|Ahead of print publication
The prophylactic antimalarial activity of Combretum nigricans hydromethanol crude extract in mice
Chinedu Enegide1, Charles C Ofili2, Uzuazokaro M Agatemor3, Dabum L Jacob4
1 Department of Pharmacology, Novena University, Ogume, Delta State; Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Nigeria
2 Department of Public and Community Health, College of Health Sciences, Novena University, Ogume, Delta State, Nigeria
3 Department of Pharmacology, Novena University, Ogume, Delta State, Nigeria
4 Department of Human Physiology, Faculty of Medical Sciences, University of Jos, Jos, Nigeria
|Date of Submission||25-Mar-2021|
|Date of Decision||24-May-2021|
|Date of Acceptance||29-Jun-2021|
|Date of Web Publication||23-Feb-2022|
Department of Pharmacology, Novena University, Ogume, Delta State
Source of Support: None, Conflict of Interest: None
Background: Malaria has maintained its historic threat to global health, with nearly 40% of the world's population being at risk of its mayhem. The increasing cases of resistance by Plasmodium parasite to the currently available antimalarial agents have led to increased scientific screening of medicinal plants for the possible development of newer agents, both for treatment and chemoprophylaxis against malaria. This study was designed to evaluate the prophylactic antimalarial activity of Combretum nigricans hydromethanol crude leaf extract in mice.
Materials and Methods: The prophylactic activity of C. nigricans hydromethanol crude leaf extract was evaluated using the method illustrated by Peters. Six groups containing five mice each were used for the study. Group 1 (control) received distilled water 10 ml/kg b.w. p.o., Groups 2–4 were treated with 200, 400, and 800 mg extract/kg b.w. p.o., respectively, Group 5 mice were treated with chloroquine 10 mg/kg b.w. p.o., whereas Group 6 mice were treated with artesunate 10 mg/kg b.w. p.o.
Results: The result showed significant (P < 0.05) chemoprophylactic activity in all treated groups compared with the control group. The chemoprophylactic activity of the extract was 55.4, 61.6, and 79.5% for 200, 400, and 800 mg/kg, respectively. A dose-related increase in mice body weight was also observed in the extract-treated groups. Phytochemical screening of the extract revealed the presence of several important active principles, including alkaloids, saponins, and flavonoids.
Conclusion: The present study revealed that C. nigricans hydromethanol crude leaf extract possesses significant chemoprophylactic activity against malaria.
Keywords: Combretum nigricans, malaria, mice, prophylaxis
|How to cite this URL:|
Enegide C, Ofili CC, Agatemor UM, Jacob DL. The prophylactic antimalarial activity of Combretum nigricans hydromethanol crude extract in mice. J Curr Res Sci Med [Epub ahead of print] [cited 2022 Jun 29]. Available from: https://www.jcrsmed.org/preprintarticle.asp?id=338246
| Introduction|| |
Malaria has maintained its historic threat to global health, with nearly 40% of the world's population being at risk of its mayhem., Africa was accountable for 90% of global malaria cases in 2016, and most of the disease cases and mortalities recorded in the sub-Saharan African region were among children <5 years old and pregnant women. Estimate shows that a child dies of malaria every 30 s in the sub-Saharan African region. Coupled with its direct health impact, malaria also elicits a huge economic burden on affected persons and nations; this is in the form of health-care cost and missed/less productive days at work, leading to reduced economic output. Although several agents are currently available both for treatment and prophylaxis against malaria, the increasing cases of resistance by Plasmodium parasite to the currently available agents present a major challenge in eradicating malaria., This challenge has led to increased scientific screening of medicinal plants for the possible development of new agents both for treatment and chemoprophylaxis against malaria. Medicinal plants have proven to be a vital source for the development of new agents, as two outstanding antimalarial agents currently in use were gotten from plant sources (quinine from cinchona plant and artemisinin from Artemisia annua). Combretum nigricans, a small tree with smooth-bark mainly found in North-Central Nigeria, is a plant whose therapeutic potentials have been explored and utilized in the traditional medicine for the management of several ailments including malaria. The stem-bark and leaves of C. nigricans are used as cough expectorant and in the treatment of rheumatism;, its aqueous macerated leaves are also used in the treatment of diarrhea and jaundice. The boiled leaves are administered orally in the treatment of prurigo, dysentery, and in the management of malaria., As part of our assessment of its antimalarial activity, this study was designed to evaluate the prophylactic antimalarial activity of C. nigricans hydromethanol crude leaf extract in mice; this is with the view of providing a scientific evidence that justifies the application of C. nigricans leaves in ethomedicine for the management of malaria.
| Materials And Methods|| |
Fresh leaves of the plant (C. nigricans) obtained from a local garden in Jos-North Local Government Area, Plateau state were identified and authenticated by a taxonomist at the Federal College of Forestry, Jos, Nigeria, Mr. Jeffrey Azila.
