- Open Access
Anti-malarial activity of a polyherbal product (Nefang) during early and established Plasmodium infection in rodent models
© Arrey Tarkang et al.; licensee BioMed Central Ltd. 2014
Received: 6 August 2014
Accepted: 15 November 2014
Published: 25 November 2014
The emerging resistance of Plasmodium species to currently available anti-malarials remains a public health concern, hence the need for new effective, safe and affordable drugs. Natural products remain a reliable source of drugs. Nefang is a polyherbal anti-malarial of the Cameroonian folklore medicine with demonstrated in vitro antiplasmodial and antioxidant activities. It is composed of Mangifera indica (bark and leaf), Psidium guajava, Carica papaya, Cymbopogon citratus, Citrus sinensis, Ocimum gratissimum (leaves). This study aimed at investigating the suppressive, prophylactic and curative activities of Nefang in Plasmodium infected rodent models.
Systemic acute oral toxicity of Nefang aqueous and ethanol extracts was assessed in mice up to a dose of 5,000 mgkg−1 body weight. BALB/c mice and Wistar rats were inoculated with Plasmodium chabaudi chabaudi and Plasmodium berghei, respectively, and treated with Nefang, the Mangifera indica bark/Psidium guajava combination and a Psidium guajava leaf aqueous extracts (75, 150, 300 and 600 mgkg−1 bwt). Their schizonticidal activity was then evaluated using the Peter’s 4-day suppressive test). The prophylactic and curative (Rane’s Test) activity of Nefang was also evaluated by determining the parasitaemia, survival time, body weight and temperature in pre-treated rodents.
Acute oral toxicity of the extract did not cause any observed adverse effects. Percent suppressions of parasitaemia at 600 mgkg−1 bwt were as follows (P. berghei/P. chabaudi): Nefang – 82.9/86.3, Mangifera indica bark/Psidium guajava leaf combination extract – 79.5/81.2 and Psidium guajava leaf – 58.9/67.4. Nefang exhibited a prophylactic activity of 79.5% and its chemotherapeutic effects ranged from 61.2 – 86.1% with maximum effect observed at the highest experimental dose.
These results indicate that Nefang has excellent in vivo anti-malarial activities against P. berghei and P. chabaudi, upholding earlier in vitro antiplasmodial activities against multi-drug resistant P. falciparum parasites as well as its traditional use. Hence, Nefang represents a promising source of new anti-malarial agents.
Despite substantial efforts to control malaria in the last few decades, it remains one of the most prevalent infectious diseases globally. The emerging resistance of Plasmodium species to currently available drugs remains a public health concern . Forty percent of the world’s population is exposed to malaria and there is a constant need for new anti-malarials. Historically, plants have had a remarkable role in therapeutics and were the principal source of drugs for many centuries. Quinine, isolated in 1820 from Cinchona species (Rubiaceae), is an illustrative example. Drugs in current use for malaria chemotherapy include artemisinin, from Artemisia annua (Asteraceae) of Chinese origin, and its semi-synthetic derivatives, artemether, artesunate and arteether . A recently introduced plant-derived anti-malarial drug is atovaquone, a synthetic naphthoquinone based on lapachol. Lapachol, a prenylnaphtoquinone, was first isolated from Tabebuia impetiginosa, a South American member of the Bignoniaceae family . Artemisinin-based combination therapy (ACT) is currently the most effective chemotherapy against Plasmodium falciparum malaria and the emergence of resistance would be a public health disaster in malaria endemic areas. Therefore, plants do not only provide valuable clues for finding new drugs, but may help to shift the drug discovery paradigm from finding new molecules to combining existing agents [4, 5].
The modern pharmaceutical industry was born from botanical medicine, but standardized synthetic combinatorial chemistry in drug discovery and high throughput screening (HTS) of potential drug targets have disconnected the historical link between plants and medicines. However, this has been rekindled by the small output of modern anti-malarial pharmaceutical research and development, which has stimulated new interest in the potential of natural compounds . Hence natural products continue to provide new starting points in drug discovery.
