|Year : 2016 | Volume
| Issue : 1 | Page : 29-35
Comparative antiplasmodial evaluation of Cymbopogon citratus extracts in Plasmodium berghei-infected mice
David Arome, Enegide Chinedu, Solomon Fidelis Ameh, Akpabio Inimfon Sunday
Department of Science Laboratory Technology, University of Jos, Jos, Nigeria
|Date of Submission||25-Feb-2016|
|Date of Acceptance||22-Apr-2016|
|Date of Web Publication||16-Jun-2016|
Department of Science Laboratory Technology, University of Jos, Jos
Source of Support: None, Conflict of Interest: None
Background: As malaria is still an important life-threatening infection in many tropical countries and drug resistance has become increasingly common to drugs used nowadays, there is a pressing need to find more drugs that may contribute to the reduction of malaria in the future. This calls for an inward look into harnessing the full potential of medicinal plants that abound around us.
Objective: To evaluate the antiplasmodial activity of aqueous leaf and root extracts of Cymbopogon citratus against Plasmodium berghei in mice.
Materials and Methods: Cymbopogon citratus extracts of 200, 400, 800 mg/kg, and 5 mg/kg of chloroquine were used. Antiplasmodial activity of the extracts was evaluated using 4-day suppressive test model.
Results: The extracts exhibited significant (P < 0.05) antiplasmodial activity in all the experimental doses used. The aqueous leaf extract produced a percentage suppressive effect of 20.83%, 55.56%, and 80.56% while that of the root extract produced a percentage suppression of 50.38%, 77.78%, and 100%. The suppressive effect of the extracts followed a dose-dependent pattern with 800 mg/kg of the aqueous root extract having the highest activity and producing the same 100% suppressive effect as chloroquine. In addition, the extracts had a mild effect on the body temperature of the infected mice; there was a significant increase only on the 2 nd day of the study.
Conclusion: The results of the study suggested that the aqueous root extract possesses a better antiplasmodial activity than the aqueous leaf extract.
Keywords: Antiplasmodial activity, chloroquine, Cymbopogon citratus, Plasmodium berghei, temperature
|How to cite this article:|
Arome D, Chinedu E, Ameh SF, Sunday AI. Comparative antiplasmodial evaluation of Cymbopogon citratus extracts in Plasmodium berghei-infected mice. J Curr Res Sci Med 2016;2:29-35
|How to cite this URL:|
Arome D, Chinedu E, Ameh SF, Sunday AI. Comparative antiplasmodial evaluation of Cymbopogon citratus extracts in Plasmodium berghei-infected mice. J Curr Res Sci Med [serial online] 2016 [cited 2021 Apr 20];2:29-35. Available from: https://www.jcrsmed.org/text.asp?2016/2/1/29/184126
| Introduction|| |
Until now, resistance to the known antimalarial drugs remains a key challenge in eradicating the scourge of malaria. Even with the current combination therapy in use, there are still issues with treatment failure, recrudescence of infection and resistance to artemisinin-based combination therapy.  Therefore, these challenges have prompted a renewed interst in the search of potent and novel antimalarial agents from medicinal plants. Malaria has been ranked as one of the five leading causes of death among developing countries of the world.  Global estimates show that about 3.3 billion people were at a risk of malaria in 2011, with people living in sub-Saharan African region having the greatest risk of acquiring malaria, and about 80% of cases and 90% of deaths occur in Africa. 
Malaria is an infectious disease cause by Plasmodium species. The parasite is transmitted to humans through the bite of infected female anopheles mosquitoes. Although more than 200 different species of Plasmodium have been identified,  five are known to infect humans: Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, and Plasmodium knowlesi. , The deadliest of all is P. falciparum, responsible for vast number of death recorded annually from malaria incidence.  Malaria is usually characterized by high spiking fevers, chills, malaise, headache, myalgias, as well as occurrence of gastrointestinal symptoms. After infection, the stages of malaria in the human body include the hepatic and erythrocyte stages.
