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Effect of chronic khat (Catha edulis, Forsk) use on outcome of Plasmodium berghei ANKA infection in Swiss albino mice

BMC Infectious Diseases volume 15, Article number: 170 (2015) Cite this article

Abstract

Background

The objective of this study was to explore effects of khat (Catha edulis) on outcome of rodent malaria infection and its anti-plasmodial activities on Plasmodium berghei ANKA (PbA).

Methods

Female Swiss albino mice were orally treated with crude khat (Catha edulis) extracts (100, 200 and 300 mg/kg) on a daily basis for 4 weeks prior to PbA infection. Physical, clinical, hematological, biochemical and histo-pathological features of the mice were assessed. In addition, in vivo anti-plasmodial activities of khat were evaluated.

Results

The finding of this study showed that khat use was strongly associated with increment of levels of liver and kidney biomarkers, leucopenia, severe anemia, rise in level of inflammation biomarkers: C-reactive protein (CRP), uric acid (UA), increased monocyte-lymphocyte count ratio (MLCR), manifestation of cerebral malaria symptoms such as ataxia, paralysis and deviation of the head but with no pulmonary edema. Significantly lower level of parasitemia (P < 0.05), rectal temperature, but, high level of hemoglobin were observed at the early stage of the PbA infection in khat treated mice than the control. With extension of the treatment period, however, drastic increments were observed in parasite load and rectal temperature although there was reduction in hemoglobin (Hb) level. Moreover, khat showed poor anti-plasmodial activity with <10% parasite suppression activity and lack protection against major malaria symptoms. The significant reduction (P < 0.01) of hematological parameters during PbA infection strengthen the notion that hematological parameters could be good predictors of severe malaria complications in human.

Conclusions

In mice model treated with khat prior to infection with the rodent malaria parasite, khat was found to worsen manifestation of most malaria complications. Furthermore, the same plant showed poor in vivo anti-plasmodial activity and protection against major malaria symptoms.

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Background

Khat (Catha edulis, Forsk), a natural stimulant, is chewed by millions of people in Yemen, Somalia, Ethiopia, Djibouti and Kenya [1]. Nowadays, the habit of khat chewing is spreading to Europe, North America, Australia and Canada [2-4]. Besides its stimulating role, the processed leaves and roots of the plant are used for treatment of various ailments including influenza, cough, asthma, gonorrhea, vomiting and headache [1,5]. In addition, some endogenous people of East Africa and Meru tribe of Kenya use khat to treat malaria [1,5-7]. Similarly, some people in Yemen use khat for treatment of obesity, suppression of appetite and to alleviate their headaches by inhaling the fumes of burning khat leaves [8].

Khat contains compounds such as alkaloids, terpenoids, flavonoids, sterols, glycosides, tannins, amino acids, vitamins and minerals [9]. However, the stimulating effect of khat is mainly associated with its alkaloids content, cathinone and to a lesser extent cathine and norephedrine [10]. Cathinone is an intermediate metabolite in the biosynthesis of cathine which is found mainly in young fresh leaves of khat plant [10]. Khat, when chewed, is rapidly absorbed after oral administration, and it reaches peak plasma levels within 1.5-3.5 hrs after the onset of chewing of khat [11,12]. Cathinone is relatively unstable and decomposes within few days of picking, or if the leaf is dried, it changes into cathine and norephedrine [13].

Several reports showed that khat could cause different health problems including increased susceptibility to cognitive impairment, cardiovascular disorders, stomach ulcer and increment in adrenocorticotrophic hormone levels. It also causes urine retention and gall bladder motility, gastro-intestinal tract constipation and hemorrhage due to tannin and norpesudoephedrine content of the plant [1,14-16]. Despite its publicized health consequences, the practice of khat use is increasing globally and becoming an everyday drug for most people of East Africa, including Ethiopia [16].

The co-existence of khat chewing habit and malaria endemicity in some parts of Ethiopia and people’s perception on medicinal value of the plant against malaria infection initiate an assumption that khat might have an effect on the outcome of malaria infection. This prompted the current study aimed to explore in vivo effect of crude khat extract on the outcome of malaria infection and its anti-plasmodial activity under controlled conditions of potential confounding factors using Swiss albino mice.

Methods

Experimental animal

In this study female Swiss albino mice, aged 7–8 weeks, weighing 25-30 g, obtained from Ethiopian Public Health Institute (EPHI), Addis Ababa, were used. The mice were housed in transparent plastic cage having SS sipper 250 ml water bottle. Wood shaving was used as bedding and it was replaced every morning after the cage was cleaned and disinfected with 70% alcohol. The animals were kept under unlimited access to commercial pellet food and fresh tap water ad libitum. The experimental room had 12/12 hrs light/dark cycles, 55% ± 5.7 relative humidity and 21°C mean temperature.

Collection and extraction of khat

Fresh khat leaves were purchased from a local market. Shoot and leaves were carefully picked and chopped into small pieces (~0.5 cm2) using grinding machine and mixed in methanol (analytical grade). For 100 g of the plant, 300 ml of methanol was used. Methanol was chosen as extraction solvent since it was known to produce an extract with appreciable levels of cathinone, cathine and norephedrine from khat [17]. First phase extraction was carried out by spinning the mixture for 15 minutes in Water Bath Shaker (RSB-12) at 120 rpm and room temperature under dark condition. The mixture was filtered using Whatmann filter paper (11 μm pore size), and the residue was re-shaked overnight under the same condition of the former filtrate. Then, the filtrate was concentrated to near dryness using Rota vapor (Heidolph Labo Rota, 4002) at 120 rpm and temperature of 40°C for 1-2 hrs. The residue was measured and dissolved in 20% Tween 80 in physiological saline solution.