There is a school of thought that biologically-derived secondary metabolites and synthetic compounds derived from them perform better as drugs than randomly synthesized compounds. Drug-derived parent molecules were present in primitive life forms and therefore co-evolved to interact with one another, thus granting direct ecological benefit to the producing organism, whether in competition for resources, avoiding predation or combating pathogens . This co-evolution between different plants and/or metabolites within the same plant brings about synergy or potentiation which has been proven to achieve favourable results, such as enhanced efficacy, decreased dosage at equal or increased level of target inhibition, reduced or delayed development of drug resistance and simultaneous reduction of toxic effects . Based on this paradigm, drug combination in anti-malarial chemotherapy has been adopted and is widely used as a strategy to monitor and prevent resistance .
Nefang is a polyherbal product composed of the aqueous extracts of Mangifera indica (bark and leaf), Psidium guajava, Carica papaya, Cymbopogon citratus, Citrus sinensis and Ocimum gratissimum (leaves). It is frequently used for the treatment of malaria in the South West Region of Cameroon. Ethnopharmacological studies confirmed its formulation and folk use . Studies have been conducted to evaluate the in vitro and in vivo antioxidant properties of this polyherbal , which may play a role in curbing oxidative stress related with malaria infection. The in vitro antiplasmodial activity of this product and solvent extracts of its constituents have also been evaluated, showing good activities and synergistic potentials of some constituent extracts .
This study aimed at evaluating the in vivo suppressive, prophylactic and curative activities of Nefang in mice and rats models. Results obtained showed that Nefang exhibited excellent in vivo anti-malarial activities in rodents, consistent with its previously observed anti-P. falciparum activities.
Extraction of plant material
Fresh parts of the constituent plants of Nefang: bark and leaves of Mangifera indica (MiB and MiL, respectively), and leaves of Psidium guajava (Pg), Carica papaya (Cp), Cymbopogon citratus (Cc), Citrus sinensis (Cs), Ocimum gratissimum (Og) were harvested from their natural habitat in Cameroon between July and August 2011. Plant identification and voucher specimen referencing were done at the Institute of Medical Research and Medicinal Plants Studies (IMPM) herbarium in Yaoundé, Cameroon by a botanist. The freshly harvested plant parts were air dried and pulverized. Aqueous extraction was performed based on the traditional knowledge of preparation. Research evidence shows that ethanol extracts are as effective as the water extracts  and, therefore, ethanol extraction was also performed. Weighed quantities (1,000 g) of each plant part were exhaustively macerated in water (2.4 L) and ethanol (2.0 L) respectively for 4 h. Each of the macerate was transferred into a conical percolator for 72 h and the extracts were filtered . Each ethanol filtrate was first concentrated using a rotary evaporator. The filtrates were then concentrated in an air oven at 60°C. The extracts were weighed and stored in labeled sealed plastic containers at 4°C until use to prevent decomposition.
Swiss albino mice (25 – 30 g) were used for the acute toxicity testing while BALB/c mice (20 – 25 g) and Wistar rats (160 – 180 g) were used for in vivo antiplasmodial activities testing. All experimental animals were housed under standard environmental conditions of temperature at 22-24°C under a 12 h dark–light cycle, and allowed free access to drinking water and standard pellet diet.
Ethical approval for the study was obtained from Kenyatta National Hospital/University of Nairobi Ethics and Research committee, Nairobi-Kenya (KNH-ERC/A/324 - 5/12/12), Institute of Medical Research and Medicinal Plants Studies Institutional Review Board (076/82-62/MINRESI/M000 – 01/06/12) and Institut Pasteur Korea-Institutional Animal Care and Use Committee (IACUC No. IPK 12009 – 29/10/12).
