Abstract
Medicinal plants produce a variety of chemical substances with varied physiological effects. They are a huge reservoir of various chemical substances with potential therapeutic properties. Allophylus spicatus is a shrub that belong to the Sapindaceae family. In this study, male albino wistar rats (18) were used for acute toxicity test. Animals were divided into six groups of three rats each. Group A served as the control group while the other groups were dosed orally with 200, 500, 1000, 2000 and 5000 mg/kg body weight of extract and were observed for 14 days. Swiss albino mice (42) were used for the antimalarial study; five groups of six infected mice per group (Groups C–G) were respectively dosed orally with 25 mg chloroquine/kg bw, 200 mg of extract/kg bw, 400 mg/kg bw of extract, 25 mg chl./kg bw + 200 mg/kg bw of extract and 25 mg chl./kg bw + 400 mg/kg bw extract with three groups serving as the control (Groups A–C) for three days. Acute toxicity test and histology analysis on the liver tissue confirmed the safety of the extract at concentrations less than 1000 mg/kg b/w. Antimalarial studies showed the highest activity in the group administered with 400 mg/kg + 25 mg chl./kg b/w. In conclusion, A. spicatus was non-toxic at doses less than 1000 mg/kg and significantly reduced parasitemia count in P. berghei infected mice, thus validating its folkloric usage.
Supplementary Information
The online version contains supplementary material available at 10.1007/s43188-020-00070-1.
Keywords: A. spicatus, Malaria, Antimalarial, Acute toxicity, Medicinal plants
Introduction
Malaria is a long-standing epidemic in tropical Africa [1]. Despite having been around since ancient times, the disease still ravages many parts of the world [2]. A whooping 219 million cases of malaria was reported across the globe by World Health Organization (WHO) in 2017 and 216 million in 2016 [3, 4]. The malaria incidence worldwide is estimated at 63 cases per 100,000 people at risk which is a decline from 76 in 2010. In 2017, fifteen countries in Sub-Saharan Africa along with India bore 80% of the disease burden worldwide, a total of 435,000 deaths was recorded worldwide [4]. Half of the world’s population is estimated to be at risk of contracting this life-threatening disease [2]. The recently released World malaria report for 2017 states that no significant progress is being made in reducing the malaria pandemic.
Malaria has a crippling effect on not just human health but also on the social and economic well-being of a nation by draining already scarce resources, reducing efficiency and increasing pain and misery [3]. The disease is spread primarily by Plasmodium falciparum, Plasmodium ovale, P. malariae and P. vivax [2]. The most prominent in tropical Africa is however P. falciparum. Artemisinin along with its derivatives discovered by Prof. Youyou Tu are the most effective antimalarial drug today. Woe betide this discovery in 2006 when studies by WHO found resistance to the drug in a clinical study. This steered the need for more effective and active compounds for disease management [5].
Approximately 92% of the total malaria cases is borne by the African continent. This continent also has the lowest investment in healthcare and the highest number of people living in extreme poverty. As a result of this, many of the residents have turned to herbal medicines for their cost effectiveness, efficacy and perceived superiority. The tropical climate of the continent which has made it a suitable space for malaria to breed has also blessed the continent with abundant vegetation with wide variety of plant species [5].
It has now been well established that medicinal plants have diverse bioactive constituents which have been used since the beginning of times to treat diverse disease conditions [6]. The current trend of multidrug resistance has revamped the search for new lead compounds from medicinal plants.
The plant Allophylus spicatus (Poir.) is of the Sapindaceae family. It is a shrub of about 4–12 ft in length. It grows commonly in the savannah, rocky areas and by the stream. A. spicatus is found mainly in West African countries such as Nigeria, Ghana and Ivory Coast, although the presence of the plant has been reported in South Africa. It is also listed in JSTOR as one of the useful plants in West Africa. It is a plant relatively new to the scientific community as not much of studies have been done on it. In folklore, the leaf of A. spicatus is used as painkillers, antimalarials, to relieve pulmonary troubles, treat diarrhea, dysentery and the fruit sap is used to cure cough and colds [7].
