Abstract: Background and Objective: Overcome the resistance of malaria parasites to most orthodox drugs requires the exploration of different drug sources for new compounds or powerful natural substances against the Plasmodium parasite. The current study was designed to investigate antiplasmodial, antioxidant properties and toxicological profile of Costus afer Ker Gawl, a medicinal plant widely used traditionally in Bénin Republic to treat malaria. Materials and Methods: Crude ethanolic and aqueous extracts were tested in vitro against both field isolates and chloroquine sensitive 3D7 strains of P. falciparum using Plasmodium lactate dehydrogenase (pLDH) assay. Hemoglobin release was measured spectrophotometrically to assess hemolytic power. Antioxidant activity of extracts was assessed in vitro by DPPH radical-scavenging, reducing power (FRAP), superoxide radical scavenging and Hydrogen peroxide scavenging assays. Acute oral toxicity of extracts was evaluated according to OECD 423 guideline. Results: Ethanolic extract exhibited very good antiplasmodial activity against both strains with IC50 values of 3.19±0.13 μg mL–1 on field isolates and 13.68±1.41 μg mL–1 on 3D7. The aqueous extract showed moderate inhibition (<50%) on both strains as well as the fractions obtained from fractionation of ethanolic extract at single dose of 100 μg mL–1. Both extracts exhibited dose-dependent antioxidant activity. Both extracts presented hemolytic power less than 3% with a LD50 greater than 2000 mg kg–1 of b.wt. There was no significant change on biochemical and hematological parameters. Conclusion: The study demonstrated the strongest antiplasmodial and antioxidant properties of Costus afer as well as an absence of acute oral toxicity. It could be used as an alternative in the management of malaria and oxidative diseases through a standard formulation.
INTRODUCTION
Historically, medicinal plants constitute an important source of satisfaction for the primary health care needs of populations, particularly in the developing regions of the world. According to the WHO, up to 80% of the population in some developing countries still continues to rely on traditional medicine for treatment of diseases1. The increasing use of medicinal plants is supported by broadly accepted beliefs and practices. To this can be added the difficulties related to the low income of these populations and the accessibility of conventional drugs. Furthermore, the discovery of quinine and artemisinin, the most worldwide used antimalarial drugs, is the best proof that medicinal plants represent a potential source of bioactive compounds, thus enhancing the belief of the population. However, it has been reported that traditional medical systems could not guarantee the efficacy of herbal medicines on the basis of traditions or supposed beliefs2. A thorough investigation of the pharmacological quality and safety issues guarantees a better knowledge of these plants.
Costus afer (Costaceae) is pantropical species encountered in Africa in the forest belt from Senegal to Ethiopia and in the East to Tanzania, Malawi and Angola, in the South and in West Africa3. Called tétégoungoun (fon, goun: Benin Republic) and tétérégoun (yoruba), It’s commonly crossed from southern to northern of Benin4. According to ethnobotanical surveys, C. afer is one of the commonly used medicinal plants in folk medicine to treat malaria in Benin5–7, in Tanzania8 and in Nigeria9. Moreover, it is also used to treat gastralgia, tuberculosis and azoospermia in Benin10, cough and rubella in Nigeria9. Previous research have reported anti-inflammatory11, hepato-protector12,13 and hypoglycaemic effect12 and antioxidant activity14–17 of C. afer.
Despite several ethnobotanicals reports on considerable use of C. afer in treatment of malaria, no scientific data was available to validate its antimalarial properties and safety. The present study was designed to assess phytochemicals, acute toxicity and in vitro antiplasmodial and antioxidant activities of this medicinal plant used in Bénin Republic.
MATERIALS AND METHODS
Plant material and extracts preparation: The present study was carried out for 18 months from May, 2015 in the laboratories of University of Abomey-Calavi, Bénin. Aerial parts of Costus afer were collected in their natural habitats in south Benin in April, 2015. Identification and authentication were done by National Herbarium, University of Abomey-Calavi, Bénin and voucher specimens (YH 354/HNB) were deposited at the same institute for further references.
