Journal of Medical and Applied Biosciences 2010 Cenresin Publications www.cenresin.

org

Volume 2, September 2010

THE ANTIPLASMODIAL ACTIVITY OF BUCHHOLZIA CORIACEA

Okoli, B.J1, Okere. O.S2 and Adeyemo.S.O3

1

Department of Chemistry, 2, 3Department of Biochemistry Bingham University, Karu, Nassarawa State, Nigeria E-mail: okolibj@binghamuni.edu.ng

ABSTRACT The antiplasmodial activity of aqueous extract of Buchholzia coriacea was investigated in intraperitoneally, malaria-induced albino mice using parasitized human group O blood. Experimental control animals were treated with chloroquine while experimental animals were treated with aqueous extracts of Buchholzia coriacea. The experimental animals that were treated with 40mg/kg and 80mg/kg of extract reduced parasitemia level from mean value for five determinations of 79parasites per field on the first day to7parasites per field on the third day and 81parasites per field to 5parasites per field respectively while that treated with 120mg/kg reduced parasitemia level from 80parasites per field on the first day to zero by the third day. During treatment also, appetite level of the experimental animals were considered and it was observed that at the initial days of treatment, when the level of parasitemia was high, correspondingly, their appetite for food was little but this changed as the level of parasitemia decreased. Keywords: antiplasmodial activity, phytochemical analysis, Buchholzia coriacea and aqueous extract INTRODUCTION Malaria is the world's most important parasitic disease especially when Plasmodium falciparum is the causative agent (Fisher and Bialek, 2002). Malaria is endemic in about 100 developing countries, accounting for about 40 to 45 million and kills an estimated 1.2 million people each year in Africa (WHO, 2001). In Sub-Saharan Africa, one in five children will die before they are five and 75 % of those deaths are attributed to malaria (Nwaka, 2005). Pregnant women and children under five years of age are the most vulnerable. The socioeconomic consequences of this disease are particularly dramatic in rural areas where poverty and malnutrition are more pronounced. In the absence of an effective vaccine, the fight against malaria depends on chemotherapy and the reduction and prevention of human/Anopheles mosquito contacts through the use of insecticides treated bed nets, insecticides and environmental care. Resistance of P. falciparum to commonly used anti-malarial drugs is increasing in Nigeria as in other parts of Africa (Mbacham et al., 2004). This has resulted in resurgence in transmission and an increase in adverse outcomes due to therapy failure. Hence, new highly efficacious anti-malarial agents are urgently needed. For thousands of years, plants have constituted the basis of traditional medicine systems and recently, natural products have been a good source of lead compounds for drug development. A good example of such compound used against malaria is quinine (1), isolated from Cinchona bark (Betti,J.L, 2004), which was used as a template for the synthesis of chloroquine and mefloquine. More recently, artemisinin (2) isolated from the Chinese plant Artemisia 21

The Antiplasmodial Activity of Buchholzia Coriacea

Okoli, B.J, Okere. O.S and Adeyemo, S.O

annua, has been used successfully against chloroquine-resistant P. falciparum strains

(Schwikkard and Van Heerden, 2006).

In West Africa, a large number of plant species have been identified as anti-malarial medicinal plants. Pure products have been isolated from some of these plants amongst which are those whose anti-malarial activities are comparable to or more active than chloroquine on sensitive and resistant strains of P. falciparum (Tane et al, 2005). It is therefore imperative that anti-malarial drug development has to be pursued further, with these highly active products, to preclinical, clinical testing, manufacture and distribution and then finally to post marketing pharmacovigilance. Buchholzia coriacea belongs to the Capparaceae family and was named after R. W Buchholz who collected plants in Cameroon in the late 19th century (Keay et al., 1989). These seeds gave the plants its common name of wonderful kola because of its usage in traditional medicine. The seeds are covered in a purple aril which is chewed in Ivory Coast and has a sharp pungent taste. Burkill (1985) reported the medical uses of this plant such as the treatment of malaria and other fevers. The grained seeds have medicinal potential and when it is mixed with palm oil and taken orally as treatment for malaria (Adjanohoun et al, 1996).

