In vivo antiplasmodial activity of the methanol leaf extract of Piliostigma reticulatum (Dc.) Hochst (Fabaceae)

Piliostigma reticulatum is a plant traditionally used to treat malaria, smallpox, neuralgia, dysentery, diarrhea, and rheumatism in Northern Nigeria. There is no scientific evidence to support the antimalarial activity of this plant. This work aims to investigate the in vivo antiplasmodial activity of the methanol leaf extract of Piliostigma reticulatum (MPR) in mice, infected with NK65 chloroquine-sensitive Plasmodium berghei. The oral lethal doses and preliminary phytochemical screening of the extract were performed. The therapeutic, suppressive, and prophylactic models were used for the antiplasmodial activity at the doses of 250, 500, and 1000 mg/kg of the MPR extract. Chloroquine and artesunate were used as the positive control drugs, while distilled water was used for the negative control group. The antiplasmodial activity was determined by comparing the mean parasite clearance in the treated groups, to the negative control group. Also the effect of the extract on the blood packed-cell volume of mice (PCV) was determined. The LD50 of MPR was found to be > 5000 mg/kg. Glycosides, saponins, tannins, flavonoids, triterpenes and alkaloids were the phytochemicals identified in the extract. The extract of MPR produced a significant reduction in the mean parasitemia level compared to the negative control in the curative test: MPR 250 (68.31%, P < 0.001), MPR 500 (76.53%, P < 0.001), and MPR1000 (83.65%, P < 0.001). The extract prolonged the survival of infected mice (18.8 days), compared to the negative control (5.2 days). The extract produced significant chemosuppression compared to the negative control; MPR 250 (73.79%, P < 0.001), MPR 500 (81.33%, P < 0.001), and MPR 1000 (78.37%, P < 0.001). The extract produced significant chemoprophylaxis compared to the negative control; MPR 250 (68.5%, P < 0.001), MPR 500 (58.7%, P < 0.001), and MPR 1000 (84.77%, P < 0.001). The extract was found to have no significant effect on the blood PCV of the treated groups compared to the negative control. The study showed that the MPR extract has significant antiplasmodial activity in mice at the doses tested, and could justify the traditional use of the plant in the treatment of malaria in Northern Nigeria.

deaths reported globally in the year 2019, and Africa has contributed 94% (215,000 million) of the reported cases (WHO 2020). In Africa, Nigeria has the highest burden of the disease, (contributing 25% of the global cases and 27% of global mortality from the disease) in 2019 (WHO 2020).
The advent of malaria parasite resistance to antimalarial drugs is another setback to eradicate the disease. The quinoline-based quinine and later chloroquine proved to be successful treatments for malaria, until quinoline resistance emerged and spread across much of the globe (CDC 2014). Artemisinin proved to be a fast-acting and efficacious remedy against chloroquine-resistant malaria in the face of this circumstance. However, artemisinin resistance in the form of delayed parasite clearance is already on the horizon (White et al. 2014), raising fears that humanity will be left without an effective malaria medication. This necessitates a continuous search for new antimalarial drugs.
Despite pharmaceutical corporations' recent breakthroughs in rational drug design and synthetic chemical techniques, natural products (particularly medicinal plants), have remained a significant source of novel medications (Kaushik et al. 2011). Classes of artemisinins and quinines (which originated from medicinal plants), stand out as the most effective drugs used in modern medicine to treat the malaria menace today. Perhaps the growing concerns of antimalarial drug resistance in certain areas, can be addressed by traditional medicines (Willcox and Bodeker 2004).
Piliostigma reticulatum belongs to Fabaceae family and Caesalpinioideae subfamily. It is a tree well distributed in the tropics, especially in northern Nigeria, popularly known as "camel's foot" and locally known as "kargo" or "kalgo." The leaves and bark are used in the treatment of malaria, smallpox, neuralgia, dysentery, diarrhea and rheumatism (Zerbo et al. 2010).

Collection of plant materials
Fresh leaves of Piliostigma reticulatum were collected from Ungogo Local Government area of Kano State, Nigeria in January 2018. The botanical identification and authentication were done by a botanist; Baha'uddeen Said Adam at the Herbarium Unit of the Department of Plant Biology, Bayero University Kano. A voucher specimen number; BUKHAN 0072 was deposited at the herbarium for future use.

