antiplasmodial activity of the ethanol leaf extract of newbouldia ... - wjpps

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Igboasoiyi et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.647

Volume 6, Issue 04, 50-61

Research Article

ISSN 2278 – 4357

ANTIPLASMODIAL ACTIVITY OF THE ETHANOL LEAF EXTRACT OF NEWBOULDIA LAEVIS Arnold C. Igboasoiyi*, Emmanuel E. Attih and Amarachi P. Egeolu Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, University of Uyo, Uyo, Akwa Ibom State, Nigeria.

Article Received on 24 Jan. 2017,

ABSTRACT Malaria is a parasitic protozoan disease caused by the protozoan

Revised on 14 Feb. 2017, Accepted on 07 March 2017

parasites belonging to the genus Plasmodium. It is a major threat to

DOI: 10.20959/wjpps201704-8716

health and constitutes medical and developmental challenge to individuals, communities and nations. Newbouldia laevis is an

*Corresponding Author

ethno-medicinal plant widely used in the treatment of malaria and

Arnold C. Igboasoiyi

other ailments. Phytochemical screening and antiplasmodial studies

Department of

were carried out on the crude and fractions of the leaf extract of the

Pharmaceutical and

plant using standard procedures. The phytochemical screening

Medicinal Chemistry,

revealed the presence of alkaloids, saponins, glycosides, tannins,

Faculty of Pharmacy, Universityof Uyo, Uyo, Akwa Ibom State, Nigeria.

terpenes, flavonoids and carbohydrates in varying proportions. The crude extract exhibited a dose-dependent antiplasmodial activity against the parasite on all the models. The fractions also exhibited

antiplasmodial activities with the ethyl acetate fraction showing the best antiplasmodial activity which is comparable to the standard drug. From the results obtained, Newbouldia laevis leaf is a good source of phytochemicals and also a potential source of potent antiplasplasmodial compounds that could be developed into drugs useful for the treatment of malaria. KEYWORDS: Newbouldia laevis, Antiplasmodial activity, Phytochemical screening. INTRODUCTION Malaria is an acute febrile illness caused by the Plasmodium protozoa transmitted through the bite of feeding female anopheles mosquitoes infected with the Plasmodium parasite. Five Plasmodium species are known to infect humans namely, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium www.wjpps.com

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knowlesi.[1] Only two of these, Plasmodium falciparum and Plasmodium vivax pose the greatest threat. P. falciparum is the most pathogenic species and responsible for greater percentage of malaria-related deaths globally. It is the dominant malaria parasite on the Continent of Africa while P. vivax is the prevalent parasite in most countries outside of sub-Saharan Africa. An estimated 1,200,000 people are at risk of malaria and half of these live in the African regions[2] Newbouldia laevis is a shrub or small tree widely distributed in West Africa. It is a medium sized angiosperm in the Bignoniaceae family which grows to a height of about 715m.[3] The leaves are large, glossy and deep green and the tree is propagated by cutting. It is regarded as a sacred and symbolic tree by some communities. It is commonly planted as a fence, thus the name boundary tree. It may be found around graves and shrines where it is easily recognized by its short branches, coarsely toothed leaflets and purple and white flowers.[4] It is known to different tribes in Nigeria by different names; Itumo in Ibibio, Ogirisi among the Igbos, Akoko in Yoruba, Aduruku in Hausa, Kontor in Tiv, Ikhimi in Bini and Ogiriki among the Urhobos. (References??) Newbouldia laevis is widely used in African folk medicine for the treatment of several diseases such as worms, malaria and fever, sexually transmitted diseases, diarrhoea and dysentery, cough, hernia, dental caries, fungal skin infections, in wound dressing and eye infections.[5,6] The leaves and roots when mixed together and boiled are used to treat fever, convulsion and epilepsy.[6] The roots and leaves are used in treating elephantiasis and convulsion, and the leaves used to treat peptic ulcer, otalgia, skin ulcer, epilepsy, hemorrhoids and constipation.[7] The leaves are used in South-Eastern Nigeria to hasten parturition and to expel the placenta after delivery.[8] Extracts of the stem bark, leaves and root of Newbouldia laevis have been extensively studied and reported to possess strong antibacterial properties.[3,6.9] The leaf and root extracts have been reported to exhibit antiplasmodial activity in Plasmodium falciparum in vitro models.[10,11] The leaf extract has been studied and reported to possess antiinflammatory activity.[12] The antioxidant activity of the stem bark and leaf extracts have been reported.[13,14,15] The anti-helmintic properties of the leaf extract was reported by Hounzangbe-Adote et al.[16] Bafor and Sanni[17] reported the uterine contractile effect of the leaf extract.

