Antimalarial Ethnobotany: In Vitro. Antiplasmodial Activity of Seven Plants Identified in the Nigerian Middle Belt

Abstract

Seven methanol extracts of seven plants from seven plant families were screened for antimalarial properties. The plants were identified and selected from Gboko and Kastina-Ala local government areas in the Tivland ethnobotany in the Middle Belt Zone of Nigeria. Methanol plant extracts were evaluated for in vitro. antimalarial properties using the lactate dehydrogenase technique, with a multiresistant strain of Plasmodium falciparum. K1. Quantification of activity was by estimation of the concentration of extracts that inhibited 50% growth of parasite (IC₅₀) in µg/ml. Of the seven plants screened, Erythrina senegalensis. DC (Leguminosae), Pericopsis elata. Harms (Papilionaceae), and Bridelia micrantha. Benth (Fabaceae) had IC₅₀ values of 99.7, 124.8, and 158.7 µg/ml, respectively. Nauclea latifolia. SM (Rubiaceae) extract exhibited the least activity in the assay with an IC₅₀ value of 478.9 µg/ml.

Keywords:

• Antiplasmodial activity
• ethnobotany
• Middle Belt
• Nigeria
• phytomedicine

Introduction

Malaria remains a major public health problem in most tropical countries, particularly sub-Saharan Africa. Annually, between 300 and 500 million individuals are infected, resulting in more than 2 million malaria-related deaths. The high burden of malaria among communities in tropical Africa supports the high priority assigned to the global effort to control the disease (Wernsdorfer & Wernsdorfer, 1992; Butler, 1997).

Ethnobotanical surveys are an important step in the identification, selection, and development of therapeutic agents for medicinal plants. The approach of retrieval of information on the folk use of plants often yields more potentially useful compounds than the empirical approach (Carvalho et al., 1991; Craigg et al., 1997). In rural populations in Africa, people often use ethnobotanical and ethnomedical alternatives for the management of health problem including malaria. Such alternative remedies have provided leads for the development of drugs useful in therapeutics as practiced in Western medicine (Phillipson, 1994; Heinrich, 2000).

The documentation of the indigenous remedies for febrile illness in non-Western communities, the identification and subsequent isolation of antimalarial compounds from plants like Cinchona. spp. and Artemisia annua. continues to stimulate the search for lead compounds that could be developed from plants (Guido et al., 1996).

Further to our study on the use of phytomedicine for prevention and treatment of fevers by the indigenous Tiv population of the Middle Belt of Nigeria (Ajaiyeoba et al., 2003) the in vitro. antiplasmodial evaluation of plants identified and selected from the region is presented as part of the effort of developing antimalarial drugs from Nigerian phytomedicine.

Materials and Methods

Plant collection and authentication

Samples of the 7 plants weighing between 0.9 and 1.5 kg were collected from various locations in Gboko and Kastina-Ala in Tivland, Benue State, in the Middle Belt Zone of Nigeria in October 2000. Plants were authenticated at the Herbaria of the Botany Department, University of Ibadan, and at the Forestry Research Institute of Nigeria, Ibadan, where voucher specimens were deposited, respectively.

Extraction of plant materials

The air-dried plant materials were ground with plant grinder, powered by a 5-hp engine. The seven plants were extracted into 95% aqueous methanol by percolation at room temperature (29°C) for 72 h each. After removal of solvent, the yield of extracts was determined. Thereafter, extracts were stored in the refrigerator until needed for analysis.

In vitro. antiplasmodial assay

Parasite strain

Plasmodium falciparum. multidrug-resistant strain K1/Thailand, obtained in culture from Dr. D.C. Warhurst, London School of Hygiene & Tropical Medicine, was used for the study.

Cultivation of asexual erythrocytes of P. falciparum.