Buchholzia coriacea has been used for years to meet a variety of illnesses; since it has been used continually over many generations it is likely that wonderful kola actually has an effect against illnesses. As a result of its supposed broad-spectrum activity, there is need to conduct studies on potential utilization of wonderful kola as an anti-malarial drug. The objectives of this study are to determine the proximate and phytochemical composition of B. coriacea and evaluate the effect of its extracts on malaria parasite.
MATERIALS AND METHODS Preparation of Crude Extracts Fresh seeds of wonderful kola (Buchholzia coriacea) were bought from a herbalist and trader in trado-medicinal herbs at Karu market, (a suburb near Abuja) and were identified by Mrs. Jemilat Ibrahim of the Herbarium department, National Institute for Pharmaceutical Research and Development, Idu. The fresh wonderful kolanuts were cleaned by the double disinfection method. They were washed thoroughly with distilled water to remove adhering particles after which they were soaked in 80% ethanol for 30 min. They were rinsed with distilled water and then washed with aqueous sodium hypochlorite (NaClO4) to reduce surface contamination. This was followed by rinsing with distilled water. The kolanuts were diced to facilitate drying at room temperature. The dried kolanuts were pulverized using a hammer mill. The powder was stored in polyethylene bags to prevent moisture absorption and contamination. 22

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Exactly 100g of the powdered seeds were dissolved in 1litre of distilled water and allowed for 72 hours. Afterwards, the mixture was then stirred vigorously and sieved to obtain the extracts. It was further centrifuged at 4000 rev/min for 10 mins to remove impurities and then stored in a refrigerator. Proximate analysis of B. coriacea Moisture content, crude protein, crude fat, ash, and carbohydrate were determined using (AOAC) 1990 method. Ash Content Determination 7.5 g of the crude extracts were put into a weighed crucible and incinerated in an oven at 300C for 3 days. The weight after incineration was taken. The Ash content was determined using the formula; %Ash content = loss in weight after incineration 100 Initial weight of crucible and extract Carbohydrate Determination This was determined using Benedicts reagent method; 5 drops of the crude extract was added to 2 ml of Benedicts reagent and place in a boiling water bath for 5minutes. A corresponding rust-brown colour was observed indicating the presence of Carbohydrate. Crude Fat Crude fat was determined by deffating a known weight of the seed sample in 25 ml petroleum ether for 30 mins. The supernatant was decanted into weighed crucibles and oven dried for 45 mins at 103C. %Crude fat = loss of weight of supernatant 100 Weight of sample used Crude Protein Presence of protein was determined by adding 1.5 ml of Biuret reagent was added to 1ml of the extract; it was mixed and allowed to stand for 15 mins. A corresponding light purple colour was observed, indicating the presence of protein. Moisture Content In determining the moisture content, exactly 10 g of the extract was dried at 103C to a known weight in an oven. %Moisture content = loss in weight after drying for 2 hrs 100. Phytochemical screening Chemical tests were carried out on the aqueous extract and on the powdered specimens using standard procedures to identify the constituents as described by Sofowara (1993), Trease and Evans (1989) and Harborne (1973). Test for tannins: About 0.5 g of the dried powdered samples was boiled in 20 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed forbrownish green or a blue-black colouration.

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The Antiplasmodial Activity of Buchholzia Coriacea

Okoli, B.J, Okere. O.S and Adeyemo, S.O

Test for saponin: About 2 g of the powdered sample was boiled in 20 ml of distilled water in a water bath and filtered. 10ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, then observed for the formation of emulsion. Test for flavonoids: A portion of the powdered plant sample was heated with 10 ml of ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. A yellow colouration was observed indicating a positive test for flavonoids. Test for steriods: Two ml of acetic anhydride was added to 0.5 g ethanolic extract of each sample with 2 ml H2S04. The colour changed from violet to blue indicating the presence of steroids. Test for terpenoids (Salkowski test): Five ml of the extract was mixed in 2 ml of chloroform, and concentrated H2S04 (3 ml) was carefully added to form a layer. A reddish brown colouration of the inter face was formed to show positive results for the presence of terpenoids. Test for cardiac glycosides (Keller-Killani test): 5ml of the extracts was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1 ml of concentrated sulphuric acid. A brown ring at the interface indicates a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer. Test for Anthraquinones : 0.5g of the extract was boiled with 10ml H2S04 and filtered while hot. The filtrate was shaken with 5ml of chloroform. The chloroform layer was pipette into another test tube and 1 ml of dilute ammonia was added. The resulting solution was observed for colour change. Test for Alkaloids: 0.5g of the powdered extracts was stirred in 5ml of 1%HClaq on a steam bath for 5mins. The mixture was then filtered using Whatmans no1 filter paper. To the filtrate, 2-4drops of Dragendoffs reagent was added to 1ml of the filtrate. An orange colour was observed indicating the presence of alkaloids. Experimental design A total number of 30 albino mice of an average weight of 30.75g, obtained from National Veterinary Research Institute, (VOM) was used for the experiment. They were distributed into 5 groups (A, B, C, D, and E) with a population of 5 mice in each group. Group A represents the group of healthy mice without treatment. Group B represents the group of mice induced with malaria and treated with chloroquine. Groups C, D and E represent the group of mice induced with malaria and treated with 40mg/kg, 80mg/kg and 120mg/kg respectively of the extract. Their level of parasitemia was also closely monitored beginning from twenty-four hours after induction for three days.