The plant material collected was dried for two weeks in laboratory (22°C) and ground to a fine powder using an electric grinder (LONG YUE LY-989, China). The aqueous extract was obtained by boiling of 150 g of powder in water (1.5 L) at 100°C for 30 min. The mixture was filtered with Whatman paper No. 1 and concentrated using rotary evaporator coupled with vacuum pump (BUCHI Rotavapor RII, Switzerland; Vacuubrand PC101NT, Germany) at 60°C. Ethanolic extract was obtained by maceration of 500 g of powder in ethanol 76% (ratio 1:10, powder: ethanol) overnight under circular agitation at 200 rpm with rotary agitator (IKA-Werke KS 260, Germany). After three washing, the filtrate was concentrated by same way as aqueous extract. Dried crude ethanolic extract was subjected to liquid-liquid partition using three solvent systems of varying polarity in sequential order. 2 g of ethanolic extract were dissolved in 100 mL of distilled water/ethanol (80:20, v/v) and extracted successively with hexane, dichloromethane and ethyl acetate using a separating funnel. The hexane (F-Hex), dichloromethane (F-DCM), ethyl acetate (F-AcOEt) and the last remaining aqueous (F-aq) fractions were concentrated using the rotary evaporator. All extracts were stored at 4°C.
Phytochemical screening: The both crude extracts were tested for the presence of alkaloids, flavonoids, triterpenes, tannins, coumarins, anthraquinone, saponins, lignanes, essential oils and anthocyanins by time layer chromatography (TLC) and colorimetric test using respectively a standard protocol of Wagner and Bladt18 and Shah and Hossain19.
Estimation of total phenolic content: Total phenolic content (TPC) was estimated by colorimetric method using Folin-ciocalteu reagent20. Briefly, 200 μL of sample (100 μg mL–1) were added to 1 mL of Folin-ciocalteu reagent (10%). After 4 min, 800 mL of saturated sodium carbonate (75 g L–1) were added. The mixture was incubated for 2 h at room temperature and the absorbance was measured at 765 nm. Standard curve for estimation of TPC was plotted with gallic acid. The essays were performed in triplicate and the results were expressed as mg of gallic acid equivalents GAE g–1 of extract.
Estimation of total flavonoid content: Total flavonoid content was measured by the aluminum chloride colorimetric assay21. About 250 μL of extract at 100 μg mL–1 were mixed with 750 μL of ethanol (96%), 50 μL of potassium acetate (1M), 50 μL of aluminium chloride (10%) and 1400 μL of distilled water. After 30 min of incubation at room temperature, the absorbance was measured at 450 nm. Quercetin was used to plot standard calibration curve and total flavonoid content was expressed as mg of quercetin equivalent (QE)/g of extract.
Antioxidant assays
DPPH radical scavenging activity assay: DPPH radical (2,2-diphenyl-1 picrylhydrazyl), scavenging activity of extracts was assessed according to the method previously described by Amoussa et al.20 with slight modification. Briefly, 1.5 mL of a freshly prepared methanolic solution of DPPH (0.04%) was mixed with 0.75 mL of extract solution diluted in methanol from 300-2.34 μg mL–1. After 20 min of incubation in dark at room temperature, absorbencies were read at 517 nm. Methanolic DPPH solution constitutes a blank and the assays were replicated three times. The DPPH radical scavenging percentage was determinate according to the formula:
| (1) |
where, Ab is absorbance of the blank and As is absorbance of the test sample.
The percentage of inhibition was plotted as a function of extracts concentration and curve fitting was obtained by nonlinear regression analysis. The IC50 value was extrapolated as the concentration that induced 50% inhibition of DPPH.