Acute (single dose) oral toxicity testing
The acute oral toxicity of Nefang aqueous and ethanol extracts was evaluated according to the procedures outlined by the Organization for Economic Co-operation and Development . Each crude extract was suspended in a vehicle (distilled water and corn oil for the aqueous and ethanol extracts respectively). Following a 4 h fasting period, mice were divided into groups of three. Extract doses were calculated in reference to mice body weight and each mouse was treated with a single oral dose of the extract.
The mice were dosed in a stepwise procedure using the fixed doses of 5, 50, 300, 1,200 and 2,000 mgkg−1 body weight (bwt) of the aqueous and ethanol extracts for each group of mice respectively. After each dose, the animals were observed for signs of toxicity for three hours. If there was no mortality or signs of toxicity at the highest dose, then the upper limit dose was used for the main test.
For the main test, a single high oral dose of 5,000 mgkg−1 bwt of each extract was administered to three male (Test 1) and three female (Test 2) mice in the treatment groups, whereas the control groups received the vehicle. Food was provided to the mice approximately an hour after treatment. The animals were observed 30 min after dosing, followed by hourly observation for 8 h and once a day for the next 13 days. Observations were systematically recorded for each animal. Surviving animals were weighed and visual observations for mortality, behavioural pattern, changes in physical appearance, injury, pain and signs of illness were conducted daily during the study period.
Parasite infection of experimental animals
The chloroquine sensitive strain of Plasmodium berghei (strain ANKA) was generously donated by the Institute of Primate Research (IPR), Nairobi, Kenya, while the Plasmodium chabaudi chabaudi was obtained from the Centre for Neglected Diseases Drug Discovery (CND3), Institut Pasteur Korea, as cryo-frozen stock of parasitized red blood cells (PRBCs). The parasites were prepared through two cycles of passage of the PRBCs in rats and mice. Donors with parasitaemia level of 20-30% were sacrificed and blood collected by cardiac puncture into heparinized tubes. The blood was then diluted with phosphate buffered saline (PBS) based on parasitaemia level of each donor and the RBC count of normal mice and rats, such that 1 mL blood contained 5 × 107 parasites. The experimental animals were each treated with 1 × 107 PRBCs by intraperitoneal (ip) injection .
Test for suppressive activity (Peter’s 4-day test)
The aqueous extracts that were selected for this study were Nefang, Psidium guajava (Pg) (i.e. the most active constituent aqueous extract) and Mangifera indica bark/Psidium guajava leaf (MiB/Pg) (i.e. the solvent extract combination that showed the most promising synergistic activity) . The suppressive activities of these extracts were evaluated in early P. chabaudi and P. berghei infection in BALB/c mice and Wistar rats, respectively, using the method described by Knight and Peters . Forty-five mice and forty-five rats were each randomly divided into fifteen groups of three each. On the first day (D0), the mice and rats were each infected with 107P. chabaudi and P. berghei, respectively. Three hours later, the experimental groups of mice and rats were each treated orally with 10 mLkg−1 bwt of the drug or extract as follows: Group 1 (negative/vehicle control) - PBS, Group 2 (positive control) - chloroquine (10 mgkg−1), Group 3 (positive control) - pyrimethamine (30 mgkg−1), Groups 4 to 7 - Nefang, Groups 8 to 11 - Pg and Groups 12 to 15 - MiB/Pg. The aqueous plant extracts were each administered orally at a dose of 75, 150, 300 and 600 mgkg−1 respectively. Treatment was carried out for four consecutive days (D0 – D3). The body weight of each mouse were measured on the first day (D0) and on the fifth day (D4) using a sensitive digital analytical balance, while the body temperature was taken before infection and three hours after infection (D0) and then monitored daily to the fifth day (D4).
where A is the average percentage parasitaemia in the negative control group and B is the average percentage parasitaemia in the test group.