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Induction of Malaria Parasite Prior to induction of malaria parasite into experimental animals, malaria parasite was absent in a thick film of their blood sample. This changed within 24hrs of intraperitoneal administration of parasitized blood to a maximum value of 80. Exactly 0.2ml of parasitized human blood of blood group O obtained from Bepos clinic and maternity in Masaka (a suburb in Abuja) was injected into the mouse of about 30.75g and observed for 3 days (72 hours) for general symptoms of malaria. The tail was punctured from which a representative quantity of blood was obtained and a thick blood film for malaria parasite was prepared. On examination under the light microscope (10 resolution), several malaria parasites were found, showing that the induced mouse was positive for malaria parasite. The parasitized mice was sacrificed and from it, blood was obtained and diluted using normal saline in the proportion of 9:1 (9 ml of blood: 1ml of normal saline), with which the rest of the mouse were induced with malaria parasite. Treatment of experimental animals After confirming the presence of malaria parasite in the experimental animals, treatment was done using chloroquine and aqueous extracts of Buccholzia coriacea as known control and experimental treated group respectively. 0.1ml of chloroquine was administered to the normal control animals and 40mg/kg, 80mg/kg, and 120mg/kg of the extract was administered to the experimental animals. The administration of both chloroquine and Buccholzia coriacea extract lasted for 3 days. Determination of parasitemia level in experimental animals The parasitemia level in experimental animals was determined using the Malaria-parasite determination technique; which requires preparing a thick or a thin film of the blood on a glass slide and viewing in oil immersion under a microscope. The parasitemia level was determined once daily and was closely monitored for 3 days. RESULTS AND DISCUSSION Table I showed the proximate composition of fresh B. coriacea. Values obtained for ash, fat crude fibre, protein and moisture content of B. coriacea showed that its incorporation in foods as an additive can improve the nutritional composition of such foods. The observed increase in food-in-take by the experimental animals is because it is a good appetizer (Table V) Phytochemical screening shows the presence of bioactive components in the plant extracts which has ethnopharmacological significance and these are tannins, saponins, alkaloids and cardiac glycosides (Table II). It has been reported that some classes of alkaloids, saponins and tannins are active antifungal agents and have strong microbial activity (Konkwara, 1976 and Sodipo, 1991). Cleone rutidosperma from the same family was investigated for its anti-malarial properties by Bidla et al (2004) and a 31.6% inhibition of P. falciparum growth was observed in vitro in the presence of 40 g/mL of chloroform/methanol (1:1) extract of the plant.

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The Antiplasmodial Activity of Buchholzia Coriacea

Okoli, B.J, Okere. O.S and Adeyemo, S.O

Table I: Proximate composition of Buchhlozia coriacea

Parameter Moisture content Crude protein Crude fat Crude fibre Carbohydrate Ash

Composition (%) 53.13 0.5 13.22 0.4 2.20 0.1 3.46 0.2 71.32 0.4 9.8 0.2

Effect of Extract on Experimental Animals All 3 dosages produced a remarkable decrease in the level of parasitemia in the animals (fig I). After 3 days of consumption of 120mg/kg body weight water extract of Buccholzia coriacea, the parasitemia level was significantly and drastically reduced(Table III).
Table 1: Qualitative analysis of the phytochemical constituents of Buchholzia coriacea

Phytochemicals Alkaloids Anthraquinone Cardiac glycosides Flavonoids Glycosides Saponin Tannin + = present

Aqueous seed extract + ++ ++ ++ + ++ + +++ + ++ +++

Table III: Level of parasitemia per field in experimental animals

Groups Day 1 Normal Nil Experimental 80 2.06 control Group C 792.32 Group D 811.72 Group E 802.28 Values are mean for five determinations SD

Day 2 Nil 152.15 251.47 192.09 172.60

Day 3 Nil 101.62 71.85 51.41 00.4

Level of parasitemia and food intake in experimental animals for 3 days of treatment The level of parasitemia alongside food intake was closely monitored for the 3 days of treatment and the level of parasitemia was highest on the first day of treatment and the least amount of food was consumed on day 1(Fig II). A bit of sluggishness or tiredness was observed in the animals also. The last day of treatment recorded the highest amount of food consumed (Table V) and the least number of parasitemia (Table IV). Experimental groups treated with 40mg/kg, and 80mg//kg recorded few troughs of parasitemia while the group treated with 120mg/kg recoded a total eradication of the parasitemia on the third day.

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Table IV: Level of Parasitemia per field in Experimental animals

Mean value day 1 Mean value day 2 Mean value day 3

Experimental control 80 15 10

Group C 79 25 7

Group D 81 19 5

Group E 80 17 0

Parasites per field

Fig I; Graph of parasitemia per field against number of days

Amount of food consumed (g)

Fig II; Graph of Amount of food consumed (g) by experimental animals against no of days.

