciparum especially has been noted to develop resistance to the majority of the drugs currently used. Hence, there is an urgent need to develop alternative therapy and cheaper drugs such as herbal therapy for malaria treatment. In malariaendemic countries of the world, natural and traditional products (plant and insect products) had been employed as arsenals to combat malaria (Gupta et al. 1993). This has necessitated many countries to change their treatment policies (Wellems 2002).
Morinda lucida, "Oruwo" (local name in Yoruba tribe), has been used as a traditional remedy for the treatment of symptomatic malaria by many people in South-West part of Nigeria. Different parts of M. lucida have been reported to possess medicinal properties. The leaf extract of the plant was reported to possess trypanocidal activities (Asuzu and Chineme 1990), antimalarial activities , and aortic vasorelaxant effect (Ettarh and Emeka 2004).
M. lucida leaf extract has also been reported to have a strong oral hypoglycaemic property, and this property was attributed to increased peripheral utilization of glucose (Adeneye and Agbaje 2008). The plant contains tannins, methylanthraquinones, and heterosides. Studies have shown that M. lucida leave extract appears to have schizontocidal and repository effects in mice infected with Plasmodium berghei (Akanmori et al. 1994).
Alstonia boonei ('Ahun' is the local name in Yoruba tribe) is among the medicinal plants that have been widely used in recipes to treat malaria (Idowu et al. 2010). It belongs to the family Apocynaceae which consists of about 50 species widely distributed in the continents of Africa, Asia, and America. The stem bark of this plant has been found to be effective in the treatment of several diseases such as fever, painful micturition, insomnia, chronic diarrhea, and rheumatic pains (Olajide et al. 2000;Odeku et al. 2008). Olajide et al. (2000) reported that the stem bark of Alstonia boonei has antiinflammatory, antipyretic, and analgesic properties.
History has revealed that plants that are eco-friendly in nature have been considered as an important source of medicine against malaria. Botanicals and products derived from them have been used for disease treatment for centuries. Presently, it has been evaluated that 80% of the world population depends on herbal preparations to meet their health needs (Shri 2003).
The array of botanicals used in the treatment or prevention of malaria in Akure Southwest Nigeria is presented in Table 1. These medicinal plants are used in most cases, in combination with other two or more plants (such as mango, guava, or cashew leaves). The two most commonly prescribed medicinal plants (M. lucida and A. boonei) by the herbalists in the study area were used for this research. The advantages of these botanicals over the synthetic drugs have necessitated their inclusion in malaria parasites and vector control campaigns. However, additional research is required to maximize the full benefits of botanicals and respond to the health needs of people, especially in endemic countries like Nigeria (Idowu et al. 2010). Hence, this study is undertaken to examine the antimalarial activities of M. lucida and A. boonei plant extracts in experimentally infected mice as an animal model.

Survey of botanicals used in malaria treatment in Akure Southwest Nigeria
The plant survey was carried out in Akure by visiting Odopetu herbal market, in Akure south L.G.A of Ondo State to find out the local names, part used, and methods of preparation of these plants.