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MATERIALS AND METHODS Plant collection and preparation Fresh leaves of N. laevis were obtained from Ibiono in Akwa Ibom State of Nigeria in the month of March, 2015 and identified at the Botany Department of University of Uyo where a Herbarium Voucher Number (UUH3540) was assigned and sample deposited at the herbarium. The fresh leaves were washed, chopped into small portions and air-dried to constant weight at room temperature. The dried leaves were pulverized to coarse powder using a manual blender. Plant extraction and partitioning The powdered plant material was extracted by maceration in 50% ethanol for 72 hours. The extract was decanted, filtered and concentrated in vacuo using a rotary evaporator at 50ºC and then dried in a water bath at 50ºC. Fractionation of the crude ethanol extract was done using the method described by Kupchan.[18] The extract (200g) was partitioned in n-hexane, ethyl acetate and butanol. The fractions alongside the aqueous residue were concentrated in vacuo using a rotary evaporator, dried in a water bath and stored at -4ºC in a refrigerator prior to use. Animal Stock Adult albino mice of either sex weighing between 18g and 23g were obtained from the Animal House of University of Uyo, Nigeria where they were maintained and fed with growers’ pellet (Bendel Feeds and Flour Mills Limited, Edo State) feed and water given ad libitum. Approval for the use of animals in this study was obtained from the animal Ethics Committee, Faculty of Pharmacy, University of Uyo, Nigeria. Malaria Parasite A chloroquine-sensitive strain of Plasmodium berghei berghei was obtained from National Institute of Medical Research, Lagos and maintained by sub-passage in mice. Phytochemical Screening Phytochemical screening was carried out to ascertain the secondary metabolites, such as alkaloids, saponins, anthraquinones, glycosides, tannins, flavonoids, terpenes and carbohydrates, present in the crude extract using standard procedures as described by Sofowora[19] and Evans.[20]

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Plasmodium Inoculum Preparation The parasitized donor mice was anaesthetized with chloroform and the parasitized blood collected by means of cardiac puncture using sterile disposable syringe into sterile heparinised bottles. The parasitaemia was determined and the blood diluted with normal saline to obtain a 5.0X107 parasitized red blood cells per 1ml of blood so that the final 0.2ml inoculum size contained 1.0X107 parasitized red blood cells. Antiplasmodial Tests A total of 162 mice were pre-screened for Plasmodium parasite by making a thin smear of the blood taken from the tail tip of each mouse. Antiplasmodial test was conducted for the crude extract and its fractions using prophylactic, suppressive and curative in vivo antiplasmodial models. The extracts and controls were administered orally using stainless feeding cannula. Thin blood smears were made from the tail blood of each mouse, stained with Leishman’s stain to reveal parasitized erythrocytes and viewed under a microscope. The parasitaemia was determined by counting the number of parasitized erythrocytes out of 500 erythrocytes in random fields of the microscope. The percentage parasitaemia was calculated in comparison with the control using the formula Percentage parasitaemia = Number of parasitized erythrocytes X 100 Total number of erythrocytes counted The percentage chemosuppression was calculated using the formula Percentage parasitaemia of negative control-Percentage parasitaemia of test group X 100 Percentage parasitaemia of negative control

