Antiplasmodial activities of the combined leaves extracts of Morinda lucida, Phyllantus Amarus, Vernonia Amygdalina and Newbouldia laevis

© Nigerian Annals of Pure and Applied Sciences Maiden Edition 2018. NAPAS re a u n P d f A o p s p l l a ie n d n S A c n ie ai n r c e e gi s N 44 | Nigerian Annals of Pure and Applied Sciences


Introduction
Approximately 3.2 billion people are at risk of malaria, with an estimated 214 million cases and 432,000 deaths annually (WHO, 2016). Malaria related deaths are caused by Plasmodium falciparum infection and most of the cases of malaria are in children under five years and pregnant women (Dondorp et al, 2009). Presently malaria chemotherapy remains the mainstay of malaria control in sub-Saharan Africa. The advent of development of resistance to chloroquine necessitates the introduction of artemisinin based combination therapy to combat resistance in malaria endemic countries, however resistance to artemisinin based combination therapy has already been reported in South East, Asia and in some parts of Africa.
Africa flora is greatly rich with a lot of medicinal plants which indigenous people are familiar with and have used over time (Dike et al., 2012). Ethnobotanical surveys have shown that these plants and herbs are effective especially in the treatment of malaria (WHO, 2002) and the use of medicinal herbs has been a common method of treating malaria among people living in malaria endemic areas (Ahmad, 2014). Different parts of plant such as leaf, bark, stem, roots and fruit are used to treat malaria. The medicinal plants used for the treatment of malaria are usually taken orally in the form of infusions (hot teas), decoctions (boiled teas), tinctures (alcohol and water extracts), paste, powder and macerations (cold-soaking) (Idowu et al., 2010;Kunle et al., 2013).
Morinda lucida is an important medicinal p l a n t i n N i g e r i a a n d t h e e v a l u a t i o n o f antiplasmodial activities of Morinda lucida extract revealed significant chemosuppression (Unekwuojo et al., 2011). Antiplasmodial activity of aqueous and ethanolic extracts of Phyllantus. amarui whole plants and leaves have been demonstrated against Plasmodium yoelii (Ajala et al., 2011;Dapper et al., 2007). Vernonia amygdalina leaves are used as green leafy vegetable and consumed either as a vegetable (leaves are macerated in soups) or aqueous extracts used as tonics for the treatment of various illnesses (Igile et al., 1995). Both aqueous and alcoholic extracts of the stem, bark, roots and leaves are reported to be extensively used as antimalarial (Kupcham, 1969;Njam, 2012). Newbouldia laevis is widely used in African folk medicine and antiplasmodial potential of N. laevis has been documented (Andrew et al., 2016) Phytochemical analysis of these leave extracts have been attributed to wide range of metabolites including , glycosides, saponins, flavonoids, anthraquinones, reducing sugar, steroids, terpenoids and alkaloids. polyphenols, terpenes, carotenoids, coumarins, volatile oils anthraquinones and steroids terpenes, steroids, coumarins, phenolic acids, lignans, (Kupcham, 1969;Odetola and Akojenu, 2000;Izevbigie, 2003;Akinmoladun et al., 2010;Amonkan et al., 2013;Adeyemi et al., 2014;Saganuwan, 2014 andAndrew et al., 2016).
In preparation of herbal recipes for malaria therapy, a single plant (monotherapy) or combination with other plants (combination therapy) could be used (Idowu et al., 2015). The combination of these different plants has been claimed to cure and ameliorate several ailments and dysfunctions associated with malaria in the body and it has been speculated that the active ingredients of each plant in the preparation complemented one another in the fight against malaria parasite (Ibrahim et al., 2012). Furthermore, most studies where combined leaves extracts have been demonstrated for antimalarial activities have failed to demonstrate the impact of these extracts on kidney and liver. The study therefore explores antimalarial suppressive and curative activities of the combined aqueous extracts of Morinda lucida, Phyllantus amarus, Vernonia amygdalina and Newbouldia laevis and assess influence of these extracts on the liver and kidney function in mice.