Potassium ferricyanide reducing antioxidant power (FRAP) assay: The ferric reducing capacity of extracts was evaluated following the potassium ferricyanide-ferric chloride method21. The reaction mixture was constituted with 2 mL of extract (100 μg mL–1), 2 mL of phosphate buffer (0.2 M, pH 6.6) and 2 mL of potassium ferricyanide (10 mg mL–1). After 20 min of incubation at 50°C, the reaction was stopped with 2 mL of trichloroacetic acid (100 mg L–1). The mixture was then centrifuged at 3000 rpm for 10 min. About 2 mL of the upper layer were mixed with 2 mL of distilled water and 0.4 mL of fresh ferric chloride (0.1%, w/v). After 10 min, the absorbances were read at 700 nm. Ascorbic acid was used to produce the calibration curve (y = 0.0069x+0.015, R2 = 0.9625). The iron (III) reducing activity assay was performed in triplicate and expressed in mmol of ascorbic acid equivalent (AAE) g–1 of extract.
Superoxide radical scavenging assay: This test consists to measure the capacity of extract to quench superoxide radical generated by alkaline DMSO22. Superoxide radical scavenging activity of extract is proportional to nitro blue tetrazolium (NBT) reduction by superoxide into formazan dye at room temperature and can be measured around 630 nm. In microplate 96 wells, the reaction mixture was constituted by 50 μL of extract (100 μg mL–1 in DMSO) and 170 μL of alkaline DMSO (1 mL DMSO, 100 μL NaOH 5 mM) and 30 μL of NBT (1 mg mL–1 in DMSO). The mixture was incubated for 5 min at room temperature and the absorbance was measured at 630 nm using microplate Reader (Rayto R 6500, China). Quercetin was used as a reference. The percentage of superoxide quenching (PI) of extracts was determinate using the following formula:
| (2) |
where, A0 is absorbance of the blank constituted by alkaline DMSO with NBT and A1 is absorbance of the tested sample.
Hydrogen peroxide radical scavenging assay: The ability of extract to scavenge hydrogen peroxide was determined according to the method of Ruch et al.23 with slight modification. 0.6 mL of hydrogen peroxide solution in phosphate buffer (0.1 mM, pH 7.4) was mixed with 0.5 mL of extract (100 μg mL–1). The mixture was incubated for 10 min at room temperature. All tests were performed in triplicate. The absorbances were determined spectrophotometrically at 295 nm against a blank containing the phosphate buffer without hydrogen peroxide and control constituted by hydrogen peroxide in phosphate buffer. The percentage of hydrogen peroxide scavenging of extracts and gallic acid (standard) was calculated using the following formula:
| (3) |
where, Ac is absorbance of control and As is absorbance of sample or standard.
Antiplasmodial activity: The plant crude extracts and fractions were evaluated in vitro for their antiplasmodial activity against a field isolates and chloroquine-sensitive (3D7) strains of Plasmodium falciparum obtained respectively from Laboratory of Infectious Vector Borne Diseases (LIVBD), IRSP/UAC, Benin and Institut Pluridisciplinaire Hubert Curien, UMR 7178-CNRS/Unistra, France. Parasites were maintained on culture in fresh O+ human erythrocytes in malaria complete medium (RPMI 1640 with 0.8% AlbumAX II, 25 mM HEPES, 1 mM L-glutamine, 0.4 mM hypoxanthine and 0.05 mg mL–1 gentamicin) according to the modified method of Trager and Jensen24. The cultures were maintained in a standard gas mixture (1% O2, 3% CO2, 96% N2) at 37°C. Parasitaemia was maintained daily between 1 and 5% using smears colored with Giemsa which are visualized on microscope (Optika Microscopes DM-25, Italy). Plasmodium falciparum sensitivity to extracts and fractions was carried out in 96-well plates as described by Desjardins et al.25 with slight modification. Crude extracts and fractions were preliminary tested at single concentration of 100 μg mL–1 and only active extracts and fractions were submitted to dose-response assay (concentration ranging from 100-0.78 μg mL–1 by two-fold dilution) to determine the 50% inhibitory concentration (IC50). In triplicate by concentration in 96-well plates, 100 μL of parasite suspension (1% parasitaemia, hematocrit, 4%) were mixed with 100 μL of each concentration of extracts and fractions previously dissolved in complete medium and the plate was incubated for 96 h at 37°C in P. falciparum culture conditions. Parasites viability was assessed by immune-dosage of Plasmodium lactate dehydrogenase (pLDH) using ELISA malaria antibody test kit (ApDia, Belgium) according to manufacturer protocol. The concentrations of pLDH in the tests well were measured at 450 nm with microplate reader (Rayto R 6500, China) against a positive control (parasite suspension only) and negative control (red blood cells only). The positive and negative controls of the kits were also plotted to access efficacy of test following the recommendations of the manufacturer. The percentage of parasite growth inhibition (PI) was extrapolated using the following formula:
| (4) |
where, ODs is the optical density generated by sample, ODnc is the optical density generated by negative control and ODpc is the optical density generated by positive control.