Pathological processes in malaria are the result of the erythrocytic cycle, and the involvement of the red cells make malaria a multisystem disease. , Common signs and symptoms of malaria as mentioned earlier in some patients may progress to severe malaria, more often seen in the cases of P. falciparum infection, where even death may occur. At the completion of schizogony within the red blood cells (RBCs), newly formed merozoites are released by lysis of the infected erythrocyte and along with them, numerous known and unknown waste substances, RBCs products, hemozoin, and other toxins such as glycosylphosphatidylinositol (GPI) anchor of parasite membrane and merozoite surface protein 1 are released into the blood. The released parasite toxin, particularly GPI activates the macrophage and epithelial cells to secrete cytokines, and other proinflammatory mediators such as interleukin 1 (IL1), IL6, IL8, interferon-γ as well as other endogenous pyrogens. The systemic manifestations such as headache, fever, diarrhea, immune suppression, and central nervous system manifestation have largely been attributed to the release of cytokines in response to parasite toxin and other membrane products.  Moreover, the parasite DNA which is highly proinflammatory can induce cytokinemia and fever. Furthermore, the hemozoin produced as a metabolic by-product by parasite digestion of hemoglobin interacts with intracellular toll-like receptor 9 leading to the release of proinflammatory cytokines which, in turn, cause the release of cyclooxygenase 2 which induces upregulation of prostaglandins leading to the induction of fever. , In severe cases, the released merozoites like in the case of P. falciparum can result in progressive and dramatic structural, biochemical, and mechanical modification that can lead to life-threatening complication. , Severe infection can also occur in non-falciparum infections as in the case of P. vivax and P. knowlesi infections,  such as cerebral malaria.
Cymbopogon citratus is a tropical perennial grass cultivated in many areas of tropical regions. C. citratus is considered by herbalists to have a wide range of medicinal values. In Nigeria, concoction preparations of lemon grass have been used in the treatment of ailments such as typhoid, fever, stomach aches,  and also in combination with other plants for the treatment of malaria.  Its folklore applications have been supported as a potential drug to control malaria. It has been established that the essential oil of the plant possesses antinociceptive and anti-inflammatory activities;  it was also found to have insect repellent and insecticide activity, ,, and neurobehavioral effect as observed with the essential oil of C. citratus in mice.  This study focused on evaluating its antiplasmodial activity and compared the activities between leaf and root extracts against Plasmodium berghei in mice.
| Materials and methods|| |
Collection of plant material
Fresh leaves and root of C. citratus were collected in March 2012 from Eto Baba, Jos, Plateau State, Nigeria. The plant material was identified and authenticated by Mr. Ikechukwu Chijioke of Federal College of Forestry, Jos. Voucher specimen was prepared and deposited in the herbarium unit of the college. The leaves and roots were separated and dried at room temperature for 3 weeks, and then crushed into coarse powder using mortar and pestle.
Extraction of plant material
Sixty grams of the leaf and root of the powdered plant materials were measured separately and dissolved in sufficient quantity of distilled water as solvent for 48 h with mechanical shaking (4h/day). At the end of 48 h, the mixture was filtered with ashless filter paper. The extract was concentrated using rotary evaporator at a temperature of 40°C. The concentrate was heated over a water bath to obtain a solvent-free extract which was later stored in the refrigerator at 4°C.
Phytochemical screening of the crude leaf and root of C. citratus extracts were carried out using standard procedure, as described below. 
Test for cardiac glycosides
About 2 ml of the extract solution was diluted with 1 ml of glacial acetic acid followed by six drops of 10% ferric chloride solution and six drops of concentrated sulfuric acid. The formation of green-blue color indicates the presence of cardiac glycosides.
Test for saponins
The extract was diluted with 20 ml of distilled water and then the test tube was shaken for about 10 min. The formation of lather or foam on top indicated the presence of saponins.
Test for tannins
The extract solution was dissolved into 4 ml of chloroform and 1 ml of acetic anhydride. About 1 ml of sulfuric acid was added to it along the wall sides of the test tube. The formation of green coloration showed the presence of tannins.
