www..mdcjournals.com Antiplasmodial Activity of Root Extracts of Napoleona imperialis Plant Using Mice

Ogu, P, Ph.D (In View)1; Njoku, S.K, Ph.D(In view)2; and Igboagi, J.O, (HND)*1

1Department of Science Laboratory and Technology, School of Industrial and Applied Sciences, Federal Polytechnic, Nekede, Owerri, Nigeria 1Department of Biochemistry, Michael Okpara University of Agriculture, Umudike, Nigeria.

*E-mail: [email protected]

ABSTRACT synthesis of useful drugs. Okwu and Ekeke (2003), Edeoga et al., (2005), Okwu (2005); and Duru et al., (2012a) noted The antiplasmodial activity of Napoleona imperialis plant was studied that the therapeutic value of medicinal plants lies in some using standard methods. Ethanol and aqueous extracts of N. chemical substances that produce definite physiological imperialis were prepared using ethanol and water respectively. Artesenate and chloroquine antimalarials were used as standards. actions on human body, against bacteria and other Twenty male albino mice were used to model the study. Results microbes. Okwu and Uchegbu (2009) went further to obtained revealed the weight reduction effects of the studied extracts isolate, characterize and screen antimicrobial compounds against the control though aqueous extract of N. imperialis had the from a medicinal plant. Osuagwu et al., (2007); Okwu and highest weight reducing effect on the mice when compared to those Josiah (2006); Okwu (2005); Bartram, (1998); Gill, (1992) of the control and the standards. The observed no of plasmodial parasites progressive reduced in mice placed on the extracts with and others have noted that the physiological action of days when compared to those of the standards and control. Observed medicinal plants is made possible by compounds such as percentage suppression or inhibition of the extracts when compared vitamins, minerals, phytochemicals, amino acids, to the standards showed antiplasmodial activity order “choloquine>N. carbohydrates, etc. Vitamins, minerals, amino acids and imperialis ethanol root extract> N. imperialis aqueous root extract carbohydrates are known as phytonutrients (Amadi et al., >artesenate” in the present study. This could be indication of N. imperialis medicinal potential against malarial disease. The present 2012a; Amadi et al., 2012b; Arukwe et al., 2012; Amadi et al., study has shown the antiplasmodial activity of root extracts of N. 2013; Duru et al., 2013a; Duru et al., 2013b) and are imperialis. responsible for plants nutrition while phytochemicals are responsible for plants protection (Amadi et al., 2012a;

Duru et al., 2013c). These compounds perform similar (Keywords: Antiplasmodial activity, Napoleona imperialis, root extract, functions in the body of animals (humans inclusive) when mice) taken into the body. Trees and shrubs with phytonutrients INTRODUCTION as well as phytochemicals abound in Nigeria. Akubugwo and Ugbogu (2007) noted that such plants and trees are From the days of our forefathers, plants have always been of nutritional and medicinal importance. employed to remedy disease conditions (Qureshi et al., 2009; Duruigbo, 2013; Agomuo et al., 2012; Duru et al., Napoleona imperialis a Lecythidaceae, used for different 2012a; Duru et al., 2012b; Duru et al., 2014). Such plants medicinal purposes is among such plants. Different tribes are called medicinal plants (Agomuo et al., 2013). in Nigeria have different names for N. imperialis. For According to Sofowora (1993), a medicinal plant is any instance, the plant is known as “nnekeloche or abakalabak plant used for the extraction of pure substances either or “utim” or ovurumgbede or akbodo” by the Igbos of for direct medicinal compounds which can be used for the Southeast, “irosun-igbo orbongibongi or boribori ” by the Yourubas in Southwest, “ukpakonrisa” by Edos, “otukuch”