Plant collection, identification, and extraction
Fresh leaves and barks of M. lucida and A. boonei were obtained, free of insecticides from Akure, Ondo State, Nigeria. The plants were authenticated by Dr. (Mrs.) B.O. Odiyi, a plant taxonomist. The leaves and barks were cleaned, air-dried at 28 ± 20°C, and crushed into a coarse powder using pestle and mortar. Five hundred grams in powder form of the stem and bark of each plant sample was soaked in 3 L of 96% ethanol for 72 h. Also, a 500-g combination of both plant samples (comprising 250 g of the individual plant powdered samples) was dissolved in 3 L of 96% ethanol for 72 h. The preparations were stirred every 2 h, thereafter decanted and filtered using a cheesecloth. The liquid filtrate obtained was filtered and concentrated using a rotary evaporator. The crude ethanolic extract was further concentrated in a vacuum oven to further remove any traces of solvent. The extract was refrigerated at 4°C prior to use (Afolabi and Abejide 2020).
Experimental design and evaluation of in vivo activities of the plant extracts against P. berghei Seventy-five mice weighing between 18 and 20 g were purchased from the Institute of Advanced Medical Research and Training (IAMRAT), College of Medicine, University of Ibadan. The animals were acclimatized for 2 weeks at 28 ± 2°C with 12-h dark/light periodicity and fed with commercial chow (purchased from Cap Feeds Ibadan, Nigeria) and water ad libitum; 2.4 inoculation of mice with P. berghei. The P. berghei (NK65) used for the in vivo study was obtained from IAMRAT.
A Swiss albino mouse (donor mouse) was intraperitoneally administered with a standard inoculum of P. berghei on the 1st day. On the 5th day (when the parasite had stabilized in the donor mouse) with a rising parasitemia of 20-30%, the mouse was sacrificed and 0.1 ml of acid citrate dextrose (ACD) was drawn into the syringe before blood was withdrawn from the heart of the donor mouse by cardiac puncture. The blood was diluted with isotonic saline to make inoculum for infecting experimental mice. The experimental mice were inoculated with 0.2 ml of the diluted parasitized red blood cell (RBC) specimen (containing about 1 × 10 7 parasitized cells). The curative activity (Rane's test) of the extracts was determined in vivo according to Iyiola et al. (2011). The 75 experimental mice were divided randomly into fifteen groups of five mice each (Table 2). Subsequently, P. berghei was injected intraperitoneally into fifty-five mice on day 1. Seventy-two hours later, group 1-9 mice were administered through oral gavage with the extracts as described in Table 2, and groups 10 and 11 were administered with 0.2 ml of 10 mg/kg chloroquine and 5 ml/kg normal saline, respectively, according to the experimental design below once a day for five consecutive days. Parasitemia levels were monitored for each mouse until 7 days.

Determination of parasitemia
The blood used for thin smear preparation was obtained from the mice via a tail cut. The smears were fixed with methanol for 5 min and stained with 10% Giemsa. The slides were observed under ×100 objective lens of the compound microscope.
Each slide was viewed at four different fields, and the infected and total number of RBCs for each field were recorded. The data obtained were used to calculate percentage parasitemia using the method described by Hilou et al. (2006). Percentage parasitemia and percentage curative were calculated using the formulae below.  Were infected with Plasmodium berghei and treated with combined recipe of Morinda lucida and Alstonia boonei extract at 400, 600, and 800 mg/kg body weight, respectively, for 5 consecutive days Combined treatment

Groups 10
Were infected with Plasmodium berghei and treated with 10 mg/kg of chloroquine phosphate solution for 5 consecutive days Drug treatment control

Group 11
Were infected with Plasmodium berghei and only given 5 ml/kg of distilled water Positive control

Groups 12
Were administered with 800 mg/kg body weight of Morinda lucida plant extract for 5 consecutive days.

Groups 13
Were administered with 800 mg/kg body weight of Alstonia boonei plant extract for 5 consecutive days.

Group 14
Were administered with 800 mg/kg body weight of combined Morinda lucida and Alstonia boonei extract for 5 consecutive days.

Group 15
Were neither infected nor treated Negative control

Data analysis
The means of the data were computed and statistical significance was tested using one-way ANOVA. The significant means were separated using Duncan's new multiple range test (DNMRT) at P < 0.05

Antiplasmodial activity of the plant extracts and chloroquine
The antiplasmodial activity of the plant extracts at graded doses of 400, 600, and 800 mg/kg body weight and the negative control group as represented in Table 3 showed that the percentage parasitemia on days 1 and 2 increased in mice treated with M. lucida at 400, 600, and 800 mg/kg. Subsequently, reduction in percentage parasitemia was noted at all doses from days 3-5. However, the least percentage parasitemia (0.40) was noted in mice treated with 400 mg/kg of M. lucida for 5 days.
In contrast, the reduction in percentage parasitemia was observed in mice treated with A. boonei at all doses of the extracts for 5 consecutive days. However, the comparative study of the individual plants with the combined recipes revealed that the antimalarial activity of the plants further increased as the two plants were combined at different doses. For instance, the percentage parasitemia range of the combined recipe of M. lucida and A. boonei extracts was noted to be 0.40-0.80 while 0.40-1.40 and 0.40-1.60 were observed for M. lucida and A. boonei, respectively. This shows that the antimalarial activity of the plants increased when both plants were combined at graded doses of 400, 600, and 800 mg/kg. Generally, it was also noted that none of the extracts and their combined recipes could completely reduce the parasitemia to zero. Meanwhile, a complete elimination of the parasites was only noted in mice treated with chloroquine (a standard drug) at 10 mg/kg at 3 days of treatment (Table 3).
The results of the percentage curative as represented in Table 4 shows that chloroquine reduced the parasitemia to zero and recorded 100% percentage curative at 10 mg/kg body weight. The percentage curatives of the plant extracts and their combination vary significantly. It was also noted that the percentage curative of the combined recipe increased from 75 to 92% as the dosage increases from 400 to 800 mg/kg. The percentage curative of A. boonei increased from 68 to 85% as the dosage increases from 400 to 600 mg/kg and subsequently decline to 75% as the dosage increased from 600 to 800 mg/kg (Table 4). In contrast, the percentage curative of M. lucida decreased as the dosage increases from 400 to 800 mg/kg. However, the optimum antiplasmodial activity of M. lucida (83%) was obtained at 400 mg/kg while that of the A. boonei (85%) was noted at 600 mg/kg. However, the antiplasmodial activities of the combined recipes of M. lucida and A. boonei performed better at each concentration than when the plant was used individually. This shows that the efficacy of the drug synergistically increased when the plants were combined.

