Dauda et al., (2016) Biological and Environmental Sciences Journal for the Tropics 13(2) September, 2016 ISSN 0794 – 9057 BEST JOURNAL 13(2): 55 - 59 Date received: 06th July, 2016 Date accepted: 25th August, 2016 Printed in Nigeria B E S T JO U R N A L IN-VIVO ANTIMALARIAL ACTIVITY OF ETHANOL LEAF EXTRACT OF GLOBIMETULA OREOPHILA (HOOK. F) DANSER AZADIRACHTA INDICA *Dauda, G1., Haruna, A. K1., Musa, A.M1., Hassan, B1., Mohammed, I.M2., Magaji, M. G2. 1 Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University, Zaria. 2 Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria. Corresponding author: dauda.garba@ymail.com ABSTRACT In this study, the antimalarial activity of ethanol leaf extract of Globimetula oreophila against Plasmodium berghei berghei infection was investigated using the suppressive and curative tests in mice. The crude ethanol leaf extract of Globimetula oreophila significantly (p< 0.05) produced a dose dependent chemo-suppression on the Plasmodium berghei in both the early and established infection at doses of 12.5, 25 and 50mg/kg per day (57%, 59.4% and 64%) respectively and 12.5, 25 and 50mg/kg per day (67.6%, 73% and 79%) respectively. The oral median lethal dose was estimated to be greater than 5000mg/kg body weight, suggesting the crude leaf extract is practically non-toxic. The finding of the study suggests that the crude ethanol leaf extract of Globimetula oreophila possesses significant in vivo antimalarial activity, which might be due to the phytochemical constituents. This provides some scientific rationale for the ethnomedicinal claim of the use of the plant in the treatment of malaria. Keywords:Anti-Malarial, suppression. Globimetula oreophila, Leaf extract, Plasmodium berghei,Chemo- plant leaves are used in the treatment of leprosy, eye problem, intestinal worm, skin ulcers and malaria (Kenichi et al., 2006).Studies have revealed that the host plant could significantly affect the chemical composition and thus, the biological activity of the hemi-parasite mistletoe (Adesina et al., 2013; Arzu et al., 2012).This study was carried out toscientifically validate the in vivo antimalarial activity and acute toxicity of the ethanol leaf extract of Globimetula INTRODUCTION Malaria is one of the most important health problems around the world, which occurs in all six World Health Organization regions. Globally, an estimated 3.2 billion people are at risk of being infected with malaria and developing the disease, and 1.2 billion are at high risk (>1 in 1000 chance of getting malaria in a year). The WHO African Region has the heaviest malaria burden with an estimated 90% of all malaria deaths with children aged less than 5 years, accounting for 78% of all deaths (WHO, 2014).In Nigeria,malariaisresponsible for 63% of all clinicattendances, 25% of infant mortalityand30% of all childhood deaths and 11% of all maternaldeathsand70.5% of morbidity in pregnantwomen (John, 2008).Globimetula oreophilacommonly called mistletoe is a parasite on trees and shrubs forming a bush to about 1m diameter.It is referred to as ‘afomo onisana’ in Yoruba,Kaúcì in Hausa and Children’s matches in Eastern Cameroon (Adesina et al., 2013).These parasitic plants, mistletoes of the Loranthaceae are widely used in various culture to treat various ailments including hypertension, cancer, diabetes and as diuretic agent (Burkill, 1985; Adodo, 2004; and Jadhav et al., 2010). For example, the tea made from Loranthaceae family is believed to cure bone fracture and body pain (Ken’ichi, et al., 2006).The leaves of Globimetula oreophila are used for the treatment of hypertension, arthritis, heart problems, infertility, and pneumonia. In Northern Nigeria, the decoction of the . oreophila. MATERIALS AND METHODS Collection and Identification The plant sample of Globimetula oreophila parasite on Azadirachta indica comprising the leaves and fruit were collected from Sokoto metropolitan, Nigeria in July 2015.The plant sample was authenticated by a Taxonomist in the Herbarium Section of the Department of Biological Sciences, Ahmadu Bello University Zaria by comparing with herbarium reference voucher specimen (Number 2839). Parasites and Animals A chloroquine sensitive Plasmodium berghei berghei parasites obtained from National Institute of Medical Research (NIMR) Lagos, Nigeria was use to assess the in vivo antimalarial activity and maintained in the Department of Pharmacology and Therapeutics, Ahmadu Bello University Zaria.A 0.2ml of blood containing approximately 1×107parasitized erythrocytes from a donor mouse was used to infect the experimental animals intra-peritoneally (Waako et al., 2005). 