Introduction

Malaria occurs due to Plasmodium infectious agents and does not propagate from individual to another. Despite all possible effort to curb the menace of malaria parasites, quite a large number of the world populace are still at risk. About 249 million malaria cases and 608 thousand malaria deaths was reported by World Health Organization (WHO) across the globe in about eighty-five countries [1]. Globally over 50% of malaria-related fatalities cases are inscribed from four African countries with Nigeria, African’s most populous country having a disproportionate highest rate of 26.8%. Other top African countries malaria death prevalence are the Democratic Republic of Congo (12.3%) trailed by Uganda (5.1%) and Mozambique (4.2%) [1]. High cases of this disease could be attributed to lack of basic infrastructural, preventive, diagnostic and treatment facilities to combat the spread of the parasite. Pregnant women and children below age five ascribed for about eighty percent of malaria-related deaths in this region [2].

The contributory action of oxidative stress during malaria infection is complex; the precise role of its relation is yet to be fully understood [3, 4]. A disproportionate ratio between the generation of oxygen-derived radicals' species and endogenous antioxidant systems results in oxidative stress [5]. Malarial infections lead to an alteration in hepatic and erythrocytic activity in the victim, and this may result in generation and loss of regulation of free radicals [6]. Studies indicates that the generation of free radicals in plasmodium infected host cell associated with oxidative stress is implicated in the pathological conditions to the victim/organism [7, 8]. Antioxidants are substances that are involved in effective scavenging or delay of the oxidation of an oxidizable substrate [9]. By effective scavenging free radicals, antioxidants can abate the harm caused by oxidants before they can damage the cells. Mosquito-borne malarial infection has been found to deplete the profile of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) peroxidase [10].

Plants with medicinal properties have been documented to be an excellent natural antioxidants source [4, 11]. The plant Spilanthes filicaulis (Schumach & Thonn) of the family Asteraceae is a small creeping plant with flowers on ascending peduncles. It is a creeping annual herb with prostrate stems rooting from the nodes. The plant is often seen in swampy soil, or very damp localities distributed in tropical Africa and South America countries [12]. Spilanthes filicaulis is used to handle malaria among the Ekiti people of the Southwest Nigeria where befitting weather conditions is highly prevalent for the survival of this disease vector. Phytochemical analysis on this plant bring to light the availability of alkaloids, steroids, triterpenes cardiac glycosides, tannins, flavonoids and saponins [13].

This research therefore focused to investigate the impact of Spilanthes filicaulis sub-fractions on biochemical markers and oxidative stress balance in P. berghei- infected mice, aiming to deduce the potential therapeutic role of S. filicaulis in alleviating Plasmodium parasite related oxidative stress and biochemical disturbances of this devitalizing disease.

Materials and Methods

Chemicals

Methanol, hexane, ethyl acetate, butanol, chloroform were obtained from BDH Poole England. Chloroquine diphosphate used in this study were from Sigma-Aldrich. Other solvents and reagents used were of analytical grades.

Collection and Identification of Plant

The aerial parts of Spilanthes filicaulis were harvested in May 2022 during the rainy season from a garden at Igbara-Odo, Ekiti State, Nigeria (Latitude 7.4333° N Longitude 5.0667° E). A plant taxonomist at Obafemi Awolowo University Ile-Ife identified the plant, with a registered voucher specimen number of IFE/17571.

Preparation of Extract and Sub-fractions

The fresh parts of this plant were obtained, washed carefully, chopped into smaller unit using a knife, dried without heat for one month, and pulverized into smooth powder with an electronic blender. The smooth powdered material (1 kg) was processed using Soxhlet extraction with a 3 L volume of 80% methanol, which was then processed with the aid of a laboratory rotary evaporator under reduced pressure at 40 °C. This was further evaporated to dryness at 22–25 °C to obtain semi-solid extract referred to as crude methanolic extract. Then 100 g of crude methanol extract was dissolved in distilled water (10 mL) and fractionated successively by solvent–solvent partitioning to pull-down the hexane, ethyl acetate, and butanol fractions. Column chromatographic separation of ethyl acetate fraction of S. filicaulis was carried out in a systematic way and eluates (5 mL each) were collected in clean test tubes labelled fractions 1 to 30. Thin-layer chromatographic method were used to pooled the eluates with similar retention factor (Rf) values together to produce three sub-fractions labelled sub-fraction A (SFA), sub-fraction B (SFB) and sub-fraction C (SFC) respectively. The sub-fractions were kept in different sealed amber glass jar in a refrigerator until needed for biological use.