Freshness of the khat extract was checked on thin layer chromatography (TLC) following the procedure developed by Lee [18]. Briefly, the plant extract was spotted directly onto a pre-coated silica gel 60 plate. Cathinone oxalate and cathine oxalate drug standards dissolved in methanol were used as references. The plate was developed in solvent composition of ethyl acetate, methanol and aqueous ammonia with respective proportion of 85:10:5, and viewed under an ultraviolet lamp (254 nm). The spots were visualized using 0.5% ninhydrin solution, and the plate was heated. Cathine appeared purple, while cathinone was a burnt orange moving spot. The Rf (retardation factor) values obtained for cathinone and cathine were 0.43 and 0.21, respectively.

Acute toxicity testing

Sixteen female Swiss albino mice were randomly divided into 2 groups of 8 mice each. After 2 hrs fasting [19,20], the first and second groups were treated with 500 and 1000 mg/kg of khat extracts, respectively. The animals were checked for two weeks daily for any sign of toxicity such as loss of appetite, hair erection, lacrimation, tremors, convulsions, salivation, diarrhoea, mortality and other signs of overt toxicity [19].

Mice grouping and treatment

After a one week period of acclimatization in the laboratory, the mice (n = 64) were grouped into eight categories (I-VIII), each containing 8 mice. The first two groups (I-II) were treated with 100 mg/kg of khat extract; the second two groups (III and IV) were treated with 200 mg/kg, while the third two groups (V and VI) were treated with 300 mg/kg of khat extracts daily for 4 weeks following the extract doses used by Girma and Engdawork [21]. The remaining two groups (VII and VIII) were exposed to 0.5 ml of 20% Tween 80 in physiological saline solution for the same duration, frequency and route of administration where one (VII) was used as positive control after infected with Plasmodium berghei ANKA (PbA) and the other (VIII) as negative control as indicated in the following section.

Parasite preparation and mice infection

PbA parasite was obtained from infected mice, carrying 30% of parasitemia, donated by EPHI. After the mice were terminally anesthetized using diethyl ether and exposure in a closed container, the infected blood was collected through cardiac puncture, and diluted to 1 × 105 PRBCs in PBS. One subgroup from the duplicates of first, second and third groups (I, III, and V) were treated with 100, 200 and 300 mg/kg of khat extract, respectively. The positive control (VII) was infected with 100 μL of the diluted PRBCs (1 × 105) in PBS through intra- peritoneal (i.p.) injection on the last day of the 4th week of the treatment period. Briefly, the experimental animals were divided into groups: (1) neither treated nor infected (n = 8 mice), (2) only treated with khat extract (100, 200 and 300 mg/kg) (n = 24 mice) daily for four weeks but not infected with PbA, (3) PbA infected, but non khat-treated mice used as positive control (n = 8 mice) and (4) PbA infected and khat treated mice (100, 200 and 300 mg/kg) (n = 24 mice) daily for four weeks. Then, the treatment of all mice exposed to methanolic khat extract was extended to day 7 of post infection (7dpi). Starting from 2dpi to the last day of survival of mice (day 7), parasite load, rectal temperature, body weight and Hb levels were monitored.

On the seventh day of post infection, rectal temperature was measured, body weight was taken and few drops of blood samples from tail snip were used for measurement of hemoglobin (Hb) level (Hb analyzer, Hemocue™ haemoglobinometer, Angelholm, Sweden). Parasite load was determined in blood smears, thin and thick, from tail snip stained in 10% Giemsa for 10 min. Percent (%) parasite load was calculated by dividing infected RBCs to total RBCs and multiplying by 100. Parasite loads of nicotine treated mice were compared with the positive control.

Assessment of severe malaria syndromes

Incidences of clinical signs of cerebral malaria (CM) that involve neurological syndromes were assessed. These were ataxia, paralysis, deviation of the head, convulsions, decrease in body temperature, loss of vascular cell integrity, tissue edema, hemorrhages in the brain of mice, and congestion of micro-vessels with parasitized erythrocytes and/or mononuclear cells [22].

After clinical and physical conditions assessment, each mouse was terminally anesthetized, and blood sample was collected through cardiac puncture for hematological and biochemical tests. Furthermore, some organs such as kidney, liver, spleen, and brain were carefully removed, weighed and processed for histo-pathological study. Lungs were removed and used for assessment of status of pulmonary edema.

Hematological and biochemical tests

About 0.2 ml of blood samples collected from mice cardiac were used for quantification of total WBCs, lymphocytes, monocytes, RBCs, Haemoglobine (Hb), Hematocrit (HCT), platelets, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH) using CBC machine (Automated CBC Analyzer: Sysmex KX-21). From 1 ml of the blood sample collected in EDTA coated tube, serum was separated by centrifugation (Centrifuge 4515R) at 10,000 rpm for 10 minutes. The supernatant was transferred into new eppendorf tube and immediately followed by measurement of liver enzymes, serum glutamic oxaloacetate transaminase (sGOT) and serum glutamic pyruric transaminase (sGPT), albumin (Alb), biomarkers of kidney functions such as creatinine (Cr) and urea, inflammation biomarkers; uric acid (UA) and C-reactive protein (CRP) using automated immunochemical analyzer (Axsym MEIA 3rd Generation).

Pulmonary edema

The status of pulmonary edema was assessed in lungs of mice infected with PbA and the positive control. The wet weight of lung was measured immediately after removal of the organ, and the dry weight was determined after overnight incubation at 80°C. Then, the ratio of wet to dry weight was calculated to determine the condition of pulmonary edema [23].