Materials and methods Plant collection and identification
The four medicinal plants used in this study were selected based on their traditional use to treat malaria locally. The leaves of the following plants, Morinda lucida (Brime stone) "Oruwo", Phyllantus amarus (Stone breaker) "Eyin olobe", Vernonia amygdalina (Bitter leaf) "Ewuro" and Newbouldia laevis (fertility tree) "Akoko" were collected from Alaba Suru market in Lagos. The identification and authentication were done by the Ethno-Survey Unit, Research and Training D e p a r t m e n t , N i g e r i a N a t u r a l M e d i c i n e Development Agency and Federal Ministry of Science and Technology, Lagos.

Plant preparation
The plant materials (leaves), were spread thinly on a flat, clean tray (to prevent spoilage by moisture condensation) and allowed to dry at room temperature for two weeks. The dried plant materials were pulverized separately into powder using an electric blender and then stored in an airtight container prior to extraction.

Plant extraction
The pulverized plants were mixed together in a ratio of 2: 1: 1: 1. Approximately 4.2 grams of Morinda lucida and 2.1 grams of Phyllantus amarus, Newboldia laevis and Vernonia amygdalina and mixed together to make a total of 10.5 grams. The combined plant was extracted 0 with 750ml of water boiled at 100 C for 24hours. The preparation was filtered and the filtrate was kept in a well-sealed container and stored in the refrigerator till it was used for the experiment.

Drugs preparation
Tablets of chloroquine phosphate, CQ (Emzor Pharmaceutical company, Nigeria), (0.05g) was dissolved in 10ml of Phosphate Buffered Saline (PBS) to final doses of 5mg/kg body weight which served as positive control.

Animals
Fifty pure strains of adult Swiss albino mice weighing between 15-30grams were obtained from the animal house, Nigeria Institute of Medical Research (NIMR), Yaba Lagos. The animals were kept in clean and well maintained cages in the Biochemistry Laboratory of NIMR. The animals were observed under 10 hours light/dark cycles in clean and well maintained cages in the Biochemistry laboratory and were fed with mice pellet diet (Ladokun Farms, Ibadan, Nigeria) and water ad libitum for one week so as for them to acclimatize to room temperature of 0 29 C. This method was carried out prior to randomization into various experimental groups of five (5) animals per group based on body weight of the mice. The groups were designated as group A (200mg), B (400mg), C (800mg), CQ and Negative control.

Preparation of parasites and innoculum
A chloroquine-sensitive Plasmodium berghei (NK65 strain) obtained from the Biochemistry Division, NIMR, was used for this study. Experimental mice were infected with blood samples from donor mouse which was obtained by ocular puncture, using a sterile capillary tube. The infected blood was diluted with phosphate buffered saline which enabled 6 innoculum of 0.1ml for each mouse containing 10 parasitized red blood cells intraperitoneally.

Antiplasmodial studies of plant extracts Suppressive treatment
The Peters' 4 days suppressive test was adopted in this study (Peter, 1965). Twenty five mice of both sexes weighing 14-19grams were i n n o c u l a t e d i n t r a p e r i t o n e a l l y w i t h 1 0 6 erythrocytes in 0.1ml phosphate buffered saline and randomized into five groups, A, B, C, D and E with each group having five (5) animals in a cage. Treatment of animals started after 2 hours on Day 0. Group A, B and C served as the experimental groups, while group D and E served as the control groups. Group D served as the positive control group which was administered with 30mg/kg chloroquine phosphate and group E served as negative control group which was not treated at all. Group A, B and C were treated with three selected doses per kg body weight (200, 400 and 800mg) of the combined extract for four (4) days and group D was treated with chloroquine phosphate for 3 days. The animals were administered with the extract 2 hours after the inoculation of the parasite on day 0 (D0) and everyday till day 3 (D3) using an oral cannular. On day 4, thin and thick films were made from the tail blood of each mouse under aseptic conditions. The thin films were prepared by spreading the blood on a clean glass slide at angle 45 degrees, and subsequently fixed with methanol. The thick and thin films were stained with 3% Giemsa stain for 45 minutes and examined with microscope under the oil immersion objective to determine the parasite density microscopically (Olympus CX, Japan). This is necessary as to monitor the level of parasitaemia. The suppression of parasitaemia in relation to the control was assessed using the recommended formular.