Test for prophylactic activity
The repository activity of Nefang was assessed using the method described by Peters . The mice were randomly divided into seven groups of three BALB/c mice each. Group 1 (negative control) was treated with 10 mLkg−1 of PBS, group 2 and 3 (positive controls) - CQ (10 mgkg−1) and pyrimethamine (30 mg/kg−1), respectively, group 4 to 7 (extract test groups) - 75, 150, 300 and 600 mgkg−1 of Nefang, respectively. Administration of the extract and standard drugs continued for three consecutive days (D0 - D2). On the fourth day (D3), the mice were inoculated with 107P. berghei infected red blood cells and the level of parasitaemia was assessed by blood smear 72 h later.
Test for curative activity (Rane’s test)
Data are expressed as mean ± standard deviation (SD) of the mean. Data were analysed using SPSS Version 20.0. Statistical significance testing was done using the one-way analysis of variance (ANOVA) followed by Neuman-Keuls multiple comparison test. P-values of less than 0.05 were considered statistically significant.
Acute (single dose) oral toxicity testing
There were no observed adverse effects at all oral dose levels (5, 50, 300, 2000 mgkg−1 bwt) for all the aqueous and ethanol extracts of Nefang and its constituents. All the mice survived. Similarly, oral administration of the aqueous and ethanol extracts of Nefang and its constituents at 5,000 mgkg−1 bwt, had no toxic effects throughout the 14-day study period. None of the mice showed any signs of toxicity, such as changes on skin, eyes and mucus membranes, behavioural patterns, trembling, diarrhoea, falling of the fur, sleep or coma. No significant changes were observed in their body weights. The estimated maximum tolerable dose (MTD) was above 5,000 mgkg−1 bwt for all extracts tested.
Evaluation of the suppressive activity (Peter’s 4-Day Test)
Evaluation of the prophylactic activity
Evaluation of the curative activity (Rane’s test)
The in vivo antiplasmodial activities of the aqueous extract of Nefang and its active components, Pg and MiB/Pg, were investigated by evaluating the chemosuppression during early infection, while Nefang alone was evaluated during established infection using standard animal models. In vivo models are usually employed in anti-malarial studies because they take into account the possible prodrug effect and probable involvement of the immune system in eradication of the pathogen . During early infection, Peter’s 4-Day suppressive test was used to evaluate schizontocidal activity while the repository test was used to study the prophylactic activity. Rane’s test was used to study curative ability during established infection. In all methods, determination of percent inhibition of parasitaemia was the most reliable parameter. A mean parasitaemia level that is ≤90% of that of the vehicle treated animals usually indicates that the test compound is active . In the 4-day suppressive activity, Nefang and MiB/Pg significantly reduced parasitaemia (in both Plasmodium spp) in animal models in a dose-dependent manner, with Nefang exhibiting anti-malarial activities comparable to that of the standard drugs tested. The repository test revealed the same dose-dependent chemosuppression by Nefang. In the curative activity, the dose-dependent activity of Nefang at the highest experimental dose was observed from Day 2 of treatment. Though its activity was lower than that of CQ, it was comparable to that of ART. Furthermore, we observed that the survival time of Nefang-treated animals was prolonged in a dose-dependent manner (Figure 7). Nefang caused a dose-dependent reduction of pyrexia and loss of body weight in infected animals. Body weight loss and temperature reduction are hallmarks of malaria infection in animal models , suggesting that an effective plant-derived anti-malarial agent should prevent body weight loss in Plasmodium infected animals. This dose-dependent preventive activity indicates that Nefang does not have any adverse effect in experimental animals at the doses tested and as observed in our oral acute toxicity studies.
In acute toxicity testing, doses higher than 5,000 mgkg−1 bwt are generally not considered as dose related, which is in accordance with the Organization for Economic Corporation and Development (OECD) Guidance Document for Acute Oral Toxicity Testing [23, 15]. Compounds with LD50 values lower than 2,000 mgkg−1 bwt are generally considered to be relatively safe, because values above this are non-classified. This signifies that Nefang and its constituent aqueous and ethanol extracts can be considered as non-toxic at acute oral administration since the extracts were well tolerated and no observed adverse effect levels were >5,000 mgkg−1. These results are consistent with earlier reports on the cytotoxicity of Nefang.