Parasite Inoculation

Chloroquine-sensitive strains of Plasmodium berghei (NK-65) were obtained from Institute of Advanced Medical Research and Training (IAMRAT), University College Hospital (UCH), Ibadan, Nigeria. Inoculation of naïve albino mice was done as described by Johnson et al. [14]. Blood was taken through cardiac puncture of a donor swiss mouse with a rising parasitaemia of about 25–35% inside a capillary tube having 0.1 mL of Acid Citrate Dextrose (ACD). A (0.9%) physiological saline was then added to the blood to dilute it based on parasitemia activity of the donor mouse to make-up a blood suspension of approximately 5 × 107 infected erythrocytes per milliliter. The experimental mice were then injected 0.2 mL of diluted parasitized red blood intra-peritoneally, containing 1 × 107 Plasmodium berghei infected red blood cells.

Grouping of Experimental Animals and Induction of Oxidative Stress

Sixty Swiss mice weighing between 20.0 ± 3.0 g were used for the in vivo antioxidant studies. Bred in the Animal Breeding Unit of IAMRAT UCH Ibadan, Nigeria. All experimental mice were housed in well ventilated cages (plastic) with soft wood shavings as bedding and acclimatized for seven days (temperature 25–30 °C, relative humidity 40–45%, and 12-h) with free access to pelletized growers mash (Top Feeds, Ibadan) and water. Evaluation of the effect of SFA, SFB and SFC on in-vivo antioxidant defense was done using the 4-day suppressive test against Plasmodium berghei (NK 65) -infected mice as described by Peters [15] to induce oxidative stress. Blood smear were prepared on Days 4 and 8 post-inoculation, fixed with methanol and stained with 5% Giemsa. Microscopic examination was performed to determine parasitaemia, following Cheesbrough's method [16]. Swiss mice were selected into five groups of 12 female mice randomly as follows. The grouping was done as follows:

Group I: Control (0.2 mL normal saline).

Group II: 10 mg/kg body weight chloroquine.

Group III: 250 mg/kg body weight fractions.

Group IV: 500 mg/kg body weight fractions.

Group V: 750 mg/kg body weight fractions.

Preparation of Plasma Erythrocytes and Organs for Antioxidant Assay

A day after the last treatment administration on day four, half of the mice in each of the five groups were humanely killed under a light diethyl ether anaesthesia. Jugular venous blood was collected into heparinized bottles and thereafter carefully centrifuged at 3000 rpm for 5 min to remove the plasma content. The erythrocytes were then washed thrice under ice-cold phosphate-buffered saline (pH 7.4) and later centrifuged at 3000 rpm for 5 min. The erythrocyte pellets obtained were thereafter re-suspended in PBS at 1:9 dilutions. The cells were disintegrated by repeated freeze–thaw method and the lysates were used for erythrocyte antioxidant assays. The liver of each mouse was carefully separated, cleansed of blood and other connective tissues. It was subsequently homogenized in ice-chilled 0.25 M solution of sucrose (1:5 w/v). The homogenates were separated by centrifugation at 10,000 rpm for four minutes in a refrigerated centrifuge and the supernatants called post mitochondrial fractions were stored at − 20 °C overnight to secure total enzyme release which will now be used for organ antioxidant assays. On day 8 post-inoculation, the remaining experimental mice were also killed and processed following similar process.

Estimation of Oxidative Stress and Antioxidant Assays

The methods of Buege and Aust [17] for malondialdehyde (MDA), Misra and Fridovich [18] for SOD, and Sinha [19] for CAT concentrations were used for antioxidant assays.