The leaves were rinsed, air-dried at the room temperature for 2 weeks and milled into fine powder with a plant miller. Using the procedure described by Handa et al., 1.6 kg of the grounded leaves were macerated in hydromethanol (methanol-water 1:1 v/v) for 48 h with intermittent vigorous shaking. The mixture was filtered after 48 h, using muslin cloth followed by Whatman filter paper (No. 1). The obtained crude extract was concentrated and stored at 4°C.
Phytochemical analysis of the crude leaf extract was carried out to test for the presence of alkaloids, glycosides, saponin, carbohydrates, tannins, flavonoids, terpernoids, proteins, resins, steroids, and reducing sugar, using standard procedures illustrated by Evans and Harborne.
Swiss albino mice of both sexes (with average weight 21 ± 0.91 g) housed at the Department of Pharmacology and Toxicology, University of Nigeria, animal unit were used. The animals were housed under the standard laboratory conditions in mice cages, at the room temperature and humidity with a 12-h light/dark cycle; and free access to feed and water. All animal experiments were conducted in conformity with the “NIH revised guidelines for laboratory animal care and use” and the University of Nigeria regulations and code of ethics for laboratory animal use.
Rodent parasite (Plasmodium berghei (NK-65))
The rodent parasite was obtained from the Faculty of Veterinary Medicine, University of Nigeria. The parasite was maintained alive in mice by serial intra-peritoneal passage of blood from donor mouse to another mouse. The re-infected mice were housed at the animal house unit, Department of Pharmacology and Toxicology, University of Nigeria; where the study was carried out.
Evaluation of prophylactic antimalarial activity (repository test)
The prophylactic activity of C. nigricans hydromethanol crude leaf extract was evaluated using the method illustrated by Peters. Thirty adult Swiss albino mice of both sexes were weighed and divided into the six groups of five mice each. The animals were pretreated daily for 4 days (D0 − D3) before inoculation. Group 1 mice (negative control) received distilled water 10 ml/kg b.w. p.o.; Groups 2–4 mice were treated with 200, 400, and 800 mg extract/kg b.w. p.o, respectively. Group 5 mice were treated with chloroquine 10 mg/kg b.w. p.o., whereas Group 6 mice were treated with artesunate 10 mg/kg b.w. p.o. On the 5th day (D4), all the mice were inoculated with 0.2 ml standard inoculum containing about 1 × 107 of Plasmodium berghei (NK-65)-infected erythrocytes through the intraperitoneal route. After 72 h of posttreatment (day 7), the mice were re-weighed. A drop of blood from the tail of each mouse was smeared on the slide of a microscope to make a thin film. Each film was stained with Giemsa stain and examined under a microscope as illustrated by Huang et al. to determine parasitemia level. The prophylactic effect was calculated (A − B/A) ×100, where A is mean parasitemia in the negative control group and B is mean parasitemia in treated group.
Body weight determination
The body weight of each mouse in the treated groups as well as the control group was taken before treatment started (D0) and 72 h after infection (D7) using a sensitive digital weighing balance. The body weight change in the control and treated groups was recorded.
The data obtained were expressed as mean and standard ± error of the mean. GraphPad Prism for Windows (version 7.0), San Diego, California, USA, was used for the analysis. One-way analysis of variance followed by Dunnet's post hoc test was used to test for significance. P < 0.05 was considered statistically significant.
| Results|| |
The hydomethanol crude leaf extract of C. nigricans consists of alkaloids, saponins, flavonoids, quinones, and other phytochemicals [Table 1].
|Table 1: Phytochemical constituents of Combretum nigricans hydro-methanol crude leaf extract|
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Prophylactic (repository) test
There was statistically significant (P < 0.05) chemoprophylactic activity in all the treated groups as compared with the control group [Table 2] and [Figure 1]. The chemoprophylactic activity of C. nigricans hydromethanol crude leave extract was 55.4, 61.6, and 79.5% for 200, 400, and 800 mg/kg, respectively; chloroquine had 72.3% while artesunate had 79.5% [Figure 1]. A dose-related increase in mice body weight was observed in the extract-treated groups [Table 3] and [Figure 2]. The increase in mice body weight was 1.7, 6.3, and 12.6% for 200, 400, and 800 mg/kg, respectively; chloroquine had 5.3% and artesunate had 2.1%. The control group, however, had 6.2% (i.e. decrease in body weight by 6.2%) [Figure 2].
