Abstract: A study of three plants traditionally used in the treatment of malaria in the Southeastern part of Nigeria was investigated to determine their efficacies as antimalarial compounds. The three herbs were collected through a traditional herbalist who uses them in his practice. Ethanolic extracts from the roots of Enyim ocha (Salacia nitida), Ovoro ilu (Nauclea latifolia) and stem bark of Erumeru (Enantia chlorantha Oliv.) were assessed for antimalarial activity against chloroquine sensitive Plasmodium berghei in mice using the 4 day suppressive test procedure. The extracts had intrinsic antimalarial properties that were dose dependent. The comparison analysis indicated that 250 mg kg-1 body weight of the root of S. nitida produced 71.15% suppression of parasitaemia and the 500 mg kg-1 body weight of the stem bark of E. chlorantha, roots of S. nitida, N. latifolia and the three herbs combined, produced 75.23, 73.28, 71.15 and 77.46%, respectively, compared with chloroquine with 71.15% suppression. The results were significant at p< 0.05 when compared to a placebo and support the traditional use of these plants for the treatment of malaria.
INTRODUCTION
Malaria represents a major cause of childhood mortality and adult morbidity in many parts of the world. Recent estimates indicate that 300 to 500 million clinical episodes of malaria occur worldwide every year and are responsible for one in five deaths in Africa (Breeman, 2001; WHO, 1997). Antimalaria drug resistance, particularly Plasmodium falciparum resistance, has been a major set back in the fight against malaria and its attendant complications (Wongsrichanalai et al., 2002). The application of herbal drugs to different human and animal disease conditions dates back to human history. Plants have been the basic source of sophisticated traditional medicine systems for thousands of years and were instrumental to early pharmaceutical drug discovery and industry (Elujoba et al., 2005). Traditional medicine practitioners, natural herbal sellers and the local indigenous people have been the source of basic information as a lead to scientific probing of medicinal plants in Africa (Baba et al., 1992). An appreciable level of studies has been done on African traditional medicinal plants. These range from ethno botanical surveys, to the actual extraction of the active ingredients in the plants (Ibeh and Nwufo, 2005; Ajaiyeoba et al., 2006; Dikaso et al., 2006). However, in Nigeria, particularly in the South Eastern part, studies on the extraction and perhaps, testing of the effects of these herbal extracts on malaria parasites have been minimal. The enormity of the malaria burden (WHO, 1997), particularly antimalaria drug resistance (Peters, 1998; Wongsrichanalai et al., 2002), demands a commensurate level of studies towards the discovery of novel but potent antimalaria drugs to combat this burden. This has become increasingly exigent, particularly in Africa, with widespread poverty and illiteracy, as neither of the antimalaria drugs in use today is indigenous to her (Milijaona et al., 2003). Therefore, the aim of the study was to investigate the antimalarial activities of some herbs used in the traditional treatment of malaria in the South Eastern part of Nigeria. This exercise would offer a scientific backing in the use of traditional herbs either separately or in combination against malarial diseases.
MATERIALS AND METHODS
Sample Collection
Three medicinal plants namely, Enantia chlorantha (stem bark), Nauclea
latifolia (root) and Salacia nitida (root), were collected from Mbutu
in Aboh Mbaise Local Government, Imo State, Nigeria. Five herbalists from randomly
selected communities in the area were interviewed on their methods of treatment
of malaria. After the interviews, 25 herbal plants were mentioned as antimalaria
herbs used in their practice. All of them were unanimous in their use of the
herbs in combination for enhanced efficacy and their mode of preparation of
the herbs (boiling or maceration in alcohol).Six herbs were discovered to be
common among the herbalists. Three of them were randomly selected for the study
and were obtained by the help of the herbalist, from the village forest. The
stem of E. chlorantha and the roots of S. nitida and N. latifolia
were collected. In addition their respective leaves were collected for botanical
identification. The identification and authentification was done at the Herbarium
of University of Port-Harcourt, Choba, Nigeria, where a voucher specimen was
deposited.
Extraction Procedure
The roots of N. latifolia and Salacia nitida and the stem
bark of Enantia chlorantha were sun-dried at ambient temperature (30±0.5°C).