Unlike in humans, increase in parasitaemia levels in rodent models usually results in decreased metabolic rates and a consequent decrease in body temperatures , which might result in death. An ideal anti-malarial agent would, therefore, prevent this occurrence, an effect observed in Nefang-treated animals. Taken together, these results confirm that Nefang has therapeutic activity against established infection and further confirm the in vitro antiplasmodial activities reported earlier. At the highest dose (600 mgkg−1) tested, Nefang exhibited an in vivo suppression of parasitaemia of >80%, prophylactic activity of 79.5% and chemotherapeutic effects of 60 – 80%. In vivo antiplasmodial activity can be classified as moderate, good, and very good if an extract displayed percentage parasitaemia suppression equal to or greater than 50% at a dose of 500, 250 and 100 mgkg−1 per day, respectively , suggesting, therefore, that Nefang has very good activity. This also suggests that the overall anti-malarial activity of the synergistic and additive components identified during the interaction studies , over-shadowed the antagonistic interactions. Synergy between different constituents of extracts has been documented for anti-malarial and other pharmacological activities [26, 27].
Earlier phytochemical screening of the constituent plant extracts of Nefang showed the presence of alkaloids, anthocyanins, flavonoids, phenols, saponins, tannins, triterpenes and sterols , suggesting that the anti-malarial activity of Nefang cannot be attributed only to the active antiplasmodial compounds in the constituent plant extracts. In our earlier in vitro antiplasmodial studies on the constituent plant extracts of Nefang, only M. indica (bark and leaf) and P. guajava showed good activities while the rest showed very weak activities or inactivity. However, there have been reports of good in vivo antiplasmodial and anti-malarial activities exhibited by C. papaya, C. citratus and O. gratissimum, which are constituent plants of Nefang. As earlier reported, many anti-malarial herbal remedies may exert their anti-infective effects not only by directly affecting the pathogen, but also by indirectly stimulating natural and adaptive defense mechanisms of the host by other mechanisms. Therefore, extracts that can stimulate innate and/or adaptive immunity may be able to contribute to prophylaxis and treatment not only for malaria but for other diseases as well [31, 32]. This suggests that a combination of the biological activities of the constituent plant extracts of Nefang results in an enhanced overall anti-malarial activity of Nefang. Therefore, an understanding of the underlying pharmacodynamic or pharmacokinetic mechanisms of this action would be very important towards understanding the anti-malarial potentials of Nefang.
This study provides evidence that Nefang is safe and possesses good in vivo suppressive, prophylactic and curative activities against Plasmodium species. These findings uphold earlier in vitro antiplasmodial activities and confirm the synergistic interactions between its constituents plant extracts. It further suggests that the observed in vivo anti-malarial activity of Nefang may result from the synergistic biological activities of its constituent plant extracts. Hence, Nefang represents a promising source of anti-malarial agents for downstream clinical development.
The authors are thankful to Institut Pasteur Korea for hosting PAT, and for providing both material and reagent support during the study. We also acknowledge the material and technical support of Dr. Ozwara HS, Institute of Primate Research (IPR), Nairobi, Kenya; Mr. Mbithi E and Ms. Muthini F, Department of Medical Microbiology; Dr. Abuga KO and Mr. Mugo HN, Department of Pharmaceutical Chemistry, University of Nairobi, Kenya.