|Table 2: Chemoprophylatic effect of Combretum nigricans hydro-methanol crude leaf extract in Plasmodium berghei-infected mice|
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|Figure 1: Percent chemoprophylaxis of Combretum nigricans hydromethanol crude extract against P. berghei in mice. CNCE: Combretum nigricans crude hydromethanol leaf extract, Chloro: Chloroquine, Art: Artesunate|
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|Table 3: Effect of Combretum nigricans hydro-methanol crude leaf extract on the body weight of P. berghei infected mice|
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|Figure 2: Percent weight change of P. berghei infected mice treated with Combretum nigricans hydro-methanol crude extract. CNCE: Combretum nigricans crude hydro-methanol leaf extract, Chloro: Chloroquine, Art: Artesunate|
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| Discussion|| |
C. nigricans is a medicinal plant whose leaves are used in ethnomedicine for the treatment of malaria in North-Central Nigeria. This study was carried out to evaluate the prophylactic activity of C. nigricans against malaria. The results from prophylactic study showed that the extract elicited a significant (P < 0.05) dose-related chemoprophylactic activity. The highest test dose (800 mg/kg) elicited the highest chemoprophylactic effect (79.5%). The extract treated groups showed increase in body weight in a dose-dependent manner, artesunate and chloroquine groups also had increase in body weight in their respective groups, while the negative control group showed decrease in body weight. Loss of body weight is one of the manifestation of P. berghei infection in mice. The decrease in body weight in the control group may be due to the depressant effect on the appetite of the mice and the consequences of disturbed metabolic function and hypoglycemic effect of the parasite., Aside from its antiplasmodial activity which may have prevented loss of body weight, the extract may also possess orexogenic (appetite stimulatory) activity that led to increased food intake and increase in mice body weight; this is hinged on the fact that that weight increase in the 400 and 800 mg/kg groups were considerably higher than those of the standard drugs [Table 3] and [Figure 2]. The phytochemical study unveiled the presence of several important active principles whose antimalarial effects have been chronicled; these include saponins, flavonoids, and alkaloids. Saponins are known to possess immunomodulatory activity, hence can augment the activity of mice immune system toward the extermination of the infection. This is a very important mechanism in the prophylaxis against malaria infection. Flavonoids chelates plasmodium nucleic acid base pairing, hence elicits plasmocidal effect., Alkaloids are also known to elicit the plasmocidal activity. Hence, the prophylactic activity elicited by the extract against malaria can be attributed to the phytochemicals present in it. However, the exact mechanism for its prophylactic antimalarial effect has not been elucidated. The oral median lethal dose (LD50) of C. nigricans hydro-methanol crude leaf extract was found to be >5000 mg/kg b.w. p.o. in another study by the authors. Therefore, the extract may serve as good herbal prophylactic antimalarial agent, due to its significant activity.
Although much work is being done to ensure effective curative treatment of malaria, there is still a call for the effective prevention of the disease. This have resulted in the increased campaigns for the use of insecticide-treated nets, increased efforts toward developing effective antimalarial vaccines and more effective chemoprophylactic agents. A lot of progress have been made in the crusade against malaria; however, to achieve the goal of eliminating the disease in the near future (as proposed by the WHO), more holistic and aggressive steps need to be taken, especially in the sub-Saharan Africa region. According to a WHO report, about 43% of population at risk to malaria in the sub-Saharan African region did not have access to conventional protection/preventive measures (including chemoprophylactic agents) in 2015. This therefore calls for the incorporation of herbal medicines (with scientifically proven activity) not only in malaria treatment, but also for prophylaxis especially in Africa. This would be of enormous gain, especially because of high accessibility to herbal medicines in Africa.
| Conclusion|| |
The present study has shown that C. nigricans hydromethanol crude leaf extract possesses significant chemoprophylactic activity against malaria. However, more studies aimed at identifying and isolating the active principles of the extract responsible for antimalarial activity as well as in-depth preclinical toxicity studies to determine the safety are needed before extrapolation to human studies.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, Guerra CA, et al
. Estimating the global clinical burden of Plasmodium falciparum malaria in 2007. PLoS Med 2010;7:e1000290.
Liu Y, Zhou RM, Zhang YL, Wang DQ, Li SH, Yang CY, et al
. Analysis of polymorphisms in the circumsporozoite protein gene of Plasmodium vivax
isolates from Henan Province, China. Malar J 2018;17:10-3.
