Auwal Adamu, Muhammad A. Saliu, Abdullahi A. Dantani, Yaqub Nimat, Hauwa T. Giaze, Najib Musa, Buba Hussena, Morik Dorathy, Ahmad M. Kandi, Musa Daniel, Mohammed Auwal Ibrahim

Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria

Keywords:Malaria Stigmasterol Chloroquine Plasmodium berghei Organ pathologies

ABSTRACT Objective: To investigate the effect of combination therapy of chloroquine and stigmasterol on Plasmodium (P.) berghei malaria-induced organ pathologies. Methods: Totally 35 mice weighing 20-30g were placed into 7 groups of 5 mice each and distributed as uninfected administered 100 mg/kg BW stigmasterol, uninfected administered only feed and water ad libitum, infected with P. berghei and administered 50 mg/Kg BW stigmasterol, 100 mg/kg BW stigmasterol, 100 mg/kg BW stigmasterol plus 5 mg/kg BW chloroquine, and 5 mg/kg BW chloroquine. The last group of mice served as P. berghei infected and not treated control. The levels of parasitemia, packed cell volume, and other biochemical parameters were measured. Results: Combination therapy of P. berghei infection with stigmasterol and chloroquine did not significantly (P>0.05) reduce parasitemia level while stigmasterol treatment alone significantly (P<0.05) reduced the parasitemia level. However, the P. berghei induced anemia was decreased significantly (P<0.05) upon treatment with a combination of chloroquine and stigmasterol as well as with stigmasterol alone. Furthermore, the combination of chloroquine and stigmasterol significantly (P<0.05) decreased the activities of serum alanine aminotransferase, aspartate aminotransferase, urea and level of spleen total proteins in P. berghei infected mice in comparison with the untreated group. Treatment of P. berghei infected mice with stigmasterol alone and in combination with chloroquine significantly (P<0.05) increased the level of serum creatinine while serum and spleen malondialdehyde levels were significantly (P<0.05) decreased. Levels of glutathione in spleen and kidney were insignificantly (P>0.05) altered upon treatment with both doses of stigmasterol as well as the combination therapy. Conclusions: This study concluded that the combination of stigmasterol and chloroquine could combat anemia and some organ pathologies associated with P. berghei infection.

1. Introduction

Malaria is one of the most severe parasitic diseases that predominantly affect pregnant women and young children in major parts of Africa, South America and Asia where it is still endemic[1], hence a critical problem to the global public health system. The disease is associated with an increase in the production of reactive oxygen species, which was linked to organ dysfunctions[2-4] including acute kidney injury and loss of liver function that might result in the death of infected patients. Furthermore, anemia results in about one-third of all deaths caused by malaria infection and therefore it is considered an important pathological feature of the disease[5].

For many decades, the 4-aminoquinoline chloroquine (CQ) remains the most successful and widely used drug for malaria treatment but widespread resistance has rendered it essentially useless in most parts of the world[6]. The consequence of CQ resistance in malaria chemotherapy has, in part, necessitated the interest in the development of newer antimalarial agents with improved chemical structures[7]. Over the last decade, World Health Organization (WHO) recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria in endemic regions of the world because of the susceptibility of the CQ-based therapies to parasite resistance. Although ACTs have been the most preferred due to the good antimalarial activity, an alarming rate of resistance to the artemisinin derivatives with the partner drugs has been reported in Africa and Asia, indicating the need for other treatment options[8,9].

In 2017, WHO discourages malaria monotherapies with newly developed antimalarial drugs in order to mitigate the rapid spread of new drug-resistant Plasmodium species[10]. Consequently, the use of hybrid drugs that combining two or more distinct pharmacophores through a covalent bond is recently considered as a viable option to retard antimalarial drug resistance[11]. This is because of their distinct biological targets making it difficult for the parasite to easily develop resistance. Moreover, the use of CQ-based hybrids is an appealing therapeutic strategy to re-discover the clinical application of CQ in malaria treatment[12]. One of the approaches to the formulation of antimalarial agents with hybrid architecture is to initially undertake in vitro and in vivo studies of a combination of the pure compounds having the tendencies to interfere with the pathogenesis of malaria. Unfortunately, only a few such studies, especially in vivo, appear in the literature. For instance, Soh et al[13] reported that ellagic acid has synergistic activity in combination with CQ, atovaquone, mefloquine, and artesunate but was slightly antagonistic with artemisinin in vitro.

