In Vivo Antiplasmodial Potentials of the Combinations of Four Nigerian Antimalarial Plants

Various combinations of Nauclea latifolia root, Artocarpus altilis stem bark, Murraya koenigii leaf and Enantia chlorantha stem bark used in African ethnomedicine as decoctions for malaria and fevers, and combinations with standard drugs, were investigated for antiplasmodial activities using Plasmodium berghei berghei-infected mice. The respective prophylactic and curative ED50 values of 189.4 and 174.5 mg/kg for N. latifolia and chemosuppressive ED50 value of 227.2 mg/kg for A. altilis showed that they were the best antimalarial herbal drugs. A 1.6-fold increase of the survival time given by the negative control was elicited by M. koenigii, thereby confirming its curative activity. Pyrimethamine with an ED50 of 0.5 ± 0.1 mg/kg for the prophylactic, and chloroquine with ED50 = 2.2 ± 0.1 and 2.2 ± 0.0 mg/kg for the chemosuppressive and curative tests, respectively, were significantly (p < 0.05) more active. Co-administrations of N. latifolia with the standard drugs significantly reduced their prophylactic, chemosuppressive and curative actions, possibly increasing the parasites’ resistance. Binary combinations of N. latifolia or M. koenigii with any of the other plants significantly increased the prophylactic and suppressive activities of their individual plants, respectively. Also, E. chlorantha with A. altilis or N. latifolia enhanced their respective prophylactic or curative activities, making these combinations most beneficial against malaria infections. Combinations of three and four extracts gave varied activities. Hence, the results justified the combinations of ethnomedicinal plants in antimalarial herbal remedies and showed the importance of the three in vivo models in establishing antimalarial activity.

dose-dependent antimalarial activity against P. berghei chloroquine-resistant NK65 strain [16]. Bioassay-guided chromatographic fractionation of its leaf ethyl acetate extract, using the in vitro and chemosuppresive in vivo antimalarial assay methods, yielded myristic acid and β-caryophyllene as its antimalarial constituents [16].
The World Health Organization [17] recommendation of ACT as a policy of malaria treatment, may be an impetus for the continued investigation of combinations of herbs with reported ethnomedicinal antimalarial usage as a promising approach for discovery of candidate drugs [4,18]. Such drugs must have reports of both in vivo and in vitro anti-plasmodial activities [19]. Hence, the potentials of A. altilis, N. latifolia and M. koenigii, as possible components of herbal decoctions, were evaluated in this study, using their aqueous-ethanolic extracts, Plasmodium berghei berghei-infected mice and the three in vivo prophylactic, chemosuppresive and curative models, with the aims of corroborating their in vitro antimalarial activities and providing scientific evidence supporting their ethnomedicinal usage. Also, the curative activity of E. chlorantha was re-evaluated.