Viral AS. Fungal, protozoal and helminthic infections. In: Laurence DR, Bennett PN, Sharma P, editors. Clinical Pharmacology. 11th
ed. New York: Churchill Livingstone; 2012. p. 232.
Sachs J, Malaney P. The economic and social burden of malaria. Nature 2002;415:680-5.
Vinetz MJ. Chemotherapy of malaria. In: Brunton LL, Hilal-Dandan R, Knollmann CB, editors. Goodman and Gilman's The Pharmacological Basis of Therapeutics. 13th
ed. New York, USA: McGraw-Hill; 2018.
Artimovich E, Schneider K, Taylor TE, Kublin JG, Dzinjalamala FK, Escalante AA, et al
. Persistence of sulfadoxine-pyrimethamine resistance despite reduction of drug pressure in Malawi. J Infect Dis 2015;212:694-701.
Chinedu E, David A, Ameh SF. Phytochemical evaluation of the ethanolic extracts of some Nigerian herbal plants. Drug Dev Ther 2015;6:11-4. [Full text]
Asase A, Oteng-Yeboah AA, Odamtten GT, Simmonds MS. Ethnobotanical study of some Ghanian anti-malarial plants. J Ethnopharmacol 2005;99:273-9.
Sacandé M, Sanon M, Schmidt L. Combretum nigricans
Lepr. Ex Guill. and Perr. West Sussex, UK: Seed Leaflet; 2007. p. 130.
Burkill HM. Useful Plants of West Tropical Africa. 2nd
ed., Vol. 1. England: Royal Botanic Gardens; 1985. p. 389-401.
Balde MA, Traore MS, Diane S, Diallo MS, Tounkara TM, Camara AE. Ethnobotanical survey of medicinal plants traditionally used in low and middle – Guinea for the treatment of skin diseases. J Plant Sci 2015;3:32-9.
Ada AG, Claffey HS. Trees of the Idoma Land. Benue, Nigeria: DIK Printers; 2003. p. 22-171.
Handa SS, Khanuja SP, Longo G, Rakesh DD. Extraction Technologies for Medicinal and Aromatic Plants. Trieste, Italy: International Centre for Science and High Technology ICS-UNIDO; 2008.
Evans WC. Trease and Evans Pharmacognosy. 14th
ed. London: WB Saunders Company Limited; 2005. p. 357-8.
Harborne JB. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 2nd
ed. London: Chapmann and Hall Publishers; 1998.
NIH. Guide for the Car and Use of Laboratory Animal (Revised). Washington: NIH Publication; 1985. p. 83-23.
Peters W. Drug resistance in plasmodium berghei. Exp Parasitol 1965;17:80-9.
Huang BW, Pearman E, Kim CC. Mouse models of uncomplicated and fatal malaria. Bio Protoc 2015;5:e1514.
Basir R, Rahiman SF, Hasballah K, Chong W, Talib H, Yam M, et al. Plasmodium berghei
ANKA infection in ICR mice as a model of cerebral malaria. Iran J Parasitol 2012;7:62-74.
Taherkhani M, Rustaiyan A, Nahrevanian H, Salehizadeh E. In vivo
antimalarial activity of Iranian flora
Artemisia oliveriana J. Gay ex DC. Extract and its comparison with other antimalarial drugs against Plasmodium berghei in mice model. J Biol Act Prod 2013;3:173-82.
Yun JW. Possible anti-obesity therapeutics from nature – A review. Phytochemistry 2010;71:1625-41.
Muniz DR, Faria RO, Benedito VA, de Fátima Â, Modolo LV. Plant as biofactories of pharmaceuticals and nutraceuticals. In: Brahmachari G, editor. Chemistry and Pharmacology of Naturally Occurring Bioactive Compounds. Broken Sound Parkway NW Suite: Taylor and Francis Group; 2013. p. 529-55.
Okokon JE, Antia BS, Azare BA, Okokon PJ. Antiplasmodial activity and cytotoxicity of ethanol extract of Zea mays
root. Avicenna J Phytomed 2017;7:275-84.
Liu KC, Yang SL, Roberts MF, Elford BC, Phillipson JD. Antimalarial activity of Artemisia annua
flavonoids from whole plants and cell cultures. Plant Cell Rep 1992;11:637-40.
Enegide C, Ofili CC, Jacob DL, Ameh SF. Preliminary studies on the toxicity profile of Combretum nigricans
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]