Phytosterols are a group of secondary metabolites in plants belonging to the triterpene family which includes stigmasterol, β-sitosterol, and campesterol. Previous studies have shown that phytosterols remarkably improved the health status of humans on account of their multiple pharmacological properties, including anti-viral, anti-mutagenic, anti-tumor, anti-inflammatory, antiosteoarthritic and anti-diabetic properties[14]. More specifically, stigmasterol was reported to have impressive in vitro antimalarial activity against CQ sensitive (D6) clones P. falciparum[15]. Furthermore, stigmasterol derivatives were shown to inhibit the growth of P. falciparum W2 strain with IC50 values ranging from 0.07 to 3.46 μg/mL in vitro[16].

In order to test the possibility of using stigmasterol to improve the efficacy and clinical application of CQ, in vivo combination therapy of stigmasterol and CQ on P. berghei induced organ pathologies were conducted in mice.

2. Materials and methods

2.1. Chemicals and reagents

Chloroquine diphosphate (100.1%) was obtained from Faculty of Pharmaceutical Sciences, Ahmadu Bello University Zaria (ABUZ), Nigeria, stigmasterol, biuret reagent, Ellman’s reagent, trichloroacetic acid, and thiobarbituric acid were procured from Sigma Chemical Company (St Louis, MO, USA), through Bristol Scientific Company Limited, Lagos, Nigeria. Assay kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), as well as urea and creatinine, were purchased from Agappe Diagnostic Laboratories., Switzerland.

2.2. Experimental animals and Plasmodium parasite

Wistar mice of six weeks old of both sexes weighing 20-30 g were obtained from the animal house of Department of Pharmacology, Faculty of Pharmaceutical Sciences, ABUZ. The animals were kept according to the rules and regulations of the experimental animal ethics committee of ABUZ and the guidelines of the Good Laboratory Practice regulations of WHO(No. ABUCAUC/2020/43). They were kept in well-ventilated cages and fed pelletized animal feed (Vital feed Jos, Nigeria) and water ad-libitum. After two weeks of acclimatization, the NK 65 CQ sensitive strain of P. berghei obtained from the Faculty of Pharmaceutical Sciences, ABUZ was subsequently injected into a donor mouse. Parasites obtained from the blood of the donor mice at peak parasitemia were diluted in physiologically cold saline for infection of the animals.

2.3. In vivo anti-P. berghei activity of co-administration of stigmasterol and chloroquine

Totally 35 mice were allocated into 7 groups with 5 mice each in order to investigate the effect of combination therapy of CQ and stigmasterol on the course of P. berghei infection. Mice in 5 of the groups were each intraperitoneally infected with 106 P. berghei parasitized red blood cells in 0.2 mL inoculum while the mice of the other 2 groups were uninfected. On day 3 after post-infection (pi) when parasitemia was established, 2 infected groups were given daily oral treatment of 50 (ISL) and 100 mg/kg BW stigmasterol (ISH) whereas another two infected groups were treated with 5 mg/kg BW CQ (ICQ) and with a combination of 100 mg/kg BW stigmasterol (ICQSH) and 5 mg/kg BW CQ respectively. The last infected group was left untreated (IUC) alongside an uninfected (normal) control group (NC) while the remaining uninfected group was treated with 100 mg/kg BW stigmasterol (USH). The choice of 100 mg/kg bw of stigmasterol was informed by the work of Hepburn PA and Kim JC et al[36,37], in which rats administered stigmasterol had no-observed-adverse-effect-level (NOAEL) at 879 and 350 mg/kg bw/day of stigmasterol respectively. Similarly, the 5 mg/kg bw of chloroquine was chosen based on standard malaria regimen with chloroquine in humans, which entails administration of 10 mg/kg bw at first instance, then 5 mg/kg bw follows, within 6 h, 24 h and 36 h after the 1st dose. The treatment lasted for 4 d and the experiment was terminated on day 7 pi. The parasites were monitored daily using the thin blood film microscopy. The pre-infection and terminal (on day 7 pi) packed cell volumes (PCV) of mice from all groups were determined by the microhaematocrit method.

2.4. Collection of blood and organs

At the end of the experimental period, all mice from the 7 groups were euthanized by anesthesia and both blood and organs were collected. The whole blood of each mouse was collected via cardiac puncture in plain containers, which was later centrifuged at 2 250 g for 15 min, and serum from each blood sample was separated and preserved at -30 ℃ for further analysis. The organs (liver, kidney, and spleen) were also collected from each mouse, washed with normal saline, dried by blotting with filter paper and weighed. Subsequently, 500 mg of each of the organs was cut and homogenized with 10 mL phosphate buffered saline (pH 7.4) for biochemical analyses.

