Research Article - Asian Journal of Biomedical and Pharmaceutical Sciences (2017) Volume 7, Issue 61

In Vivo Antiplasmodial Evaluation Of Methanol Leaf Extract and Fractions of Chrysophyllum albidum G. Don (Sapotaceae)

Ezenwa CJ¹, Onyegbule FA², Umeokoli BO² and Osonwa UE³

¹Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria

²Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria

³Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria

*Corresponding Author:

Onyegbule FA
Department of Pharmaceutical and Medicinal Chemistry
Faculty of Pharmaceutical Sciences
Nnamdi Azikiwe University, Awka, Nigeria
E-mail: af.onyegbule@gmail.com

Accepted on June 07, 2017

Abstract

Malaria is a disease of major concern that has affected millions of people especially in the Sub-Saharan Africa. There is an urgent need for development of alternative treatment means for malaria gives the emergence of resistance and adverse effects to current agents. This study evaluated the in vivo antiplasmodial activity of the methanol leaf extract and fractions of Chrysophyllum albidum. Dried C. albidum leaves were cold macerated in methanol. The median lethal dose (LD50) was evaluated in mice. 54 albino mice grouped into 9, were used for the study. Animals were treated with 125, 250 and 500 mg/kg of methanol leaf extract of C. albidum, 500 mg/kg each of n-hexane, ethyl acetate, butanol and aqueous fractions of C. albidum leaf; Arthermeter:lumefantrine (0.73:4.4 mg/kg) and 5 ml of distilled water respectively. Biochemical and hematological effects of C. albidum leaf extract were also evaluated. The LD50 was calculated to be 2739 mg/kg body weight. The results revealed that the methanol extract, ethyl acetate, butanol and aqueous fractions exhibited significant (P<0.05) reduction in percentage parasiteamia. There were no significant (P<0.05) changes in the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphate, bilirubin, creatinine, urea, white blood cell count, red blood cell counts, heamoglobin and packed cell volume. The result of this study validates the use of C. albidum in ethnomedicine for malaria treatment. However, further studies need to be done to identify and characterize the active principles presence. This study may be useful as lead in future development of antimalarial drugs.

Keywords

Antiplasmodial; Chrysophyllum albidum; Toxicity; Biochemical parameters; Hematological analysis

Introduction

Malaria is an infectious disease that is usually caused by a parasite (protozoan) of the genus plasmodium (WHO, 2014). Although the disease is life threatening, it can be controlled and treated if diagnosed early. Five species of plasmodium are implicated in the infection and spread of malaria parasite and these include Plasmodium vivax, Plasmodium ovale, Plasmodium falciparium, Plasmodium malariae and Plasmodium knowlesi [1]. P. vivax, P. ovale, P. malariae causes severe malaria, and most malaria death is due to them [1]. Malaria risk is peculiar to some groups in the population, including infants, children below 5 years, pregnant women, non-immune travellers from non-malaria endemic regions to malaria endemic regions and immune compromised patients (HIV) [2-4]. Malaria is prevalent in tropical and subtropical regions because of rainfall and many favourable factors [5,6].

In Nigeria, malaria accounts for most cases death with 97% of the population being at risk. There is an estimate of about 100 million malaria cases with over 300,000 deaths per year in Nigeria. It contributes to an estimated 11% of maternal mortality and accounts for 60% of outpatient visits and 30% of hospitalizations among children under five years of age in Nigeria [7].

Malaria has a negative impact on the economy [8-10]. Despite the rising deaths due to malaria, no effective vaccine is in existence for the treatment of malaria, prevalence of toxicities associated with current agents, emergence of resistant strains of parasite to several drugs such as chloroquine resistant P. falciparium and artemisinin resistance in some parts of the world like southern Asia [1]; abound.

In the last few years, there has been an exponential growth in the field of herbal medicine, and these drugs are gaining popularity both in developing and developed countries. This increase in popularity may be due to their natural origin, insufficient drug supply, increase in population size, high cost of orthodox drugs, side effects and also increase in the rate of development of resistance to currently used medication [11]. The importance of plants in traditional medicine cannot be overemphasized and these plants remain valuable for many people in Sub-Saharan Africa.