Histopathological analysis

For histological analysis, liver, kidney and brain of sacrificed mice were collected in 10% buffered neutral formaldehyde; paraffin-embedded brain, liver and kidney tissues were sectioned and stained with hematoxylin and eosin. Slides were coded and scored blind for histological evidence of cerebral syndromes and liver and kidney damages.

Anti-plasmodial activity of khat extract

Mice infection and follow up

For this assay, mice grouping and treatment procedures different from the earlier protocol were used, in that mice infection was commenced after four weeks of oral khat treatment. Accordingly, chloroquine sensitive female Swiss albino mice were grouped into five categories (n = 40), each containing eight mice. Here, all mice were initially infected with 1x105 PRBC in PBS intraperitonally. Then, plant extract was solublized in 20% Tween 80 in physiological saline solution and tested in three doses; 100, 500 and 1000 mg/kg administered to the infected mice (each group contained 8 mice) orally and once daily for four consecutive days. Other two groups: a negative control (n = 8) and a positive control (n = 8) received 20% Tween 80 in saline solution and 10 mg/kg base body weight of chloroquine sulphate, respectively.

Suppressive test

Chemo-suppressive activity of the plant crude extract on parasitemia was evaluated following four day suppressive test protocol. Blood sample was taken from tail vein of each mouse on the fourth day p.i. Methanol fixed and 10% Giemsa stained (pH = 7.2 for 10 min) thin film was examined microscopically. Percentage parasitemia and average percentage suppression of parasitemia by the extract were determined following the formula described below [24]:

$$ \mathrm{Mean}\ \%\ \mathrm{parasitemia} = \frac{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{pRBCs}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{RBCs}}\times 100 $$

Percent parasitemia suppression was calculated by using the following formula [25].

$$ \%\ \mathrm{parasitemia}\ \mathrm{suppression} = \frac{\mathrm{Mean}\ \mathrm{parasitemia}\ \mathrm{of}\ \mathrm{control}\ \hbox{--}\ \mathrm{Mean}\ \mathrm{parasitemia}\ \mathrm{of}\ \mathrm{test}}{\mathrm{Mean}\ \mathrm{parasitemia}\ \mathrm{of}\ \mathrm{control}}\times 100 $$

Mean survival time of each mouse in all groups was determined by calculating the average survival days of mice in each group over 30 days. Furthermore, body weight reduction and temperature rise were monitored every 48 hours p.i. using digital balance and thermometer, respectively.

Data analysis

Data were checked for their completeness, correctness, and then double entered into Microsoft Office Excel (2007) sheet and analyzed using SPSS version 20.0 software. The analyzed data were expressed in mean ± standard error of mean (SEM), otherwise indicated. One-way analysis of variance (ANOVA) followed by Tukey’s HSD post-hoc test was employed to compare the effect of crude khat extract on different variables. Mantel-Cox Log-rank test was also used to analyze the survival time between groups. Values of p < 0.05 were considered statistically significant. All assay results were mean of triplicate analysis.

Ethical consideration

The study was ethically approved by the Ethical Review Committee of the College of Health Sciences of Jimma University, Ethiopia. Detail experimentation procedures involving mice were managed following the Ethiopian Public Health Institutes (EPHI) animal handling and treatment guidelines.

Results

Acute toxicity study

The acute toxicity study made using the current doses of khat extracts did not cause any mortality in all mice within the first 24 h and beyond for period of 14 days. Physical and behavioural observations of the experimental mice also revealed no visible signs of overt toxicity like lacrimation, loss of appetite, tremors, hair erection, salivation and diarrhea.

Hematological and biochemical features

According to the result of the in vivo study, mice exposed to khat extract followed by PbA infection had significantly higher risk of jaundice or liver and renal impairments. This was evidenced by increased level of liver enzymes such as serum GPT and GOT, which were significantly higher (P < 0.001) in blood of mice exposed to the highest dose of khat extract (300 mg/kg) followed by PbA infection than the positive control, while albumin level was significantly reduced (P < 0.001). However, creatinine and urea levels did not show any difference between mice treated with khat followed by PbA infection and the positive control.

Influence of khat on the status of liver biomarkers was further observed in mice treated with khat but without PbA infection, in a dose dependent manner. Serum GOT and GPT were significantly higher (P < 0.05) in mice treated with higher dose of khat (300 mg/kg) although albumin level did not show significant differences (P > 0.05) between mice treated with khat extract and the negative control. Likewise, in mice treated with higher dose of khat extract (200 and 300 mg/kg) without PbA infection, significantly higher (P < 0.05) level of urea was observed than the negative control, while creatinine level was only affected at the highest dose (300 mg/kg) of the khat extract, though it was not detected after PbA infection (Figure 1). However, necrosis of kidney and liver tissue were observed in the positive control mice treated with khat followed by PbA infection under all doses.

Figure 1
figure 1

Levels of liver and kidney biomarkers(mean ± SEM) of Swiss albino mice (n = 8) treated with khat extracts (100, 200, and 300 mg/kg) daily for 4 weeks, and khat treatment followed byPbAinfection [(100, 200 and 300) mg/kg plusPbAinfection] and the controls. Values with asterisk are significantly different (ANOVA, Tukey’s HSD post-hoc test) from values of the controls.

Full size image

Inflammation biomarkers such as CRP and UA levels measured in mice treated with khat extract (100, 200 and 300 mg/kg) did not show significant differences (P > 0.05) from the negative control. However, in mice treated with khat extract, under all doses, followed by PbA infection, significant increment (P < 0.05) in level of UA than the positive control was observed, while significant rise (P < 0.05) of CRP level was only observed in mice which received 300 mg/kg doses of crude khat extract (Figure 2).