Intraspecific Journal of Biodiversity and Environmental Sciences –1– Website link: http:ijbe.mdcjournals.com Volume 3, Issue 2, 2016 by Igalas,and “mabungi” by Hausas of Northern, Nigeria Experimental Animals: A total of twenty albino mice (Mus (Umberto, 1999; Burkill, 1985; Iwu, 1993). An infusion of musculus) were purchase from the animal colony of Napoleona imperialis leaves is used to dissolve clotted Biochemistry Department, Nnamdi Azikiwe University, blood in freshly delivered women; but used as vermifuge Awka, Anambra State, Nigeria. The mice were allowed to for children. Its stemis used to cure gonorrhoea while the acclimatize for a period of two weeks with access to clean roots are used againstfevers (Bartram, 1998; Burkill, water and standard animal feeds (pelletized animal feeds 1985; Iwu, 1993). from a dealer in Owerri main market). Five mice out of the twenty mice were injected with plasmodium gotten from Since the acceptance of forkric, herbal or traditional Abuja, Nigeria. This infection was done by injecting the medicine at Alma Ata, USSR by World Health Organisation plasmodium into the mice through intraperitoneal route. in 1978 (WHO, 1978), the interest on plants of medicinal After forty-eight hours of injection, the malaria parasite importance became renewed hence the desire to discover count was tested in the mice that received the the potency of variety of such plants against many plasmodium injected. disease conditions increased (Kadiri et al., 2010). Previous studies have revealed the nutritional composition The remaining fifteen mice were divided into five groups (Alaekwe and Mojekwu, 2013) and potency of N. imperialis of three mice each and treated as follows; on some diseases (Esimone et al., 2005), but not much has been with the plant on a disease such as malaria Group 1: Control rats that received only normal saline which is ravaging the people of African continent. (0.34/34.00±0.82g).

Sequel to this, there is need to extend the studies on N. Group 2: Mice treated with root ethanol extract of imperialisplant on a disease such as malaria. This will help Napoleona imperialis. to discover the efficacy of the plant against malaria parasites. Group 3: Mice treated with root aqueous extracts of Napoleona imperialis. MATERIALS AND METHODS Group 4: Mice treated with arteseunate (Dose; Sample Collection: The samples of Napoleona imperialis 0.31/31.00±0.82g) . roots used in this study were collected fromIsuochi village in Umunneochi L. G. A of Abia State, Nigeria. The roots Group 5: Mice treated with chloroquine (Dose; were properly identified by a Botanist in the Department 0.31/31.00±0.82g). of Biotechnology, Imo State University, Owerri, Nigeria as roots of Napoloena imperialis. The identified root samples Groups 4 and 5 mice placed on arteseunate and were ground using electric blender into powder and used chloroquine antimalarials were used as the standards. The for preparation of extracts used in the present study. weights of the mice were taken appropriately with help of weighing balance. Methanol Extract: 200g ground sample of Napoleona imperialis was dissolved in 500ml methanol and extracted Producedure Used to Infect the Experimental Mice: From with the help of soxhlet extract for 72 hours. the extract the tails of the infected mice, blood samples were obtained was concentrated using rotary evapourator. collected into sterile EDTA tubes containing normal saline. 0.2ml of infected blood in normal saline (solution mixture Water Extract: 200g of the ground sample of Napoleona formed) was then injected into the test mice through imperialis was dissolved in 500ml water and extracted intraperitoneal route. The procedure of infecting the test with the help of soxhlet extract for 72 hours. the extract obtained was concentrated using rotary evapourator.

Intraspecific Journal of Biodiversity and Environmental Sciences –2– Website link: http:ijbe.mdcjournals.com Volume 3, Issue 2, 2016 mice was repeated after forty-eighty hours and then the 100 - [(meanparasitaemia treated/mean parasitaemia malaria parasite load was tested to ensure infection. control) × 100].

Blood Sample Collection and Malaria Parasite Count: After Statistical Analysis: Each group was compared directed five days, the mice were weighed and sacrificed by making with the control and standards using students’ t- incision at their regions and blood was collected in EDTA distribution at 5% significant level. bottle and used for parasite count. The malaria parasite count was carried out using the high power field (HPF) RESULTS AND DISCUSSION method as described by Moody and Chiodini (2000). This method used a defined mount of whole blood to make a Table 1 above shows the weight changes of albino mice thick smear. The parasites present in 100 high-power field given the extracts. The Table reveals that mice placed on (HPF) was counted .One parasite/100 HPF was assumed to root aqueous extract had the highest weight lost (-6.40g); be the equivalent to 50 parasites/ml. A thick smear was followed by mice on chloroquine (-2.00g); the next was considered negative if no parasites were seen. mice on ethanol root extract (-1.67g) and the least was mice on artesenate (-1.00g) against the control mice (- Percentage suppression of parasite for the plant extracts 6.00g). The obtained results are presented in Figure 1 for was calculated using method as described Gessler et al., better understanding. (1995).

