Evaluation of the efficacy of Syzygium aromaticum-derived Oleanolic acid on malaria parasites in Plasmodium berghei-infected male Sprague-Dawley rats and effects on blood glucose and renal electrolyte handling.
Introduction Malaria continues to be one of the major causes of morbidity and mortality worldwide even though the disease is treatable. This is as a result of malaria parasites developing resistance to current antimalarial drugs (WHO 2001a). Malaria treatment has been changed in the previous years due to the emergence of resistant strains. WHO currently recommends artemisinin combination therapies (ACT’s) as first line treatment for uncomplicated malaria (WHO 2001b). Following this recommendation many countries changed their treatment policies into using ACT’s as first line treatment for malaria. Although this treatment is effective, the emergence of resistance to artemisinin has been reported in countries like Cambodia and Vietnam (WHO 2013). This poses a threat to halt the effort to eradicate malaria since there is currently no treatment that provides the same level of efficacy as the ACT’s. Another shortfall of the ACT’s is that they are quite expensive and therefore unaffordable to the poorest which are the most burdened by malaria. Also the demand and supply is not balanced. Hence there is an imperative need to develop more effective, affordable and easily accessible antimalarial drugs. Oleanolic acid (OA) is a pentacyclic triterpene derived from the Syzygium aromaticum cloves amongst other sources. Various therapeutic effects of OA have been reported including anti-inflammatory, anti-diabetic, and hepatoprotective effects but the antimalarial effects of this triterpene have not been reported (Gao et al., 2009; Ngubane et al., 2011; Lee et al., 2013). Maslinic acid (MA), a triterpene also derived from S. aromaticum, has been shown to possess antimalarial effects in vitro (Moneriz et al., 2011). These results suggest that OA may also possess antimalarial properties. OA has also been reported to possess anti-oxidant effects, and can therefore alleviate the oxidative stress that is manifested during the malaria infection. Hence this study investigated the effects of OA on the malaria parasites. Some of the antimalarial drugs such as quinine (QN) and chloroquine (CHQ) have been shown to possess hypoglycaemic effects (Musabayane et al., 2010; Elbadawi et al., 2011). The hypoglycaemic effects of CHQ are mediated in part via an increase in insulin secretion (Musabayane et al., 2010). Hence the effects of OA on blood glucose were also investigated in this study. Studies have shown that orally administered CHQ is also deposited in organs such as the kidneys therefore altering the functions of these organs (Musabayane et al., 1994). Acute renal failure, pulmonary oedema and metabolic acidosis have been observed in patients with malaria. A study conducted in our laboratory reported that OA increases GFR in streptozotocin (STZ) induced diabetic rats (Mapanga et al., 2009). Another study reported that OA improved kidney function by increasing metabolic function of the kidney cell lines (Madlala et al., 2012). These results indicate that OA may be useful in alleviating renal function disturbances exerted by the malaria infection as well as other antimalarial drugs. Hence we evaluated the effects of OA on kidney function. The mechanisms for the development of kidney dysfunction are mediated, at least in part, via abnormal electrolyte handling by the kidney due to oxidative stress. Therefore, we investigated the effects of OA on oxidative stress in the kidney and liver. Materials and methods The studies were carried out over a period of 3 weeks and were divided into pre-treatment (days 0-7), treatment (days 8-12) and post treatment (days 13-21) periods. The non-infected control group was monitored for 21 days. In the infected control group, malaria was induced by a single intraperitoneal injection of P. berghei infected red blood cells. These rats were also monitored and sacrificed at day 14 for ethical reasons. To evaluate the effects of OA on malaria parasites, blood glucose homeostasis and renal function, separate groups of non-infected and P. berghei-infected male Sprague-Dawley rats (90g-120g) were used. These rats were treated with either OA (40, 80 and 160 mg/kg, p.o) or CHQ (30 mg/kg, p.o). The animals were housed individually in Makrolon polycarbonate metabolic cages. Percentage parasitaemia, mean body weight changes, food and water intake, blood glucose concentrations, haematocrit, oxidative stress, 24 hour urine volume voided, Na⁺, K⁺, Clˉ and creatinine levels were monitored every third day during the pre-treatment