Discussion
Malaria is a fatal parasitic infection especially in the tropical African and Asian continents, and it is believed that it kills more than AIDS and the most vulnerable are children below 5 years (WHO 2014). Herbal remedy, which in most cases, is a combination of two or more plants is usually taken ad libitum for malaria treatment or prevention in Africa (Oludele and Olufunso 2018). The curative antiplasmodial tests indicated that the mice treated with M. lucida, A. boonei, and their combined recipes at graded doses of 400, 600, and 800 mg/kg body weight resulted in reduced parasite load as compared to the untreated mice. The comparative curative antiplasmodial activity of M. lucida and A boonei, the combined recipe and chloroquine at 10 mg/kg body weight from this study shows that chloroquine, the standard drug, cleared the parasites by 100% compared to the percentage of clearance by plant extracts which varies at graded doses of 400, 600, and 800 mg/kg against P. berghei infection in mice on the fifth day of treatment. For the plant extracts, antiplasmodial activity was highest with the combined recipe of the extracts at 800 mg/kg and 600 mg/kg, 600 mg/kg of A. boonei, and 400 mg/kg of M. lucida plant extracts compared to other graded doses of M. lucida at 600 mg/kg, A. boonei at 800 mg/kg, and the combined recipe of the two extracts at 400 mg/kg. Parts of A. boonei plant are employed in the treatment of a variety of ailments in Africa, and the stem bark has been used as useful plant agent for the treatment of malaria, intestinal helminths, muscular pain, insomnia, hypertension, fever, painful micturition, chronic diarrhea, and rheumatic pains, as anti-venom for snake bites and in the treatment of arrow poisoning (Faparusi and Bassir 1972;Oliver-Bever 1986;Kweifo-Okai et al. 1995). In addition, the antimalarial property of the stem bark of A. boonei elucidated in this research concurred with the work of other authors (Bello et al. 2009;Iyiola et al. 2011). The authors in their separate studies reported the antimalarial activities of different fractions of the stem bark extract of A. boonei. Also, Odugbemi and Akinsulire (2007) and Gbadamosi et al. (2011) confirmed the indigenous use of A. boonei for malaria treatment in southwestern Nigeria. In West Africa, M. lucida is an important plant in traditional medicine. In Nigeria, M. lucida has also been noted as one of the four most important plants in the preparation of traditional medicines against fever  reported in vitro activity of M. lucida leaf extract against P. falciparum and activity of M. lucida against P. berghei in mice. Awe and Makinde (1998) also been reported that the stem bark of M. lucida inhibited the maturation of a drug-sensitive strain of P. falciparum in rabbit in vivo technique The in vivo curative antiplasmodial test results of this study indicated that the mice treated with M. lucida, A. boonei, and combined recipes of the plant extracts at graded doses of 400, 600, and 800 mg/kg showed no significant difference (P < 0.05) against P. berghei infected mice on the day 5 of the treatment. Chloroquine reduced the parasites by 100% at 10 mg/kg body weight compared to the plant extracts with varying levels of percentage curative.

Conclusions
The study indicates that the combined recipe of M. lucida and A. boonei plant extracts at 800 mg/kg and 600 mg/kg has a synergistic effect on the parasite and hence produced more curative effects than when the plants were used individually. However, M. lucida when recommended at 400 mg/kg of body weight produced optimal curative effect than when used at higher doses. In contrast, A. boonei produces optimal antimalarial activity when recommended at 600 mg/kg of body weight. Therefore, medicinal plants should be considered veritable alternatives in malaria control and prevention.