After drying, 1 kg each of the samples was pulverized to coarse powder using
sterile mortar and pestle to avoid contamination. Two hundred and eighty-two
grams each of the pulverized herbs was measured using Mettler PN 163 electric
weighing machine and transferred into a conical flask. Ninety-four grams each
of the three herbs was weighed and mixed together to give Two hundred and eighty-two
grams of the combined herbs and transferred into the fourth flask. The respective
herbs and their combination were extracted for 72 h using 1 L of 98% ethanol.
The herb-ethanol mixtures were shaken daily to ensure proper extraction (Abosi
and Raseroke 2003). After 72 h, the extracts were filtered using clean, white
cotton handkerchief. The filtrates, were concentrated under vacuum using a vacuum
rotary evaporator, to yield, 3.885, 14.035, 5.517 and 7.537 g of gummy oily
residues of E. chlorantha, S. nitida, N. latifolia and
their combination, respectively and were stored in screw capped vials at 4°C
until tested (Abosi and Raseroke, 2003). Prior to use, 1 g mL-1 of
each extract was prepared in ethanol to ensure adequate dissolution and then
4 mL of sterile water was added to give a stock of 1000 mg in 5 mL. Subsequently,
125, 250 and 500 mg kg-1 doses were prepared using distilled water.
The extraction was carried out in the Microbiology Research Laboratory, Rivers
State University of Science and Technology, Port Harcourt.
Mouse Strain
Healthy albino mice, 5 weeks old and weighing 18-24 kg, were obtained from
the animal house of the Department of Pharmacology, University of Nigeria Nsukka.
They were kept in plastic cages and adequately fed with livestock feed and water
for optimal health. Before the experiment, the animals were bred for one week
at the Malaria Research Laboratory, University of Port Harcourt where the research
took place, for proper acclimatization.
Malaria Parasite
Chloroquine-sensitive P. berghei (NK-65), used in the experiment
was obtained from the Malaria Research Laboratories, Institute for Advanced
Medical Research and Training, (IAMRAT), College of Medicine, University of
Ibadan. Parasites were maintained through weekly passage in mice, by inoculation
of known amount of parasite into healthy mice every week.
Inoculation and Treatment
Peter's 4 day test (Peter and Anatoli, 1998; David et al., 2004)
was followed to evaluate the blood schizontocidal action against Plasmodium
berghei. Donor albino mice previously infected with chloroquine-sensitive
P. berghei and with a rising parasitaemia of 30% as determined using
thin blood film, were sacrificed and the blood collected using EDTA bottle.
The blood sample was diluted using phosphate buffered saline (concentration
of 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4) such that 0.1 mL contained
eleven million parasites. To avoid variability in parasitaemia, all the animals
used were infected from the same source. Forty-two mice were used to assess
the effect of the herbal extracts. On day 0, experimental as well as control
groups of animals were inoculated with eleven million P. berghei-infected
red blood cells. The mice were then randomly divided into groups of three per
cage and groups of experimental animals were given different doses of test materials
consecutively from day 0 to day 3. Three of the groups were given 500, 250 and
125 mg kg-1 of the plant extract per day, orally. The initial treatment
started about two hours after infection. The other two groups received either
5 mg kg-1 chloroquine per day (positive control), or 0.2 mL of phosphate
buffered saline (concentration of 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,
pH 7.4) (negative control). All experiments were carried out in triplicate.
The animals were adequately fed and no death was recorded throughout the duration
of the experiment.
On day four of the test, thin blood smears were prepared using well-labeled and properly cleaned slides (Devi et al., 2000). Blood was collected from the tail vein of each animal using heparinized capillary tube. The dry blood films were fixed with methanol and subsequently, stained with Giemsa for 25 min. They were then washed with phosphate buffer, pH 7.2 (Dikaso et al., 2006) and allowed to dry. To ensure optimal film quality, a total of eighty-four slides were made, each animal in duplicate. The slides were then microscopically examined using x100 magnification in oil immersion (Model Olympus microscope) (Dikaso et al., 2006). The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in infected controls with those of treated mice. Average percentage suppression of parasitaemia was calculated using the formula:
Where:
| A | = | Average percentage suppression |
| B | = | Average percentage parasitaemia in the placebo group |
| C | = | Average percentage parasitaemia in the test group (Abosi and Raseroke 2003) |
Student's t-test was used to compare the differences in the results between the groups.