55 Dauda et al., (2016) Biological and Environmental Sciences Journal for the Tropics 13(2) September, 2016 Extraction of Plant Material The leaves were air dried under shade and crushed into powder. One thousand five hundred grams (1500g) of the powdered plant materialwas macerated with 70% ethanol with occasional shaking for 92 hours. The mixture was concentrated using rotary vacuum evaporator to afford the crude extract. The crude extract was stored in a desiccator until used. Acute toxicity The method of Lorke (1983) was used in the LD50 determination. In the first phase, mice were divided into three groups each consisting three mice and were treated with the geometric doses of the crude extract at 10, 100 and 1000mg/kg body weight via the oral route and observed for 24hours for signs of toxicity and death. In the second phase, based on the outcome of the first phase, three groups with one mouseeach were treated with doses at 1600, 2900 and 5000mg/kg of the extract. The median lethal dose was calculated using the formula: LD50 In vivo Determination of Antimalarial Activity Test on early malaria infection (4-day suppressive test) The method previously described by Peter(1965) was adopted. After 3hrs of infection with the parasite, twenty-five mice were randomly selected and assigned into treatment groups of five containing five mice in each group. Onday 1 of the experiment, groups 1-3 mice received 12.5, 25 and 50mg/kg body weightof the extract respectively, while the 4th and 5thgroups received 5ml/kg of chloroquine and 0.2ml/kg of distilled water and served as positive and negative controls respectively. The extracts were administered i.proute once daily for four consecutive days. On the fifth day, thin smear was made on microscopic slide by taking a drop of blood from the animal caudal vein. The smears were air dried, fixed in methanol and stained with Giemsa (3%) at pH of 7.2 and viewed under the microscope. The average percentage (%) evaluated as: % Suppression = ISSN 0794 – 9057 parasitaemia suppression was Test on established infection (curative or Rane test) The method previously described by Ryley and Peter (1970) was adopted in this study.A total of twentyfive (25) mice were infected with the parasite and left for 72 hours for parasitaemia to developand they were randomly divided into five groups each containing five mice in each group. On the fourth day of the experiment,groups 1-3 mice received 12.5, 25 and 50mg/kg body weight of the extract respectively, while the 4th and 5th groups received 5mg/kg of chloroquine and 0.2ml/kg of distilled water and servedas positive and negative control respectively. The extract was administered i.p once daily for four consecutive days. On the fifth day, thin smear was made on microscopic slide by taking a drop of blood from the animals caudal vein. The smears were air dried, fixed in methanol and stained with Giemsa (3%) at pH of 7.2 and viewed under the microscope. Percentage parasitaemia suppression was calculated as discussed above. Data Analysis Chemosuppression means obtained from the fourth day suppressive and curative assays were analyzed using one-way Analysis of Variance (ANOVA) (Morgan et al., 2004) followed by post hoc Dunnett test for multiple comparisons.Differences were considered significant atp˂0.05. RESULT Preliminary Phytochemical Screening The preliminary phytochemical screening of the ethanol crude leaf extract of Globimetula oreophila revealed the presence of alkaloids, flavonoids, saponins, carbohydrate (reducing sugar), glycosides and tannins showed in Table 1 Table 1:Phytochemical Screening of the Crude Ethanol Extract (CEE) Leaf Extract of Globimetula oreophila Constituent Carbohydrates Anthraquinones Steroids/Terpenes Glycosides Saponin Tannins Flavonoids Alkaloids Test Molisch Bontrager Liebermann-Burchard Keller-Kiliani Frothing Ferric Chloride Lead acetate Sodium Hydroxide Shinoda Dragendoff Observation + + + + + + + + + Mayer + + = present, - = absent 56 Dauda et al., (2016) Biological and Environmental Sciences Journal for the