- WHO: World Malaria Report. 2013, Geneva,http://www.who.int/malaria/publications/world_malaria_report_2013/en/, . World Health Organization,Google Scholar
- Oliveira AB, Dolabela MF, Braga FC, Jacome RL, Varotti FP, Povoa MM: Plant-derived antimalarial agents: new leads and efficient phythomedicines. Part I Alkaloids Acad Bras Cienc. 2009, 81: 715-740. 10.1590/S0001-37652009000400011.View ArticleGoogle Scholar
- Castellanos JRG, Prieto JM, Heinrich M: Red Lapacho (Tabebuia impetiginosa)– a global ethnopharmacological commodity?. J Ethnopharmacol. 2009, 121: 1-13. 10.1016/j.jep.2008.10.004.View ArticleGoogle Scholar
- Kong DX, Li XJ, Zhang HY: Where is the hope for drug discovery? Let history tell the future. Drug Discov Today. 2009, 14: 115-119. 10.1016/j.drudis.2008.07.002.View ArticlePubMedGoogle Scholar
- Wagner H, Ulrich-Merzenich G: Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine. 2009, 16: 97-110. 10.1016/j.phymed.2008.12.018.View ArticlePubMedGoogle Scholar
- Ginsburg H, Deharo E: A call for using natural compounds in the development of new antimalarial treatments - an introduction. Malar J. 2011, 10 (Suppl 1): S1-10.1186/1475-2875-10-S1-S1.PubMed CentralView ArticlePubMedGoogle Scholar
- Ganesan A: The impact of natural products upon modern drug discovery. Curr Opin Chem Biol. 2008, 12: 306-317. 10.1016/j.cbpa.2008.03.016.View ArticlePubMedGoogle Scholar
- Ma XH, Zheng CJ, Han LY, Xie B, Jia J, Cao ZW, Li YX, Chen YZ: Synergistic therapeutic actions of herbal ingredients and their mechanisms from molecular interaction and network perspectives. Drug Discov Today. 2009, 14: 579-588. 10.1016/j.drudis.2009.03.012.View ArticlePubMedGoogle Scholar
- Nosten F, White NJ: Artemisinin-based combination treatment of falciparum malaria. Am J Trop Med Hyg. 2007, 77: 181-192.PubMedGoogle Scholar
- Tarkang PA, Okalebo FA, Agbor GA, Tsabang N, Guantai AN, Rukunga GM: Indigenous Knowledge and folk use of a polyherbal antimalarial by the Bayang Community, South West Region of Cameroon. J Nat Prod Plant Res. 2012, 2: 372-380.Google Scholar
- Tarkang PA, Atchan APN, Kuiate J, Okalebo FA, Guantai AN, Agbor GA: Antioxidant potential of a polyherbal antimalarial as an indicator of its therapeutic value. Adv Pharmacol Sci. 2013, 2013: 678458-Google Scholar
- Tarkang PA, Franzoi KD, Lee S, Lee E, Vivarelli D, Freitas-Junior L, Liuzzi M, Tsabang N, Ayong LS, Agbor GA, Okalebo FA, Guantai AN: In vitro antiplasmodial activities and synergistic combinations of differential solvent extracts of the polyherbal product. Nefang BioMed Res Int. 2014, 2014: 835013-Google Scholar
- Willcox M: Improved traditional phytomedicines in current use for the clinical treatment of malaria. Planta Med. 2011, 77: 662-671. 10.1055/s-0030-1250548.View ArticlePubMedGoogle Scholar
- Handa SS, Khanuja SPS, Longo G, Rakesh DD: Extraction Technologies for Medicinal and Aromatic Plants. 2008, Trieste, Italy: ICS-UNIDO International Centre for Science and High Technology, 266-Google Scholar
- Organization for Economic Cooperation Development: Environment, Series on testing and Assessment N° 24. Guidance Document on Oral Toxicity Testing 423 and 425. 2001, Paris, France: Health and Safety Publications, 24-Google Scholar
- Basir R, Fazalul Rahiman SS, Hasballah K, Chong WC, Talib H, Yam MF, Jabbarzare M, Tie TH, Othman F, Moklas MAM, Abdullah WO, Ahmad Z: Plasmodium berghei ANKA infection in ICR mice as a model of cerebral malaria. Iranian J Parasitol. 2012, 7: 62-74.Google Scholar