The PI was plotted as a function of extract or fraction concentration and the IC50 was estimated by nonlinear regression analysis. Chloroquine diphosphate and artesunate were used as antimalarial drug.
Toxicity assays
Hemolytic power essay: Hemolytic power assay was performed to assess toxicity effects of extracts and fractions on human red blood cells. The test was assessed following the method used by Sarr et al.26 with slight modification. Briefly, 100 μL of extracts or fractions dissolved in RPMI (ranging from 200-1.56 μg mL–1 by two-fold dilution) were mixed with 100 μL of non-infected erythrocytes (4% hematocrit) in 96-well plate. The mixture was incubated at 37°C under circular agitation. After 1 h, the plate was centrifuged at 2000 rpm for 5 min and 150 μL of supernatant was transferred into new 96-well plate. Haemoglobin content in the supernatants was determined by absorbance measurements at 450 nm in microplate reader. The 100% hemolysis (positive control) was obtained with 5% sodium dodecyl sulfate (SDS) and non-infected erythrocytes suspension was used as the blank. Hemolysis percentage was expressed according to the formula:
| (5) |
where, As is absorbance of the sample, Ab is absorbance of the blank and Ac is absorbance of positive control.
Acute oral toxicity test: The acute toxicity of ethanolic extract was carried out using27 Organization for Economic Cooperation and Development (OECD) guidelines N°423. Healthy, nulliparous and non-pregnant females Albinos Wistar rats aged 10-12 weeks (180-200 g body weight) were used. Animals were kept in animal house and maintained under laboratory conditions (22-25°C, 12 h light/dark cycle) with diets and water available ad libitum. A total of 6 rats divided in 2 groups of 3 animals each were ranged in different cage during experiments. The animals were deprived of food over-night before administration of extract. The control group (group 1) has received the vehicle (distilled water) and second group has received plant extract at a single dose of 2000 mg kg–1 b.wt. The rats were observed in detail during the first 8 h and daily for a period of 14 days. The monitoring was based on general toxicity signs, behavior changes, body weight evolution (days 0, 7 and 14) and mortality. The animals were anesthetized with ether on day 15 after an over-night food privation and blood samples were collected into tubes with EDTA and tubes without EDTA respectively for hematological and biochemical analysis.
Hematological analyzes were carried out using an automated hematology analyzer (Sysmex XP-300, Japan). Parameters as hematocrit (HCT), haemoglobin (Hgb), Mean corpuscular haemoglobin concentration (MCHC), red blood count (RBC), leukocytes formula (lymphocytes), mean corpuscular volume differential (MCV), mean corpuscular haemoglobin (MCH), platelet count (PLT), white blood cells count (WBC) were examined. Biochemical parameters including blood glucose, blood urea, creatinine (Crea), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed using clinical chemistry analyzer (CHEM-7 ERBA diagnostics mannheim Gmbh, Germany).
Statistical analysis: All data were presented as Mean±SD. The IC50 values were estimated by nonlinear regression analysis using Graphpad prism version 8.0.2 (GraphPad Prism software Inc., San Diego CA).The graphical representations of the data were performed using Microsoft Excel 2010 and Graphpad prism version 8.0.2. The difference was considered statistically significant when p<0.05.