Chrysophyllum albidum, commonly known as white star apple, belongs to the family of Sapotaceae [12-14]. It is a forest tree species that usually grows up to 25 to 37 m in height [15]. The fruit is ovoid to sub-globose, pointed at the apex, and up to 6 cm long and 5 cm in diameter (Figure 1), the skin or peel, is orange to golden yellow when ripe and the pulp within the peel may be orange, pinkish, or light yellow when it is ripe [16]. Chrysophyllum albidum has many ethnomedicinal uses. The bark is for treating yellow fever and malaria in folklore medicine [14]. Its leaves are used as emollient and for the treatment of malaria, stomach ache and diarrhoea. Also, its leaves and seed cotyledons are used as ointments in treating vaginal and dermatological infections in Western Nigeria [17]. Seed and root extracts of C. albidium are used to arrest bleeding from fresh wounds, and to inhibit microbial growth of known wound contaminants and also enhance wound healing process as they have astringent characteristics [18].

Figure 1. pictures of Chrysophyllum albidum fruit, tree and seed

There is a high level of treatment failure to conventional antimalarial drugs. Despite significant progress in the treatment of malaria, this disease has staged a huge comeback in large areas of the world, due to the development of drug resistant parasites. The progressive spread of P. falciparium resistance to antimalarial drugs poses a serious threat to malaria control program. In addition, there is lack of scientific information on effect of C. albidum as anti-malarial drug. There is therefore urgent need for alternative approach. Thus, Chrysophyllum albidium used in traditional treatment of malaria was evaluated for efficacy against the Plasmodium berghei infected mice. The usefulness of this medicinal plant may hold the key to new and effective alternative antimalarial medicine.

Materials and Methods

Materials

Solvents: Analytical grades of n-Hexane (JHD, Guangdong), Ethyl acetate (JHD, Guangdong), Methanol (JHD, Guangdong) and Butanol (JHD, Guangdong) were used. Fresh distilled water was used when required.

Parasites: Chloroquine-sensitive P. berghei was obtained from the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka.

Drugs: Arthemether: lumefantrine, Lonart^®, (Bliss GVS Pharma, Ltd, India) was used.

Plant material: The fresh leaves of Chrysophyllum albidum were used.

Equipment: These include oven (Costab Sci – Tech, China), hot plate (Jenway, UK), rotary evaporator (Balloworld Scientific Limited, UK), vacuum pump (GEC Electromotors, UK), furnace (Vestor Furnace, Germany), weighing scale (Ohaus, China) and UV/visible spectrophotometer, Jenway).

Animals: Albino mice (25–30 g) were used for the study. The animals were obtained from the animal house of Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka. Animals were allowed free access to food and water. Animals were maintained in standard laboratory animal conditions [19].

Methods

Collection of plant material

Fresh leaves of C. albidum were collected from its natural habitat at Adagbe Avomimi village, Enugwu-ukwu, Njikoka Local Government area of Anambra State, Nigeria in August 2015. It was identified by Mrs. Anthonia Emezie, a senior technologist in Department of Pharmacognosy, of Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka. A voucher specimen PCG474/A/043 was deposited at the Herbarium of the Department of Pharmacognosy, of Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka.

Preparation of plant material

The plant material was dusted and air dried for 3 weeks and then ground to powder using a dry laboratory electric milling machine. The powdered material was stored in air tight container before use.

Extraction and fractionation of plant material

The powdered plant material (2.0 kg) was cold macerated in methanol for 48 h with intermittent shaking. The resulting solution was filtered using Whatman No 1 filter paper and the filtrate concentrated using rotary evaporator at 40°C. A solid residue yield was obtained and referred to as the extract. Then 40 g of the extract was subjected to liquid-liquid partitioning successively with n-hexane, ethyl acetate, butanol and distilled water. The fractions were filtered and concentrated using rotary evaporator at a temperature of 40°C.

Acute toxicity test

Acute toxicity of the leaves extract was carried out using 32 mice grouped into eight groups A-H (n=4). Groups A, B, C and D received 100 mg/kg, 500 mg/kg, 1000 mg/kg and 2000 mg/kg of methanol extract of C. albidum leaf respectively, while groups E, F and G respectively received 3000 mg/kg, 4000 mg/kg, 5000 mg/kg of methanol extract of C. albidum leaf, while group H received 5 ml of distilled water as a control. The extract was dissolved in distilled water and was orally administered to the different groups after an overnight fast. The number of deaths in each group within 24 h was recorded. The animals were left for further 14 days for any delayed signs of toxicity. The LD₅₀ was calculated according to Lorke [20].