Although herbal plants are considered to be safe for consumption, they are not completely free from having adverse effect which makes toxicological evaluation important in accessing their safety limits [8]. Toxicological evaluations are widely used to assess the toxicity in animals which is then used as a guide to choose a safe dose in humans during preclinical and clinical trials. The aim of this study is to scientifically validate its antimalarial efficacy and also assess acute toxicity studies on the leaf extract of A. spicatus in animal models.
Materials and methods
Collection and identification of plants
A. spicatus leaf samples were collected from Agbegi-odofin, Osun State, Nigeria. Identification was done by Dr. J.O. Popoola, a certified botanist from Biological Science Department, Covenant University, Nigeria. Samples were prepared and deposited in the Herbarium at the Forest Research Institute of Nigeria (FRIN) with voucher no. AS/BIO/H808.
Preparation of leaf extract
The leaves (488.5 g) were processed using methods by Adebayo et al. [9]. Fresh leaves of A. spicatus was dried with ambient air at 25 °C for 8 weeks, after which they were pulverized using an electric blender into fine powder. Weighed amount (488.5 g) of leaves was extracted by cold maceration with 95% ethanol (1:4 w/v) for 72 h. The extract was filtered and concentrated at 50 °C using a rotary evaporator to give 51.8 g of extract and a percentage yield of 10.6%.
Phytochemical screening
Standard procedure was used to qualitatively determine the phytochemicals present in the leaf extract as described by Sofowara and Harborne [10, 11]. Phytochemicals screened for include; include tannins, saponin, anthocyanin and betacyanin, flavonoids, alkaloids, quinine, terpenoids, phenol, coumarins, carbohydrates, glycosids, cardiac glycosides, acids and steroids.
Acute toxicity studies
Animals
Male albino Wistar rats (18) with an average weight of 164 g and 42 albino mice with an average weight of 25 g were purchased from the University of Lagos, Idi-Araba, Nigeria and used for the study. Animals were accommodated in cages at the animal house of the Department of Biochemistry, Covenant University and maintained at a temperature of 25 °C with an alternating 12 h light and dark cycle. Experimental procedures were carried out in compliance with the guidelines for the care and use of laboratory animals documented by Covenant University Health Research and Ethics Committee (CHREC /016 /2019). They were also treated following the National Institutes of Health (NIH) guidelines for the usage and care of laboratory animals.
Toxicity tests
Acute toxicity studies were carried out according to OECD [12]. Albino Wistar rats (18) with average weight of 165 g and 10 weeks old were divided into six groups of three (3) each. The first group served as control while the other five groups were administered 200, 500, 1000, 2000 and 5000 mg/kg body weight of extract respectively (Table 1). They were given a single dose and observed for 14 days for adverse reactions (Tables 2, 3).
Table 1.
Grouping of animals for acute toxicity test
| GROUP | DOSAGE (mg/kg body weight) |
|---|---|
| Group A | Distilled water |
| Group B | 200 |
| Group C | 500 |
| Group D | 1000 |
| Group E | 2000 |
| Group F | 5000 |
Table 2.
Grouping of animals for antimalarial test
| Groups | Class | No. of mice | Treatment mode | Dosage |
|---|---|---|---|---|
| A | Uninfected + no treatment | 6 | None | |
| B | Negative control (parasitized) | 6 | Distilled water | 0.2 ml |
| C | Parasitized (positive control) | 6 | Chloroquine | 25 mg/ kg body weight/ day |
| D | Parasitized | 6 | Extract | 200 mg/kg body weight/ day |
| E | Parasitized | 6 | Extract | 400 mg/kg body weight/ day |
| F | Parasitized | 6 | Extract + chloroquine | 200 mg/kg body weight extract + 25 mg/kg body weight chloroquine |
| G | Parasitized | 6 | Extract + chloroquine | 400 mg/kg body weight extract + 25 mg/kg body weight chloroquine |
Table 3.