Prophylactic Test The repository test was done using the method described by Ryley and Peters.[21] A total of 54 mice were weighed and divided into nine groups of six mice each. Groups I, 2 and 3 received 200mg/kg, 400mg/kg and 600mg/kg of the crude extract respectively. Groups 4, 5, 6 and 7 received 400mg/kg each of the different fractions. Group 8 received 1.2mg/kg of pyrimethamine as positive control while group 9 received 10ml/kg of distilled water as negative control. The crude extract, its fractions, pyrimetamine and distilled water were administered orally to the mice for three consecutive days and on the fourth day the mice were inoculated with 0.2ml of the infected blood intraperitoneally. Thin films of blood obtained from the tail of each mouse were made on microscopic slides after 72 hours of

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inoculation, stained with Leishman’s stain and viewed under the microscope to determine the percentage parasitaemia and chemosupression. Suppressive Test The suppressive test was done using the method described by Knight and Peters.[22] On the first day, a total of 54 mice were each inoculated with 0.2ml of the infected blood. The mice were weighed and divided into nine groups of six mice each. Group I, 2 and 3 received 200mg/kg, 400mg/kg and 600mg/kg of the crude extract respectively. Group 4, 5, 6 and 7 received 400mg/kg each of the different fractions. Group 8 received 5mg/kg of artesunate as positive control while group 9 received 10ml/kg of distilled water as negative control. The crude plant extract, fractions, artesunate and distilled water were administered to the mice orally for four consecutive days. On the fifth day, thin films of blood obtained from the tail of each mouse were made on microscopic slides, stained with Leishman’s stain and viewed under the microscope and percentage parasitaemia and chemosuppression determined. Curative Test The curative test was done using the method described by Lui et al. [23]. On the first day, a total of 54 mice were each inoculated with 0.2ml of the infected blood. After 72 hours, the mice were weighed and randomly assigned into nine groups of six mice each. Group I, 2 and 3 received 200mg/kg, 400mg/kg and 600mg/kg of the crude extract respectively. Group 4, 5, 6 and 7 received 400mg/kg each of the fractions. Group 8 received 5mg/kg of artesunate as positive control while group 9 received 10ml/kg of distilled water as negative control. The crude plant extract, fractions, artesunate and distilled water were administered to the mice with the aid of an oral cannula for five consecutive days. Thin films of blood obtained from the tail of each mouse were made on microscopic slides every alternate day, stained with Leishman’s atain and viewed under the microscope and percentage parasitaemia and chemosuppression calculated. The survival of all the groups was determined over a period of 30 days. The mean survival time (MST) was calculated using the formula, MST = Number of days survived X 100 Total number of days Where MST = Mean Survival Time

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Statistical Analysis Results were expressed as Multiple Comparison of Means ± Standard Error of Mean (SEM). Significance was carried out by one-way analysis of variance followed by Turkey-Kramer Multiple Comparison Post Test. A probability level of less than 5% was considered significant (p˂0.05). RESULTS Table 1: Summary of phytochemical content of Newbouldia laevis leaf extract.

Key:

TESTS FOR SECONDARY METABOLITES Alkaloid a. Meyer’s Reagent b. Dragendorff Reagent c. Hagger’s Saponins a. Frothing test b. Emulsion test c. Fehlings test Anthraquinones a. Bontrager’s Glycosides a. Hydrolysis b. Salkowski’s Tannins a. Ferric chloride Terpenes a. Libermann Burchard’s test Favonoids a. Shinoda test Carbohydrates a. Molisch’s test +++ abundant ++ moderate + trace - absent

CONCLUSION ++ ++ ++ +++ +++ +++ ++ ++ + ++ +++ +++

Table 2: Prophylactic activity of Newbouldia laevis leaf extract and fractions. Treatment Distilled water

