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ORIGINAL ARTICLE
Year : 2021  |  Volume : 11  |  Issue : 1  |  Page : 31-37  

In vitro antiplasmodial activity of Phyllanthus amarus against Plasmodium falciparum and evaluation of its acute toxicity effect in mouse model


1 Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, Bayero University, Kano, Nigeria
2 Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, Bayero University, Kano; Department of Medical Laboratory Science, Faculty of Allied Health Science, College of Medical Science, Ahmadu Bello University, Zaria, Nigeria
3 Department of Community Medicine, Faculty of Clinical Sciences, Bayero University, Kano, Nigeria

Date of Submission15-Jul-2020
Date of Decision23-Nov-2020
Date of Acceptance01-Dec-2020
Date of Web Publication13-May-2021

Correspondence Address:
Yusuf Mohammed
Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, Bayero University, Kano
Nigeria
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DOI: 10.4103/tp.TP_78_20

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   Abstract 


Background: The emergence of widespread resistance of Plasmodium species to most antimalarial drugs has led to a more vigorous and concerted research on traditional medicinal plants for the treatment of malaria.
Objective of Study: The study was aimed to investigate the in vitro antiplasmodial activity of crude ethanolic and aqueous extracts of Phyllanthus amarus against clinical isolates of Plasmodium falciparum in Northwestern Nigeria.
Materials and Methods: The plant was extracted using two solvents, water and ethanol, where a high yield was obtained from the aqueous extracts (11.9%) as compared to the ethanolic extract (9.64%). The extracts were evaluated in vitro at concentrations of 6.25, 12.5, 25, 50, and 100 μg/ml, and the level of potency in each case was expressed as the concentration of the extract that exhibited a 50% reduction of the parasites relative to control (100%) parasitemia. Artemether-lumefantrine was used as a positive standard in the assay.
Results: All extracts showed a significant reduction in parasite growth relative to control (P ≤ 0.05). Ethanolic extract exhibited a higher antiplasmodial activity of 76.8%, half-maximal inhibitory concentration (IC50) of 5.80 μg/ml, and aqueous extract had an activity of 75.3%, IC50 of 7.94 μg/ml. Both extracts exhibited very active antiplasmodial activity. Oral acute toxicity test in the doses of 500, 1000, and 1500 mg/kg showed no sign of toxicity on albino mice after 48 h.
Conclusion: Although there was an increase in appetite after 24 and 48 h, the findings from this study show that P. amarus possesses a promising antimalarial activity which can be exploited for malaria therapy and justifies the traditional use of the plant in malaria treatment.

Keywords: Anti-malaria, ethnomedicine, Nigeria, Phyllanthus amarus


How to cite this article:
Aliyu K, Mohammed Y, Abdullahi IN, Umar AA, Bashir F, Sani MN, Kabuga AI, Adamu AMY, Akande AO. In vitro antiplasmodial activity of Phyllanthus amarus against Plasmodium falciparum and evaluation of its acute toxicity effect in mouse model. Trop Parasitol 2021;11:31-7

How to cite this URL:
Aliyu K, Mohammed Y, Abdullahi IN, Umar AA, Bashir F, Sani MN, Kabuga AI, Adamu AMY, Akande AO. In vitro antiplasmodial activity of Phyllanthus amarus against Plasmodium falciparum and evaluation of its acute toxicity effect in mouse model. Trop Parasitol [serial online] 2021 [cited 2021 Jun 14];11:31-7. Available from: https://www.tropicalparasitology.org/text.asp?2021/11/1/31/315937




   Introduction Top


Malaria is an infectious disease caused by protozoa belonging to the genus Plasmodium. This illness is still a major public health problem, especially in tropical and subtropical regions.[1] It has been estimated to cause 266,000 deaths globally, and children under 5 years of age are more at risk.[2] Malaria remains a major public health concern in Nigeria, with about 76% of the population at risk.[3] It is still one of the leading causes of death in the country, with the country having one of the greatest numbers of malaria cases in the world.[3]