The mice after four days of treatment looked very healthy and active. They were then monitored for 40 days. All of the treated mice were alive and active. On the 31st day of observation, one of the extract treated mice had obstructed labor and died two days later. Two other extract treated ones had normal delivery of 4 and 5 young ones on the 35th and 38th days, respectively. The chloroquine treated mice did not die within 40 days but were less active than the extract treated animals. However, all of the untreated mice died within 14 days after the experiment.
RESULTS AND DISCUSSION
The results of the study indicated that in vivo, ethanolic extracts of the roots of Salacia nitida, Nauclea latifolia and stem bark of Enantia chloranta Oliv. displayed a very good activity against P. berghei malaria parasite when given at doses of 125, 250 and 500 mg kg-1, respectively. The comparism analysis indicated that 250 mg kg-1 of the root of S. nitida and the herbal mixture, as well as the 500 mg kg-1 of the stem bark of E chloranta, root of S. nitida and the herbal mixture, showed statistically significant difference on day four parasitaemia level compared to the negative control (p<0.05). A high level of inhibition was observed with 500 mg kg-1 of E. chloranta, N. latifolia, S. nitida and the herbal mixture (75.23, 71.15, 73.28 and 77.46%), as well as 250 mg kg-1 of S. nitida and herbal mixture (71.15 and 69.11%). The result indicated that, the highest level of inhibition (77.46%) was obtained in 500 mg kg-1 of the herbal mixture (Table 1). The overall result showed a higher reduction in mean percentage parasitaemia with increased herbal concentration for each herb and their combination. The positive control, chloroquine induced the same level of chemosupression (71.15%) with 500 and 250 mg kg-1 of N. latifolia and S. nitida, respectively Plants have undoubtedly been a rich source of new drugs in use today. It is estimated that over 1200 plant species from 160 families are used to treat malaria and fever (Wilcox and Gerald, 2004). A number of studies have been done to evaluate the inhibitory effect of various plant extracts on P. falciparum (Milijaona et al., 2003; Sanon et al., 2003). Similarly, the in vivo antimalarial properties of several plant extracts have been studied in mice (Devi et al., 2000; Abosi and Raseroke, 2003; Ajaiyeoba et al., 2006). Following the trend, this study presents the results got from the evaluation of the in vivo antimalarial activity of three herbs namely: Enantia chloranta, Nauclea latifolia and Salacia nitida, commonly used in the Eastern part of Nigeria against malaria.
N. latifolia (Pin cushion tree) is a staggering shrub or small tree, native to tropical Africa and Asia. It is commonly used in the Eastern part of Nigeria to treat malaria. The root extract of this herb, in the present study was observed to show some intrinsic antimalarial activity. This is evident from its percentage chemosupression when compared with the positive control, chloroquine in the 4 day suppression test (Peter and Anatoli, 1998; David et al., 2004). Treatment of mice infected with P. berghei with ethanolic extracts of N. latifolia showed a dose dependent chemosupression with the 500 mg kg-1 treated group of mice showing the highest percentage suppression. Similar findings were reported by Benoit-Vical et al. (1998) that showed that aqueous extracts obtained from the root and stem of N. latifolia when tested on two strains of P. falciparum (fcbi strain), inhibited the parasites. Other medicinal ingredients have also been found in this herb. Gidado et al. (2005) discovered that the aqueous extracts of N. latifolia at 200 mg kg-1 body weight of mice, significantly lowered glucose levels (p<0.05) of diabetic rats by 45% within four hours, but showed no similar effect in normoglyceamic rats.
E. chlorantha (Annonaceae) is an ornamental tree of up to 30 m high with dense foliage and spreading crown. It is a component of fever preparations by traditional medicine practitioners in the Eastern part of Nigeria. The ethanolic extract of the stem bark of E. chlorantha displayed high level of suppression of parasitaemia compared with the positive control.
| Table 1: | In vivo anti-malarial effect of three medicinal plants and their combination |
| |
|
| n: No. of animals in each group; *: Significance at p<0.05; Values indicate mean percentage parasitaemia; Values in parenthesis indicate percentage reduction in parasitaemia compared to control; CQ: Chloroquine; PBS: Phosphate Buffered Saline | |
The level of activity was discovered to be dose dependent, with the dose of 500 mg kg-1 having the highest activity (75.23%). Agbaje and Onabanjo (1991) reported that aqueous extract of the plant E. chlorantha when tested in vivo against Plasmodium youlii parasite in mice, was found to be effective in suppressing Plasmodium youlii infection in mice if given orally in drinking fluid at 0.2-150 mg mL-1. The animals given the extracts in their drinking fluid survived for over 60 days. They also observed that the ethanolic extract was effective in eliminating the parasites when administered subcutaneously in doses of 0.05-0.5 mg mL-1. They noted that the aqueous and ethanolic extracts had ED50 of 6.9 and 0.34 mg g-1, respectively and were schizontocidal in action. Its antimicrobial activity was also confirmed by Atata et al. (2003).
