Evaluation of the efficacy of maslinic acid on malaria parasites in Plasmodium berghei-infected male Sprague-Dawley rats : effects on blood glucose and renal fluid and electrolyte handling.
Introduction Malaria remains a major socio-economic burden in Africa despite the numerous global efforts to control and manage the disease through prevention and drug intervention. Clinical studies have shown that malaria infection cases reach a total of 300-500 million every year of which 1 million occur in infants and children. Malaria infection often presents with metabolic complications which include impairment in glucose homeostasis, cardiovascular and kidney functions whose underlying causes can partly be ascribed to Plasmodium infection and/or drugs used manage malaria. Malaria cases have increased rapidly due to Plasmodium resistance to conventional treatment. The World Health Organisation (WHO) recommended the use of artemisinin combination therapy (ACT) as the first line of defense against malaria. However ACTs are expensive and not accessible to African countries which are the most affected by the disease. Chloroquine (CHQ), therefore, remains the mainstay therapy in some parts of Africa despite the developed P. falciparum resistance. Plasmodium resistance to CHQ may be due to the inconvenient dosing schedule and bitter taste of the drug which often causes patient non-compliance. The partial use of the drug has been suggested to be a major cause of the rapid development of P. falciparum resistance. Furthermore, the high plasma CHQ concentrations following oral administration lead to accumulated deposition of the drug in various organ systems eliciting adverse effects and organ damage. Reports suggest that CHQ accumulates in the kidney (Mc Chesney et al., 1967), heart (Baguet and Fabre, 1999) and adrenal glands (Gustafsson et al., 1987) to alter the physiological functions of these organs (Rodrigues et al., 1994; Nord et al., 2004). Studies have reported that maslinic acid (MA) possesses anti-parasitic, antioxidant properties and averts kidney dysfunction in diabetic rats (Mkhwanazi et al., 2014). The present study evaluated the effects of MA on malaria parasites, glucose homeostasis, renal fluid and electrolyte handling of P. berghei-infected male Sprague-Dawley (SD) rats in an effort to further investigate the pharmacological properties of the triterpene. We envisaged that the anti-oxidant effects of MA may ameliorate the malaria and/treatment induced impairment of glucose homeostasis and renal function. Materials and methods The extraction of MA was conducted at the University of KwaZulu-Natal (UKZN) Pietermaritzburg chemistry laboratory under the supervision of Prof Van Heerden. MA was extracted using a validated protocol in our laboratory. The effects of MA and CHQ on malaria parasites, blood glucose concentrations, renal fluid and electrolyte handling were investigated in male SD rats (90-120 g). Studies were carried out on non-infected and P. berghei-infected animals for a period of 3 weeks divided into pretreatment (days 0-7), treatment (days 8-12) and post treatment (days 13-21) periods. During treatment period separate groups of non-infected and P. berghei-infected animals were administered MA (40, 80 and 160 mg/kg, p.o.) or CHQ (30 mg/kg, p.o.) twice daily at 9h00 and 17h00 for 5 consecutive days. The selected doses were based on our preliminary findings. % parasitaemia was monitored daily in the P. berghei-infected control and P. berghei-infected animals treated with MA or CHQ. All groups of animals were individually housed in Makrolon polycarbonate metabolic cages to monitor 24 h food consumption, water intake, % body weight, blood glucose concentrations and urine volume output. These parameters were monitored daily during the treatment period and every third day during the post-treatment period. Overnight urine samples were collected daily during the treatment period and every third day during the post-treatment period for the measurement of Sodium (Na⁺), potassium (K⁺)and chloride (Cl⁻). All groups of animals were anaesthetized in an anaesthetic chamber with 100 mg/kg of isofor inhalation anaesthetic for 3 min for terminal studies. % parasitaemia in the untreated P. berghei-infected animals peaked at day 12 of the study. These animals were therefore sacrificed on day 12 as per ethics guidelines. Separate groups of animals (n=6) were sacrificed during pre-treatment days 0 and 7, treatment period days 1, 5 and post-treatment period day 14 to assess the effects of MA and CHQ on biochemical parameters. Blood was collected by cardiac puncture for plasma insulin, arginine vasopressin (AVP) and aldosterone analyses. The collected liver and kidney were used for the measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and GPx concentrations. Results Percentage (%) parasitaemia in the untreated P. berghei-infected control was 55 ± 8 % by day 12 of the experimental period. The P. berghei-infected control animals were therefore sacrificed on day 12 of the experimental period and as such all the subsequent results showing the untreated P. berghei-infected control animals have no post treatment period (days 13 - 21). The lower dose of MA (40 mg/kg, p.o.) significantly reduced the malaria parasite in comparison with the P. bergei-infected control at corresponding time periods. However, this dose did