Statistical Analysis

The one-way analysis of variance (ANOVA) followed by Duncan’s Post-hoc multiple comparisons were utilized in analyzing and comparing the results at a 95% confidence activity. A p-values less than 0.05 were considered to indicate significance. Values were shown as mean ± standard error of mean (SEM). Graph of the data obtained was done with the aid of GraphPad Prism version 9.1.

Results

4-Day Suppressive In-vivo Antimalarial Studies

SFA of Spilanthes filicaulis showed inactive antimalarial activity on day 4 post-inoculation causing less than 30% chemosuppression but improved partial antimalarial activity was observed at all doses on day 8 post inoculation. The highest chemosuppression on day 8 was 45.74% at dose of 500 mg/kg body weight. SFB exhibited inactive antimalarial activity at the lower dose of 250 mg/kg body weight but an improved partial activity at the higher dose of 500 and 750 mg/kg body weight on day 4 post inoculation. However, on day 8 post-inoculation an improved dose dependent antimalarial activity was exhibited with highest chemossuppression of 57.32% at the highest dose of 750 mg/kg body weight. SFC showed dose dependent partial activities on day 4 post inoculation. An improved chemosuppression activity greater than 50% was observed on day 8 post-inoculation whose activity increased dose dependently. Highest chemosuppression was 68.27% at the highest dose (Table 1).

Table 1 4-day suppressive treatment for SFA, SFB and SFC from ethyl acetate partition extract of Spilanthes filicaulis aerial parts on Plasmodium berghei (NK65)-infected mice
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Malondialdehyde (MDA) Concentration

Figure 1 shows that SFA caused a dose dependent reduction in the MDA levels in erythrocyte and liver which showed a significant effect on day 4 and 8 post inoculation at all doses (p < 0.05), when compared to untreated control group. Most of the SFA treated groups showed no significant (p > 0.05) difference in both erythrocyte and liver, when compared to chloroquine treated group. Conversely, a slight increase (p < 0.05) was noticed across day 4 to day 8 post-inoculation for SFA in both erythrocyte and liver relative to the chloroquine administered group. Figure 2 shows that SFB treated groups significantly (p < 0.05) decrease in the MDA levels in both erythrocyte and liver, at all doses, when compared to untreated control group. Most of the treated groups showed no significant (p > 0.05) difference in liver, but showed a slight decrease (p < 0.05) in erythrocyte on day 4 post inoculation which was significant when compared to chloroquine administered group. The SFC treated groups showed a significant (p < 0.05) decrease in the MDA levels in both erythrocyte and liver at all doses, when compared to untreated control. Also, most of the SFC treated groups showed no significant (p > 0.05) difference in both erythrocyte and liver, when compared to chloroquine treated group (Fig. 3).

Fig. 1
figure 1

Effects of sub-fraction A (SFA) of ethyl-acetate partitioned extract of S. filicaulis on MDA level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Fig. 2
figure 2

Effects of sub-fraction B (SFB) of ethyl-acetate partitioned extract of S. filicaulis on MDA level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Fig. 3
figure 3

Effects of sub-fraction C (SFC) of ethyl-acetate partitioned extract of S. filicaulis on MDA level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Superoxide Dismutase (SOD) Activity

SFA treated groups caused a dose dependent increase in the SOD activity in the erythrocyte and liver which was significant at (p < 0.05) at all doses on day 4 and 8 post inoculation when compared to untreated control group. Nevertheless, a substantial drop in SOD activity (p < 0.05) was observed across day 4 to day 8 post-inoculation in both erythrocyte and liver of the untreated control group. Likewise, chloroquine treated groups significantly (p < 0.05) increase the SOD activities in both erythrocyte and liver when compared to the untreated group (Fig. 4). The SFB treated group showed a significant (p < 0.05) increase in the SOD activity in both erythrocyte and liver at all doses, when compared to untreated control group. In addition, most of the treated groups showed no significant (p > 0.05) difference in erythrocyte, but showed a slight increase (p < 0.05) in liver at most doses on day 8 post inoculation (Fig. 5). SFC treated group showed a dose-dependent significant (p < 0.05) increase in the SOD activity in both erythrocyte and liver at all doses relative to the control. Conclusively, most of the treated groups with SFC showed no significant difference (p < 0.05) in SOD activities in both erythrocyte and liver when compared to chloroquine treated group (Fig. 6). SFC caused the highest percentage increase in the activity of SOD when compared to other sub-fractions.