Extraction of plant materials
Fresh leaves of Piliostigma reticulatum were air-dried. The leaves were size reduced into a fine powder using pestle and mortar. One thousand grams (1000 g) of the plant material were macerated with 4 L of 70% methanol for 72 h, with frequent stirring to facilitate the extraction process. After 72 h, the mixture was filtered using Whatman filter paper number 1. The filtrate was concentrated using a water bath at 45 °C and a percentage yield of 10.41% w / w was obtained. The extract was packed in airtight containers, protected from light and stored in a desiccator.

Preliminary phytochemical screening
The phytochemical screening of methanol leaf extract of Piliostigma reticulatum was done using standard procedures (Sofowora 1993;Trease and Evans 2002).

Experimental animals
Swiss albino mice of either sex weighing 18-25 g were obtained and maintained in the Animal House, Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Bayero University Kano. The animals were kept based on the guidelines of the National Institutes of Health (NIH 2011).They were fed with standard animal feeds and allowed free access to water ad libitum.
Ethical approval was obtained from the College of Health Sciences, Bayero University Kano, with the Protocol no: BUK/CHS/REC/VII/45.

Rodent Plasmodium parasite
Chloroquine-sensitive Plasmodium berghei (P. berghei) NK65 strain was used to induce malaria in the experimental mice. The parasite was obtained from the National Institute of Medical Research (NIMR), Yaba, Lagos State, Nigeria. It was maintained via intraperitoneal (i.p) sub-passage in mice.

Parasite inoculation
Parasitized blood was collected from donor mouse with rising parasitemia (34%) retro-orbitally. The blood was diluted with 0.9% saline in EDTA containing sample bottle, such that every 0.2 ml of blood contained 1 × 10 7 infected erythrocytes. Each mouse used in the study was inoculated with 0.2 ml of infected blood intraperitoneally.

Acute toxicity test
The Median lethal dose (LD 50 ) of the extract was determined using the Lorke method (Lorke 1983). The protocol consists of two phases. In the first phase, three groups of three mice were treated orally with graded doses of the extract at 10, 100 and 1000 mg/kg. The mice were observed for signs of toxicity and/or death for the first 4 h and then over 24 h. In the second phase, three mice were divided into three groups of one mouse each, and treated with 1600, 2900 and 5000 mg/kg of the extract, based on the outcome of the first phase. The animals were also observed for signs of toxicity and/or death over 24 h. The LD 50 was calculated as the geometric mean of the lowest lethal dose and that of the highest non-lethal dose.

Antiplasmodial screening Curative test (Rane's Test)
The method of Ryley and Peters (1970) was employed in this study. The inoculum containing 1 × 10 7 P. bergheiinfected RBCs (red blood cells) was injected intraperitoneally (i.p) into each of the thirty-six (36) mice on the first day (day 0). The mice were maintained for 72 h (day 0-day 3) for the infection to be established. Afterward, the mice were randomly divided into six groups; Group I (negative control) were given 10 ml/kg of distilled water, groups II-IV were treated with the graded doses of the extract (250, 500 and 1000 mg/kg body weight) and groups V and VI (positive controls) were treated with chloroquine 10 mg/kg, and artesunate 5 mg/kg, respectively. The treatment was carried out daily via oral route for 5 days (day 3-day 7). Twenty-four hours after the last treatment, blood was collected from the tail of each mouse and smeared onto a microscope slide to make a thin film. The blood films were fixed with methanol, stained with 10% Giemsa, and examined microscopically to determine the mean parasitemia level (by counting the number of parasitized RBCs in three random microscopic fields).
The Mean Survival Time (MST) for animals in the curative test group, was also determined by calculating the average survival time (in days) of mice after infection with P. berghei over a period of 28 days (i.e., from d 0 to d 27), as given by the formula below;