Results and Discussion
Antiplasmodial activities of the four plants were evaluated against P. berghei berghei, using the in vivo prophylactic, chemosuppressive and curative test models, in order to validate their ethnomedicinal claims and determine the potentials of their various combinations, and also of combinations with standard drugs. Considering the ED 50 and ED 90 values of the extracts, N. latifolia had the best prophylactic and curative activities, while A. altilis was the best chemosuppressive drug. In all the models, the activities of the standard drugs, pyrimethamine (PYR) or chloroquine (CQ) were significantly better than those of the extracts (Table 1). Hence, N. latifolia and M. koenigii would be effective at all stages of malaria treatment. Antimalarial drugs with good prophylactic activity, when absorbed into the blood stream, should prevent the invasion of the liver by the sporozoites, while those with good chemosuppressive activity should suppress the development of the merozoites to schizonts in the liver and their release into the blood as trophozoites. Those with good curative activity should destroy the trophozoites in the blood and/or prevent formation of gametocytes, thereby preventing re-infection of the mosquitoes and man [20].
The rural dwellers of the African, Asian and Latin America continents consult traditional practitioners as their first choice of medical care and only visit the hospital or qualified medical personnel when there is no relief. Even on the hospital beds, herbal drugs are secretly taken together with their prescribed drugs. Hence, the investigation of the effects of co-administering herbal and orthodox drugs was imperative, using the extracts' median doses (ED 50 ), and CQ or PYR at 10.0 or 1.2 mg/kg, respectively, in order to ascertain the possible effects of such practice on the activities of standard drugs employed for the treatment of malaria in the hospitals.
Generally, the co-administrations of the individual extracts with standard drugs gave activities that were similar (p > 0.05) or lower than those of CQ or PYR. The co-administration of CQ with N. latifolia or A. altilis or E. chlorantha significantly reduced the curative activity of CQ while only N. latifolia inhibited the prophylactic activity of PYR and suppressive activity of CQ ( Table 2), indicating that combining these herbal drugs with CQ or PYR would increase the development of the parasites' resistance. Only M. koenigii did not significantly affect the prophylactic, chemosuppressive and curative effects of CQ (Table 2). The co-administration of standard drugs with N. latifolia in the prophylactic and suppressive models, with A. altilis in the prophylactic model, and with E. chlorantha in the prophylactic and curative models significantly extended the survival times of the mice compared to those given the individual extracts. The percentages of these elongations over those given by their respective negative controls were also comparable to those elicited by the standard drugs (Table 3). In the curative model, only co-administration of E. chlorantha with CQ gave 168.2% ± 50.9%, that was more than double [21] the 67.3% ± 20.0% prolongation of the lives of the mice given by its extract alone, although this was just 1.6 times the 100.0% ± 8.2% elicited by normal saline (Table 3). Also, the percentages of the survival times given by N. latifolia and M. koenigii as well as the combinations of CQ with either N. latifolia or E. chlorantha or M. koenigii were comparable to those elicited by the standard drugs (Table 3). There was a correlation only between the curative activity and elongation of the survival times in the mice co-administered with CQ + M. koenigii while the discrepancies observed in the combinations of CQ with other extracts (Tables 2 and 3) may indicate that other properties of the extracts, such as immune enhancement, may be responsible for their increased survival times [4]. Three decoctions consisting of barks of E. chlorantha, Alstonia boonei and Diospyros mespiliformis; E. chlorantha bark with rhizomes of Curcuma longa; and barks of E. chlorantha and A. boonei are usually taken as antimalarial recipes in Oke-Igbo, Nigeria [3], implying that E. chlorantha, like some other plants, has the potential of being combined in an antimalarial remedy. Therefore, various combinations of the extracts of two, three or four of the plants used in this study were given to the mice. The dosage of each extract in each combination was that equal to its individual ED 50 value in that particular model.
Co-administration of N. latifolia (NL) with either A. altilis (AA) or M. koenigii (MK) or E. chlorantha (EC) significantly improved their prophylactic activity (Table 4) over the individual extracts (Table 2) while NL combined with EC significantly gave higher curative activity (Tables 2 and 4). Similarly, MK + NL, MK + EC and MK + AA gave significantly enhanced suppressive activities over their individual plants. These increased effects observed with the decoctions over the activities of the individual herbal extracts may suggest synergism in their activities. Similar synergistic effects have recently been reported for some decoctions of Nigerian herbs [4]. On the other hand, AA + NL, AA + MK or AA + EC combinations elicited significantly reduced curative activities than their individual extracts, while combining MK with EC resulted in reduced prophylactic and curative activities (Tables 2 and 4). Hence, caution must be exercised in the choice of ethnomedicinal plants to be combined in traditional decoctions for the treatment of malaria, as not all combinations have increased benefits to the users as antimalarial drugs [4].
The tripartite combination of NL + AA + EC gave increased prophylactic and suppressive activities over those of the NL + AA, NL + EC and AA + EC dual combinations, confirming synergism in the antimalarial actions of the plants. Also, addition of either NL to MK+EC or EC to NL + MK to obtain the NL + MK + EC combination significantly increased their curative activities to 80%, which was the same value given by NL + EC (Table 4). Therefore, in this case, the addition of MK to the latter combination had no curative antimalarial beneficial effect. Furthermore, the combination of the four plants (NL + AA + MK + EC) gave significantly lowered suppressive activity compared to the individual plants, their 2-and 3-combinations The survival times elicited by these numerous combinations in the treated mice were also significantly lower than those of animals treated with the standard drugs (Table 5), confirming their lower antimalarial activities. Hence, the totality of these results showed that the plant components of a decoction are crucial to its ethnomedicinal usefulness in preventing, suppressing or curing malaria infection. Similar observations of synergism and the effects of the ratio of the plant components on the prophylactic, chemosuppressive and curative activities of MAMA decoction, an antimalarial herbal remedy, commonly prepared and used in Nigeria from 1:1:1:1 ratio of Mangifera indica, Alstonia boonei, Morinda lucida, and Azadirachta indica leaves and its four combination variants, MAMA-1, -2, -3 and -4, consisting of 1:2:2:2; 2:1:2:2; 2:2:2:1 and 1:1:2:2 ratios of the above plants, respectively were recently made [4]. Finally, the combinations of the extracts of the two, three and four plants gave prophylactic, suppressive and curative activities that were significantly lower than those of the standard drugs. Despite their lowered potencies, these results justified the traditional usage of these plants as antimalarial remedies. Also, some cases of synergistic or inhibitory effects between the plant-drugs were demonstrated (Table 4).