Fig. 4
figure 4

Effects of sub-fraction A (SFA) of ethyl-acetate partitioned extract of S. filicaulis on SOD level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Fig. 5
figure 5

Effects of sub-fraction B (SFB) of ethyl-acetate partitioned extract of S. filicaulis on SOD level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Fig. 6
figure 6

Effects of sub-fraction C (SFC) of ethyl-acetate partitioned extract of S. filicaulis on SOD level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Catalase (CAT) Activity

The SFA-treated group caused a dose dependent increase in the activities of CAT in the erythrocyte and liver which in most cases was significant (p < 0.05) at all doses on day 4 and 8 post inoculation when compared to the untreated control group. Moreover, all the treated groups revealed a significant (p < 0.05) decrease in both erythrocyte and liver when compared to chloroquine treated group (Fig. 7). The SFB-treated group showed a significant (p < 0.05) increase in the CAT activity in both erythrocyte and liver at all doses, in comparison to untreated control dose-dependently. Likewise, most of the treated groups displayed a significant difference in erythrocyte (p > 0.05), and a slight increase (p < 0.05) was observed in liver at most doses on day 8 post inoculation, when compared to chloroquine treated group (Fig. 8). The SFC-treated group showed a significant (p < 0.05) increase in the CAT activity in both erythrocyte and liver, at all doses, when compared to untreated control dose-dependently. Consequently, most of the SFC treated groups showed a significant (p < 0.05) higher increase in erythrocyte and liver CAT activity making it similar to the activity of chloroquine treated group (Fig. 9).

Fig. 7
figure 7

Effects of sub-fraction A (SFA) of ethyl-acetate partitioned extract of S. filicaulis on CAT level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Fig. 8
figure 8

Effects of sub-fraction B (SFB) of ethyl-acetate partitioned extract of S. filicaulis on CAT level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Fig. 9
figure 9

Effect of sub-fraction C (SFC) of ethyl-acetate partitioned extract of S. filicaulis on CAT level of P. berghei infected mice in erythrocyte and liver on day 4 and 8 post inoculation. CQ chloroquine, bwt body weight. Values are expressed as mean ± S.E.M (n = 6). Bars with different alphabets are significantly different (p < 0.05)

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Discussion

In this study, we assessed the oxidative stress reducing potential of ESSF on Plasmodium berghei on infected female mice. The 4-day suppressive test revealed that all the treatment doses of SFB and SFC were considered to be active in a dose-dependent manner, while those of SFA were considered partially active on day 8 post-inoculation. However, SFC produced the best activity with the highest maximum parasitemia inhibition of 68.27% on day 8 post-inoculation (Table 1). These findings are consistent with previous reports by Ofeniforo et al. [20], and Chinedu et al. [21]. The superior chemo-suppressive effect of SFC particularly at the highest dose, may be attributed to its high antioxidant content from its phytoconstituents which counteracts the depletion and exploitation of host antioxidants by Plasmodium parasites for their own survival.

Peroxidation of lipids is an auto-catalytic process, mediated by free-radicals, whereby membranes polyunsaturated fatty acids (PUFAs) form lipid hydroperoxides through a degradation process [22]. Reactive oxygen and nitrogen species degrade polyunsaturated lipids, forming MDA [23]. Lipid peroxidation is a major cause of disorders that occurs during infection of the erythrocytes by malaria parasites [24], and MDA is the biomarker of lipid peroxidation [25]. Antioxidant enzymes like SOD and CAT shield the biological system by mitigating oxidative stress in maintaining a normal activity of free radicals [26]. Approaches in combating of malaria infer that the control of oxidative stress may be of therapeutic advantage in infected host organism [4].