Peter's 4-day suppressive test
This test was conducted as described by Peters (1965). Thirty-six (36) mice were infected with P. berghei inoculum (1 × 10 7 ) i.p and distributed into six groups; Group I (negative control) were treated with 10 ml/kg of distilled water (negative control), groups II-IV were treated with graded doses of the extract (250, 500 and 1000 mg/kg), groups V and VI (positive controls) were treated with chloroquine 10 mg/kg and artesunate 5 mg/kg, respectively. Treatment with the extract commenced 4 h after infection (i.e., on Day 0) and continued daily for four days (i.e., from day 0 to day 3). On the 5th day (i.e., day 4), the blood was collected from the tail of each mouse and the parasitemia level was determined microscopically, as done with the curative test. Prophylactic test (Repository test) This test was done as described by Peters (1965). Thirty mice (30) were randomly distributed into 5 groups. In this test, thirty mice were used as only one positive control was employed (i.e., Pyrimethamine). Group I were treated with 10 ml/ kg of distilled water (negative control), groups II-IV were treated with graded doses of the extract (250, 500 and 1000 mg/kg) and group V was treated with Pyrimethamine 1.2 mg/kg (positive control). Treatment continued orally for five days (from day 0 to day 4). On the 6th day (i.e., day 5), the mice were inoculated intraperitoneally with 1 × 10 7 P. berghei infected erythrocytes. After 72 h, blood was collected from the tail of each mouse and the parasitemia level was determined.

Packed cell volume (PCV)
The packed cell volume (PCV) of mice in the curative test group was measured to predict the effectiveness of the extract in preventing hemolysis due to a rise in parasitemia caused by the infection. Blood samples were collected in heparinized capillary tubes by tail bleeding each mouse, and each capillary tube was filled up to ¾ of its length. The end of the capillary tubes was sealed with sealing clay. The tubes were placed in a microhematocrit centrifuge (Sigma-Aldrich, India) with the sealed end of the capillary tubes facing outwards. The blood was centrifuged for 5 min at 11,000 revolutions per minute (rpm). After centrifuging, the capillary tubes were removed from the machine, the PCV was measured using a microhematocrit reader, and the results were recorded. The PCV was calculated using the formula below; In this study, the PCV was determined during the curative test on the third day i.e., the day the infection was expected to be established and on the seventh day i.e., after treatment.

Euthanization of mice
The mice used in the study were euthanized as described by Jung et al. 2019. The mice were injected intraperitoneally with Ketamine at 100 mg/kg and Xylazine at 10 mg/kg. Peak serum ketamine level is achieved 10-20 min after the injection (Jung et al. 2019). Therefore, the mice were monitored within that time, until they were unconscious.

Statistical analysis
The statistical analysis was done using SPSS Statistics for Windows, version 16.0 (SPSS Inc., Chicago, Ill., USA). Data were presented as mean ± standard error of mean (mean ± SEM). The difference in mean among the PCV = Volume of erythrocytes in blood × 100 Total volume of blood

Acute toxicity test
The oral median lethal dose (LD 50 ) of methanol leaf extract of Piliostigma reticulatum in mice was estimated to be greater than 5000 mg/kg body weight.

Preliminary phytochemical screening
The preliminary phytochemical screening of methanol leaf extract of Piliostigma reticulatum revealed the presence of glycosides, saponins, tannins, flavonoids, triterpenes, and alkaloids.

Curative test
The methanol leaf extract of Piliostigma reticulatum produced a significant dose-dependent antiplasmodial effect, compared with the distilled water group (P < 0.001). At doses of 250 500 and 1000 mg/kg, the extract produced parasite clearance of 68.31%, 76.53% and 83.65%, respectively. The standard drugs chloroquine (10 mg/kg) and artesunate (5 mg/kg) produced parasite clearance of 89.09% and 91.13%, respectively. The mice in the extract-treated groups survived longer than those in the negative control group; those treated with MPR 1000 mg/kg survived for 19 days, while those in the negative control group survived for 5 days only. The mice in the chloroquine and artesunate-treated groups survived for 26 and 27 days, respectively (Table 1).

Prophylactic test
The Piliostigma reticulatum extract showed significant activity as compared with the distilled water group (P < 0.001). At the doses of 250 mg/kg, 500 mg/kg and 1000 mg/kg, the extract produced a chemoprophylactic effect of 68.5%, 58.70% and 84.77%. The standard drug Pyrimethamine at 1.2 mg/kg had a chemoprophylactic effect of 80.7% (Fig. 2).

Packed cell volume (PCV)
The Piliostigma reticulatum methanol extract produced no significant effect on the packed cell volume of the extract-treated mice on days 3 and 7 as compared with the distilled water group (i.e., P > 0.05). A similar result applies to the chloroquine-treated group. A statistical significance was however observed on day 7 amongst the artesunate-treated group (P < 0.05), which had a higher PCV compared with the other groups (Fig. 3).

