Antiplasmodial activity of Indigofera spicata root extract against Plasmodium berghei infection in mice

Background In addition to pharmacovigilance and pharmaco-economic concerns, resistance to anti-malarial medicines has been documented in all classes of anti-malarials and this is further worsened by resistance to common insecticides by malaria vector, which is a major threat to malaria control. As a means of facing the challenges of searching for new anti-malarial agents, the current study focused on evaluation of anti-malarial activity of root extract of Indigofera spicata. Methods Chloroquine-sensitive rodent malaria parasite, Plasmodium berghei (ANKA strain) was used to infect the Swiss Albino mice in 4-day suppressive and curative models. The crude hydromethanolic root extract of I. spicata at 200, 400 and 600 mg/kg doses was administered to a group of five mice. Important parameters, such as level of parasitaemia, packed cell volume (PCV), survival time, and body weight were determined and the significance of the differences between mean values of the five groups was analysed by one-way ANOVA followed by post hoc Tukey’s Multiple Comparison test. Results In both the suppressive and curative models, 400 and 600 mg/kg doses of the extract suppressed the level of parasitaemia significantly (p < 0.001) compared to the vehicle-treated groups, 34.93 and 53.42%, respectively. However, only the mice which were treated with the 600 mg/kg dose of the extract had significant difference in their mean survival time. In other parameters, namely PCV and mean body weight, there was no statistically significant difference between the extract-treated groups when compared to the negative control. Conclusions This study revealed that the root extract of I. spicata possesses anti-malarial activity and necessitates further scientific validation.

transmission coincides with the planting and harvesting season. Historically, malaria has forced people to inhabit the less agriculturally productive highlands. About 75% of the country is malarious, with about 68% of the country's total population living in areas at risk of malaria [5,6].
The malaria vector, the female Anopheles mosquito, which has developed resistance against insecticides, such as dichlorodiphenyltrichloro-ethane (DDT), and chemoprophylaxis has not often yielded the expected results. Additionally, the disease-causing protozoans have developed resistance against most of the drugs currently used to treat malaria. New, highly effective anti-malarial drug candidates, based on new mechanisms of action or with new structures, are urgently needed to overcome the problem of rapid emergence of drug resistance and achieve long-term clinical efficacy [7,8].
Traditional preparations have been the main source of treatment of malaria in Africa and other continents where the disease is endemic and resources are limited [9]. They have, in the past, been the source of some of the most successful anti-malarial agents, such as the quinolines and the endoperoxide artemisinin. One of the areas to search for new anti-malarials is the traditionally claimed anti-malarial plants from the African flora. It has been claimed that about 80% of the Ethiopian population rely on medicinal plants for treating various illnesses, including malaria [10]. However, traditional medicine is not without its limitations, such as lack of sciencebased efficacy and safety evaluations [11]. Although several plants that have potential anti-malarial properties have been studied, there still exists innumerable potentially useful medicinal plants awaiting evaluation and exploitation for therapeutic applications against various groups of pathogens [7,8]. Furthermore, the possibility that some chemicals could serve as a prodrug and immunomodulatory agents in managing infectious disease will make this study justifiable when conducted in vivo antimalarial models [12,13].
The root of Indigofera spicata have been used orally in the treatment of malaria [14,15] and traditional healers use the plant material locally. Yet systemic pharmacological studies have not been reported to support its claim. This study was designed to evaluate the in vivo antimalarial effect of root extract of I. spicata.

Collection and preparation of plant materials
Fresh roots of I. spicata were collected from the surrounding area of Shawra town, South West Gondar Province, North West Ethiopia in December, 2014. The plant was botanically identified and a voucher specimen is already deposited in the Department of Biology, National Herbarium, Addis Ababa University.

Animals used
Male Swiss albino mice were used, 22-30 g weight and 6-8 weeks old, from the Department of Pharmacology, College of Medicine and Health Sciences, University of Gondar animal house. The housing and feeding conditions were maintained as per recommended standards. All mice were acclimatized to the working environment 1 week before the beginning of the experiment. They were also handled according to the international animal care and welfare guidelines [16,17].

Parasites inoculation
Chloroquine-sensitive Plasmodium berghei (ANKA strain) was used for induction of malaria in experimental mice. Mice previously infected with P. berghei were used as donor. The donor P. berghei-infected mice were obtained from Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Ethiopia. The parasites were subsequently kept alive by continuous serial intraperitoneal (IP) passage of blood from donor mouse to uninfected mouse on a weekly basis.

Extraction of plant material
The fresh roots of I. spicata were thoroughly washed with distilled water to remove dirt and soil, and dried under shade and optimal ventilation for 2 weeks. The dried roots were further chopped into small pieces and reduced to powder using an electronic miller. The powdered roots of the plant were weighed before maceration with 80% methanol. The coarsely powdered roots were subjected to maceration extraction procedure using 80% methanol (for 72 h at room temperature) as a menstruum and this was done three times. The respective extract was filtered using Whatman No-1 filter paper and the solvent was evaporated in an oven under reduced pressure at 40 °C. Finally the dried extract was stored at 4 °C in refrigerator until used.