Animals and Parasites
Healthy Wistar albino mice (18-22 g) of either sex, maintained in separate cages at 22 °C under natural 12 h daylight/night conditions in the animal houses of University of Uyo and Obafemi Awolowo University and acclimatized for at least 5 days preceeding the experiments, were fed on standard pellet diet (Bendel Feeds, Benin , Nigeria) and water was given ad libitum. The "principle of laboratory animal care" (NIH publication No. 85-23, 1985) guidelines and procedures [22] were followed for the experiments. Rodent parasites used were P. berghei berghei NK65 obtained from the Institute of Medical Research and Training, University College Hospital, Ibadan, Nigeria.

In Vivo Antiplasmodial Activities of the Individual Plants
Suspensions of the extracts and pyrimethamine were prepared by triturating weighed samples in tragacanth and diluting with distilled water to give a 1% final concentration of tragacanth, while chloroquine phosphate was dissolved in distilled water. A total of three hundred mice were used for the ten experiments (Tables 1-3). The prophylactic, chemosuppressive and curative antiplasmodial activities of the aqueous-ethanolic extracts of N. latifolia, A. altilis and M. koenigii as well as the curative activity of E. chlorantha were assessed by the methods described by Peters, and Ryley and Peters [23,24]. Extracts of N. latifolia, or A. altilis, or M. koenigii or E. chlorantha (100, 200, 400 mg/kg), or standard drugs of CQ (10 mg/kg) or PYR (1.2 mg/kg), or the vehicle (negative control), were administered (p.o.), using oral cannula, to groups of five mice each, daily for 3 days before infection in the prophylactic model. Extracts/drugs were also given p.o. two hours after infection and thereafter daily for 3 days in the chemosuppressive model while in curative model, administration was done daily for 5 days, starting from the third day after infection. The blood films of the mice were respectively taken at 3 and 4 days and daily after infection for these 3 models. Percentage parasitaemia, determined by counting 10 fields of the blood smear in a view under the microscope, was used to calculate percentages reduction in parasitaemia, chemosupression and clearance for each dose, using the formula:

APU-APT × 100 APU
where APU is average percentage parasitaemia in the untreated mice and APT is average percentage parasitaemia in the treated mice [23]. Microsoft Excel 2007 was used to forecast the ED 50 and ED 90 values of the extracts and standard drugs, as a measure of their activities.

In Vivo Antiplasmodial Activities of the Various Combinations of the Plants with Standard Drugs
The doses that gave 50% of the respective activities (ED 50 ) of the extracts were administered p.o. in quick succession with the standard drugs to the groups of mice and their prophylactic, chemosuppressive and curative antiplasmodial activities were determined, as given above. Also, the prophylactic, chemosuppressive and curative antiplasmodial activities of combinations of the aqueous-ethanolic extracts of the two plants as: NL + AA, NL + EC, NL + MK, AA + EC, AA + MK, MK + EC; three plants as: NL + AA + EC, NL + AA + MK, NL + MK + EC, AA + MK + EC, and of four plants as: NL + AA + MK + EC were assessed by administering each in quick succession to the groups of mice, the median effective doses (ED 50 ) in the three models. A total of one hundred and ninety-five mice were used for the thirteen experiments (Tables 4 and 5).

Survival Times
The mean survival time of the mice in each treatment group was also determined arithmetically by finding the average survival time (days) of the mice post inoculation in each group over a period of 28 days.

Statistical Analysis
The percentage parasitaemia, percentage clearance, percentage chemosupression, percentage reduction in parasitaemia, ED 50 and ED 90 values and survival times were expressed as mean ± SEM. The significance of comparative difference was determined between the extracts/standard drugs/combinations and the negative control using One-Way Analysis of Variance (ANOVA), followed by Student-Neumann-Keuls post-hoc test. Values of p < 0.05 were considered statistically significant.

Conclusions
In conclusion, although the activities of these four plants appear low in comparison with the standard drugs, they show significant antimalarial effects in the various models investigated in this study, which can be interpreted as a justification for their antimalarial ethnomedicinal claims. Studies on the chemical constituents responsible for these effects will be interesting and might lead to new chemical entities with antimalarial potential. The present results obtained by three complementary in vivo models furthermore show the importance of the choice of plant components used to prepare herbal remedies to prevent, suppress and cure malaria infestation.