Tropics 13(2) September, 2016 ISSN 0794 – 9057 Acute toxicity The oral median lethal dose of the extract in mice was found to be greater than 5000mg/kg body weight showed in Table 2. Table 2: Determination of oral median lethal dose (LD50) of the crude extract of Ethanol Extract of Globimetulaoreophila First Phase Dose (mgkg-1) Number of mice used Mortality 10 3 0/3 100 1000 3 3 0/3 0/3 Second Phase Doses (mgkg-1) 1600 2900 5000 Number of mice used 1 1 1 Mortality 0/1 0/1 0/1 The crude ethanol leaf extract of Globimetula oreophila produced a dose dependent activity against Plasmodium berghei. At 12.5, 25 and 50mg/kg of body weight, the crude extract significantly (p<0.05) showed parasitaemia suppression (57, 59.4 and 63.7%) respectively. The standard agent, chloroquine at dose of 5mg/kg produced the hig7hest parasite suppression (84%) compared to the negative control showed in Table 2 for early malarial infection in mice. In the established infection test, the highest parasitaemia inhibition of the crude extract was seen with a dose of 50 mg/kg (79.3%) and the lowest parasitaemia inhibition was with dose of 12.5mg/kg (67.6%) while the standard chloroquine has the highest inhibition of 95.1% at dose of 5 mg/kgcompare to the negative control as shown in Table 3. Table 3: Suppressive Effect of Crude Ethanol Leaf extract of Globimetula oreophila in Early Malarial Infection in Mice Treatment (mg/kg) Average Parasitaemia ± SEM % Suppression Effect CEE (12.5) 11.54±1.10** 57.0 CEE (25) 10.90±0.86** 59.4 CEE (50) 9.73±0.74** Chloroquine (5) Normal Saline (0.2) 4.30±0.96** 26.80 ± 1.77 64.0 84.0 - Data presented as Mean ± SEM, CEE: 12.5, 25, 50mg/kg; n=5, p<0.05*, p<0.01**, Dunnett post hoc test (multiple comparison), CEE (Crude ethanol extract). Table 4:Curative Effect of Crude Ethanol Leaf Extract of Globimetula oreophila in Established Infection in Mice Treatment (mg/kg) Average Parasitaemia ± SEM % Curation Effect CEE 12.5 12.28±2.10** 67.6 CEE 25 10.84±0.91** 73.0 CEE 50 8.16±1.15** 79.3 Chloroquine 5 1.93±1.45** 95.1 Normal Saline 0.2 39.45 ± 2.27 Data presented as Mean ± SEM, CEE: 12.5, 25, 50mg/kg; n=5, p<0.05*, p<0.01**, Dunnett testpost hoc (multiple comparison), CEE (Crude ethanol extract). 57 Dauda et al., (2016) Biological and Environmental Sciences Journal for the Tropics 13(2) September, 2016 Discussion The crude ethanol leaf extract of Globimetula oreophila produced a dose dependent activity against Plasmodium berghei. At 12.5, 25 and 50mg/kg of body weight, the crude extract significantly (p<0.05) showed parasitaemia suppression (57%, 59.4% and 63.7%) respectively (Table 3). The standard agent, chloroquine at dose of 5mg/kg produced the highest parasite suppression (84%) compared to the negative control since chloroquine is a pure antimalarial compound.The ability of the extract to suppress malaria in the study indicates that the extract possesses blood schizonticidal effect. In the established infection test, the highest parasitaemia inhibition of the crude extract was seen at dose of 50 mg/kg (79.3%) and the lowest parasitaemia inhibition was with dose of 12.5mg/kg (67.6%) while the standard chloroquine has the highest inhibition of 95.1% at dose of 5 mg/kg compare to the negative control (Table 4). This further established the presence of bioactive constituents in the leaves of the Globimetula oreophila plant. The percentageparasitaemia suppression exerted by the crude ethanol extract at the lowest dose in curative test was higher than that in the suppressive test perhaps due to the fact that the crude extract was non- selective to proliferative process of the parasite (Salawu et al., 2010). The antiplasmodial effect of the extract in established infection may also be due to its inhibitory effect of the processes arising from high parasitaemia level within the system (Carvalho et al., 1991; Salawu et al., 2010). The pharmacological properties attributed to most medicinal plants have been found to be due to the REFERENCES Adesina, S. K., Illoh, H. C., Imoh I. J. and Imo E. J. (2013). African mistletoes (Loranthaceae); ethnopharmacology, chemistry and medicinal values: an update. African Journal Traditional Complementary to Alternative Medicine. 10(3):161-17 Adodo, A. (2004). Nature Power, A Christian Approach to Herbal Medicine. 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