Infecting, grouping and dosing of animals
Blood was taken from a donor mouse with approximately 30% parasitaemia and diluted in physiological saline to 5 × 10 7 parasitized erythrocytes per ml. Swiss albino mice (male) weighing 24-29 g were infected with 0.2 ml blood (1 × 10 7 P. berghei parasitized erythrocytes) intraperitoneally and randomly divided into three test groups and two control groups (each for 25 mg/kg chloroquine [18] as a standard drug and normal saline as a negative control). The extract doses 200, 400, 600 mg/kg were determined based on the previous acute toxicity evaluation reports [19] and preliminary anti-malarial activity evaluation done on this extract. For all animals, volume of administration was 1 ml/100 g [16] and as the plant material is traditionally used [14] to treat malaria, the oral route of administration was used in all cases [20,21].

4-day suppressive test
It is the most widely used preliminary test, in which the efficacy of a compound is assessed by comparison of blood parasitemia and mouse survival time in treated and untreated mice [22]. Treatment of infected mice was started after 3 h of infection on day 0 and continued daily for 4 days (i.e., from days 0-3). On the fifth day (day 4) blood samples were collected from tail snip of each mouse. Thin smears were prepared and stained with 10% Giemsa solution. Then, each stained slide was examined under the microscope with an oil immersion objective of 100× magnification power to evaluate the percent suppression of each extract with respect to the control groups. Average percent parasitaemia and suppression was calculated by using the following formula [19,23]. RBCs were injected intraperitoneally into 25 Swiss albino mice. Seventy-two hours later the mice were randomly divided into five groups of five mice each. Subsequently different doses of the extract (200, 400 and 600 mg/kg/ day), chloroquine phosphate (25 mg/kg/day) and the vehicle (10 ml/kg/day) were given orally to the respective groups for 5 consecutive days. For each mouse, thin blood films stained with 10% Giemsa was prepared from tail blood of each mouse daily for 5 consecutive days to monitor the levels of parasitaemia. Starting from the date infection (day 0) the mice were observed for 15 days and any death that occurred during this period was recorded and used to determine the mean survival time. Average percentage parasitaemia suppression will be calculated: where, A = average percentage suppression of parasitaemia, D = average percentage parasitaemia before treatment and E = average percentage parasitaemia after treatment.

Determination of packed cell volume
Determination of packed cell volume (PCV) was measured to predict the effectiveness of the test extracts in preventing haemolysis resulting from increasing parasitaemia associated with malaria. Blood was collected from the tail of each mouse in heparinized microhematocrit capillary tubes by filling three-quarters of its volume. The tubes were sealed by sealant and placed in a microhematocrit centrifuge with the sealed ends outwards. The blood was centrifuged at 12,000 rpm for 15 min. The PCV of each mouse was then measured before infection and on day 4 after infection using the formula: PCV = Volume of erythrocytes in a given volume of blood/total blood volume × 100

Curative test
Curative test is the evaluation of the efficacy of a chemical substance against an established malaria infection by using level of parasitaemia suppression and survival time as important parameters. Thus, evaluation of curative anti-malarial potential of the extract was done by using a method described by Ryley and Peters [21]. On the first day, standard inoculum of 1 × 10 7 P. berghei-infected

Determination of level of Parasitaemia
Tail blood smearing of each mouse was made for both the suppressive and curative tests. After fixation with absolute alcohol and staining with 10% Giemsa at pH 7.2 for 15 min, the slides were washed gently with distilled water followed by air drying at room temperature. By using Olympus microscope oil immersion objective of 100× magnification power, the number of parasitized erythrocytes in random fields of the microscope was counted. Three separate fields on each of the slides were used to calculate percent parasitaemia and percent suppression using the following formula, respectively.

Determination of mean survival time
From the time of infection until death, mortality of each mouse was monitored and recorded regardless of the group in which the mouse was allocated throughout the follow-up period (15 days). Mean survival time of mice of each group was determined using the following formula:

Determination of body weight
For Peter's test, body weight of each mouse was measured before infection (day 0) and on day 4 using sensitive electronic balance.

Data quality control
Each of the microscopic slides was coded and the measurements were determined blindly by a laboratory technician. In addition, for greater reliability each measurement was done in triplicate.

Ethical clearance
The study protocol was ethically reviewed by the Institutional Review Board of the University of Gondar. All protocols were performed in accordance with the international animal care and welfare guidelines [16].

Statistical analysis
All data values are expressed as mean ± SEM (standard error of means) for five mice per group. Statistical analyses were carried out by using SPSS Statistical software version-21. Statistical significance of mean of parasitaemia suppression, weight, PCV, and survival time differences between groups was computed by one-way ANOVA, followed by post hoc Tukey's Multiple Comparison Test. p value of less than 0.05 was considered statistically significant.