RESULTS
Phytochemical constituents: Preliminary phytochemical analysis of extracts of C. afer revealed the presence of various phytoconstituents such as flavonoids, anthraquinones, coumarins, triterpenes and tannins. Alkaloids, saponins, anthocyanins, lignans and essential oil were not detected in both extracts as well as tannins in aqueous extract (Table 1). Total phenolic content (TPC) and total flavonoid content (TFC) were presented in Fig. 1. Ethanolic extract showed the highest TPC (10.51±0.16 mg GAE g–1 of extract) compared to aqueous extract (8.07±0.07 mg GAE g–1 of extract) with significant different (p<0.05). However, both extracts presented similar amount of TFC with respective values of 20.17±0.36 and 20.38±2.59 mg QE g–1 of extract.
Antioxidant activity: Antioxidant activities of C. afer ethanolic and aqueous extracts assessed in vitro against DPPH free radical, iron III, superoxide anion and hydrogen peroxide anion are presented in Fig. 2.
| Fig. 1: | Total phenolic (TPC) and flavonoids (TFC) contents crude extracts of C. afer |
| *Statistically significant at p<0.05, each value represents a Mean±SD (n = 3) | |
| Table 1: | Phytochemical contents of Costus afer crude extracts |
| +: Presence, -: Absence | |
The results showed that DPPH radical scavenging activity of both extracts was dose-dependent (Fig. 2a). Additionally, the both extracts exhibited interesting DPPH radical scavenging activity with respective IC50 values (p>0.05) of 181.90±26.99 and 159.60±15.62 μg mL–1. The ferric reducing antioxidant capacity of both extract was respectively 1272.88±50.29 and 1294.83±34.26 μM AAE g–1 of extract (p>0.05) indicating strong reducing capacity of extracts when compared to Ascorbic acid (1004.24±62.03 μM AAE g–1) with significant difference (p<0.05) (Fig. 2b). According to superoxide method, it was observed that extracts quench superoxide anion more than 90% at 100 μg mL–1 where quercetin exhibited 83.58% (Fig. 2c). Thus, the both extracts possess strong superoxide anion scavenging capacity significantly different from quercetin (p<0.05). At the same concentration, both extracts exhibited moderate hydrogen peroxide quenching capacity (inhibition <60%) when compared to gallic acid (inhibition >70%) with significant difference (p<0.05) on the one hand between both extracts and on the other hand between extracts and quercetin (Fig. 2d).
| Fig. 2(a-d): | Antioxidant activity of crude ethanolic and aqueous extracts of C. afer, in vitro free radicals scavenging capacity of both extracts obtained against (a) DPPH radical, (b) Iron III, (c) Superoxide anion radical and (d) Hydrogen peroxide |
Each value represents mean for 3 experiments±SD (standard deviation), *Statistically significant difference (p<0.05), Ascorbic acid, quercetin and gallic acid were used as reference compounds, Et-ex: Ethanolic extract, Aq-ex: Aqueous extract |
|
Antiplasmodial activity: Crude ethanolic and aqueous extracts of aerial part of C. afer and fractions obtained by bioassay-guided fractionation were tested in vitro on asexual stage of both field isolates and chloroquine sensitive (3D7) strains of P. falciparum using pLDH assay (Fig. 3). At single dose of 100 μg mL–1, the crude ethanolic extract (Et-ex) showed the highest activity with a parasites growth inhibition more than 60% on 3D7 strain and 80% against field isolates. The aqueous extract exhibited moderate inhibition (<50%) on both strains (Fig. 3a). Additionally, the Et-ex showed interesting dose-response-inhibition effect on both strains and mainly on field isolates with IC50 values of 3.19±0.13 μg mL–1 and 13.68±1.41 for 3D7 strain (Fig. 3b). However, among the fractions obtained from partition of Et-ex, ethyl acetate fraction (F-AcOEt) exhibited moderate activity against both strains (40%<inhibition<50%) while the others inhibited at less than 40% at the single dose (Fig. 3c). The chloroquine and artesunate used as control drugs are very active on chloroquine sensitive (3D7) and field isolates strains with respective IC50 values of 1.87±0.32 and 2.57±0.38 μg mL–1. However, the field isolates is less sensitive to chloroquine with an IC50 value of 19.85±2.71 μg mL–1 (Fig. 3b).