Phytochemical constituents of Ethanolic leaf extract
| Phytochemicals | Carbohydrates | Tannins | Saponin | Flavonoids | Alkaloids | Ant. & Beta | Quinine | Glycoside | C. Glycoside | Terpenoids | Phenol | Coumarins | Steroids | Acids |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Crude Extract | − | + | + | + | + | + | + | − | − | + | + | + | + | − |
In vivo antimalarial studies
Swiss albino mice (42) were used in the in vivo antimalarial assessment. Antimalarial studies were carried out according to the protocol described by Adebayo et al. [13] and Adebayo et al. [14] with modifications. The strain of Plasmodium berghei (NK-65) parasite was obtained from National Institute for Medical Research (NIMR), Lagos, Nigeria and sustained via constant intraperitoneal re-infestation of parasitized erythrocytes which were sourced from a donor-infected mouse by the tail via a heparinized syringe and made up to 20 ml with normal saline. The animals were inoculated with 0.2 mL of infected blood suspension having 1.0 × 107 parasitized erythrocytes intraperitoneally. On day 0, pre-treatment parasitemia was done and infected animals were then distributed randomly to groups of six (6) animals each. To determine post-treatment parasitemia, blood smears were made, films fixed in methanol and stained with Giemsa before being examined microscopically.
where A is the mean percentage parasitaemia in the negative control group and B is the mean percentage parasitaemia in the test group.
Statistical analysis
Data obtained was presented as mean ± Standard Error of Mean (SEM). Analysis of Variance was used for group comparisons using SPSS Version 25. Values with p < 0.05 were considered significant.
Results
Phytochemical assessment of A. spicatus
After qualitative assessment of the ethanolic extract, the leaves of A. spicatus was found to contain ten of the fourteen phytochemicals tested for. They include; tannins, saponin, anthocyanin and betacyanin, flavonoids, alkaloids, quinine, terpenoids, phenol, coumarins and steroids.
Acute toxicity test results
Acute toxicity studies of the leaf extract of A. spicatus resulted in no death even at the highest concentration of 5000 mg/kg body weight. Observation over the 14-day period showed increased appetite in experimental animals which resulted in an increased body weight (Fig. 1). Animals in groups D and E had a significant (p < 0.05) weight increase after the treatment period. The animals maintained normal daily activities and showed no sign of weakness or illness until the point of sacrifice. The histology of the liver (Plate 1) showed an increase in vascular congestion as the dosage increased. Although the extract had no visible effect on the animals’ health.
Fig. 1.
Body weight of experimental animals before and after administration of A. spicatus leaf extracts. Group A served as control with Group B, C, D, and E receiving 200, 500, 1000, 2000 and 5000 mg/kg body weight respectively. *Significant as compared with weight before treatment (p < 0.05)
Antimalarial test
Parasitemia suppression test
The parasitemia was done using the 3-day suppressive test method. Results showed that animals treated with 200 mg/kg body weight of the extract exhibited the highest suppression rate which is similar to the drug control group ( 25 mg/kg body weight of chloroquine) (Table 4, Fig. 2). The other treatment groups showed suppressive rates similar to that of chloroquine treated animals. Co-administration of the extract and- chloroquine were more efficacious when compared with single administration (Plate 2) (Figs. 3, 4).
Table 4.
Parasitemia count before and after administration of extract of A. spicatus in mice infected with Plasmodium berghei
| Groups | Pre-treatment Parasitemia Count | Post-treatment Parasitemia Count (Day 3) |
|---|---|---|
| Grp A (No treatment) | – | – |
| Grp B (0.2 ml distilled water) | 4.86 ± 0.17 | 2.66 ± 0.06b |
| Grp C (25 mg/ kg body weight/ day chl) | 5.09 ± 0.77 | 1.21 ± 0.02a |
| Grp D (200 mg/kg body weight/ day) | 5.62 ± 0.25 | 1.73 ± 0.16a |
| Grp E (400 mg/kg body weight/ day) | 5.04 ± 0.27 | 1.39 ± 0.09a |
| Grp F (200 mg/kg body weight extract + 25 mg/kg body weight chloroquine) | 5.47 ± 0.30 | 1.43 ± 0.12a |
| Grp G (400 mg/kg body weight extract + 25 mg/kg body weight chloroquine) | 5.43 ± 0.14 | 1.10 ± 0.04a |
Values are expressed as mean ± standard error of mean
aSignificant as compared with negative control (grp B) Significant as compared with positive control (grp C); p < 0.05
Fig. 2.