and post-treatment periods for all the groups. During the treatment period all these parameters were monitored daily. To assess the effects of OA on AVP, aldosterone and insulin concentrations, separate groups of non-infected and P. berghei-infected rats were sacrificed at 0, 12 and 24 hours for acute studies as well as on day 14 and 21 for chronic studies. Blood was collected through cardiac puncture and organs such as heart, liver and kidneys were collected. Results There was a continuous increase in percentage parasitaemia of the P. berghei-infected control throughout the study and as such the animals were sacrificed at day 14. OA was able to clear the malaria parasites, with the most potent dose (160 mg/kg, p.o.) eliminating the parasites after 6 days of treatment. CHQ (30 mg/kg, p.o) cleared malaria parasites after 5 days of treatment. There was a continuous decrease in blood glucose concentrations of the P. berghei-infected control until the rats were sacrificed at day 14. Rats treated with OA showed an increase in glucose concentrations during treatment period when compared to the infected control as well as CHQ-treated rats. CHQ-treated rats displayed a significant decrease in blood glucose concentrations during treatment period when compared to the non-infected control as well as OA-treated rats. There was an improvement in blood glucose levels of these animals during the post treatment period, but glucose levels still remained significantly low when compared to the non-infected control as well as OA-treated animals. There was no significant change in plasma insulin concentrations of rats treated with OA when compared to the non-treated control. There was an increase in plasma insulin concentration of rats treated with CHQ when compared to the non-infected control as well as the OA treated groups. OA was also able to increase haematocrit levels during treatment and continued to maintain these levels at normal ranges throughout the post-treatment period. CHQ also increased heamatocrit levels in P. berghei-infected rats during treatment period. OA increased urinary Na⁺ output as well as GFR when compared to the non-infected control. OA also decreased urinary K⁺ output significantly during the treatment period when compared to the infected control. CHQ increased urinary Na⁺ output in both non-infected and P. berghei-infected rats during the treatment period. OA-treated animals exhibited significantly low malondialdehyde (MDA, a marker of lipid peroxidation) and increased activity of the anti-oxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) in renal and hepatic tissues. However, an increase in MDA levels was observed in CHQ-treated animals as well as a decrease in activities of SOD and GPx. Discussion S. aromaticum derived-OA demonstrated antimalarial properties by completely clearing the malaria parasites in P. berghei-infected rats. OA was able to alleviate malarial hypoglycaemia by improving blood glucose levels in P. berghei-infected rats during the treatment period. These anti-hypoglyceamic effects may in part be due to the eradication of the malaria parasites from the red blood cells (RBC’s) hence no utilisation of the blood glucose by the parasites. The hypoglycaemia observed in CHQ treated rats may be attributed to the increase in insulin secretion that has been shown in rats orally treated with CHQ. We speculate that an increase in haematocrit levels observed in P. berghei-infected rats treated with either OA or CHQ is due to both these drug’s ability to clear the malaria parasites from the systemic circulation hence preventing the lysis of RBC’s. OA improved renal function by increasing Na⁺ output and GFR as well as decreasing K⁺ output. The results suggest that effects of OA on the kidney function are mediated, in part, via increased improved oxidative status. Conclusions The results of the current study demonstrate the ability of OA to clear malaria parasites, maintain glucose homeostasis as well as improve kidney function in P. berghei-infected rats. We conclude that this triterpene can play a crucial role in the formulation of more effective antimalarial drugs. Recommendations This study has demonstrated the ability of orally administered OA to clear malaria parasites. Future studies should therefore investigate the effects of the transdermally delivered OA via the pectin hydrogel patch. The mechanisms responsible for antimalarial as well as hypoglycaemic effects of OA should also be investigated.
M. Med. Sc. University of KwaZulu-Natal, Durban 2014.
Malaria--Treatment., Clove tree--Physiological effect., Kidneys., Blood sugar., Medicinal plants., Malaria--Control., Theses--Human physiology.