Antimalarial study

This was carried out according to the method described by Knight et al. [21]. Fifty four albino mice grouped into nine groups A - I (n=6) were used for the study. Groups A, B, C, received 125, 250 and 500 mg/kg of methanol leaf extract of C. albidum respectively, groups D, E, F and G received 500 mg/kg n-hexane fraction, 500 mg/kg ethyl acetate fraction, 500 mg/kg butanol fraction and 500 mg/kg aqueous fractions of C. albidum leaf, group H received 0.73:4.4 mg/kg of arthermeter: lumefantrine (ACT) as positive control, while group I received 5 ml of distilled water as negative control. All animals were infected with P. berghei by single intra peritoneal administration of 0.2 ml of infected blood diluted with normal saline such that it contains 1 × 10⁶ infected red cells. The animals were left for 72 h for the infection to be established. A thin blood film was made from the tail vein of each animal to confirm parasitaemia. These mice were treated with C. albidum methanol extract and fractions, arthemether: lumefantrine (Lonart^®) (both dissolved in distilled water) and an equivalent volume of distilled water (negative control) for 4 consecutive days. The treatments were given orally once daily to the appropriate group. The level of parasitaemia was examined on the 4^th and 7^th day post treatment. Percentage parasitaemia was determined using standard laboratory procedures described by Knight et al. [21].

Serum biochemistry and hematological analysis

Blood samples from animals were collected into plain tubes to obtain sera for biochemical analysis as follows; Serum activities of alanine aminotransferase (ALT), alkaline phosphate (ALP), aspartate amino transferase (AST) and urea were measured as described Teco laboratory kit, serum creatinine was estimated as described by Randox kit while haemoglobin was estimated using Sahli’s method. Packed cell volume and white blood cell counts were also evaluated.

Alanine aminotransferase (ALT) and aspartate amino transferase (AST)

R1 and R2 (in the kit) were mixed at the ratio of 5:1. Fresh tubes were labeled samples and blanks respectively. To all the tubes, 1.0 ml of working reagent was dispensed and incubated for 5 min at 37oC. To the samples, 100 μl of each sample was added respectively and to the blank, 100 μl of distilled water was added. The absorbance of was read at 1 min and at 3 min using a spectrophotometer at 340 nm. Then the ALT and AST were calculated as follows:

The same procedure was used to determine AST.

Alkaline phosphate (ALP)

Fresh tubes were labeled standard, sample and blank respectively. 0.5 ml of alkaline phosphate substrate (in the kit) was dispensed in all the tubes and was equilibrated to 37oC for 3 min using water bath. To the standard, 50 ug of the standard reagent (in the kit) was dispensed to the sample, 50 μg of the sample was dispensed and to the blank 50 μg of de-ionized water was dispensed. The whole tubes were mixed gently and incubated for 10 min at 37°C. Then 2.5 ml of alkaline phosphatase colour developer (in the kit) was dispensed in all the tubes and was mixed properly. The absorbance was read at 600 nm using spectrophotometer. Then the concentration of ALP was calculated as follows:

Estimation of white blood cell count

The fresh blood sample was pipetted to the 0.5 mark on the white blood cell count pipette. It was made up to the 11 mark by sucking up the white blood cell diluting fluid (Turk’s fluid). The content of the pipette was mixed to assure even distribution of cells by rocking the pipette. Four to five drops of the fluid was discarded. The two sides of a clean hemacytometen were filled by touching the pipette to the edge of the cover glass. The cells were allowed to settle for about two minutes before counting. The cells were counted using low power (X10) objective microscope, counting in four large corner squares. The white cells lying within each of the four corner squares were counted using the left to right pattern. The values were expressed as x10³/mm³

Estimation of packed cell volume (PCV).

Capillary tube was placed in the EDTA container containing the blood sample and was allowed for the blood to suck up to 70% of the entire length of the tube by capillary action. Then one end of the tube was sealed with plasticine. Then the tubes were placed in the microhematocrite centrifuge making sure the sealed end was placed against the rubber gasket. Then the centrifuge was closed, put on and was allowed to centrifuge at 1200 rmp for 10 min. then the value was read using microhematoctite reader.

Estimation of heamoglobin (Sahli’s Method)

The dilution tube in the Sahli’s apparatus was filled to mark 10 with fresh 0.1N HCl. Blood was sucked up to 20 mm³ of the pipette. Then the blood was blown into the HCl and was mixed well and then allowed to stand for 5 min. Then distilled water was added drop by drop from a pipette stirring with a glass rod after each drop until the colour was just like that of the standard. Distilled water was added again until the colour became paler than that of the standard. The average of the two readings were taken, the values are expressed in mg/dl.