Figure 2
figure 2

Level of C-reactive protein (CRP) (a) and uric acid (UA) (b) in Swiss albino mice (n = 8) treated with khat extracts (100, 200, and 300 mg/kg) daily for 4 weeks, and khat treatment followed byPbAinfection [(100, 200 and 300) mg/kg plusPbAinfection] and the controls. Values with asterisk are significantly different (ANOVA, Tukey’s HSD post-hoc test) from values of the controls.

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WBCs and Hb levels significantly reduced (P < 0.05) in mice exposed to the highest doses of crude khat extract (300 mg/kg) followed by PbA infection compared to the positive controls. In addition, platelets count significantly reduced (P < 0.05) in mice treated with khat extract of the two upper doses of this study (200 and 300 mg/kg), while lymphocyte and RBCs counts were not significantly different (P > 0.05) between the two groups. Effect of khat extract on hematological parameters without PbA infection also demonstrated the same pattern as in mice treated with khat extract followed by PbA infection. In mice treated with the highest dose of crude khat extract (300 mg/kg), the levels of WBC, Hb, MCV and platelets counts were significantly affected (P < 0.05) (Table 1).

Table 1 Hematological analysis of mice treated with khat followed by Pb A infection with reference to controls (n = 8)
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Mice treated with higher dose (300 mg/kg) of crude khat extract showed significant increment (P < 0.05) of monocytes to lymphocyte count ratio (MLCR) compared to the positive control. But, neutrophils to lymphocyte count ratio (NLCR) of the two groups (khat treated and positive control) did not show significant differences (Figure 3).

Figure 3
figure 3

Box-plots showing distribution of neutorphil-lymphocyte count ratio (NLCR) (a) and monocyte-lymphocyte count ratio (MLCR) (b) of Swiss albino mice treated with crude khat extract (100, 200, and 300 mg/kg) followed byPbAinfection. There was significant difference in the MLCR distribution between the positive control and mice treated with 300 mg/kg dose of khat (One way ANOVA, Tukey’s HSD post-hoc test, P = 0.024).

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Assessment made to explore in vivo effect of malaria infection on hematological parameters showed that there were significant changes on almost all RBC and WBC indices in PbA infected mice. HCT, mean corpuscular volume (MCV), platelet and lymphocyte level were significantly reduced (P < 0.001), while WBC, mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were significantly increased (P < 0.05) in PbA infected mice compared to uninfected or negative control (Figure 4).

Figure 4
figure 4

Hematological parameters (mean ± SEM) ofPbAinfected andPbAuninfected Swiss albino mice. Values with asterisk indicate significant difference from values of the uninfected mice (ANOVA, Tukey’s HSD post-hoc test).

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Effect of khat on outcome of PbA infection

Mice exposed to different doses of khat extract (100, 200 and 300 mg/kg) did not show significant difference (P > 0.05) in mean body weight from the control mice during the four weeks of treatment period. Body weight of the animals showed an increment starting from the first to the fourth week of the follow-up period (Figure 5a). Rectal temperature was <36°C for all groups of mice on the day of PbA infection (Day 0). Although gradual rising was observed in all groups, in mice treated with 300 mg/kg of khat extract, significant increment (P < 0.01) of rectal temperature was recorded from day 3 to 7p.i. (Figure 5b). Similarly, there was a gradual decline in Hb level in mice treated with khat extract followed by PbA infection. However, a significantly higher reduction (P < 0.05) than the positive control mice was observed after day 5 of p.i. (Figure 5c). Parasite load in mice exposed to different doses of crude khat extract (200 and 300 mg/kg) was significantly lower (P < 0.05) than the control group on the third and fifth day (Figure 5d), but significant increment (P < 0.001) was observed on the seventh day of p.i.

Figure 5
figure 5

Body weights of Swiss albino mice treated with khat extract daily for four weeks (a), mean rectal temperature (°C) recorded (b), mean Hb level (g/dL) measured (c), and percentage of parasitemia observed (d) on different days of p.i. of PbA infection.

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Based on the incidence of pulmonary edema assessment, khat extract did not show significant association with increased susceptibility to respiratory problems during Plasmodium berghei ANKA infection. This is, there was no significant difference (P > 0.05) between pulmonary edema status in khat exposed mice prior to infection and the positive control (Figure 6).

Figure 6
figure 6

The status of pulmonary edema (mean ± SEM) of Swiss albino mice (n = 8) treated with crude khat extract followed by PbA infection and positive control.

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Cerebral malaria (CM) state of mice

On the 7th day of p.i. (last day of survival), cerebral malaria symptoms such as limb paralysis, convulsions, shivering, neurological syndromes including ataxia, paralysis, deviation of the head, convulsions, and necrosis of brain tissue observed in all mice irrespective of treatment dose. Due to intense brain necrosis, detection of adherence of parasitized RBC to the endothelial cells was difficult.

Anti-plasmodial activity of khat

Crude khat extract displayed lower chemo-suppressive activity against PbA malaria. Percentage inhibition or suppression analysis showed that under almost all doses of the khat extract (100-1000 mg/kg), the observed percentage parasitemia reduction due to the extract was not significantly different (P > 0.05) from the positive control mice. The percentage suppression observed for all dose of the extract did not exceed 10%. Moreover, maximum survival time of mice infected with PbA and treated with khat extract observed was at1000mg/kg, which was only 14 days. This survival time was significantly lower (P < 0.05) than the chloroquine sulphate treated mice (had >30 days survival time). However, the log rank (Mantel-Cox) analysis showed that the overall survival time of mice treated with khat extract was significantly longer (P < 0.001) than the positive control or vehicle mice (Table 2).