Photomicrograph of rat liver a control group showing normal liver (H and E, × 400), b group dosed with 200 mg/kg bw extract showing normal liver histology (H and E, × 400) c group dosed with 500 mg/kg bw extract showing normal histology with processing artifact (H and E, × 400) d group dosed with 1000 mg/kg bw extract showing congested blood vessels (H and E, × 400) e group dosed with 2000 mg/kg bw extract showing congested blood vessels (H and E, × 400) (f) group dosed with 5000 mg/kg bw extract showing severe vascular congestion (H and E, × 400). Black arrows indicate areas with liver damage
Fig. 3.
Percentage parasitemia suppression after administration of extract of A. spicatus in mice infected with Plasmodium berghei after treatment
Fig. 4.
Photomicrograph of mice liver tissues of mice a uninfected negative control showing normal liver (H and E × 400), b infected with P. berghei and treated with distilled water showing congested blood vessels (H and E × 400) c treated with 25 mg/kg bw chl. showing no liver abnormalities (H and E × 400) d treated with 200 mg/kg bw extract showing congested blood vessels (H and E × 400) e treated with 400 mg/kg bw extract showing mild vascular congestion (H and E × 400) f treated with 25 mg/kg chl + 200 mg/kg b/w extract showing congested blood vessels (H and E × 400) g treated with 25 mg/kg chl. + 400 mg/kg bw extract showing congested blood vessels (H and E × 400) Black arrows indicate areas with liver damage
Haematological study
Upon assessment of haematological parameters (Table 5), there was a significant (p < 0.05) increase in the White Blood cell (WBC) count of animals treated with 25 mg/kg bw chl. when compared with the negative control while the other groups showed no significant difference. Packed cell volume (PCV) of Groups D and G showed a significant (p < 0.05) increase while Group C showed a statistically significant (p < 0.05) decrease. Platelet count of Groups F and G showed a significant (p < 0.05) increase when compared with the negative control group. There was a statistically significant (p < 0.05) increase in lymphocyte count in Group E and a significant (p < 0.05) decrease in Group G when compared with the negative control group. The Mean Cell Volume (MCV) was statistically increased (p < 0.05) in Group E and reduced in Group F and G. Mean cell haemoglobin (MCH) of Groups F and G were significantly (p < 0.05) reduced when compared with negative control. Haemoglobin ( Hb) count and percentage mean corpuscular haemoglobin concentration (MCHC) showed no statistically significant (p > 0.05) change in the groups when compared with the negative control group.
Table 5.
Effect of extract of A. spicatus on hematological markers of mice infected with Plasmodium berghei
| Parameters | Grp A | Grp B | Grp C | Grp D | Grp E | Grp F | Grp G |
|---|---|---|---|---|---|---|---|
| WBC (× 103/L) | 4.07 ± 0.35 | 5.80 ± 0.76 | 8.57 ± 0.99a | 3.97 ± 0.90a | 6.47 ± 0.93 | 4.37 ± 0.87 | 5.20 ± 0.13 |
| Hb (g/dl) | 13.30 ± 0.32 | 12.23 ± 0.32 | 9.83 ± 1.12 | 14.27 ± 0.06 | 13.33 ± 1.79 | 13.57 ± 0.43 | 13.13 ± 1.19 |
| PCV (%) | 43.60 ± 3.10 | 39.27 ± 3.80 | 30.37 ± 2.50a | 45.13 ± 2.48a | 39.77 ± 5.21 | 33.00 ± 12.81a | 47.23 ± 4.18a |
| RBC (× 103/L) | 6.27 ± 2.55 | 5.53 ± 2.07 | 3.86 ± 0.42 | 6.38 ± 2.27 | 4.40 ± 1.12 | 7.46 ± 2.68 | 8.35 ± 1.52 |
| Platelet/L (104) | 52.10 ± 2.40 | 28.10 ± 4.23 | 36.07 ± 6.08 | 25.83 ± 1.39 | 23.83 ± 5.70 | 89.97 ± 3.29a | 99.33 ± 7.52a |
| Neutrophil (%) | 44.00 ± 1.53 | 49.67 ± 4.67 | 52.33 ± 6.17 | 41.00 ± 5.57 | 43.67 ± 2.33 | 40.33 ± 3.93 | 44.00 ± 4.93 |
| Lymphocyte (%) | 34.67 ± 13.53 | 31.00 ± 10.44 | 36.00 ± 5.50 | 38.00 ± 6.66 | 45.33 ± 6.66a | 34.67 ± 6.43 | 21.00 ± 1.73a |