Estimation of urea

Fresh tubes were labeled samples, blank and standard. To all the tubes, 1.5 ml of urea enzyme reagent was added, allowed to equilibrate to room temperature. To the tubes labeled samples, 10 μl of serum from treated animal was added. To the blank, 10 μl of de-ionized water was added, and to the standard, 10 μl of the standard reagent (in the kit) was added. The tubes were mixed properly and incubated for 10 min at room temperature. Then 1.5 ml of urea colour developer (in the kit) was added to all the tubes and mixed gently. Then the tubes were again incubated for 10 min at room temperature. The absorbance of the samples and standard were read at 600 nm against the blank using spectrophotometer. Then the concentration was calculated using the formula below.

Estimation of creatinine (Randox Kit)

Equal volumes of R1a and R1b in the Randox^®kit were mixed and termed reagent. Fresh tubes were labeled samples and standard. 1.0 ml of the reagent was pipetted to all the tubes. Then to the tubes labeled sample, 0.1 ml of sample was added, and to the standard, 0.1 ml of standard reagents was added. The mixture was mixed properly and the absorbance was taken after 30 seconds (A1) and after 3 min (A2) for both samples and standard at wavelength of 600 nm. Then the concentration of the creatinine was calculated as follows:

Statistical analysis

Data obtained from the study were expressed as the mean ± SEM. Statistical comparisons between the groups were made using the one way analysis of variance (ANOVA). The level of significant difference between the groups was evaluated at P<0.05 at each level.

Results

Result of acute toxicity test

One mouse in the groups treated with doses from 3000-4000 mg/kg of C. albidum died. The oral LD₅₀ of the plant extract in mice was calculated to be 2739 mg/kg.

The result of the LD50 of 2739 mg/kg of mice signifies that the plant may be toxic at high doses. The result of this present study is similar to those reported by Adebayo et al. [15].

The results of the antimalarial study as shown in Figures 2 and 3, showed that the methanol extract of leaf of C. albidum exhibited a dose dependent significant reduction in percentage parasiteamia (P<0.05) in the groups treated with 125 mg/kg, 250 mg/kg and 500 mg/kg of C. albidum methanol leaf extract as compared to positive and negative controls. The groups treated with 500 mg/kg body weight of C. albidum methanol leaf extract showed the highest reduction in parasiteamia level on both 4th and 7th days post treatment, while the groups treated with 125 mg/kg body weight of C. albidum methanol leaf extract showed the least reduction in level of parasitaemia. The groups treated with 500 mg/kg of ethyl acetate fraction, 500 mg/kg of butanol fraction and 500 mg/kg of aqueous fraction of C. albidum methanol leaf extract also showed a significant reduction in percentage parasiteamia (P<0.05) as compared to positive and negative controls. The groups treated with 500 mg/kg of n-hexane fraction of C. albidum leaf did not show any significant reduction of level of parasiteamia compared to the negative and positive control. ACT (Lonart^®); used as the positive control also showed significant reduction in level of parasiteamia (P<0.05).The negative control group showed an increase in parasiteamia on 4th, reaching 11.5% on the 7th day post treatment. Also, the ethyl acetate fraction appears to possess the highest activity followed by butanol fraction and then aqueous fraction. The percentage parasiteamia reduction shown by the fractions were similar, however, the methanol leaf extract of C. albidum at different doses had a very significant antimalarial activity compared to the fractions. Thus it may be that the reduction in parasiteamia shown by the various doses of the crude methanol extract may be as a result of synergism compared to when fractionated.

Figure 2. Effects of methanol extract of C. albidum leaf on P. berghei berghei induced malarial in mice (Values are expressed as Mean ± SEM, n=6 in each group, * indicate P<0.05 compared tocontrol).

Figure 3. Effects of fractions of C. albidum leaf on P. berghei berghei induced parasiteamia in mice (Values are expressed as Mean ± SEM, n=6 in each group, * indicate P<0.05 compared tocontrol).