Despite the availability of several antimalarial drugs, their efficacies are limited due to the development of resistance by the parasites, especially Plasmodium falciparum.[4] The emergence of drug resistance to the current antiplasmodial agents necessitates urgent research for new antimalarial drugs.[5] The antimalarial potential of compounds derived from plants is proven by examples such as quinine obtained from Cinchona species and artemisinin from Artemisia annua.[6] Although many drugs such as quinine and artemisinin originated from medicinal plants, it is likely that several other plants described in the traditional medicine literature would yield other plant-derived antimalarials.[7] The need to urgently develop alternative antimalarials that are not only effective against resistant malaria but also relatively inexpensive for those who need them is important. One area that seems to offer promising prospects concerns the use of plant extracts.[7] However, considering the economic cost of synthesizing new drugs, scientists interested in the development of antimalarials of plant origin need information about the status of in vitro effects of antiplasmodial herbs.[8]

P. amarus (Schum and Thonn) is a widely distributed, small, erect, tropical annual plant whose stem has a green capsule and grows up to 10–60 cm in length. The plant is bitter and possessed the following effects: astringent, cooling, diuresis, stomachic, febrifuge, and antisepsis.[9] It grows in moist, shady, and sunny places and is widely distributed in the tropics.[10] Some of the medicinal usages have been proven in experimental models, which suggest that the extracts of the plant possess various pharmacological activities.[11] It can be used as a potential source of antioxidants which can be used to cure various ailments and as an antibacterial agent that can inhibit the growth of various pathogenic and antibiotic-resistant bacterial strains.[12]

The plant extracts have been evaluated in human trials for the treatment of hypertension, jaundice, diabetes, hypercalciuria, and urolithiasis.[13] An extract of the leaves showed antioxidant activity.[13] It is a powerful kidney stone deterrent and helps cleanse the ureter after lithotripsy to help kidney stones pass.[14] The formulated capsules of both the aqueous and ethanol extracts of the plant have demonstrated chemotherapeutic effects on Plasmodium yoelii in Swiss albino rats with results comparable to chloroquine.[10],[14]

Pharmacologically, various extracts of P. amarus have been shown to exhibit antidiabetic, anticarcinogenic, antimicrobial, anti-inflammatory, and antioxidant activities.[15] Essentially, P. amarus herb has a number of ethnobotanical uses against malaria parasite, diarrhea, dysentery, urinary tract infection, asthma, appendix, and inflammations.[15]

P. amarus consists of different classes of organic compounds of medicinal importance including alkaloids, flavonoids, hydrolysable tannins (ellagitannins), major lignans, polyphenols, triterpenes, sterols, and volatile oil.[16] In the last decade, antimalarial drug resistance appeared to be a major challenge in the fight against malaria. This has underscored the need to search for alternative herbal-based products with efficacy against chloroquine-resistant and other multiple drug-resistant strains of malaria parasites.[17] In view of these, P. amarus is one of the medicinal plants traditionally used to treat malaria in Nigeria. However, very little scientific information is available about its activity against P. falciparum, though it is extensively used to treat malaria. In this study, the antiplasmodial activity of P. amarus was evaluated against P. falciparum in vitro including its acute toxicity profile.


   Materials and Methods Top


Collection and extraction of plant material

Fresh plants of P. amarus were collected in August 2019 from different locations of Aminu Kano Teaching Hospital (AKTH), Kano State, Northwestern Nigeria. The plant was identified at the herbarium section, Department of Plant Biology, Bayero University, Kano, with accession number BUKHAN 278. Ethical clearance was obtained from the Research and Ethical Committee of AKTH, Kano, Nigeria (AKTH/MAC/SUB/12A/P-3/VI/1951).

The plant was thoroughly washed with distilled water and air-dried for 2 weeks. The dried plant was pulverized into fine powder using a laboratory mortar and pistol. Aqueous and ethanolic extracts of the plant were prepared using maceration method by dissolving 80 g of the powdered plant material in 1 L of distilled water and ethanol, respectively, for 72 h with constant shaking, and it was then filtered using Whatman No. 1 filter paper.[18] They were evaporated using rotary evaporator at 40°C and dried using water bath. The crude extracts were stored in sterile airtight containers and refrigerated at 4°C to protect from sunlight and moisture until use.[18]

Blood sample collection for in vitro assay

The study was conducted at AKTH, Kano, Nigeria. The study was carried out between August 1 and November 30, 2019, among patients referred to the laboratory section for diagnostic tests. The inclusion criteria used in enrolling subjects in this involved, fresh samples collected less than 2 weeks and provision of informed consent. Patients who were not on anti-malaria treatment were screened for P. falciparum infection. Rapid diagnostic test strips (CTK Biotech Inc., California, USA) were used to screen for the parasites, and then, 5 mL of the blood was collected using a Vacutainer syringe into sterile citrate, phosphate, dextrose, and adenine tubes to confirm the presence of the parasites using thin and thick films stained with Giemsa's staining microscopy.