Test for steroids
The extract was dissolved in 10 ml of chloroform, and 1 ml of concentrated sulfuric acid was added into the test tube. The formation of red color in the upper layer and yellow color in the sulfuric acid layer showed the presence of steroids.
Test for carbohydrates
About 2 ml of the extract solution was diluted with two drops of Molisch's test reagent and mixed thoroughly. Then, 4 ml of concentrated sulfuric was added. The formation of purple color indicates the presence of carbohydrates.
Test for flavonoids
A few drops of diluted sodium hydroxide were added to the extract solution. The formation of intense yellow color which becomes colorless upon the addition of a few drops of diluted sulfuric acid showed the presence of flavonoids.
Test for alkaloids
One gram of the dry extract was taken into the test tube and 3 ml of ammonia was added to it. It was allowed to stand for few minutes. Later, 10 ml of chloroform was added and shaken, then filtered to remove powder extract. Chloroform was evaporated using the water bath and Mayer's reagent was later added. The formation of cream coloured precipitate showed the presence of alkaloids.
Swiss albino mice of either sex weighing 20-28 g were obtained from the Nigerian Institute for Trypanosomiasis Research, Jos, Nigeria. The mice were acclimatized for 2 weeks to laboratory condition in the Animal Unit of the University of Jos, Nigeria. The mice were housed in plastic cages in a ventilated room at a temperature of 20 ± 0.6°C, fed with standard rodent chow and allowed free access to potable water. All experiments were carried out in accordance with the experimental procedure of the animal unit of the university.
Body temperature of the mice were measured with digital thermometers inserted into the rectum and read when the readout stabilized (20 s). The body temperatures of the mice were measured regularly throughout the 4-day study period.
Acute toxicity test
The modified method of Lorke's  was used in the LD 50 test of C. citratus extracts. This test was carried out in two phases. In the first phase, nine mice randomized into three groups of three mice each were given 10, 100, and 1000 mg/kg of the prepared extract orally. The mice were observed at 4 h and subsequently, daily for 7 days for any behavioral sign of toxicity. The same procedure as used in the first phase was adopted in phase two, but with different dose levels of 1600, 2900, and 5000 mg/kg. The acute toxicity test was carried out separately for the aqueous extracts of C. citratus. LD 50 was calculated as the square root of the product of the lowest lethal dose and highest nonlethal dose.
Then, the LD50 is calculated by the formula: √ D0 × D100. Where, D0 = Highest dose that gave no mortality, D100 = Lowest dose that produced mortality.
Chloroquine-sensitive P. berghei (NK65) rodent parasite was sourced from the National Institute for Medical Research, Lagos, Nigeria. Parasitemia was maintained by continuous intraperitoneal reinfection of healthy mice in the animal unit of the University of Jos.
One week after parasite inoculation, a Giemsa-stained thin blood smear was prepared from the tail vein of the donor mouse on a glass slide, and the level of parasitemia was determined using Neubaur hemocytometer. The withdrawn blood sample was diluted with normal saline such that 0.2 ml contained approximately 10–5 parasitized RBCs. Each experimental mouse was inoculated with 0.2 ml of the diluted infected blood.
Antiplasmodial assessment of Cymbopogon citratus in early infection (4-day suppressive test)
Four-day suppressive test model was employed in the study,  against chloroquine-sensitive P. berghei (NK65). 40 mice were randomized into eight groups of five mice each such that each group had approximately the same mean weight. On day 0, within 4 h after inoculation of mice with the parasite, treatment of all the groups was initiated orally. Groups 1 and 2 served as negative and positive control that received distilled water and chloroquine (5 ml and 5 mg/kg body weight, respectively), Groups 3, 4, and 5 received the aqueous leaf extract at doses of 200, 400, and 800 mg/kg orally via intragastric cannula while Groups 6, 7, and 8 were given the aqueous root extract at the same dose levels as the aqueous leaf extract. Treatment lasted for 4 days and treatment was administered at the same time each day. On the 4 th day, thin films of tail vein blood were prepared and stained with Giemsa stain. The film was examined microscopically and parasitemia was expressed as the mean number of parasitized erythrocytes counted in ten fields of approximately 250 erythrocytes per field.