2.5. Biochemical assays

The serum AST and ALT concentrations were measured according to Clim and Clin[17] and Thefeld et al[18], respectively using commercial reagent kits. Urea and creatinine concentrations were measured according to Kassirer[19] and Artiss et al[20] respectively, using commercial reagent kits.

Total proteins (TP) concentration in the serum and organ (liver, kidney, and spleen) homogenates were determined using the biuret method according to Wokes and Still[21]. Briefly, 2.5 mL of biuret reagent was added to all required test tubes (sample blank, standard and test sample), 0.05 mL of the sample and standard reagent was added to the test sample tube and standard test tube respectively. The mixture was allowed to stand at room temperature for 10 min and absorbance of all tubes was read at 540 nm against the blank. The concentration of sample total proteins was obtained from a standard curve.

Glutathione (GSH) concentration in the serum and some organs (kidney and spleen) homogenates was determined according to Ellman[22] as described by Rajagopalan et al[23]. To 150 μL of serum or tissue homogenate, 1.5 mL of 10% trichloroacetic acid was added and centrifuge at 1 500 g for 5 min. 1 mL of the supernatant was treated with 0.5 mL of Ellman’s reagent and 3 mL of phosphate buffer. The absorbance was read at 412 nm and the quantity of GSH was obtained from a GSH standard curve.

Malondialdehyde (MDA) concentration in the serum and some organs (kidney and spleen) homogenates was determined as thiobarbituric acid reactive substances (TBARS) according to Okhawa et al[24] with slight modification by Atawodi et al[25]. Exactly 2 mL of 15% trichloroacetic acid was measured into a test tube, 2 mL of thiobarbituric acid was added and 100 μL of the serum or tissue homogenate was added. The mixture was incubated at 80 ℃ for 30 min in a water bath and allowed to cool for some time, followed by centrifugation at 2 250 g for 10 min. Clear supernatant was collected and the absorbance was determined at 535 nm in a spectrophotometer. TBARS concentrations were expressed in nmol/mg protein.

2.6. Statistical analysis

All values are expressed as mean ± SD of five mice. The significance of differences between the means of the tests and controls was determined by one-way ANOVA and Tukey's-HSD multiple range posthoc test. P<0.05 were considered as statistically significant.

3. Results

3.1 Chemotherapeutic effects of oral administration of stigmasterol alone and in combination with CQ on P. berghei infection in mice

The treatment of P. berghei infected mice commenced on day 3 pi. Although the level of parasitemia in the ICQSH group did not significantly (P>0.05) differ from the IUC group, the treatment with stigmasterol alone impeded the progressive increase in parasitemia, and the effect was dose-dependent (Figure 1). This is confirmed especially from day 5 to 6 pi by the significantly (P<0.05) lower parasitemia level of ISH and ISL groups compared to the IUC group (Figure 1). Essentially, it was observed that parasitemia level was significantly (P<0.05) lessened in ICQ, ISH and ISL groups but the ICQSH group instead had its parasitemia level significantly (P>0.05) increased from day 5 to day 7 (Figure 1).Values bearing different letters (a-c) for the daily parasitemia across the groups are significantly different (Tukey's HSD multiple range post hoc test, P < 0.05). NC = Normal Control; IUC = Infected Untreated Control; USH = Uninfected Stigmasterol High-Dose; ISL = Infected Stigmasterol Low-Dose; ISH = Infected Stigmasterol High-Dose; ICQSH = Infected Chloroquine-Stigmasterol High-Dose; ICQ = Infected Chloroquine

Fig 1 Chemotherapeutic effects of oral administration of stigmasterol alone and in combination with chloroquine on the course of Plasmodium berghei infection in mice.

3.2 Effects of oral administration of stigmasterol alone and in combination with CQ on PCV levels of P. berghei infected mice

The effect of stigmasterol treatment alone and in combination with CQ on PCV is shown in (Figure 2). Mice in all groups had the same pre-infection PCV levels which were maintained to the end of the experiment in all groups with exception of IUC and ICQ groups, however, the terminal PCV of ICQSH, ISH and ISL were significantly (P<0.05) increased compared to that of IUC group (Figure 2).

Fig 2 Efects of oral administration of stigmasterol alone and in combination with chloroquine on packed cell volumes (PCV) levels of Plasmodium berghei infected mice.