Curative treatment
Ryles and Peters procedure was adopted in this study (Ryles and Peters, 1970). On day 0, twenty five (25) Swiss albino mice were randomized into A, B, C, D and E with each group h a v i n g fi v e a n i m a l s . I n o c u l a t i o n w a s intraperitoneal and all mice were inoculated with106 parasitized red blood cells in 0.1ml of phosphate buffered saline. Treatment started 72 hours after inoculation. Group A, B and C served as the experimental group and were administered with three selected doses per kg body weight (200, 400 and 800mg) of the combined extract orally for five (5) days using an oral canular. Group D served as the positive control administered with a curative dose of 30mg/kg of Chloroquine for three (3) days through oral canular. The preparation of thick blood films commenced 72 hours after inoculation and lasted for ten days. The level of parasitaemia was calculated based on parasite density. Number of parasite counted against 200 white blood cells (WBC), multiply by 8000 Parasite density = No of parasite X 8000 200 WBC

Determination of biochemical parameters of the liver and kidney
On the 10th day post inoculation, 5ml of fresh blood was collected from three mice from each group by ocular puncture. The blood was used for the biochemical analysis of the liver and kidney, for quantitative determination of alanine a m i n o t r a n s f e r a s e ( A LT ) , a s p a r t a t e aminotransferase (AST) and alkaline phosphatase (ALP), urea and creatinine (Cr).
ALT and AST levels were measured with commercially available standard blood ALT and AST kits by Randox, Reitman and Frankel (1957). The plasma urea level was measured with commercially available standard blood urea kit by (Randox, United Kingdom). The plasma creatinine level was measured with commercially available standard blood creatinine kit (Randox United Kingdom) (Bartels et al., 1972)

Statistical analysis
Mean and standard deviation of the results of suppressive and curative treatment were calculated using Excel package, which is expressed in mean ± Standard deviation. Results of biochemical analysis of kidney and liver were subjected to One-way Analysis Of Variance (ANOVA), using SPSS. The results were also expressed in mean ± Standard deviation (SD) and the level of significance was estimated at P<0.05. Differences among groups were determined by Duncan multiple range test.

Results
The result of this work shows that the combined extract did not cause mortality within 24hours at all doses. In the suppressive test, two mortalities were recorded in 800 and 400mg/kg. Two death were recorded in 400mg/kg, one in 800mg/kg, one in positive control (Chloroquine) and three in negative control in the curative test.

Parasitaemia suppression
There was significant reduction of parasitaemia (P<0.05) in the treatment group compared to the negative control. Chloroquine (CQ) (30mg/kg) gave an absolute (100%) reduction (Table1). The parasitaemia reduction was dose dependent with 800mg/kg having the highest reduction (5.3±3.66). The percentage suppression of the groups treated with 200, 400 and 800mg/kg were 9.8, 58.3 and 59.8% respectively, indicating that suppression increases with increasing dosage of the combined extract. CQ gave 100% suppression ( Table 1).

Clearance of parasitaemia
The result of this study signifies that the combined extract showed a potent antiplasmodial activity against Plasmodium berghei rodent malaria in vivo at doses of 200, 400 and 800mg/kg. Total clearance of the parasites was not achieved by the combined extract but the clearance was dose dependent (Table 2).
Two hundred milligram had a higher parasite clearance compared to the negative control on D1 (27.89±30.05) but the parasitaemia count shot higher on D7 (147.9±125.35). Four hundred milligram maintained a higher level of parasite clearance throughout the study compared to the negative control, but the parasite count shot a bit higher on D8 (55.53±19.44) compared to the negative control, 800mg had a higher parasite c l e a r a n c e w i t h t h e h i g h e s t c l e a r a n c e (29.51±29.14) on D7 (Table 2). Table 3 showed no significant difference in the level of ALP and Urea at all doses (P<0.05). AST had significant decrease (P>0.05) at 200mg/kg (35.7±6.9), ALT showed significant increase at 800mg/kg (81.2 ±6. 2) compared to the positive control and creatinine had significant increase at doses 400mg/kg (1.4±0.2) and 800 mg/kg (0.9±0.5).