Table 2 Parasitemia status, survival time and percentage suppression of parasitemia in infected Swiss albino mice treated with khat extract in the 4 day suppressive test
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Rectal temperature measurement made during the in vivo anti-plasmodial activity study indicated that mice treated with higher dose of khat extract (500 and 1000 mg/kg) had significantly reduced (P < 0.05) rectal temperature than the control. Also, on the 4th day, the level of hemoglobin and body weight of khat treated mice were comparably reduced in the same manner the positive control mice (Table 3). This indicates that during PbA infection in mice, the crude khat extract did not show preventive property against weight loss and anemic condition.

Table 3 Hemoglobin level, rectal temperature, body weight and parasite load of infected Swiss albino mice treated with crude khat extract
Full size table

Discussion

Most of the findings of this in vivo study were in agreement with our earlier epidemiological study conducted on P. falciparum infected khat chewers. Accordingly, a significant change in liver biomarkers in mice treated with khat extract followed by PbA infection than the control mice was observed. Change in liver function tests is a hallmark of pathological change in liver during malaria infection [26]. Malaria associated liver dysfunction is usually characterized by a rise in serum bilirubin along with the rise in serum GOT and GPT levels from mild abnormality to more than three times the upper limit of normal [27]. Besides, liver and kidney tissues necrosis observed in a histopathological study further illustrate the possible involvement of these two organs in metabolism and elimination of khat, respectively. Thus, khat use could increase the risks of liver and renal impairment, severe malaria pathologies.

Anemia is a hallmark of malaria infection that occurs as a result of intense hemolysis (destruction) of infected RBCs due to higher parasitemia caused mainly by P. falciparum [28]. The remarkable reduction of Hb and HCT level after day 5 of p.i. in mice treated with khat followed by PbA infection in this study could be an implication for the relative risk of khat user malaria patients to severe anemia. Thus, the incidence of anemia in mice treated with higher dose of khat was mainly due to hemolysis of infected RBCs as parasite load on 7dpi which was significantly higher. This implies that khat was one of the aggravating factors for incidence of malaria associated anemia among khat users who lack protective activity against hyperparasitemia pathology.

Pulmonary edema, one of the most severe forms of pathophysiology of malaria that involve lung and characterized by increased alveolar capillary permeability leading to intravascular fluid loss into the lungs [29], was not affected by exposure to khat. Usually, this pathology is associated with non-immune individuals with P. falciparum infections as part of a severe systemic illness [30]. On the other hand, lower platelet count, a condition known as thrombocytopenia, was manifested in mice chronically exposed to khat prior to the infection irrespective of dose of khat extract. This has a remarkable implication for its possible frequent manifestation among khat user malaria patients. Such reduction in platelet count usually results from apoptosis or activation of platelet [31,32] and their phagocytosis following platelet adherence in the spleen during malaria infection [33,34].

Although WBC counts do not directly involve in malaria infection, it is generally low to normal due to localization of the WBCs from the peripheral circulation to the spleen and other internal organs [35]. Thus, significant reduction in total WBC count observed in mice treated with highest dose of khat (300 mg/kg) followed by PbA infection could be attributed to the cytotoxic property of the khat extract [36].

The PbA infection in Swiss albino mice was reported to cause neurological problem (the so-called experimental cerebral malaria) [37] and to cause nearly 100% lethality [38]. There is accumulating evidence that the pathogenesis of cerebral malaria is due to an immune-pathological reaction giving rise to excessive production of cytokines such as tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) [39]. As all mice treated with khat (100 to 300 mg/kg) prior to PbA infection developed neurological symptoms in the same way as the positive control, khat had neither blood brain barrier (BBB) enhancement nor protection potential against cerebral malaria. The elevated level of inflammation biomarkers such as CRP and UA, which triggers secretion of pro-inflammatory cytokines [40], mediators for incidence of cerebral malaria pathology, observed in mice treated with khat extract followed by PbA infection strengthen the relative risk of khat users to severe malaria pathologies. Substantial evidences indicated that long term or chronic use of khat increases the level of uric acid [41-45]. Furthermore, the necrosis of brain tissue observed during histopathological study, and clinical manifestations such as ataxia, paralysis, deviation of the head and convulsions in khat treated mice prior to PbA infection could strengthen the fact that khat lacks protection against severe malaria pathologies.

In our epidemiological study, the observed reduction in parasite burden among khat chewer malaria patients seems that khat has anti-plasmodial activity. However, following khat treatment extension, the drastic increment of parasite load in mice treated with higher dose of khat extract (200 and 300 mg/kg) followed by PbA infection observed certainly question anti-plasmodial activity of the khat. Moreover, the result from anti-plasmodial activity evaluation revealed that except slight reduction on parasite load in khat treated mice, significant parasite suppression was not observed. In support of this, rise of rectal temperature, physical deterioration and severe clinical symptoms appeared in respect to increased parasite load and reduction of Hb level, starting from day 5 p.i. in khat treated mice prior to PbA infection. According to Ingyang et al. [46], in Swiss albino mice infected with PbA, parasitemia started progress on the 2nd day of p.i and reached significant levels from day 4th - 6th and all untreated animals died by the 6th day of infection. Thus, lack of significant effect of khat on some clinical and parasitological state of khat treated mice on early stage of infection cannot be due to absolute suppressive effect of khat, but due to anti-inflammatory activity of the khat [47,48] that can suppress the effect of inflammatory reactions or due to cytotoxicity effect of the plant on the parasite [31]. However, as the parasite load increased and kidney function affected by the parasite and the khat, the inflammation reaction mediated by the uric acid and some parasite’s components [glycosylphosphatidylinositol (GPI)] and toxin released from infected RBC (hemozine) during malaria infection [39] incidence of some pathologies such as cerebral malaria and other could be exacerbated.