Toxicity effect
Hemolytic power: The cytotoxic effect of C. afer crude extracts assessed on human red blood cells is shown in Fig. 4. It has been observed that both extracts possess very low hemolytic power less than 3%. This indicates non cytotoxic effect of both extract against human red blood cells.
Acute oral toxicity: The acute oral toxicity effect of C. afer ethanolic extract assessed at single dose of 2000 mg kg–1 b.wt., caused no death in rats.
| Fig. 3(a-c): | Antiplasmodial activity of C. afer against chloroquine sensitive (3D7) and field isolates strains (Iso) of P. falciparum, (a) In vitro inhibition of parasites by the crude extracts at single dose, (b) IC50 values of crude extracts and control drugs (artesunate and chloroquine) and (c) In vitro inhibition of parasites by the fractions at single dose |
Data represent means for three experiments±SD (standard deviation), Artesunate and chloroquine diphosphate are positive drug control, *Statistically significant difference (p<0.05), Et-ex: Ethanolic extract, Aq-ex: Aqueous extract, F-Hex: Fraction hexane, F-DCM: Fraction dichloromethane, F-AcOEt: Fraction ethyl acetate, F-Aq: Aqueous residual fraction |
|
| Fig. 4: | Hemolytic activity of crude ethanolic and aqueous extracts of C. afer |
Data represent means for 3 experiments±SD (standard deviation), Et-ex: Ethanolic extract, Aq-ex: Aqueous extract |
|
The rigorous monitoring of behavior (respiration, food consumption, contortion and coma) of the rats showed no toxicity signs during the experiment period. The body weight of treated animals in comparison to control showed progressive increasing without significant change (p>0.05) (Fig. 5).
Among the hematological parameters analyzed, only platelet count of treated group decrease significantly (p<0.05) when compared to control group. No significant difference was observed between the others hematological parameters of treated and control groups (Table 2). Similarly, C. afer ethanolic extract not induces significant changes in biochemical markers of liver and renal functions as well as serum glucose of tested compared to control rats (Table 2).
| Fig. 5: | Effect of C. afer crude ethanolic extract on body weight of Wistar rats |
Data represent means for 3 experiments±SD (standard deviation), p<0.05 indicates statistically significant difference, J1: Day 1, J7: Day 7, J14: Day 14 |
|
| Table 2: | Effect of the extract on biochemical and hematological parameters |
EE-CA: Ethanolic extract of Costus afer, WBC: White blood cells, ALAT: Alanine aminotransferase, ASAT: Aspartate transaminase, MCV: Mean corpuscular volume, MCH: Mean corpuscular haemoglobin, MCHC: Mean corpuscular hemoglobin concentration, *p<0.05 indicate statistically significant difference when compared to control, each value represents mean for 3 experiments±SD (n = 3) | |
DISCUSSION
The use of medicinal plants in the treatment of malaria is a very old practice which for decades has served as a track record for the search for new anti-malarial drugs. In this study, in vitro antiplasmodial potential of C. afer against field isolates and chloroquine sensitive 3D7 strains of P. falciparum have been investigated. According to the appreciation grid of natural substances28,29, C. afer ethanolic extract is highly active on field isolates strain and active on 3D7 strain while aqueous extract is moderately active against both strains. The difference in activity could be explained by the variability of their secondary metabolites which depends on the extraction solvent. Otherwise, the fractions obtained from the liquid-liquid partition of the ethanolic extract are weakly active on both strains. This result indicated that the strong antiplasmodial activity demonstrated by the ethanolic extract could result from the synergistic action of secondary metabolites. The qualitative analysis of secondary metabolites of both extracts revealed the presence of diverse phytoconstituent groups that are known to have antiplasmodial activity including flavonoids30, tannins, coumarins31, triterpenes32 and anthraquinones33. These groups of secondary metabolites are also known to act either alone or in synergy against P. falciparum30. The interesting antiplasmodial activity observed with ethanolic extract in comparison to the aqueous extract could be attributed to the presence of tannins that are absent in aqueous extract. The same extract presented the high amount of phenolic compounds which is also associated with the antiplasmodial activity of extract34. Although no antiplasmodial activity report of C. afer exists in the literature, interesting antimalarial activity has been reported with related species, Costus Lucanusianus extract35.