Dose % Parasitaemia % Chemosuppression 10ml/kg 17.53±0.82 a 200mg/kg 9.23±0.75 47.34 Crude extract 400mg/kg 6.20±0.89a 64.64 a 600mg/kg 2.57±1.17 85.36 a n-Hexane fraction 400mg/kg 5.67±0.82 67.68 Ethyl acetate fraction 400mg/kg 3.53±0.82 a 79.85 a Butanol fraction 400mg/kg 5.87±0.82 66.54 Aqueous fraction 400mg/kg 4.13±1.03 a 76.43 a Pyrimethamine 1.2mg/kg 2.37±0.98 86.50 Values are expressed as mean ± SEM. Significance relative to control ap˂0.05; n=6

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Table 3: Suppressive activity of Newbouldia laevis leaf extract and fractions. Treatment Distilled water

Dose % Parasitemia % Chemosuppression 10ml/kg 24.3±1.05 200mg/kg 15.23±0.75 37.31 Crude extract 400mg/kg 12.33±0.82 a 49.24 a 600mg/kg 7.17±0.98 70.51 n-Hexane fraction 400mg/kg 6.37±1.17 a 73.80 a Ethyl acetate fraction 400mg/kg 2.57±0.75 89.44 a Butanol fraction 400mg/kg 7.13±0.82 70.64 Aqueous fraction 400mg/kg 2.70±0.84 a 88.89 a Artesunate 5mg/kg 1.76±0.72 92.73 Values are expressed as mean ± SEM. Significance relative to control ap˂0.05; n=6

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Table 4: Curative activity of Newbouldia laevis leaf extract and fractions. Day 3 Day 5 Day 7 Day 9 Treatment (mg/kg) Parasitemia Parasitemia Chemosupression Parasitemia Chemosupression Parasitemia Chemosupression Distilled water 19.43±1.33 25.33±1.21 29.53±1.51 31.3±0.84 (10) Crude extract 17.10±0.84 12.67±1.03 50.00 8.03±0.98 a 72.80 4.77±0.98 a 84.77 (200) (400) 17.63±0.75 11.43±0.98 a 54.86 7.13±0.82 a 75.85 4.57±0.98 a 85.41 (600) 17.60±0.89 11.23±0.75 a 55.66 6.90±1.05 a 76.64 3.03±0.75 a 90.31 n-Hexane fraction 16.67±2.25 12.83±3.19 49.34 6.80±0.89 a 76.98 4.77±1.60 a 84.77 (400) Ethylacetate 16.47±0.82 10.37±0.75 a 59.08 5.50±2.43 a 81.38 3.10±1.05 a 90.09 Fraction 400 Butanol fraction 17.80±0.89 13.57±0.98 46.45 11.03±0.98 62.64 7.60±2.19 a 75.72 400 Aqueous fraction 16.73±0.82 11.43±0.98 a 54.87 6.47±1.03 a 78.10 3.83±0.98 a 87.75 400 Artesunate 5 15.80±1.26 8.77±1.17 a 65.39 4.80±0.89 a 83.75 2.83±0.75 a 90.95 a Values are expressed as mean ± SEM. Significance relative to control p˂0.05; n=6