Researchers of epidemiological studies reported the effect of malaria infection on hematological parameters differently. Some stated that hematological changes are unreliable laboratory indicators of malaria in acute uncomplicated P. falciparum malaria [49], while others reported that lower level of some hematological parameters such as platelet, WBCs, and lymphocyte counts WBCs, eosinophils, RBCs and Hb level when used in combination with other clinical and parasitological method could be the most important predictors of malaria infection [50-53]. Moreover, higher monocytes to lymphocytes count ratio (MLCR) measured in peripheral blood have directly correlates with risk of clinical malaria [54]. This might be due to the major role of the monocytes in the initial stage of innate immune response by releasing cytokines to respond to malaria infection [55]. In agreement to the second notion, our in vivo analysis made on association between PbA infected mice and haematological parameters showed that almost all, except RBCs count and Hb level, haematological parameters such as WBC, platelet, HCT, MCV, MCH, MCHC and lymphocyte were more significantly reduced in PbA infected than in uninfected mice. This finding supports the possible predictor relevance of malaria risks using haematological indicators. Moreover, the high MLCR observed in mice treated with highest dose of crude khat extract (300 mg/kgBW) could be an evidence for increased risk of severe malaria than the positive control [54].

Conclusions

The finding of this study strengthen an assumption that khat could exacerbate manifestation of most severe malaria symptoms in mice and lacks chemo-suppressive property on Plasmodium berghei ANKA parasite.

Abbreviations

Alb:

Albumin ANOVA analysis of variance

BBB:

Blood brain barrier

CBC:

Complete blood cells count

CM:

Cerebral malaria

Cr:

Creatinine

EPHI:

Ethiopia Public Health institute

HCT:

Hematocrit

MLCR:

Monocyte-lymphocyte count ratio

NLCR:

Neutrophil-lymphocyte count ratio

p.i.:

Post infection

PbA:

Plasmodium begrhei ANKA

pRBC:

Parasitized red blood cells

RBC:

Red blood cells

Rf:

Retardation factor

sGOT:

Serum glutamic oxaloacetate transaminase

sGPT:

Serum glutamic pyruvic transaminase

WBC:

White blood cells

References

  1. Al-Hebshi NN, Skaug N. Khat (Catha edulis)-an updated review. Addict Biol. 2005;10:299–307.

    Article  CAS  PubMed  Google Scholar 

  2. Advisory Council on the Misuse of Drugs (ACMD). Khat (Qat): assessment of risk to the individual and communities in the UK. London: Home Office; 2005. http://www.homeoffice.gov.uk/publications/drugs/acmd1/khat-report2005/KhatReportpdf?view=Binary. (Accessed on 03 February, 2015).

    Google Scholar 

  3. Fitzgerald J. Khat: a literature review. Centre for culture, ethnicity & health, and Louise Lawrence research. Louise Lawrence Pty Ltd; 2009. http://www.ceh.org.au/downloads/khat_report_finalpdf (Accessed on 03 February, 2015).

  4. Odenwald M, Warfa N, Bhui K, Elbert T. The stimulant khat -another door in the wall? A call for overcoming the barriers. J Ethnopharmacol. 2010;132:615–9.

    Article  PubMed  Google Scholar 

  5. Balint EE, Falkay G, Balint GA. Khat, a controversial plant. Wien Klin Wochenschr. 2009;121:604–14.

    Article  PubMed  Google Scholar 

  6. Graeme B. Garden of Eden: the Shamanic use of psychoactive flora and fauna and the study of consciousness. Aust J Med Herbal. 2010;22(3):107. http://search.informit. com.au/documentSummary;dn=407056871720658;res=IELHEA> ISSN: 1033-8330.

    Google Scholar 

  7. WHO (World Health Organization). Assessment of khat (Catha edulis Forsk). WHO Expert Committee on Drug Dependence (ECDD). 34th report, Geneva, Switzerland; 2006.

  8. Christian R. The encyclopedia of psychoactive plants: ethnopharmacology and its applications. Rochester: Park Street Press; 1998.

    Google Scholar 

  9. Cox G, Rampes H. Adverse effect of khat: a review. APT. 2003;9:456–63.

    Google Scholar 

  10. Kalix P. Cathinone, a natural amphetamine. Pharmacol Toxicol. 1992;70:77–86.

    Article  CAS  PubMed  Google Scholar 

  11. Toennes SW, Harder S, Schramm M, Niess C, Kauert GF. Pharmacokinetics of cathinone, cathine and norephedrine after the chewing of khat leaves. Br J Clin Pharmacol. 2003;56:125–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Feyissa AM, Kelly JP. A review of the neuropharmacological properties of khat. Neuro-Psychopharmacol Biol Psychiat. 2008;32:1147–66.

    Article  CAS  Google Scholar 

  13. Al-Motarreb A, Baker K, Broadley KJ. Khat: pharmacological and medical aspects and its social use in Yemen. Phytother Res. 2002;16:403–13.

    Article  CAS  PubMed  Google Scholar 

  14. Hassan NA, Gunaid AA, Abdo-Rabbo AA, Abdel-Kader ZY, Al Mansoob MA, Awad AY, et al. The effect of Qat chewing on blood pressure and heart rate in healthy volunteers. Trop Doct. 2000;30:107–8.