Table 1: Weight changes of albino mice given extracts. Group Root Ethanol Root Aqueous Artesenate Chloroquine Control

Parameters Weight after 19.33±0.82 32.60±7.73 30.00±0.82 28.00±2.16 28.67±2.87 Inducement (g) Weight before 21.00±0.82 39.00±0.82 31.00±0.82 31.00±0.82 34.00±0.82 inducement (g) Weight changes ( - 1.67 -6.40 -1.00 -2.00 -6.00 Weight lost) (g) Results are means ± standard deviations of triplicate determinations.

Intraspecific Journal of Biodiversity and Environmental Sciences –3– Website link: http:ijbe.mdcjournals.com Volume 3, Issue 2, 2016 weight changes of mice given extracts 1 0 -1 -2

-3 weight changes of mice given -4 extracts -5

-6 Weight of mice(g) -7 -8

Fig.1: Weight changes of mice given various extracts.

8000 Day zero 7000 Day one 6000 5000 Day two 4000 Day three 3000 Day four 2000 Day five 1000 Day six 0 Day seven Ethanol Root Aqueous Root Artesenate Chloroquine Control Extract Extarct Day eight

Fig. 2: No of antiplasmodial parasites with days of study.

Table 2: Percentage plasmodial parasites suppression (inhibition) by the extracts and standards with days of study. Group Day zero Day one Day two Day three Day four Day five Day six Day Day seven eight Root ethanol 22.92 20.00 44.59 48.64 41.89 34.61 38.61 66.66 23.51 extract Root -5.18 30.00 42.42 42.62 51.35 63.54 54.55 68.75 23.51 aqueous extract Artesenate 6.30 -1.25 22.51 49.18 37.83 34.61 36.36 58.34 17.64 Chloroquine -2.96 23.13 50.65 75.15 35.13 19.23 25.00 72.91 11.75

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80

60 Root ethanol extract 40 Root aqueous extract 20 Artesenate 0 Chloroquine Day Day Day Day Day Day Day Day Day -20 zero one two three four five six seven eight

Fig. 3: Graphical representation of percentage plasmodial parasites suppression (inhibition) by the extracts with days of study.

Malaria is a major parasitic disease in the world, antimalarial drugs in use today such as quinine and especially in Africa. WHO (2008) noted that malaria is artemisinin were either obtained directly from plants or responsible for 500 million cases and 2 or 3 million developed using chemical structures of plant-derived deaths every year. WHO went further to state that most of compounds as templates. these deaths include children under the ages of five years as well as pregnant women. Although a report by the The studied plants inhibited weight in mice (Table 1 and same WHO in 2010, noted a significant reduction in overall Figure 1). Mice placed on N. imperialis aqueous root disease burden, with mortality due to malaria estimated extract had higher weight loss (-6.40g) against the to have declined from 2 or 3 million, to 660,000 deaths in control (-6.00g). Those on ethanol extract had lower 2010 due to global efforts and improved antimalarial weight loss (-1.67g) when compared to the control (- drugs. The continents of African and her countries’ 6.00g). When results obtained in terms of weight loss, especially those within the sub-Saharan Africa were not were compared to those of standard drugs (Table 1 and left out in the significant decrease (Lubell et al., 2008; Figure 1), the observed weight loss followed the order; D’Acremont et al., 2010; Mbando et al., 2010;). mice on aqueous extract >control >mice on chloroquine> mice on ethanol extract > mice on artesenate. The Aside the observed decrease, the disease called malaria observed results could therefore mean that aqueous is still of public health concern, especially now that it has extract of plant understudy may be good for weight been discovered that some Plasmodium species are reduction. become increasingly resistance to some of these improve drugs. For instance Cátia et al., (2008) noted that Figure 2 shows the antiplasmodial activity of the plant. Plasmodium falciparum the mostly widespread etiological From the observed result, infected mice placed on the agent for human malaria has become increasingly studied extracts had reductions in no of plasmodial resistant to standard antimalarials such as chloroqiune parasites as day’s progresses. Though mice placed on and antifolates. ethanol extract had a slight increase of parasite load on day three (Figure 2) while mice on aqueous extract has In view of the problems associated with antimalarial drug increases on day two and day three (Figure 2). The resistance, new drugs or drug combinations are urgently observed increase could due to stimulation of the immune required for treatment of malaria. Basco et al., (1994) system of the mice. The observed parasite load in day noted plants have always been considered to be a possible zero was significantly (p<0.05) reduced in mice given alternative and rich source of new drugs and most of the ethanol extract of N. imperialis when compared to the