Values with different alphabets (a–d) over the respective bars are significantly different (Tukey's HSD multiple range post hoc test, P < 0.05). NC = Normal Control; IUC = Infected Untreated Control; USH = Uninfected Stigmasterol High-Dose; ISL = Infected Stigmasterol Low-Dose; ISH = Infected Stigmasterol High-Dose; ICQSH = Infected Chloroquine-Stigmasterol High-Dose; ICQ = Infected Chloroquine; PCV = Packed Cell Volume

3.3 Effects of stigmasterol alone and in combination with chloroquine on some serum biochemical indices and organ homogenates total protein in P. berghei infected mice

As a consequence of P. berghei infection in the IUC group, the activities of serum ALT and AST, as well as the level of total protein in spleen homogenate significantly (P<0.05) increased while the infection significantly (P<0.05) reduced the level of liver total proteins, but the level of serum and kidney total proteins remain insignificantly (P>0.05) altered in comparison with NC group (Table 1). However, the activities of serum ALT and AST, as well as the level of total protein in the spleen, were significantly (P<0.05) decreased in ICQSH, ISL, and ICQ groups compared to the IUC group. While serum total protein levels in ISH and ISL groups significantly (P<0.05) increased compared to the IUC group, the level of kidney total proteins in all treated groups and serum total proteins of the ICQSH group were not significantly (P>0.05) affected (Table 1). The levels of serum urea were significantly (P<0.05) elevated in P. berghei infected mice while serum creatinine was significantly (P<0.05) lowered (Table 1). However, serum urea significantly (P<0.05) decreased in ICQSH, ISH, and ISL groups while serum creatinine significantly (P<0.05) increased in ICQSH, ISH and ICQ groups when compared with the IUC group (Table 1).Data are expressed as mean ± SD. Values bearing different superscript alphabets (a–d) along the respective horizontal rows are significantly different (Tukey's HSD multiple range post hoc test, P < 0.05). NC = Normal Control; IUC = Infected UntreatedControl; USH = Uninfected Stigmasterol High-Dose; ISL = Infected Stigmasterol Low-Dose; ISH = Infected Stigmasterol High-Dose; ICQSH = Infected Chloroquine-Stigmasterol High-Dose; ICQ = Infected Chloroquine; ALT = Alanine Amino Transferase; AST = Aspartate Amino Transferase; CREA = Creatinine; TP = Total Protein

Tab 1 Effects of stigmasterol alone and in combination with chloroquine on some serum biochemical indices and organ homogenates total protein in Plasmodium berghei infected mice.

Tab 2 Effects of stigmasterol alone and in combination with chloroquine on serum and organ-homogenates antioxidant status in Plasmodium berghei infected mice.

3.4 Effects of stigmasterol alone and in combination with chloroquine on serum and organ-homogenates antioxidant status in P. berghei infected mice

The investigation into the effects of stigmasterol alone and in combination with CQ on antioxidant status of P. berghei infected mice showed significant (P<0.05) increase in the levels of serum and spleen MDA while GSH was insignificantly (P>0.05) altered (Table 2). Compared with the IUC group, the levels of serum MDA in ICQSH, ISH and ISL as well as spleen MDA levels in ISH and ISL groups significantly (P<0.05) decreased. Contrariwise, the levels of GSH in the spleen and kidney in ICQSH and ISH groups had insignificantly (P>0.05) altered(Table 2).

Data are expressed as mean ± SD. Values bearing different superscript alphabets (a–f) along the respective vertical columns are significantly different (Tukey's HSD multiple range post hoc test, P < 0.05). NC = Normal Control; IUC = Infected Untreated Control; USH = Uninfected Stigmasterol High-Dose; ISL = Infected Stigmasterol Low-Dose; ISH = Infected Stigmasterol High-Dose; ICQSH = Infected Chloroquine-Stigmasterol High-Dose; ICQ = Infected Chloroquine; MDA = Malondialdehyde; GSH = Glutathione

4. Discussion

A multitude of plant-derived pure compounds have been isolated but only a few were tested against CQ sensitive and resistant strains of malaria parasites via the in vitro system. It is appalling to discover that many of these compounds with good antimalarial potentials including stigmasterol have not been subjected to an in vivo examination, which is usually the next step in drug discovery. The present investigation demonstrated the in vivo activity of stigmasterol treatment alone and in combination with CQ against P. berghei malaria induced organ pathologies.