- Knight DJ, Peters W: The antimalarial action of N-benzyloxy dihydrotriazines. The action of cycloguanil (BRL50216) against rodent malaria and studies on its mode of action. Ann Trop Med Parasitol. 1980, 74: 393-404.PubMedGoogle Scholar
- Peters W: Drug resistance in Plasmodium berghei. Vincke and Lips, 1948; 1. Chloroquine resistance. Exp Parasitol. 1965, 17: 80-89. 10.1016/0014-4894(65)90012-3.View ArticlePubMedGoogle Scholar
- Ryley JF, Peters W: The antimalarial activity of some quinone esters. Ann Trop Med Parasitol. 1970, 84: 209-222.Google Scholar
- Waako PJ, Gumede B, Smith P, Folb PI: The in vitro and in vivo antimalarial activity of Cardiospermum halicacabum and Momordica foetida. J Ethnopharmacol. 2005, 99: 137-143. 10.1016/j.jep.2005.02.017.View ArticlePubMedGoogle Scholar
- Peter IT, Anatoli VK: The Current Global Malaria Situation. Malaria Parasite Biology, Pathogenesis, and Protection. 1998, Washington DC, USA: ASM Press, 11-22.Google Scholar
- Langhorne J, Quin SJ, Sanni LA: Mouse models of blood-stage malaria infections: immune responses and cytokines involved in protection and pathology. Malaria Immunology. Edited by: Perlmann P, Troye Blomberg M. 2002, Stockholm: Karger, 204-228. 2View ArticleGoogle Scholar
- Hayes AW: Guidelines of Acute Oral Toxicity Testing. 1987, New York: Raven Press Ltd, 185-2Google Scholar
- Chinchilla M, Guerrero OM, Abarca G, Barrios M, Castro O: An in vivo model to study the anti-malaria capacity of plant extracts. Rev Biol Trop. 1998, 46: 1-7.Google Scholar
- Deharo E, Bourdy G, Quenevo C, Munoz V, Ruiz G, Sauvain M: A search for national bioactive compounds in Bolivia through a multidisciplinary approach. Part V. Evaluation of the antimalarial activity of plants used by the Tecana Indians. J Ethnopharmacol. 2001, 77: 91-98. 10.1016/S0378-8741(01)00270-7.View ArticlePubMedGoogle Scholar
- Williamson EM: Synergy and other interactions in phytomedicines. Phytomedicine. 2001, 8: 401-409. 10.1078/0944-7113-00060.View ArticlePubMedGoogle Scholar
- Houghton PJ: Synergy and polyvalence: paradigms to explain the activity of herbal products. Evaluation of Herbal Medicinal Products. Edited by: Houghton PJ, Mukherjee PK. 2009, London: Pharmaceutical Press, 85-94.Google Scholar
- Ofori-Attah K, Oseni LA, Quasie O, Antwi S, Tandoh M: A comparative evaluation of in vivo antiplasmodial activity of aqueous leaf exracts of Carica papaya, Azadirachta indica, Magnifera indica and the combination thereof using Plasmodium infected BALB/c mice. Int J Applied Biol Pharm Tech. 2012, 3: 372-378.Google Scholar
- Melariri P, Campbell W, Etusim P, Smith P: In vitro and in vivo antiplasmodial activities of extracts of Cymbopogon citratus Staph and Vernonia amygdalina Delile leaves. J Nat Prod. 2011, 4: 164-172.Google Scholar
- Murithi CK, Fidahusein DS, Nguta JM, Lukhoba CW: Antimalarial activity and in vivo toxicity of selected medicinal plants naturalised in Kenya. Int J Edu Res. 2014, 2: 395-406.Google Scholar
- Masihi KN: Immunomodulatory agents for prophylaxis and therapy of infections. Int J Antimicrob Agents. 2000, 14: 181-191. 10.1016/S0924-8579(99)00161-2.View ArticlePubMedGoogle Scholar
- Muniz-Junqueira MI: Immunomodulatory therapy associated to antiparasite drugs as a way to prevent severe forms of malaria. Curr Clin Pharmacol. 2007, 2: 59-73. 10.2174/157488407779422285.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.