The asexual erythrocytic stages of P. falciparum. used for the study were cultivated based on the in vitro. technique described by Jensen and Trager (1977) and that modified by Fairlamb and co-workers (1985). All experiments involving parasites were carried out aseptically in a Cytox 11 laminar flow hood (Envair, Lancashire, UK).

Maintenance of culture

Parasites were preserved under liquid nitrogen to ensure a continuous supply of parasites. Cultures so cryopreserved contained at least 5% ring form parasites. P. falciparum. parasites obtained from the preserved stock were maintained at 2–4% parasitemia in sterile culture flasks with filter caps and human A+ washed erythrocytes at 5% hematocrit. A prefiltered mixture of 3% oxygen, 4% carbon dioxide, and 93% nitrogen was run through the suspension, sealed, and incubated at 37°C.

Parasite lactate dehydrogenase (pLDH) assay

The method of Markler and co-workers (1993) was used in the estimation of parasite growth inhibition. Cultures were cryopreserved to contain at least 5% ring-form parasite and were maintained at 2–4% in sterile culture flask with filter caps and complete human A+ washed erythrocytes at 5% hematocrit. This was used in preparing 2% hematocrit by washing with phosphate-buffered saline (PBS) three-times. Stock solutions of extracts were prepared by dissolving known quantities of dried extracts in 1:1 dimethyl sulfoxide and distilled water. Serial dilutions of the extracts/fractions were made in quadruplicate in 96-well microtiter plates. Each well contained 50 µl of the diluted extracts, 50 µl of the complete medium, and 50 µl of infected blood. The extract concentrations tested ranged from 0.5 to 500 µg/ml (10 dilutions). Rows of blank (dimethyl sulfoxide/water, 1:1) and standard antimalarial drug (chloroquine diphosphate, obtained from Sigma Chemical Co., Bradford, UK) were included in the drug plate. The drug plate was placed in the chamber with sterile water in a Petri dish. The plate was placed in the laminar flow chamber (Envair), gassed with a prefiltered mixture of 3% O₂, 4% CO₂, and 93% N₂, then swiftly sealed and incubated at 37°C for 48 h.

Estimation of P. falciparum. growth inhibition

After the incubation period, 50 µl of 3-acetyl pyridine dinucleotide (APAD) reagent were added to each well, followed by 50 µl of National Blood Service (Midlands) (NBS) reagent and finally, after removal of air bubbles, the drug plate was incubated at 37°C for 20 min. Then, optical density was measured in a microplate reader at 550 nm, with which IC₅₀ values were estimated using the Microsoft Excel program. Chloroquine diphosphate was included in the assay as standard reference drug. The IC₅₀ values of the seven plant extracts were determined.

Results and Discussion

The seven plant materials that were evaluated in this study, their plant families, and percentage yields of methanol extracts are listed in Table 1. The in vitro. antiplasmodial properties of all the extracts, using the multidrug-resistant strain of P. falciparum. (K1) expressed as IC₅₀ values in µg/ml, are also presented in Table 1.

Table 1. Antiplasmodial activities of plant methanol extract from the Nigerian Middle Belt using multiresistant P. falciparum. (K1) strain.

Plant/part^a.	Family	Yield (%)	IC50 (µg/ml) ± SEM^b.
Erythrina senegalensis. (s)	Leguminosae	10.4	99.6 ± 1.25
Pericopsis elata. (l)	Papilionaceae	10.9	124.7 ± 4.73
Lophira alata. (s)	Ochnaceae	7.5	156.6 ± 5.70
Bridelia micrantha. (l)	Euphorbiaceae	5.1	158.7 ± 7.82
Cassia alata. (l)	Fabaceae	12.1	407.4 ± 7.24
Adansonia digitata. (s)	Bombaceae	8.2	423.9 ± 8.50
Nauclea latifolia. (s)	Rubiaceae	7.4	479.9 ± 8.12
Chloroquine	—	—	0.21 ± 0.002

^a.s, stem bark; l, leaves.