    CAS  PubMed  Google Scholar 

  15. Dhaifalaha I, Santavy J. Khat habit and its health effect; a natural amphetamine. Biomed Papers. 2004;148:11–5.

    Article  Google Scholar 

  16. Selassie SG, Gebre A. Rapid assessment of drug abuse in Ethiopia. Bull Narc. 1996;48:53–63.

    CAS  PubMed  Google Scholar 

  17. Dimba EA, Gjertsen BT, Bredholt T, Fossan KO, Costea DE, Francis GW, et al. Khat (Catha edulis)induced apoptosis is inhibited by antagonists of caspase-1 and −8 in human leukaemia cells. Br J Cancer. 2004;91:1726–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lee MM. The identification of cathinone in khat (Catha edulis) a time study. J Forensic Sci. 1995;40:116–21.

    CAS  Google Scholar 

  19. Center for Drug Evaluation and Research. Guidance for industry single dose acute toxicity testing for chemicals. Fishers Lane, Rockville 20857, Maryland, USA; 1996.

  20. Guidance document on acute oral toxicity testing. The Organization of Economic Co-operation and Development (OECD), France; 2001.

  21. Girma A, Engidawork E. The effect of acute and sub-acute exposure to crude khat (Catha edulis F.) extract on learning and memory in rodents. J Addict Res Ther. 2014;5:3.

    Google Scholar 

  22. Curfs JH, Schetters TP, Hermsen CC, Jerusalem CR, van Zon AA, Eling WM. Immunological aspects of cerebral lesions in murine malaria. Clin Exp Immunol. 1989;75:136–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Hansen DS, Evans KJ, D’Ombrain MC, Bernard NJ, Sexton AC, Buckingham L, et al. The natural killer complex regulates severe malarial pathogenesis and influences acquired immune responses to ANKA Plasmodium berghei. Infect Immun. 2005;73:2288.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hilou A, Nacoulma G, Guiguemde TR. In vivo antimalarial activities of extracts from Amaranthus spinosus and Boerhaavia erecta in mice. J Ethnopharmacol. 2006;103:236–40.

    Article  CAS  PubMed  Google Scholar 

  25. Kalra BS, Chawla S, Gupta P, Valecha N. Screening of antimalarial drugs. Ind J Pharmacol. 2006;38:5–12.

    Article  CAS  Google Scholar 

  26. Kochar DK, Agarwal P, Kochar SK, Jain R, Rawat N, Pokharna RK, et al. Hepatocyte dysfunction and hepatic encephalopathy in Plasmodium falciparum malaria. Oxford J Med. 2003;96:505–12.

    CAS  Google Scholar 

  27. Anand AC, Ramji C, Narula AS, Singh W. Malarial hepatitis: a heterogeneous syndrome? Natl Med J India. 1992;5:59–62.

    CAS  PubMed  Google Scholar 

  28. Dhaliwal G, Cornett PA, Tierney LM. Hemolytic anemia. Am Fam Physician. 2004;69:2599–607.

    PubMed  Google Scholar 

  29. Lee SH, Looareesuwan S, Chan J. Plasma macrophage colony stimulating factor and P selectin levels in malaria associated thrombocytopenia. Thrombostat. 1997;77:289–93.

    CAS  Google Scholar 

  30. Piguet PF, Kan CD, Vesin C. Thrombocytopenia in an animal model of malaria is associated with an increased caspase-mediated death of thrombocytes. Apoptosis. 2002;7:91–8.

    Article  CAS  PubMed  Google Scholar 

  31. Cox D, McConkey S. The role of platelets in the pathogenesis of cerebral malaria. Cell Mol Life Sci. 2010;67:557–68.

    Article  CAS  PubMed  Google Scholar 

  32. Yamaguchi S, Kubota T, Yamagishi T, Okamoto K, Izumi T, Takada M, et al. Severe thrombocytopenia suggesting immunological mechanisms in two cases of vivax malaria. Am J Hematol. 1997;56:183–6.

    Article  CAS  PubMed  Google Scholar 

  33. McKenzie FE, Prudhomme WA, Magill AJ, Forney JR, Permpanich B, Lucas C, et al. White blood cell counts and malaria. J Infect Dis. 2005;92:323–30.

    Article  Google Scholar 

  34. Al-Meshal I, Qureshi S, Ageel AM, Tariq M. The toxicity of Catha edulis in mice. J Subst Abuse. 1991;3:107–15.

    Article  CAS  PubMed  Google Scholar 

  35. Vestbo J, Hurd SS, Agustí AG, Jones PW, Vogelmeier C, Anzueto A, et al. Global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease. GOLD executive summary. Am J Respir Crit Care Med. 2013;187:347–65.

    Article  CAS  PubMed  Google Scholar 

  36. Taylor WR, Canon V, White NJ. Pulmonary manifestations of malaria: recognition and management. Treat Respir Med. 2006;5:419–28.

    Article  CAS  PubMed  Google Scholar 

  37. Amani V, Boubou MI, Pied S, Marussig M, Walliker D, Mazier D, et al. Cloned Lines of Plasmodium berghei ANKA differ in their abilities to induce experimental cerebral malaria. Infect Immun. 1998;66:4093–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Carvalho LJ, Ferreira-da-Cruz MF, Daniel-Ribeiro CT, Pelajo-Machado M, Lenzi HL. Germinal center architecture disturbance during Plasmodium berghei ANKA infection in CBA mice. Malar J. 2007;6:59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Rasti N, Wahlgren M, Chen Q. Molecular aspects of malaria pathogeneisis. FEMA Immunol Med Microbio. 2004;41:9–26. 31.