not eliminate the malaria parasite by the end of the 21 days experimental period. The higher doses of MA (80 and 160 mg/kg, p.o.) cleared the parasite from systemic circulation by day 9 following treatment. The effects of MA (80 and 160 mg/kg, p.o.) on % parasitaemia were statistically significant compared to MA (40 mg/kg, p.o.). However, the effects of MA (80 and 160 mg/kg, p.o.) on % parasitaemia were not dose dependent. CHQ (30 mg/kg, p.o.) eliminated the malaria parasites by day 5 following treatment. Blood glucose concentrations, food consumption, water intake and % body weight changes in the non-infected control animals were not altered throughout the experimental period. The untreated P. berghei-infected control animals exhibited a significant reduction in the above mentioned parameters when compared with the non-infected control at corresponding time periods. Plasma insulin concentrations in P. berghei-infected animals remained at values comparable with the non-infected control. MA (40, 80 and 160 mg/kg, p.o.) had no significant effects on blood glucose concentrations, food consumption, water intake and % body weight changes of the non-infected animals when compared with the non-infected control. Treatment with MA significantly increased the above mentioned parameters in P. berghei-infected. Following treatment with MA plasma insulin concentrations in the noninfected and P. berghei-infected animals remained unchanged at values comparable with the non-infected control. When compared with the non-infected control, CHQ (30 mg/kg, p.o.) significantly increased plasma insulin concentrations with a concomitant decrease in blood glucose concentrations in non-infected and P. berghei-infected animals. Food and water intake as well as % body weight changes were significantly reduced in these animals. Plasma AVP and aldosterone concentrations in the non-infected animals served as baseline values. When compared with the non-infected control, untreated P. berghei-infected animals exhibited increased plasma AVP concentrations while aldosterone concentrations remained unchanged. However urinary sodium (Na⁺) and urine volume outputs of the untreated P. berghei-infected were significantly reduced. Treatment with MA did not alter plasma AVP concentrations and urine volume outputs in the non-infected animals however plasma AVP concentrations in the untreated P. berghei-infected animals were significantly increased when compared with the non-infected control. The urine volume outputs in the P. berghei-infected animals treated with MA were restored to values comparable with the non-infected control. Plasma aldosterone concentrations of the non-infected and P. berghei-infected animals remained unchanged following the administration of MA. However urinary Na⁺ outputs were significantly increased in these animals by comparison with the non-infected control. Urinary K⁺ and Cl⁻ outputs in the non-infected and P. berghei-infected animals remained unchanged following treatment with MA. CHQ administration significantly increased Na⁺ outputs in the non-infected and P. berghei-infected animals on day 1 of the treatment period. However urinary Na⁺ outputs were significantly reduced in these animals from day 2 of the treatment period until post-treatment day 14. Urinary K⁺ outputs were significantly increased during the treatment period while Cl- outputs remained unchanged throughout the study. Liver and kidney MDA, SOD and GPx concentrations of the non-infected animals served as baseline. MDA concentrations of untreated P. berghei-infected and CHQ treated groups were significantly increased with diminished activity of SOD and GPx when compared with the non-infected control. Interestingly, MDA concentrations following treatment with MA were comparable with those of the non-infected control. MA significantly (p<0.05) increased the activity of SOD and GPx in the liver and kidney of P. berghei-infected animals to values comparable with the non-infected control. Discussion MA eliminated malaria parasites in P. berghei-infected animals. Our findings were in agreement with previous studies which reported that MA possesses anti-plasmodial activity in vitro. Furthermore De Pablos and colleagues (2010) reported that MA possesses on antiparasitic effects against the tachyzoites of Toxoplasma gondii. MA maintained blood glucose concentrations of non-infected animals at physiological levels. This could be due to the fact that MA did not alter food intake in these animals. Our results indicate that MA does not influence insulin secretion. This could therefore be one of the mechanisms by which MA maintains glucose homeostasis. MA significantly increased blood glucose concentrations in P. berghei-infected animals to values comparable with the noninfected control. This could be attributed to increased food intake by the P. berghei-infected animals. Furthermore, MA administration showed no significant effect on plasma insulin concentrations in these animals. This is another mechanism by which MA could have maintained the blood glucose concentrations at physiological levels. The ability of MA to maintain physiological blood glucose concentrations of indicates that MA may avert the adverse effects on glucose homeostasis that are often observed following infection with malaria and/treatment with CHQ. Indeed treatment with MA showed a significant