Discussion
The LD 50 of Piliostigma reticulatum was found to be > 5000 mg/kg, this indicates that the plant is practically non-toxic and therefore safe to the mice (Lorke 1983;Aliyu et al. 2015). This result is similar to the findings of Dosso et al. (2012) and Dosso et al. (2014). Related results were also reported by Ajayi et al. (2019) and Fayanju et al. (2021).
The antiplasmodial activity of the plant could be due to the presence of pharmacologically active principles (glycosides, saponins, tannins, flavonoids, triterpenes, and alkaloids), identified in the plant. Our findings could be supported by earlier studies that reported that alkaloids, flavonoids, triterpenoids, glycosides and tannins possess antiplasmodial activities (Kirby et al. 1989;Phillipson and Wright 1991;Christensen and Kharazmi 2001;Saxena et al. 2003;Willcox and Bodeker 2004;Inbaneson et al. 2012). Further studies supporting our data that the phytochemicals present in Piliostigma reticulatum have antiplasmodial activity, includes; Ahmed et al.  Thakur and Kumari (2021).
The mechanism of antiplasmodial activity of this plant could be explained by the mode of action of the identified secondary metabolites. The plant contains alkaloids, which was reported to act by inhibiting heme transformation into hemozoin in the parasite food vacuole (as Table 1 The curative effect of methanol leaf extract of Piliostigma reticulatum against P. berghei in mice (n = 6) Results are expressed as mean ± SEM. n = 6 DW, distilled water; MPR, Piliostigma reticulatum methanol extract * Significantly different from control at P < 0.001 using One-way ANOVA and Dunnett's post hoc test  . Several alkaloids of different classes (Bisindole, indole, terpenoidal, quinolone, etc.) have been identified in medicinal herbs, to possess weak, moderate, and significant antiplasmodial activity (Uzor 2020). Generally, the terpenes (monoterpenes and sesquiterpenes e.g., artemisinins) exert their activity through heme cleavage of the endoperoxide bridge (Okokon et al. 2017). Also, the triterpenes act via inhibition of protein synthesis (Kirby et al. 1989;Inbaneson et al. 2012). Furthermore, the antimalarial actions of the plant could be due to the flavonoids. This bioactive metabolite interferes with protein biosynthesis and chelates the nucleic acid base pairing of the plasmodium parasite (Okokon et al. 2017;Abdussalam et al. 2018). Flavonoids also inhibit the formation of hemozoin from heme released by digestion of hemoglobin, generating free radicals which elicit the death of the parasites (Marliana et al. 2018). Therefore, the antiplasmodial activity of Piliostigma reticulatum extract could be due to the presence of phytochemicals acting singly or in combination to produce a lethal effect on the parasite.
The results of the antiplasmodial screening in this study showed that the methanol extract of Piliostigma reticulatum produced significant activity in an established plasmodial infection (curative model), suppressed early form of the plasmodial infection (suppressive model) and protected the mice from the plasmodial infection (prophylactic model).
The mean survival time (MST) is another relevant tool for measuring the activity of antiplasmodial agents. The literature consistently demonstrates that an agent that extends the survival of experimental animals beyond 12 days is considered an effective antiplasmodial agent (Peter and Anatoli 1998;Ural et al. 2014;Widyawaruyanti et al. 2017;Mulisa et al. 2018;Abdullahi et al. 2021). The extract of Piliostigma reticulatum achieved a mean survival time of up to 18.8 days, thus, confirming the plants antiplasmodial activity.
The packed cell volume (PCV) was evaluated to measure if the extract can prevent hemolysis in infected animals. Malaria infection is associated with lysis of erythrocytes which could result in anemia, especially in areas of high malaria transmission (Sumbele et al. 2015). Also, some antimalarials may affect the packed cell volume. A drug such as primaquine causes hemolysis (Braga et al. 2015), while therapy with artesunate results in post-artemisinin delayed hemolysis a few days after use (PADH) (Salehi et al. 2018). The case of PADH has been reported with artemether and oral artemisinins (White 2018). In this study, the PCV result showed no significant difference between the extract-treated groups and the control. The phytochemical saponin may be responsible for the reduction in red blood cells caused by the extract (evident from the PCV result). Saponins cause eryptosis by increasing calcium permeability into cells, causing cell shrinkage and cell membrane scrambling, thereby affecting the red blood cells (Bissinger et al. 2014).