Compared with chloroquine, S. nitida was discovered to have a high suppressive ability that was equally dose dependent with the 250 and the 500 mg kg-1 having significant difference at (p<0.05) when compared with the placebo. The overall result shows that the crude extracts of the three herbal plants have intrinsic antimalarial components, which are dose dependent. The extract dose of 500 mg kg-1 displayed the highest level of inhibition of parasitaemia with that of the drug mixture maintaining the lead (77.46%). This drug mixture demonstrated synergistic inhibitory effect on the in vivo plasmodial development. This result justifies their use in combination in traditional medicine (Hilda et al., 1994; Azas et al., 2001). Chloroquine at a dose of 5 mg kg-1 gave a percentage suppression of 71.15%, a value equal to that of 250 mg kg-1 of S. nitida and lower than the 500 mg kg-1 of the individual herbs and their combination (Table 1). Since chloroquine sensitive parasites were used, it could be possible that the potency of the respective herbs and their combination at 500 mg kg-1 were greater than that of chloroquine at a dose of 5 mg kg-1. Devi et al. (2000) and Ajaiyeoba et al. (2006) got a clearance rate of 100% parasitaemia with chloroquine sensitive P. berghei parasites in their respective studies. However, Ajaiyeoba et al. (2006) used a total dose of 25 mg kg-1 of chloroquine in their study while Devi et al. (2000) used a total dose of 20 mg kg-1. Both used 1.0x107 parasites whereas, this present study was done with a total dose of 20 mg kg-1 and 1.1x107 parasites. In untreated mice, the parasite count increased as observed in previous studies (Ajaiyeoba et al., 2006; Devi et al., 2000). The high levels of chemosupression produced at high doses of extracts, is in line with previous studies (Ajaiyeoba et al., 2006; Devi et al., 2000). At 125 mg kg-1 per day, the stem bark extract of E. chlorantha and the root extract of N. latifolia decreased parasitaemia by 53.62 and 48.42%, respectively. This indicated that the minimum effective dose of these extracts is approximately 125 mg kg-1 day-1. However, under the experimental conditions employed, the extracts failed to eliminate P. berghei parasites completely.
CONCLUSIONS
The development of antimalarials from indigenous plants depends to a large extent on the screening of appreciable number of these herbs, particularly those that have been in use by the indigenous people from different geographical areas, especially, from the different tribes where they are endowed by nature. Once the preliminary anti plasmodial effect has been established, further studies to ascertain the active ingredients that exert these effects could then be carried out. This will ultimately help in discovering potent and novel antimalarials to counter the threat posed by malaria in recent times.
There is increasing concern by Medical practitioners as the resistance of malaria parasites to available drugs continues to grow, increasingly limiting our ability to control this serious disease. However, it is reassuring that many new approaches to antimalarial drug discovery are now under evaluation as carried out in this research. Recent increases in the pace of progress in the search especially in the area of herbal medicine, suggest that, if support for antimalarial drug discovery is adequate, the development of novel, but potent antimalaria is underway.
From the present study, it can be concluded that the ethanolic extracts of the roots and stem bark of N. latifolia, S. nitida and E. chlorantha have shown parasite suppressive effects on P. berghei-infected albino mice in a dose-related fashion. This result therefore, offers a scientific basis for the traditional use of these herbs separately and in combination against malaria parasite. Further studies on the herbs, especially on the refined extracts, to among other things, assess their pharmacokinetic properties, is recommended.
ACKNOWLEDGMENTS
We thank Professor Omotayo Ebong of University of Port Harcourt, for making available her Laboratory for the Research and Dr. O.G. Ademowo of the Malaria Research Laboratory, Institute of Advanced Medical Research and Training, University of Ibadan, for the provision of the Malaria parasite.