The blood samples were centrifuged at 2500 rpm for 5 min, and the supernatant was discarded. The packed red blood cells (RBCs) were washed thrice in Roswell Packed Memorial Institute 1640 Medium before use for parasite cultivation.[19]

Preparation of culture medium and extract solutions

Roswell Park Memorial Institute (RPMI) powder 10.43 g was dissolved in 960 mL distilled water supplemented with 0.5 mL gentamicin, sterilized using 0.45 and 0.22 membrane filters in a biosafety cabinet, and then kept in the refrigerator at 4°C until use. Before cultivation, it was supplemented with 5% albumin II and sodium bicarbonate to maintain the pH of the medium.[19]

Aqueous and ethanolic crude extracts of P. amarus were dissolved in distilled water to produce 1 mg/ml each of the solutions. The 1 mg/ml solution was further diluted in 9 ml of malaria culture medium giving a stock solution of 100 μg/ml which was sterilized by filtration through 0.22 μm membrane (Millipore).[20] Both extracts were tested in 5 serial dilutions in duplicates (6.25, 12.5, 25, 50, 100 μg/ml).

In vitro antiplasmodial assay

The assay was performed in triplicate in a 96-well microliter plate. Using a micropipette, a total of 200 μl of 1% parasitemia, 5% hematocrit aliquots of the culture were placed into each well (a total of 15 wells). About 20 μl of the test extract prepared in 200μl RPMI 1640 was made in different concentrations (6.25, 12.5, 25, 50, and 100 μg/ml). These were incorporated into the 15 wells to give a final volume of 220 μl. Control I: negative control was maintained with fresh RBCs and 1% parasitized P. falciparum diluted to 5% hematocrits without drug. Control II: positive control was maintained with 100 μl parasitized blood culture treated with 100 μg/ml artemisinin-based combination therapy (ACT) (artemether-lumefantrine). The plates were incubated in 5% CO2 conditions at 37°C in a glass desiccator for 48 h. After 48 h, the contents of the wells were harvested and a thin blood film was prepared for parasitemia estimation. The films were fixed in methanol, stained for 10 min in 10% Giemsa, and viewed under a light microscope after washing and drying. The percentage of parasite inhibition was calculated using the following formula:[21]



The threshold for the in vitro antimalarial activity of the plant extracts was obtained according to Shaa et al.[16] They were classified as follows: extracts with half-maximal inhibitory concentration (IC50) <10 μg/ml were considered very active, 10–50 μg/ml considered moderate activity, and above 50 μg/ml considered low activity.

Experimental animal

Adult albino rats were used. They were obtained from the farmhouse of the Department of Biological Sciences, Bayero University, Kano. These animals were acclimatized for 7 days at room temperature before use. They were housed in standard cages and maintained on standard animal pellets and water.

Acute toxicity test

The acute toxicity test was carried out on albino rats. Twenty-one rats were divided into 7 groups of 3 rats each. The first group served as a control, while the other 6 groups were administered 500, 1000, and 1500 mg/kg of the aqueous and ethanolic extracts, respectively. The control groups were kept under the same condition without any treatment. The animals were routinely inspected for (0, 6, 12, 24, and 48 h) for signs of toxicity such as tremors, weakness and refusal to feed, falling off of hair, coma, or even death after 48 h.

Statistical analysis

Microsoft Excel 2007 was used to calculate mean parasite growth and percentage parasite inhibition. The student t-test was used to statistically analyze the data, and P ≤ 0.05 was considered a significant difference between the two groups. The IC50 values were obtained from the log-linear regression analysis of log-dose response curves using GraphPad Prism version 8 (San Diego, USA).