Results from this study revealed that MDA values significantly increase (p < 0.05) in the erythrocyte and liver homogenate of P. berghei NK65-infected untreated group compared to groups administered chloroquine, SFA, SFB and SFC (Figs. 1, 2, 3) on day 8 post-inoculation. Increase in MDA value signifies increased in lipid peroxidation which could have caused injury as a result of oxidative stress caused by H2O2 thus resulting from the depletion of in vivo antioxidant enzymes concentration. In contrast, the reasonable reduction in erythrocyte and liver MDA activity of the group co-treated with SFA, SFB and SFC could be attributed to the antioxidants (flavonoids and polyphenols) mitigating lipid peroxidation in the treated groups. Results of this study collaborates prior research findings of Nwonuma et al. [27] and Ajiboye et al. [28]. The increased rate of lipid peroxidation caused by the breakdown of polyunsaturated fatty acids in the liver and red blood cells during the process of xenobiotic biotransformation and pathophysiology of Plasmodium infection must have been responsible for the increased MDA activity of the parasitized untreated control group [29].

SOD forms the first line of enzymatic antioxidant defence, as systemic enzymatic antioxidant defense against reactive species occurs in two steps. The first step entails conversion of superoxide anions by SOD to molecular oxygen (O2) and H2O2, while the other step involves conversion of H2O2 to H2O by CAT. The results obtained from this study showed that SOD activities on day 4 post-inoculation, for SFA, SFB and SFC at various gradient doses of our administration significantly increased the erythrocytes and liver of infected mice compared to untreated control. However, SFC had the highest increase at all doses, when compared to other sub-fractions (Figs. 4, 5, 6). On day 8 post-inoculation there was a slight increase in activities of the erythrocytes and liver of infected mice when compared to the activities elicited on day 4 post-inoculation suggesting an enhanced protective effect on host cells against oxidative stress. Result from our study shows that extract treatment enhances cellular antioxidant capacity thus eliminating reactive oxygen species. Chloroquine treated group also increased the SOD activities in the erythrocytes and liver of infected mice compared to untreated control both on day 4 and 8 post-inoculation. The result of our investigation corroborates with reports’ by Ezzi et al. [30], Reginald and Kavishe [31], Sandro et al. [32], that untreated control group showed a decreased antioxidant activity thus confirming oxidative stress.

CAT are enzymatic antioxidant widely distributed in all animal tissues that catalyze the enzymatic conversion of hydrogen peroxide to water and oxygen, using either an iron or manganese cofactor. The detoxification of H2O2 in living organism by its conversion to H2O by the action of catalase requires high concentration of H2O2 for its activities [33], thus preventing the accumulation of H2O2.

CAT activities in the erythrocyte and liver homogenate of P. berghei NK65-infected treated group increased on day 4 and 8 post-inoculation in SFA, SFB and SFC compared to untreated control. Also, SFC produced the best activities (Figs. 7, 8, 9). Treatment with the subfractions restored and raised the activities of CAT in the erythrocyte and liver studied, indicating that the extracts scavenged peroxide radicals. However, decreased activities of CAT in the untreated control group may be due to the overuse of these enzymatic antioxidants triggering an elevation in the activity of peroxide radicals which in turn increases lipid peroxidation during haemoglobin degradation by the malaria parasite [34].

The results of this study is in agreement with the report of Agbafor et al. [3] who reported that all antioxidant defenses are interlinked in such that disruption of one may disrupt the whole microenvironment and vice versa. The improved antioxidant activities of the treated sub-fractions against P. berghei might be due to its secondary metabolites acting singly or synergistically, possibly leading to modification of parasite protein structure and compromise its function resulting to inhibition of erythrocytic stage of the parasites.

Conclusion

Oxidative stress generated from Plasmodium berghei infection resulted in an increased MDA level in the untreated mice group. However, treatment of parasitized mice with S. filicaulis sub-fractions reversed the reduced damage caused by MDA which was evident with improve antioxidant enzymes activities of SOD and CAT. Consequently, S. filicaulis sub-fractions could be a promising valuable tool in combating mosquito-borne malaria and oxidative stress-related diseases. It is recommended that SFC bioactive compounds needs to be identified, isolated and characterized which might give insight on the possible proposed mechanism of action of the subfractions.