Stigmasterol was selected for this study, partially because phytosterols have been shown to promote programmed cell death (apoptosis)[26], which presently serves as an important lead pathway for malaria chemotherapy[27]. The co-treatment of P. berghei infection in mice with stigmasterol and CQ was un-suppressive on parasite level but stigmasterol treatment alone reduced the parasite levels in a dose-dependent manner. The reduction in parasitemia level caused by stigmasterol treatment shows its potential as an antiplasmodial agent. This result is in line with the in vitro findings of Yamthe et al. in which stigmasterol-3-O-β-D-glucopyranoside of Annona muricata stem bark extract (IC50 > 10 μg/mL) manifested an impressive antiplasmodial effect[16]. It is possible that CQ reacts with stigmasterol and renders it inactive in suppressing parasite level which leads to the observed outcome in the combination therapy. Similarly, this finding may inform the need for structural modification of stigmasterol and/or combination with the chemical compound(s) other than CQ for a potentiating antiplasmodial activity in order to hamper malaria parasite resistance linked to malaria monotherapies and ensure sustainable antiplasmodial activity as a function of the half-life of the compound(s).

Anemia is a common feature of all forms of malaria linked to the destruction of infected erythrocytes with the concomitant production of toxic free haem (Ferri/ferroprotoporphyrin IX; FP) and reactive oxygen species as well as the clearance of uninfected erythrocytes alongside erythropoietic suppression and dyserythropoiesis[28,29]. The present study showed the capability of combination therapy of CQ and stigmasterol as well as stigmasterol alone to combat malaria parasite-induced anemia in mice. The exhibited antianemic properties of either the combination therapy or stigmasterol alone could be that the compounds are able to prevent the infected erythrocytes from destruction. Amazingly, the restored PCV observed was not in agreement with the recorded parasitemia level in the combined CQ and stigmasterol treatment but agreed with the recorded parasitemia level upon treatment with stigmasterol alone. Hence, these findings might suggest the ability of the combination therapy of CQ and stigmasterol to rescue parasitized erythrocytes membrane from the damaging effects of reactive oxygen species attack and also stigmasterol might not reduce the parasite killing ability of CQ but could contribute to ameliorating other P. bergheiinduced organ pathologies.

Liver, kidney, and spleen pathologies are cardinal manifestations of acute malaria infection[30]. The observed increases in liver and kidney biochemical indices following acute infection of P. berghei in mice could be linked to the parasite-induced hepatic, renal and splenic lesions, which culminate in the abnormal release of liver function enzymes, urea, and creatinine into the systemic circulation because of leakages from these tissues. Although the 100 mg/kg BW of stigmasterol showed tendency to slightly perturb protein turnover and intoxicate hepatic and renal functions in the absence of infection, the present findings indicated repair of hepatic and renal tissues damages with either CQ and stigmasterol combination therapy or stigmasterol alone during P. berghei infection, in which the restoration to normal function could be linked to the reported anti-oxidative properties of stigmasterol[31]. Oxidative stress plays a vital role in the pathogenesis of malaria infections[32,33] and can wreak havoc in biological systems, altering biomolecules and destroy cells[34]. The observed increase in the MDA level across the tissues of P. berghei infected mice is an indication of the massive generation of reactive oxygen species[30,33]. Similarly, the oxidative stress afflicted on the 100 mg/kg BW stigmasterol control group might suggest the ability of the compound to serve as a pro-oxidant in the absence of P. berghei malaria . The findings are further supported by the drop in levels of GSH across the tissues of P. berghei infected mice alluding to the fact that the available defense system has been overwhelmed by the massively produced reactive oxygen species. However, the treatment with either CQ in combination with stigmasterol or stigmasterol alone invigorated these antioxidant defenses. This was probably achieved via an anti-oxidative process in which the piled up oxygen radicals were being engulfed and detoxified by stigmasterol alone and also boosting CQ action when combined together. Overall, it appears that the anti-oxidative activity of stigmasterol might have potentiated the ameliorative effects of CQ toward the malaria-induced organ pathologies.

In conclusion, stigmasterol exhibited antimalarial potential in P. berghei infected mice but could not boost the parasite killing ability of CQ. Interestingly, however, stigmasterol has increased the ameliorative potential of CQ against P. berghei-induced anemia and organ pathologies.

Conflict of interest statement

The authors declare there is no conflict of interest.

Acknowledgment

The authorities of Ahmadu Bello University, Zaria, Nigeria are acknowledged for providing the equipment used for the study.