Malaria infection has been associated with the production of reactive oxygen and nitrogen species, responsible for the oxidative stress observed in the pathophysiology of the disease36. Antioxidants, mainly from natural sources, are important for minimizing or reversing the oxidative damage that may occur during malaria. Moreover, antioxidants can block the conversion of free heme to hemozoin, thus making the host environment toxic to the survival of the parasite37. For this purpose, the antioxidant potential was evaluated from both crude extracts of C. afer using different in vitro antioxidant models. The results showed that both extracts have interesting antioxidant potential and can act as scavengers, electron or hydrogen donors, or as reducing agents38. These antioxidant capacities could be attributed to phenolic compounds, mainly the flavonoids present in these extracts39. Costus afer, through his antioxidant activity, could be useful in the management of malaria mainly oxidative damages and red blood cell lysis induced by the infection. These findings are supported by reports of several studies15-17,40.
Red blood cells membrane is a delicate structure that can be easily altered by plant extract41 resulting in erythrocyte rupture. Thus, hemolytic power of extracts was assessed to know whether they have cytotoxic effect on red blood cells. The results showed that C. afer ethanolic and aqueous extracts do not have hemolytic effect on human red blood cells. These results confirmed that the antiplasmodial activity observed with the extracts is induced by the direct effect of secondary metabolites on P. falciparum.
In the acute oral toxicity assessed with ethanolic extract (most active extract against strains of P. falciparum), LD50 is above 2000 mg kg–1 b.wt., as indicated in the OECD Guidelines. The absence of changes in behavior, of an overt sign of distress, of death and the normal increase of body weight in the test animals during the experimentation period is good evidence for lack of acute toxicity as set by Institute for Laboratory Animal Research (ILAR)42. Biological parameters, mainly serum biomarkers that provide information on the health status of vital organs including the liver (ALT and AST)43 and kidneys (creatinine and urea)44 and serum glucose were also investigated in this study. The results revealed no statistically significant change in these biological parameters indicating that C. afer ethanolic extract does not induces alteration of renal and liver functions as well as glycaemia. Hypoglycaemic and organ protective properties of C. afer have been also reported12,13. Moreover, no statistically significant variation between hematological parameters of group treated with C. afer ethanolic extract and control group (Table 2) indicated normal physiological condition of treated animals.
CONCLUSION
The present study demonstrated the greatest antiplasmodial activity of ethanolic extract of Costus afer against field isolates and chloroquine-sensitive (3D7) strains of P. falciparum with moderate activity exhibited by aqueous extract as well as fractions from the ethanolic extract. This suggested a probable synergetic action provided by phytoconstituents observed in ethanolic extract. Strong antioxidant activity as well as the lack of toxicity was found with this extract. However, further in vivo antiplasmodial investigations in order to better interpret some physiological factors and immune responses in the use of Costus afer need to be carried out.
SIGNIFICANCE STATEMENT
This study discovers the possible synergistic effect ofsecondary metabolites ofCostus aferethanolic extract, acombination that can be beneficial forthe management of malaria as well as oxidative damages resulting from the pathophysiologyof the disease. This study will help the researcher to uncover the critical area of Plasmodium parasites resistance and malaria transmission-blockingthat many researchers were not able to explore.
ACKNOWLEDGMENTS
The authors thank Professor Hounanpkon yedomonhan, Head of National Herbarium of Benin, University of Abomey-Calavi, for helping us to identify this species. We also thank Professor Anatole LALEYE, Head of (Training and Research Unit in Human Biology, Faculty of Health Sciences, University of Abomey-Calavi, Benin), for their collaboration for the assessment of the acute toxicity.