| MCV (fl) | 74.37 ± 10.23 | 74.33 ± 7.74 | 78.63 ± 5.95 | 74.87 ± 9.72 | 90.87 ± 2.60a | 62.47 ± 11.52a | 56.83 ± 1.89a |
| MCH (pg) | 23.40 ± 4.81 | 23.80 ± 4.01 | 25.43 ± 2.66 | 24.07 ± 4.20 | 30.40 ± 0.75a | 20.00 ± 4.54a | 15.73 ± 0.37a |
| MCHC (g/dl) | 30.80 ± 2.51 | 31.57 ± .2.34 | 32.17 ± 1.13 | 31.77 ± 1.70 | 33.47 ± 0.14 | 31.67 ± 1.27 | 27.77 ± 0.24 |
Values represent mean ± SEM of three replicates
White blood cell count (WBC), haemoglobin (Hb), red blood cell count (RBC), mean cell haemoglobin (MCH), mean cell volume (MCV), percentage mean corpuscular haemoglobin concentration (MCHC)
Grp A (No treatment), Grp B (0.2 ml distilled water), Grp C (25 mg/ kg body weight/ day chl), Grp D (200 mg/kg body weight/ day), Grp E (400 mg/kg body weight/ day), Grp F (200 mg/kg body weight extract + 25 mg/kg body weight chloroquine), Grp G (400 mg/kg body weight extract + 25 mg/kg body weight chloroquine)
a p < 0.05 compared with negative control
Histopathological studies
Histology results (Plate 2) showed that the group treated with 25 mg/kg body weight of chloroquine has normal liver architecture. Other groups however showed mild (Group E, Plate 2) to severe vascular congestions (Groups B, D, F & G).
Discussion
The fight to develop an efficacious, non-toxic, resistant proof antimalarial is still on till today. Ever since the discovery of quinine in the eighteenth century, many other drugs have since been developed. These discoveries have however been faced with the issue of resistance. As a result of this, there is a need to keep discovering innovative ways of fighting this old but ever-changing parasite. The WHO currently lists 14 drugs for treating malaria. Of the 14, artemisinin combination therapy (ACT) is the most efficacious. This combination therapy was however hit with the problem of resistance in 2001 [15]
The phytochemical result of A. spicatus showed that the leaves contain diverse phytochemicals such as tannins, phenols, terpenoids, flavonoids, anthocyanins, glycosides, alkaloids etc. The presence of these secondary metabolites has been linked to the medicinal activities exerted by plants [16]. These secondary metabolites contribute significantly to biological activities of medicinal plants such as antimicrobial, antioxidant, antimalarial etc. [17]. A. spicatus was found to contain phytochemical groups which have previously been reported to have antimalarial properties [18, 19]
Toxicity tests are important for many reasons. Apart from helping to know how safe a substance is for use, they also help to establish a dose response curve, establish side effect of substances, validate new methods of testing, and explain the mechanism of toxicity of substances amongst others [20]. Before an extract can be scientifically validated for use, it is therefore important for toxicity tests to be carried out. The acute toxicity test involves a single dose of the crude extract at specific concentration followed by observation over a 14-day period [12]. During this period, animals treated with the extract showed increased food consumption, less physical activity. No form of convulsion, tremor or itching was observed during these 14 days. From the results, there was a significant increase in the animals’ weights before and after administration in groups B, D, E, and F (Fig. 1). This may be due to an increase in appetite of animals exposed to a higher concentration of the extract [21]. The increase in weight suggests that the plant extract was not toxic enough to impair normal body function and allows normal metabolic processes go on as normal [21].