Percentage suppression of parasitemia was calculated using the following equation: mean parasitemia control group - mean parasitemia treated × 100/mean parasitemia control.
The results of the study were expressed as mean ± standard error mean. Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test, and values of P < 0.05 were considered significant. The analysis was performed using instant GraphPad Software, Inc. (Version 5.02).
| Results|| |
Results obtained from the phytochemical screening of the aqueous leaf and root extracts of C. citratus showed the presence of the following constituents: alkaloids, carbohydrates, tannins, flavonoids, cardiac glycosides, steroids, saponins, and anthraquinones as shown in [Table 1].
|Table 1: Phytochemical screening of aqueous leaf and root extracts of Cymbopogon citratus|
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No mortality was recorded at the experimental dose levels used in both phases in the acute toxicity study, but mice showed some behavioral signs of toxicity at 5000 mg/kg of the aqueous leaf and root extracts, which includes sedation, weakness, and restlessness. The LD 50 of the aqueous extract was estimated to be >5000 mg/kg [Table 2].
|Table 2: Acute oral toxicity test of aqueous leaf and root extracts of Cymbopogon citratus|
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The aqueous leaf extract of C. citratus exhibited a dose-dependent suppressive effect against rodent malaria parasite as shown in [Table 3]. The suppressive effect of the extract was statistically significant (P < 0.05) at all the dose levels used when compared to the control group. The suppressive effects of the extract were 20.83%, 55.56%, and 80.56% at 200, 400, and 800 mg/kg, respectively. The standard drug chloroquine caused 100% parasitemia suppression which was higher than the extract-treated groups.
|Table 3: Suppressive effect of aqueous leaf extract of Cymbopogon citratus and chloroquine against Plasmodium berghei-infected mice|
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The suppressive effect of aqueous root extract C. citratus also exhibited a dose-dependent pattern and its effect was significant (P < 0.05) at all the experimental dose levels, with suppressive effect of 58.33%, 77.78%, and 100% when compared to the control group. The highest experimental dose level of the extract (800 mg/kg) as well as the standard drug chloroquine produced the same suppressive effect of 100% as shown in [Table 3] with decreased level of parasitemia.
The effects of the leaf and root extract on the body temperature of the infected mice are summarized in [Table 5] and [Table 6], respectively. The aqueous leaf and root extracts of C. citratus have a mild effect on the body temperature of P. berghei-infected mice with slight increase in body temperature. This rise in temperature was significant (P<0.05) only on the 2 nd day with the extract-treated group, while there was a significant rise on the 2 nd and 3 rd days with the positive control group, as shown in [Table 5] and [Table 6].
| Discussion|| |
As malaria is still an important life-threatening infection in many tropical countries and drug resistance is increasingly common to drugs used nowadays, it has become necessary to find more drugs that may contribute to the reduction of malaria in the future. This calls for an inward look into harnessing the full potential of medicinal plants that abound around us. Medicinal plants are general assumed to be safe, although many of them are potentially toxic.  In general, substances or compounds with LD 50 >5000 mg/kg are regarded as being safe or generally free from toxicity. ,,, No mortality was recorded in all the experimental dose levels that were used in the oral acute toxicity test. Thus, the aqueous leaf and root extracts may be regarded as acutely safe for use. The phytochemicals results showed that alkaloids, carbohydrates, tannins, flavonoids, cardiac glycosides, steroids, saponins, and anthraquinones were present in the leaf and root crude extracts of C. citratus. These phytochemicals and other chemical constituents might account for their medicinal values.
P. berghei, a rodent malaria parasite, is commonly used to assay antimalarial activity of medicinal plant extracts as well as conventional antimalarial drugs. The common strains of P. berghei are ANKA, K173, NK65, SP11, and LUKA.  P. berghei provides a well-established experimental model of malaria infection,  producing pathological symptoms which closely mimic those of human malaria.  The rodent malaria model was employed to study the antiplasmodial activity of aqueous leaf and root extracts of C. citratus against P. berghei in mice. This study focused on evaluating its antiplasmodial activity and compared the activities between leaf and root extracts against P. berghei in mice.