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The Antiplasmodial Activity of Buchholzia Coriacea

Okoli, B.J, Okere. O.S and Adeyemo, S.O

Table V; level of food intake

Normal Experimental Group control C Average amount 10 consumed/mouse(g) day 1 Average amount 10 consumed/mouse(g) day 2 Average amount 10 consumed/mouse(g) day 3 0.9 1.2 6.1 0.8 2.0 5.6

Group D 0.7 2.4 7.1

Group E 0.6 2.8 8.2

The observation in table IV may be as a result of the presence of alkaloids which has been reported to have anti-malarial property (Adjanohoun et al, 1996). The first day of treatment recorded the highest number of parasitemia per field of about 80. This figure dropped to about 0 parasitemia per field on the third day (Table IV). Animals treated with chloroquine, 40mg/kg and 80mg/kg of extracts were seen to still have few troughs (Fig I) of parasitemia on the third day, which means that the dosages were not potent enough to clear the parasitemia. Animals treated with 120mg/kg of extract were seen to have no deposits of parasitemia in them on the third day which means that the dosage was the most effective. Nevertheless, none of the animals treated with the plant extract died within the three days of treatment which means that Buchholzia coriaceais not toxic at the administered dose. CONCLUSION

Buchholzia coriace demonstrated antiplasmodial activity in the experimental animals (Table IV).The implication of this is that B. coriacea can serve as a potential anti-malarial
drug source with a strong appetite disturbance (Table V) and immunogenic properties. This study also indicates clearly that B. coriacea possesses an invaluable but yet to be tapped potentials which, if exploited, will benefit the pharmaceutical industry. The development of new anti-malarial drug from highly active natural products, which have already been discovered, is crucial in order to overcome the increasing resistance of Plasmodium to available anti-malarial drug. Therefore, there is a need to advance the work on B. coriacea which have already been shown to have anti-malarial activity through further in vitro and in vivo testing in animal models of malaria followed by sub acute and chronic toxicity tests. This is likely to reveal suitable candidate molecules which may serve as leads which can be optimized followed by development into new anti-malarial drug. This task will require capacity building in the various facets of drug development required integrated, inter-disciplinary approaches. REFERENCES Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enoworock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu, K. J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.C., Mbome, J.C., Muberu, N.K., Nancy, W.L., Kongmeneck, B., Satabie, B., Sowora, A., Tamze, V. and Wirmum, C.K.

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(1996).Traditional Medicine and Pharmacopoeia: Contribution to ethno botanical and floristic studies in Cameroon. Ed. Organization of African Unity; Scientific, Technical and Research Commission. AOAC (1990). Official methods of analysis, (13th edition). Association of Official Analytical Chemists. Washington, DC. Betti, J.L. (2004). An ethnobotanical Study of medicinal plants among the Baka pymies in the Dja biopher reserves, Cameroon. African Studies Monographs. .25 (1): 127. Bidla, G., Titanji, V.P.K., Jako, B., Ghazali, G.E. Bolad, A. and Berzins, K. (2004). Antiplamodial activity of seven plants used in African folk medicine. Indian J. Pharmacol. .36: 245-246. Fisher, P.R and Bialek, R. (2002). Prevention of malaria in children. Clin. Inf. Dis. 34: 493-498 Fluck, H. (1973). Medicinal Plants and Their Uses. Feulsham and comp ltd, New York, 715. Harborne, J.B., (1973). Phytochemical methods, London. Chapman and Hall, Ltd. pp. 49-188. Konkwara, J. O., (1976), Medicinal Plant of East Africa. Literature Burea, Nairobi;3-8. Mbacham, W., Roper, C., Targett, G. and Grenwood, B. (2004). Antimalarial drug resistance in Cameroon. Nwaka, S. (2005). Drug discovery and beyond: The role of public-private partnerships in improving access to new malaria medicines. Trans.Royal Soc. Trop. Med. Hyg. 99: 20-29. Schwikkard, S. and Van Heerden, F.R. (2006). Antimalrial activity of plant metabolites. Nat. Prod. Rep. 19: 675-692. Sodipo, O.A, Akanji, M. A., Kolawole, F .B,. Odutuga A.A (1991), Saponin is the active anti- fungal principle in Garcina kola ,heckle seed ,Biological science research community 3(1);171 Sofowara,A.,(1993). Medicinal plants and Traditional medicine in Africa. Spectrum Books Ltd, Ibadan, Nigeria. p. 289. Tane, P., Wabo, H., Tchimene, M., Okunji, C., Iwu, M., Schuster, B. and Connolly, J. (2005). Lead antiplasmodial substances from some Cameroonian medicinal plants. Abstract/Acta tropica 96S (2005), pp S1-S506. Trease GE, Evans WC (1989). Pharmacognsy. 11th edn. Brailliar Tiridel Can. Macmillian publishers. WHO. World Health Report 2001. 29