   Results Top


[Table 1] shows the yields of crude extract and solvent extraction of the plant. Water solvent yielded more extracts than the ethanol. The crude extracts were tested mainly on the trophozoites of P. falciparum. [Table 2] shows the mean number of parasitized red blood cells at various concentrations. There was a significant (P ≤0.05; P ≤0.0001) reduction in the number of parasitized cells compared to control. The basic measurement of antimalarial activity used in this study was reduction in the number of parasitized cells in the test cultures after 48-h incubation. Among the two crude extracts, the ethanol extract gave a higher antimalarial activity of 76.8%, with an IC50 of 5.80 μg/ml. This was followed by aqueous extract with parasite growth inhibition of 75.3%, IC50 of 7.94 μg/ml. However, the difference in antimalarial activity of extracts was not statistically significant (P >0.05, P = 0.3223, and P = 0.281).
Table 1: Yield of extract of Phyllanthus amarus

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Table 2: Mean number of parasitized red blood cells at various concentrations

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The oral administration of aqueous and ethanolic extracts of P. amarus in the doses of 500, 1000, and 1500 mg/kg body weight did not cause any sign of acute toxicity. No deaths of albino mice were recorded even up to 72 h after oral administration. All other parameters such as tremors, refusal to feed, falling of hair, and coma were not observed, however, there was an increase in appetite at all concentrations of the extract at 24 and 48 h [Figure 1].
Figure 1: Percentage inhibition of parasite growth by aqueous and ethanolic extracts of Phyllanthus amarus at various concentrations (6.25–100 μg/ml)

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   Discussion Top


The emergence of widespread resistance of Plasmodium species to most antimalarial drugs has led to a more vigorous and concerted research in traditional medicinal plants for the treatment of malaria.[18],[22] The plant evaluated in this study was identified through an ethnobotanical approach provided by a Botanist. P. amarus is a plant that is traditionally used to treat malaria in Nigeria but with no scientific proof of efficacy. This study provided scientific evidence for the claim.

The result of this study revealed that P. amarus has an excellent antimalarial activity against P. falciparum isolates in vitro. The aqueous extract exhibited the highest extraction yield (9.53%) as compared to the ethanolic extract (7.71%), hence indicating that the extraction efficiency favors the highly polar solvent. This agrees with the findings of Truong et al.[22] which suggested that the more polar a solvent, the more yield it results. It also corroborated with the work of Dhawan and Gupta[23] where the most polar solvent gave more yield of the extracts, although Dhawan and Gupta[23] reported that the difference in yield could be due to the extraction method used.

The in vitro antiplasmodial assay was done at 1% parasitemia and 5% hematocrit to mimic in vivo infection of P. falciparum, since in 10 mL of in vitro P. falciparum culture, over 10,000 are infected RBCs. This is comparable to the percentage parasitemia in the human bloodstream 7 days after incubation.[24] The IC50 values of the plant extracts were solely calculated following 48 h exposure because the life cycle of P. falciparum in the erythrocytes takes about 48 h to complete.[24]

P. amarus ethanol and aqueous extracts were not significantly different at 48 h (P >0.05), which means that the two crude extracts are equally potent and can be used for inhibiting P. falciparum. The highest antimalarial activity was observed with ethanol extract which had parasite growth inhibition of 76.8% while the aqueous extract had parasite growth inhibition of 75.3%. Water is the main and preferred solvent used in traditional settings. This is not surprising because some previous studies showed similar patterns of crude aqueous plant extracts less potent than their corresponding organic extracts.[18],[20],[25]

The possible reasons are that, first, water is unable to extract lipophilic phytochemicals that are extracted by ethanol.[20] Second, aqueous extracts are not prepared the same way as obtainable in traditional settings. In traditional settings, different plants and their parts are mixed into concoctions, which might enhance their antimicrobial activity.[26] Third, perhaps the phytochemicals extracted by water are more potent in vivo than in vitro or active against another stage of parasites or other plasmodium species.[20] These patterns of antimalarial activity suggest that extracts or drugs would exhibit higher antimalarial activities at higher concentrations.