The suppressive activity of the aqueous leaf extract followed a dose-dependent pattern as shown in [Table 4] with varying degree of percentage parasitemia suppression. 200, 400, and 800 mg/kg of the aqueous leaf extract caused 20.83%, 55.56%, and 80.56% suppressive effect, respectively, but was less when compared to the reference standard (chloroquine) which completely abolished the parasite to an undetectable level. Standard antimalarial drugs can suppress parasitemia to nondetectable level.  The suppressive effect of the aqueous leaf extract may be due to the presence of phytochemical constitutes present in it. The suppressive activity of the extract is expressed as a reduction in the parasite load post treatment, which is characterized by an increase in percentage parasitemia suppression.
|Table 4: Suppressive effect of aqueous root extract of Cymbopogon citratus and chloroquine against Plasmodium berghei-infected mice|
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The root extract displayed chemosuppression activity against P. berghei in a competitive dose-dependent fashion with 100% chemosuppressive effect at 800 mg/kg similar to the standard drug chloroquine (5 mg/kg). Genetic background of a mouse can affect the course of parasitemia and disease.  Chemosuppression activity has previously been reported with the essential oil derived from C. citratus against P. berghei with the highest parasitemia suppression activity put at 86.6%.  This clearly shows that the aqueous root extract has better suppressive effect than essential oil obtained from the same plant.
The presence of diverse phytochemicals such as alkaloids, anthraquinones, and flavonoids has been implicated in antiplasmodial activity of some herbal preparations, , making the extract a potential antiplasmodial candidate. These phytochemicals present in the extract may probably act synergically to suppress the level of parasitemia as shown in [Table 3] and [Table 4]. This synergistic pharmacological activity can be beneficial by eliminating the problem of unwanted side effects associated with a single xenobiotic compound in the body. 
The extracts produced appreciable effect on the body temperature of the infected mice, significant (P < 0.05) only on the 2 nd day with the extracts-treated group (800 mg/kg), while there was a significant rise on the 2 nd and 3 rd days with the chloroquine-treated group, though a mild effect was observed all through the 4-day test period. There was a consistent drop in the body temperature of the negative control group as shown in [Table 5] and [Table 6]. This may be probably due to the high level of parasitemia which tends to disrupt the body's temperature-regulating center.
|Table 5: Effect of aqueous leaf extract of Cymbopogon citratus and chloroquine on body temperature of Plasmodium berghei-infected mice|
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|Table 6: Effect of aqueous root extract and chloroquine on body temperature of Plasmodium berghei-infected mice|
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The reason for the decrease in the body temperature of infected mice is still unclear.  This is in contrast to humans where malarial fever is characterized by increased body temperature; mice show a decrease in body temperature upon infection with malaria parasite (rodent malaria parasite). Decrease in body temperature (hypothermia) is not only associated with experimental malaria; it is a general marker for moribund mice suffering from infectious diseases.  Body temperature of reference standard-treated group remained fairly constant throughout the 4-day test period. Result of the experimental findings showed that the aqueous root extract exhibited a better antiplasmodial suppressive activity than the aqueous leaf extract. The plant extracts also have a mild effect on the body temperature of the infected mice.
| Conclusion|| |
In line with the current global reality of high prevalence of antimalarial drug resistance and recrudescence of malaria infection, the use of C. citratus could be a better choice or a substitute used as a supportive therapy for malaria treatment. The antiplasmodial effect of C. citratus can further be investigated to verify the active ingredients responsible for this effect. In addition, further research work needs to be carried out to see the interactive effect of C. citratus with other antimalarial drugs.
The authors would like to appreciate the support and advice of Pharmacist Ezenyi Ifeoma and Division of the Animal unit of the University of Jos toward the completion of this research work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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