reduction in blood glucose with concomitant increases in plasma insulin concentrations in non-infected and P. berghei-infected animals. Renal fluid and electrolyte handling in the non-infected animals remained unchanged throughout the study. P. berghei-infected control animals showed a significant decrease in urinary Na⁺ excreation and urine volume outputs when compared with the non-infected control. This could be attributed to inappropriate plasma AVP concentrations following malaria infection. Treatment with MA significantly increased urinary Na⁺ outputs of the noninfected and P. berghei-infected rats. These findings correlate with previous studies in our laboratory which showed that MA increases urinary Na+ outputs in streptozotocin-induced diabetic animals. The increased urinary Na⁺ output can be attributed in part to increased plasma AVP concentrations. Results from the present study show that chronic administration of CHQ causes reduced urinary Na⁺ outputs and urine volume outputs in non-infected and P. berghei-infected animals. Indeed previous studies in our laboratory reported that CHQ causes renal Na+ retention via increased plasma aldosterone concentration and reduced glomerular filtration rate (GFR). Urinary K⁺ outputs of the P. berghei-infected control were significantly increased in comparison with non-infected animals. The P. berghei-induced hyperkalaemia is thought to be due to the increased release of K⁺ from RBCs due to cell lysis. Indeed, the present study recorded a concomitant reduction in haematocrit levels of the P. bergheiinfected control. The reduced haematocrit values suggested that there was a significant reduction of RBC’s due to malaria parasite-induced hemolysis of infected and non-infected cells. Urinary K⁺ output of the P. berghei-infected animals decreased following MA administration however this decrease was not of statistical significance. The slight decrease in urinary K⁺ output could be due to parasite reduction by MA which led to decreased cell lysis with concomitant improvement of haematocrit values. Malaria infection increased MDA concentrations and diminished the activity of SOD and GPx in the liver and kidney of P. berghei-infected animals. The malaria parasite induced oxidative stress has been attributed to degradation of haemoglobin by the Plasmodium parasite as well as increased utilisation of antioxidants to counteract the malaria parasite induced oxidative stress. CHQ has been found to decrease the availability of reduced glutathione to pathways involved in detoxification and reacts with ferriprotoporphyrin IX which produces highly reactive radicals that generate oxidative stress in the host. Indeed finding from the current study show increased MDA concentrations and decreased SOD and GPx in the liver and kidney of non-infected and P. berghei-infected animals. Interestingly treatment with MA restored MDA concentrations in the liver and kidney of P. bergheiinfected animals to normal values. The reduction of MDA levels could be due to increased antioxidant activity. Indeed our results show that MA increases the activity of SOD and GPx. Findings from the current study are in agreement with a previous study in our laboratory which reported that MA improves renal function of diabetic animals by increasing antioxidant activity in the liver, kidney and heart. The antioxidant activity of MA has been suggested to be due to the ability of MA to inhibit NO and reducing susceptibility of plasma to lipid peroxidation. Furthermore MA has been found to possess peroxyl radical scavenging activity and metal chelating effects. In summary, findings from the present study confirm the previously reported anti-plasmodial activity of MA on malaria infected cell lines. Furthermore our results show that MA sustains blood glucose concentrations at physiological levels with a concomitant improvement of renal function of P. berghei-infected animals. Findings from the current study not only validate our endogenous knowledge systems but provide scientific evidence that contributes to current knowledge about the therapeutic effects of plant-derived MA in malaria. This study introduces the first in vivo evidence of antihypoglycaemic and antioxidant effects of MA on malaria and/treatment-induced adverse effects. Conclusions S. aromaticum-derived MA possesses anti-plasmodial activity with concomitant improvement of glucose homeostasis and renal fluid and electrolytes handling which are associated with malaria infection and/treatment. Our findings suggest that MA has potential to serve as an alternative anti-malaria compound in malaria management. Recommendations for future studies In the present study we have reported decreased haematocrit levels in P. berghei-infected rats. Measurements of hepcidin, a recently discovered peptide hormone that plays a major role in iron regulation could provide a mechanism by which malaria anaemia. This could provide information that would aid in the development of therapeutic strategies in the future. In addition to the present study, the anti-plasmodial activity and therapeutic effects of MA have been demonstrated in a number of in vitro and in vivo studies. Therefore, further studies are required to elucidate the mechanisms of action by which MA improves blood glucose homeostasis and renal function which are associated with malaria infection and/treatment.