Histological findings however showed signs of vascular congestion on animals administered 2000 and 5000 mg/kg body weight (Plate 1). The derangement observed may be due to the higher concentration of the extract administered to these groups of animals. A chronic state of vascular congestion in the liver could progress to hepatic injury as a result of decreased hepatic blood flow, decreased arterial oxygen saturation, or increased hepatic venous pressures [22]. High doses of the plant extract may therefore be unsafe for chronic consumption.
Suppressive antimalarial test was carried out during this study. Animals were induced with malaria parasites using Plasmodium berghei and treated for 3 days. After the treatment period, the extract showed varying suppressive capacity at different concentrations with 400 mg/kg + 25 mg/kg bw of chloroquine being the most effective treatment regimen (Table 4, Fig. 2). Other treatment groups (200, 400, 200 mg/kg bw + 25 mg/kg bw chl.) showed similar effectiveness as the chloroquine standard. There was also a higher survival rate amongst the group treated with 400 mg/kg bw + 25 mg/kg bw chl. after the 3-day treatment period.
Our findings from the suppressive experiment is very promising and means that upon testing for curative ability, the plant could also give high parasitemia suppression. This is similar to results obtained by Maje et al. [23] on the ethanolic leaf extract of Paullinia pinnata a member of the Sapindaceae family which showed good activity against P. berghei in mice. The activity was not dose-dependent and at increased concentration, some toxicity was also observed. Animals treated with 50 mg/kg of the extract had a reduced survival day when compared with animals treated with lower doses.
Haematological parameters assessment showed a significant increase (Table 5) in the total white blood cell count of infected animals in the chloroquine treated animals (Grp C), while others had no significant change. There was also an increase although not significant in haemogblobin levels amongst treatment groups. This is in line with research conducted by [24] where they assessed the feasibility of using haematological parameters in predicting malaria infection. The study showed that people infected with malaria parasite are likely to experience decreased haemoglobin, leukocyte and platelet counts. They also concluded from their study that people with this haematological profile were more likely to be infected with malaria.
The value of haemoglobin (Hb), an intracellular protein in the red blood cells, usually decrease in malaria patients due to the fact that they are consumed by growing malaria parasites [25]; the value of Hb was significantly reduced in the chloroquine treated group while others remained the same when compared with the untreated group (Table 5).
Packed cell volume (PCV) is also used to assess the anaemic state of malaria patients. Consequently, in this study, PCV was measured to evaluate the efficacy of chloroquine and the extract in preventing haemolysis as a result of increased parasitemia. The group administered with 400 mg/kg bw of extract (Grp D) and 400 mg/kg bw extract + 25 mg/kg bw Chl.(Grp G) had their PCV significantly increased while Grp C (25 mg/kg bw) had significantly reduced PCV. The increase in PCV level across all treatment groups may be linked to the anti-haemolytic effect of phytochemicals present in the leaf extract [26]. White blood cell (WBC) helps to fight diseases and are thus involved in the body’s immune system [27]. The result from this study shows a significant increase of WBC in the negative control group when compared with the normal control and the reduction of WBC across all treatment groups (Table 5). Significant increase in WBC level has been reported to be associated with severe malaria state [28].
A. spicatus leaves were found to be non-toxic at concentrations less than 1,000 mg/kg body weight. It was also discovered from the acute toxicity studies that the plant may induce weight gain. Our findings from the antimalarial suppressive tests showed promising results as the suppression rates of the parasites by the extract of A. spicatus were similar to that of mice administered with chloroquine and this has validated its folkloric usage.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We acknowledge the effort of the technical staff of the Department of Biochemistry for the laboratory support. We also thank Covenant University Center for Research Innovation and Development (CUCRID) for funding the publication of this paper.
Compliance with ethical standards
Conflict of interest
The authors have no conflict of interest to disclose.
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