Intraspecific Journal of Biodiversity and Environmental Sciences –5– Website link: http:ijbe.mdcjournals.com Volume 3, Issue 2, 2016 control and the standards, while the parasite load of mice imperialis may be effective against malaria disease hence given aqueous extract of the studied plant increased it can be used as a malaria herb or its components significantly (p<0.05) against the control as well as the extracted, modeled and used as synthetic drugs. standards. In day one, the observed parasite loads in mice placed on the studied extracts reduced significantly REFERENCES (p<0.05) against the control and the standards. Day two also had significant (p<0.05) reduction on parasite loads 1. Achan, J, (2009): Effectiveness of quinine versus in mice given the studied extracts when compared to artemether-lumefantrine for treating uncomplicated control and artesenate standard but the loads increased falciparum malaria in Ugandan children: Randomised trial. significantly (p<0.05) with respect to chloroquine BMJ 339: b2763. 2. Agomuo, E. N., Amadi, B.A and Duru M. C. (2012): Some standard (Table 2). The day three reduction followed the biochemical studies on the leaves and fruits of Persea same order as in day two. The extracts reduced the Americana.. IJRRAS 11 (3):565-569. www. Arpapress parasite loads significantly (p<0.05) as observed in day .com/Volumes/Vol11Issue3/IJRRAS_11_3_21.pdf four when compared to the control as well as the 3. Agomuo, E. N., Duru, M.K.C and Amadi, B.A. (2013): Some standards. Mice placed on ethanol extract of N. imperials bioactive constituents of Asmina triloba (paw paw) leaf had parasites load reduction relatively to mice on variety. International Science Research Journal 4(2):18-22. artsenate in day five. The parasite loads in the infected 4. Akubugwo, E.I and Ugbogu, A. E. (2007): Physiochemical mice significantly reduced (p<0.05) against the control studies on oils from five selected Nigerian plants seeds. and the standards in day five. The studied extracts Pakistan Journal of Nutrition 6(1): 75-78. continued to have reduced effect on the parasite loads in 5. Alaekwe, I. o and Mojekwu, O. E. (2013): Proximate infected mice against the control and the standards on composition and utilization of Napoleona imperialis fruits. the fifth, sixth, seventh and eighth day (Figure 2). Journal of Natural Research 3(6):160-165. 6. Amadi, B.A., Arukwe, U., Duru, M. K.C., Amadi, C.T., Adindu,

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Ogu P is a Lecturer in the Department of Science Laboratory and State, Nigeria. Her research interests are in the areas of Medical Technology, Federal Polytechnic Nekede, Owerri, Imo State, Nigeria. Laboratory Sciences/Biochemistry. She is a member of the parent body of Laboratory Scientists in Nigeria, and her research interests are in the areas of malaria and How To Reference This Article medicinal plants. Ogu, P., Njoku, S, K., and Igboagi, J.O, (2016): Antiplasmodial Activity of Njoku S. K is a researcher and a Ph.D student in the Department of Root Extracts of Napoleona imperialis Plant Using Mice Intraspecific Biochemistry, Michael Okpara University of Agriculture, Umudike, Journal of Biodiversity and Environmental Sciences 3(2): 001-009. Nigeria. He is a member of NSBMB and his research interests are in areas of Medical Biochemistry. www.ijbe.mdcjournals.com

Igboaghi J. O is graduate student from the Department of Science Laboratory and Technology, Federal Polytechnic Nekede, Owerri, Imo

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