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DISCUSSION Phytochemical studies carried out on the crude extract of the plant revealed the presence of alkaloids, saponins, glycosides, tannins, terpenes, flavonoids and carbohydrates in varying proportions. Alkaloids have been reported to play a protective role in plants and animals and are known to be good antiplasmodial agents.[24,25] Some alkaloids are used clinically for medicinal purposes. A notable example is quinine which is a powerful antimalaria agent still used in clinical practice today as well as its other counterparts quinidine, cinchonine and cinchonidine all isolated from the bark of the cinchona plant. Frederich et al.[26] have studied and reported the high antiplasmodial activity of indole alkaloids isolated from natural sources. The presence of cardiac glycosides in the extract could be responsible for its folklore use in cardiovascular diseases as well as its cardioprotective effect reported by Agbafor et al.[27] Tannins have been reported to possess good antioxidant activity which could further result in good antiplasmodial activity.[28] Terpenes are known to exhibit good antiplasmodial activity as they are commonly implicated in the antiplasmodial activity of medicinal plants.[29] A typical example of a terpene used in antimalarial therapy is the sesquiterpene dihydroartemisinin isolated from Artemissia annua of which its structural modifications have resulted in various antimilarials used today in the mainstay treatment of malaria. Goulart et al.[30] has investigated and reported the antiplasmodial activity of several terpenes such as the farnesol, nerolidol, limonene and linalool. De Monbrison et al.[31] has tested several flavonoid derivatives and reported their antiplasmodial activity. The antiplasmodial activity of the crude extract carried out showed that the leaves of Newbouldia laevis possess good antiplasmodial activity in a dose-dependent manner which is comparable to the standards used. The result compared favourably with that obtained by Gbeasor et al.[10] where they tested the leaf extract of the plant in vitro. The fractions were tested for antiplasmodial activity. The fractions showed good antiplasmodial activity with the ethyl acetate fraction exhibiting the highest antiplasmodial activity and mean survival time comparable to the standard drugs. CONCLUSION From the research carried out, it can be concluded that the leaf of Newbouldia laevis contains various phytochemical compounds of which saponins, flavonoids and carbohydrates were in abundance. The plant was found to be relatively non-toxic. The antiplasmodial property of

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the leaf was confirmed thereby reaffirming its ethnopharmaceutical use in the treatment of malaria. The antiplasmodial activity was found to be highest in the ethylacetate fraction. REFERENCES 1. World

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23. Lui KC, Yang SC, Roberts MF. Antimalarial activity of Artemisia annua flavonoids from whole plants and cell cultures. Plants Cell, 1992; 637-640. 24. Qui S, Sun H, Zhang A, Xu HY, Yan GL, Han Y, Wang XJ. Natural alkaloids: Basic aspects, biological roles and future perspectives, Chinese Journal of Natural Medicine, 2014; 12(6): 401-406. 25. Cushnie TP, Cushnie B, Lamb AJ. Alkaloids: An overview of their antibacterial, antibiotic-enhancing and antivirulence activities. International Journal of Antimicrobial Agents, 2014; 44(5): 377-386. 26. Frederich M, Tits M, Angenot L. Potential antimalarial activity of indole alkaloids. Transaction of the Royal Society of Tropical Medicine and Hygiene, 2008; 102(1): 11-19. 27. Agbafor KN, Ezeali C, Akubugwo EI, Obiudu IK, Uraku AJ, Ogbanshi ME, Edwin N, Ugwu OPC. Cardioprotective effect of leaf and root extracts of Newbouldia laevis against carbon tetrachloride-induced cardiotoxicity in albino rats. European Journal of Medicinal Plants, 2015; 9(3): 1-7. 28. Boyle MA, Long S. Personal nutrition, 6th Ed., Thompson Wadsworth, Belmont, 2007; pp.191-202. 29. Asase A, Akwetey GA, Achel DIG. Ethnopharmacological use of herbal remedies for the treatment of malaria in Dangme West district of Ghana. Journal of Ethnopharmacology, 2010; 129: 367-376. 30. Goulart HR, Kimura EA, Peres VJ, Couto AS, Duarte FAQ, Katzin AM. Terpenes arrest parasite development and inhibit biosynthesis of isoprenoids in Plasmodium falciparum. Antimicrobial Agents and Chemotherapy, 2004; 48(7): 2502-2509. 31. De Monbrison F, Maitrejean M, Latour C, Bugnazet P, Peyron F, Barron D, Picot S. In vitro antimalarial activity of flavonoid derivatives dehydrosilybin and 8-(1;1)-dmakaempferide, Acta Tropica, 2006; 97 (1): 102-107.

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