The observed reduction in parasiteamia level with methanol leaf extract of C. albidum and fractions, except n-hexane fraction, may be as a result of some of the phytochemicals present in the plant as earlier stated. The effects of these phytochemicals such as saponins, alkaloids and flavonoids may be attributed to a single or combination of their effects. In addition, the mechanism of antiprotozoal activity of triterpenoids and steroidal saponins may be due to their toxicity to protozoa, which may be as a result of their surfactant effect on the cell membrane [22]. Alkaloids have been reported to be toxic to cells of microorganisms such as bacteria, virus and protozoa to which malaria parasite belongs [23], and alkaloid is one of the major phytochemical found in C. albidum leaves. This may be the possible mechanism of antimalarial activity attributed to alkaloids. Unlike in humans, increase in parasitaemia levels in rodent models usually results in decreased metabolic rates and a consequent decrease in body temperatures [24], which might result in death. An ideal antimalarial agent would, therefore, prevent this occurrence, an effect observed in C. albidum treated animal.

The hematology and serum biochemical parameters of the experimental mice are as shown on Tables 1 and 2. No significant changes were observed in the hematological parameters of the groups infected and treated with n-hexane, ethyl acetate, butanol and aqueous fractions of C. albidum leaf extract and ACT as compared to basal (P<0.05). However, the infected and treated mice with distilled water developed moderate anemia (P<0.05) as compared to basal. The result showed that there was no significant change in most of the parameters in the experimental mouse groups. No significant changes (P<0.05) were observed in the WBC in all the groups as compared to basal. Leukocytosis may be related to the presence of infection and the severity of the stress condition [25]. This non-significant change indicates that C. albidum is not infectious to the mice. The non-significant decrease in the levels of RBC, Hb, PCV and non-significant increase in WBC may be an indication that the extract does not affect the hematopoietic system when administered orally and at the doses used in this study.

Table 1: Effects of C. albidum leaf extract on hematological profile in mice (Values are expressed as Mean ± SEM. n = 6 in each group, “*” indicate P<0.05 compared to basal. NH=n- Hexane fraction, EAF=Ethyl Acetate Fraction, BF=Butanol Fraction, AF=Aqueous Fraction, ACT= Arthemeter: Lumefantrine, DW= Distilled Water)

Table 2: Effects of C. albidum leaf extract on serum biochemical profile in mice (Values are expressed as Mean ± SEM. n=6 in each group, “*” indicate P < 0.05 compared to basal. NH=n- hexane fraction, EAF=ethyl acetate fraction, BF=butanol fraction, AF=aqueous fraction, ACT=arthemeter: lumefantrine, DW=distilled water)

The elevation of various liver markers may indicate the alteration in the structural integrity of the liver. ALT is a cytoplasmic enzyme found in very high concentration in the liver and an increase of this specific enzyme indicates increase in an induction of mild injury caused by drugs to the liver. AST is less specific than ALT as an indicator of liver function [26]. In this study the fractions of C. albidum did not show any significant changes in the levels of ALT, AST, ALT and bilirubin at the doses used as compared to their basal levels (P>0.05). This may indicate that C. albidum leaves may not be harmful to the liver at the doses used. The findings of this study may correlate with the findings of [15], where ethanol leaf extract of C. albidum was also found not to show any significant changes in the levels of AST, ALT, ALP and bilirubin as compared to the basal levels (P<0.05).

The result of the study showed that there was no significant increase in the level of urea and creatinine in all treated groups. The kidney is the major excretory and osmo regulatory organ of mammals. This makes it a target for most toxic and harmful chemicals by concentrating metabolites to high level, leading to immediate organ failure or delayed malfunctioning [27]. The lack of significant increase of creatinine and urea levels in blood (P>0.05) may indicate that C. albidum leaf may not cause dysfunction of the kidney. The insignificant changes in the hematological and biochemical parameters observed may be attributed to some of the phytochemicals present in the plant. However, these effects may be as a result of the individual or combined effects of these phytochemicals.

Conclusion

In conclusion, the findings of this study support the use of this plant in the traditional treatment of malaria in Nigeria. The ethyl acetate fraction had the highest antimalarial activity. All the fractions and crude extract showed significant antimalarial activity except n-hexane fraction (P<0.05). It was also observed that the extract, at the dosages used, were non-toxic to mice and did not show any significant effects in hematological and serum biochemical parameters.

Recommendation

Isolation and characterization of the active constituents of the ethyl acetate and butanol fractions, to determine their specific mechanism of action, is recommended. Further investigation of the toxicity of this plant to evaluate its effects during long-term administration is recommended.

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