^b.SEM, n = 10.

In Nigerian ethnomedicine, plants are frequently used in the prevention and treatment of febrile illnesses. A compilation of such plants, used commonly in management of the illnesses in Tivland in the Middle Belt Zone, has been done in a previous study (Ajaiyeoba et al., 2002). Seven of these plant extracts were selected and screened for their in vitro. antimalarial properties, using P. falciparum. (K1 strain; Markler et al., 1993). In the antiplasmodial assay, Erythrina senegalensis. DC (Leguminosae) stem bark possessed the highest activity with an IC₅₀ value of 99.6 µg/ml, whereas Nauclea latifolia. SM (Rubiaceae) had the lowest activity with an IC₅₀ value of 479.9 µg/ml. The details of the antiplasmodial activities of the other plants are presented in Table 1.

E. senegalensis. has been reported to contain isoflavones and prenylated isoflavonoids (Fomum et al., 1994; Wandji et al., 1995; Oh et al., 1999). The stem bark of E. senegalensis. has been reported to show weak antimalarial activity in the rat (Saidu et al., 2000).

From Pericopsis elata. Harms (Papilionaceae), α-hydroxy dihydrochalcones have been reported (Augustyn et al., 1990).

Chalcones from Lophira alata. Bank ex. Gaertn F (Ochnaceae) have been reported as being responsible for possible anti–tumor-promoting properties (Murakami et al., 1992). A variety of steroids, including friedelin, taxarone, and tannins (gallic, ellagic, and caffeic acids) have been isolated from Bridelia micrantha. Benth (Euphorbiaceae) (Pegel & Rogers, 1968).

Cassia alata. Lam (Fabaceae), like all plants of this genus, is known to contain anthraquinones. Of the Cassia. spp. only C.. occidentalis. L. had been reported to show antiplasmodial properties (Benoit et al., 1996; Tona et al., 1999). In another study, the use of C. siamea. bark and leaves, respectively, was mentioned as an antimalarial in the ethnomedicine of southwestern Nigeria (Ajaiyeoba et al., 2003).

Adasonia digitata. syn. A. sphaerocarpa. L. (Bombaceae), commonly known as the baobab tree, has been shown to exhibit antifungal, anti-HIV type 1, and anti-Herpes simplex viruses activities, respectively (Locher et al., 1996; Hudson et al., 2000). The acetone extract of the leaf also displayed weak in vitro. antiplasmodial activity on P. falciparum. (Gessler et al., 1994). The plant has been reported to contain steroids, flavonoids, and other primary metabolites (Shukla et al., 2001).

N. latifolia. had been investigated for antiplasmodial properties in Mali (Benoit-Vical et al., 1998; Traore-Keita et al., 2000). Several indoloquinolizidine alkaloids were isolated from the root bark of the plant (Hotellier et al., 1975).

Currently, no data are available regarding the in vitro. antimalarial properties of the plant materials, except A. digitata. and N. latifolia., although the in vivo. animal antimalarial activity of E. senegalensis. has been reported.

In conclusion, this study has identified a number of plant extracts (Table 1) that have shown in vitro. antiplasmodial activities, based on identification and selection of plants used in the ethnomedicine in the Middle Belt of Nigeria. Though the IC₅₀ values of the methanol extracts of the plants indicated moderate to weak activities for a good number of them using the multidrug-resistant strain of P. falciparum., other bioassay-guided studies are needed to confirm selectivity to malaria parasites. The bioassay-guided fractionation and isolation of active compounds from the active extract is in progress. In addition, toxicological evaluations of active plant compounds in animal models are being assessed.

Acknowledgments

This study received financial support from WHO/TDR/MIM Africa research capability strengthening grant ID 980046. We are grateful to Mr. G. Ibhanesebhor of FRIN, Ibadan, Nigeria, and to Mr. A. Ogunduyilemi of the Botany Department, UI, Ibadan, for plant identification.