P. amarus is used by traditional healers for the treatment of diabetes, jaundice, gonorrhea, polymenorrhea, skin ulcers, sores, swelling, and itchiness.[9],[26] Both the aqueous and ethanolic extracts of P. amarus have a high antimalarial activity. It is prepared as a decoction by boiling 50 g of plant in 1 L of water. The patient drinks a cupful of these 3 times daily after meals until recovery. Children take half of the dose.[27] In Nigeria, a decoction of the leaves is taken 4 times daily for 5 days as a treatment for malaria.[28] Traditional healers in Nigeria usually use decoctions of P. amarus in combination with other plant parts to treat malaria and fever which results in enhanced antimalarial effect.[26],[28] Occasionally, some people administer it alone as an antimalarial therapy.[28]

The in vitro antiplasmodial activity of P. amarus had similarly been reported by Donkor et al.[21] who reported antimalarial activity of 80.09% at 100 μg/ml which is comparatively higher than what we obtained (76.8% at 100 μg/ml). The findings of this work also agree with Uchenna et al.[25] in which the plant extract was tested against Plasmodium berghei in a 5-day suppressive in vivo test. The differences in results might be due to the different methods employed.

Comparatively, the ethanolic extract exhibited a higher antiplasmodial activity unlike the aqueous extract (ethanolic: 76.8%–5.80 μg/ml; aqueous: 75.3%–7.94 μg/ml) although the difference was not significant. It is interesting to note that the in vitro antiplasmodial activity of P. amarus is being reported for the first time in this study in the northern part of Nigeria.

Based on Shaa et al.[18] threshold for in vitro antiplasmodial activity, the aqueous and ethanolic extracts of P. amarus with IC50 7.94 and 5.80 μg/ml are considered to have an excellent antiplasmodial activity. All the two extracts showed dose-dependent antimalarial activity against P. falciparum isolates. A similar dose-dependent pattern of activity has been reported against P. falciparum and P. amarus.[25]

Acute systemic toxicity evaluates adverse effects or safety of a substance that occurs following exposure of organisms to a single or multiple doses of a test substance within 24 h by a known route (oral, dermal, or inhalation).[29],[30],[31] It estimates the dose of a test substance that produces 50% death in a given species of animals. It is usually the first test conducted for every chemical before further toxicity tests are carried out.[30] The result obtained in the acute toxicity test implies that aqueous and ethanolic extracts of P. amarus in the doses of 500, 1000, and 1500 mg/kg body weight (orally) were not toxic to the mice, and the extract is therefore considered safe. No death or major signs of toxicity were observed. This finding agrees with Adolor et al.[31] where the plant extracts at higher doses of 2000 and 5000 mg/kg did not cause any death or signs of toxicity. Furthermore, the findings agree with Umar et al.[32] whose report showed that P. amarus is nontoxic. There was an increase in appetite of the animals after 24 and 48 h. A report showed that hot water extract of the plant is taken orally to increase appetite.[33],[34] A change in weight of albino rats was also reported which was said to be as a result of an increase in feed intake.[34],[35]

This study is not without limitation. Unfortunately, the present study did not check the activity of our P. amarus extract against the chloroquine-resistant strains which would have provided more information on its efficacy as a new antimalarial compound. However, a study had previously reported that the antiplasmodial effects of the extracts were comparable to the standard prophylactic and chemotherapeutic drugs used in chloroquine resistant.[10] Although P. amarus antimalarial activity depends on the dose of the extract administered, the chemotherapeutic effect of P. amarus extracts is good at higher doses (1 600 mg/kg/day).[10] Finally, the absence of in vivo antimalarial evaluation in this study limited the authentication and consideration of the P. amarus extracts for immediate application for treating human malaria. Hence the need to explore the in-vivo potentials of this plant extracts.


   Conclusion Top


The result of this study shows that both aqueous and ethanolic extracts of P. amarus pose an excellent antimalarial activity. The ethanolic extract exhibited a higher antiplasmodial activity than the aqueous extract. Both the aqueous and ethanolic extracts of P. amarus have been found to be nontoxic and improve appetite; therefore, it is potentially safe for malaria therapy. Further work on phytochemical properties and anti-inflammatory and analgesic activities of the plants should be carried out to give the plant wider therapeutic advantage.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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