4-day suppressive test
As it is shown in In the 4-day suppressive test, none of the extract dosetreated groups was significantly protected from PCV reduction. However, the standard drug induced significant protection against PCV reduction compared to both the normal saline and the 200 mg/kg extract dose-treated groups (p < 0.01). In addition, neither the extract doses nor the standard drug-treated group demonstrated statistically significant difference in body weight compared to the control group (Table 2).

Curative activity evaluation
The plant material suppressed the parasite level of mice by 58.18, 78.04 and 85.88% at day 7 compared to the normal saline-treated group, while the suppression induced by the standard drug was 100%. In parallel, compared to the negative control at day 7, those treated with the extract (all doses) and chloroquine had significantly (p < 0.001) reduced level of parasitaemia. Furthermore, like the positive control on day 7, both 400 and 600 mg/kg extract dose-treated groups had significantly (p < 0.001) controlled level of parasitaemia compared to the 200 mg/ kg extract dose-treated groups. Except the 600 mg/kg dose, none of the extract doses suppressed the parasite level significantly compared to the normal saline at day 4.
In the next days, all the extract doses reduced the level of parasitaemia considerably to various extent compared to the control group. In mean survival time, only the 400 mg/kg (p < 0.05) and 600 mg/kg (p < 0.01) extract doses substantially prolonged survival time compared to normal saline. However, the mean survival time of mice treated with chloroquine was significantly (p < 0.001) higher than not only the negative controls but also the extract dosetreated groups (Table 3).

Discussion
In addition to the claimed traditional use, an advantage of the mouse model is that it may detect compounds requiring bioactivation and/or which have immunomodulatory activity [21]. More often, in the early in vivo anti-malarial activity evaluation, use of P. berghei as an infecting parasite is recommended. Similarly, the 4-day suppressive model and the Raye's test are also widely acceptable in order to evaluate the anti-malarial potential of natural or synthetic products on early infection and late infections, respectively [19,21].
Anti-malarial test materials with more than 30% suppressive effect on the level of parasitaemia [7,24] or that can prolong the survival date of treated mice [25] compared to the control group are often considered effective in standard screening tests, which is in agreement with this study finding. As is depicted in Table 1, in the 4-day suppressive test the 400 and 600 mg/kg plant extract doses had significant anti-malarial activity (p < 0.05) in a dose dependent manner which was further demonstrated by the mean survival time of the mice. Similarly, many previously reported plant materials have shown dose-dependent anti-malarial effect [26]. In the 4-day suppressive model, none of the extract dose-treated groups of mice illustrated significant change in level of PCV compared to the negative control and that is why this evaluation parameter was not considered in the curative test of the plant material. Lack of significant PCV protection from the extract doses might be due to the presence of saponins in the crude extract, which are known to cause haemolysis by increasing the permeability of the plasma membrane [27,28]. Active anti-malarial agents have been expected to protect weight loss associated with malaria infection but in this study the protective effect of the extract doses on loss of body weight was not found to be significant. The discrepancy might be explained by the imbalance of the protective effect of the extract and the cumulative pathophysiologic changes associated with the infection.
In the curative test, the significant (p < 0.001) suppression of level of parasitaemia across all doses was also revealed in a dose-dependent manner. Accordingly, this is confirmation of the plant material having effective antiplasmodial activity in the late stages of the infection. Hopefully, a plant with such anti-malarial activity at crude extract level may serve as a promising lead compound for further optimization and development of new therapeutic agents. Complementing the demonstrated parasitaemia suppression of the plant material, this study also revealed that animals that were treated with the extract live longer than animals treated with the vehicle. Even if it was not statistically significant, there was similar reflection in terms of PCV and body weight parameters. This might be explained by the presence of various secondary plant metabolites in the crude extract that may have a complex biochemical effect and lack of very strong anti-malarial activity that could ameliorate all pathologic changes. Furthermore, this might be associated with the impurity of the extract and if the active principle(s) were isolated and/or optimized the activity of the plant material might statistically sound in all evaluation parameters.
Substances with antioxidant activity have known antimicrobial activity, i.e., associated with the elevated level of oxidative stress in a person who is being infected. As it was confirmed in previous reports [29], this plant has antioxidant activity which might support its antidiabetic, anticancer, antidiarrhoeal activities [19,30] and the current anti-malarial activity of the extract. Furthermore, the antiplasmodial activity of the plant material is supposed to be due to the presence of secondary plant metabolites, such as alkaloids, flavonoids and tannins. Those metabolites may work independently or synergistically [31,32].
In summary, according to the anti-malarial activity classification of Deharo et al. [33], the methanolic crude extract of the root of I. spicata showed moderate antimalarial activity in both the 4-day suppressive and curative models.

Conclusions
The finding of this study supports the claimed traditional use of I. spicata in treating malaria. From this, there may be a possibility of developing new anti-malarial lead compound with rigorous molecular mechanism and characterization studies.
Abbreviations IRBC: infected red blood cells; NS: normal saline; PCV: packed cell volume; WHO: World Health Organization.