    Article  CAS  Google Scholar 

  40. Gallego-Delgado J, Ty M, Orengo JM, van de Hoef D, Rodriguez A. A surprising role for uric acid: the inflammatory malaria response. Curr Rheumatol Rep. 2014;16:401. doi: 10.1007/s11926-013-0401-8.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Al-Meshal IA. Effect of (−)-cathinone, an active principle of Catha edulis forssk. (Khat) on plasma amino acid levels and other biochemical parameters in male wistar rats. Phytother Res. 1988;2:63–6.

    Article  CAS  Google Scholar 

  42. Al-Qirim TM, Shahwan M, Zaidi KR, Uddin Q, Banu N. Effect of khat, its constituents and restraint stress on free radical metabolism of rats. J Ethnopharmacol. 2002;83(3):245–50.

    Article  CAS  PubMed  Google Scholar 

  43. Al-Ashwal RH, Al-maqtari MA, Naji KM, Alwsabai NA, Al Hazmy SM. Potential health effects of daily khat leaves chewing: Study on the biochemical blood constituents changes among adults in Sana’a city Yemen. Inter J Biochemist Biotechnol. 2013;2(6):461–3.

    Google Scholar 

  44. Masoud A, Al-Assar A, Abu-zeed H, Albem S, Al-Hubaishi S. Chewing Catha edulis with amphetamine-like effect alters liver and kidney functions of female chewers. Inter J Pharmaceut Sci Invention. 2014;3(2):34–9.

    Google Scholar 

  45. Alam MS, Bin-Jerah AA, Nabi G, Husain Q. Effect of khat (Catha edulis) consumption on the functions of liver, kidney and lipid profile in male population of Jazan Region of Kingdom of Saudi Arabia. Inter J Applied Natural Sci. 2014;3(2):9–14.

    Google Scholar 

  46. Ingyang AL, Okpako DT, Essien EM. Platelet reactions in acute Plasmodium berghei infection in Swiss albino mice. Haematologia. 1987;20:101–8.

    Google Scholar 

  47. Al-Meshal IA, Tariq M, Parmar NS, Ageel AM. Anti-inflammatory activity of the flavonoid fraction of khat (Catha edulis Forsk). Agents Actions. 1986;17:379–80.

    Article  CAS  PubMed  Google Scholar 

  48. Murdoch C, Aziz HA, Fang HY, Jezan H, Musaid R, Muthana M. Khat (Catha edulis) alters the phenotype and anti-microbial activity of peripheral blood mononuclear cells. J Ethnopharmacol. 2011;138:780–7.

    Article  PubMed  Google Scholar 

  49. Muwonge H, Kikomeko S, Sembajjwe LF, Seguya A, Namugwanya C. How Reliable Are Hematological Parameters in Predicting Uncomplicated Plasmodium falciparum Malaria in an Endemic Region? ISRN Trop Med. 2013. http://dx.doi.org/10.1155/2013/673798.

  50. Erhart LM, Yingyuen K, Chuanak N, Buathong N, Laoboonchai A, Miller RS, et al. Hematologic and clinical indices of malaria in a semi-immune population of western Thailand. Am J Trop Med Hyg. 2004;70:8–14.

    PubMed  Google Scholar 

  51. Bakhubaira S. Hematological parameters in severe complicated Plasmodium falciparum malaria among adults in Aden. Turk J Haematol. 2013;30:394–9.

    Article  PubMed  PubMed Central  Google Scholar 

  52. van Wolfswinkel ME, Vliegenthart-Jongbloed K, De Mendonca MM, Wever PC, McCall MB, Koelewijn R, et al. Predictive value of lymphocytopenia and the neutrophil-lymphocyte count ratio for severe imported malaria. Malar J. 2013;12:101.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Kotepui M, Phunphuech B, Phiwklam N, Chupeerach C, Duangmano S. Effect of malarial infection on haematological parameters in population near Thailand-Myanmar border. Malar J. 2014;13:218.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Warimwe GM, Murungi LM, Kamuyu G, Nyangweso GM, Wambua J, Naranbhai V, et al. The ratio of monocytes to lymphocytes in peripheral blood correlates with increased susceptibility to clinical malaria in Kenyan children. PLoS One. 2013;8:e57320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Rhee MS, Akanmori BD, Waterfall M, Riley EM. Changes in cytokine production associated with acquired immunity to Plasmodium falciparum malaria. Clin Exp Immunol. 2001;126:503–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgment

The authors would like to thank officials of EPHI for mice donation (infected and non-infected). The work was financially supported by Jimma University, DAAD, and L’Oreal UNESCO, a fellowship to TK.

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Authors and Affiliations

  1. Department of Environmental Health Sciences and Technology, College of Health Sciences, Jimma University, Jimma, Ethiopia

    Tsige Ketema & Argaw Ambelu

  2. School of Civil and Environmental Engineering, Jimma University Institute of Technology, Jimma, Ethiopia

    Esayas Alemayehu

  3. Department of Biology, College of Natural Sciences, Jimma University, Jimma, Ethiopia

    Tsige Ketema

  4. Department of Microbiology and Veterinary Public Health, College of Agriculture and Veterinary Medicine, Jimma University, Jimma, Ethiopia

    Moti Yohannes

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  1. Tsige Ketema
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Correspondence to Tsige Ketema.

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The authors declare that they have no competing interests.

Authors’ contributions

TK was fully involved in all phases of the study; EA and AA involved in design of the study project, supervision and critical revision of the manuscript. MY involved in mice experimentation and laboratory work. All authors read and approved the final manuscript.

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Ketema, T., Yohannes, M., Alemayehu, E. et al. Effect of chronic khat (Catha edulis, Forsk) use on outcome of Plasmodium berghei ANKA infection in Swiss albino mice. BMC Infect Dis 15, 170 (2015). https://doi.org/10.1186/s12879-015-0911-2

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BMC Infectious Diseases

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