Discussion
The emergence of Plasmodium falciparum multi-drug resistant malaria and drug resistance to artemisinin-derivatives and to other drug combination therapies makes the development of new potent antimalarial drugs an alternative therapy. Traditional medicinal plants have proved to be rich sources of new drugs coupled with the fact that antimalarial drugs in use presently were either obtained directly from plants or developed using chemical structures of plant-derived compound as templates. The feasibility of discovering new potent antimalarials from traditional medicinal plants is very promising.
This study demonstrated the in vivo antiplasmodial activities of aqueous leaves extracts of a combination of Morinda lucida, Phyllantus amarus, Vernonia amygdalina and N e w b o u l d i a l a e v i s . T h e s i g n i fi c a n t chemosuppression demonstrated in the extract treated group is in agreement with the traditional use of combined extract herbal medication against malaria in many part of Nigeria (Igoli et al., 2005;Adebayo and Krettli, 2011). The extract was effective and dose dependent. The observed efficacy of the standard antimalarial drug, chloroquine (100%) which was higher than the extract treated groups may be due to nonselectivity of the extract or slow absorption and poor bioavailability of crude extract (Adzu and Haruna, 2007).
The curative test revealed that the extract   has a considerable high antiplasmodial effect. The extracts produce a dose dependent parasitaemia levels in the extract treated groups. The parasite reduction at 800mg/kg treated group was similar to that of chloroquine which established that combined extracts of Morinda lucida, Phyllantus amarus, Vernonia amygdalina and Newbouldia laevis has therapeutic efficacy against malaria parasites. This finding is consistent with previous studies that have confirmed the antiplasmodial potency of the four individual plants (Abosi and Raseroka, 2003;Ajala et al., 2011;Unekwuojo et al., 2011;Njam, 2012;Andrew et al., 2016). The antimalarial activity could be attributed to the presence of some phytochemicals like alkaloids, saponins, flavonoids, taninis, terpenes and cardiac glycosides present in the plants. These constituents have been found in other natural products which exhibite antimalarial activity (Ayoola et al., 2008). The compounds could be acting singly or in synergy with one another to exert the antimalarial activity observed in this study. Saponins, flavonoids, alkaloids and tannins have been suggested to act as primary antioxidants or free radicals scavengers that can counteract the oxidative damage induced by malaria parasite (Alli et al., 2011;David 2004 andOkonkon et al., 2008).
Aspartate aminotransferase (AST) and Alanin aminotransferase (ALT) level increased in 400mg/kg and 800mg/kg respectively. The increase significantly differ from the negative control but are not significantly different from the values of the positive control. This suggests that the impact of the extract was masked by the hepatoprotective effect of Morinda lucida (Oduola, et al., 2010). The level of alkaline phosphate (ALP) showed no significant difference in all the experimental groups, suggesting the leakage in AST and ALT at 400 and 800mg/kg respectively was from the liver and not the bile duct (Idowu et al., 2015). Increased serum level of AST and ALT are reported to be associated with liver damage (Mukherje, 2003).
There was no significant difference in the values of urea between the extract treated groups and chloroquine treated group, but were significantly different from the negative control. However, creatinine showed significant difference only at the lowest dose (200mg/kg) which was not significantly different from the negative control. This implies that the extract impacted the kidney at 200mg/kg, but the impact was suppressed at higher doses (400 and 800mg/kg). This could be attributed to the hepatoprotective effect of Morinda lucida (Oduola, et al., 2010). Urea and creatinine are markers of kidney function, elevation of these markers indicates an impairment of the kidney.
Recent successes recorded in malaria control can be jeopardized by malaria drug resistance and the WHO aim of eliminating malaria 2030, local plants that have showed antiplasmodial activities should be fully assessed for their effectiveness and the possibilities of using them as new antimalarial drugs.