In vitro and in vivo analysis of the anti-plasmodial activity of ethanol extract of Phyllanthus nivosus W. Bull leaf

Abstract

Antimalarial agents are necessary tools in the global malaria eradication agenda and plants used traditionally in the treatment of malaria are indispensable sources of antimalarial compounds. The aim of this study was to evaluate the antiplasmodial potential of Phyllanthus nivosus leaf. In vitro antiplasmodial assay was conducted using Plasmodium falciparum infected erythrocytes incubated at 37 °C in modified RPMI 1640 culture media. The inhibitory effect of the ethanol extract on plasmodium lactate dehydrogenase (pLDH) activity was determined as a measure of antiplasmodial activity. In vivo study was done using mice infected with chloroquine sensitive P. berghei (NK-65 strain). Parasitemia, packed cell volume (PCV), hemoglobin (Hb) and liver lipid peroxidation (MDA) levels were determined after a 4 day treatment. Chloroquine was used as standard drug for both assays. The extract reduced pLDH activity by 39.52, 42.07 and 43.87% at 12, 25 and 50 μg/mL respectively. 100 and 200 mg/kg body weight of extract and 10 mg/kg chloroquine suppressed parasitemia of infected mice by 82.76, 81.11 and 86.87% respectively. Furthermore, the extract significantly reduced (p < 0.05) the elevated MDA level and reversed PCV and Hb levels of infected mice to normal values. Phytochemical screening of the extract revealed the presence of alkaloids, tannins, flavonoids, cardiac glycosides, anthraquinones, steroids and terpenes. Gas chromatography–mass spectrometry (GC–MS) analysis showed the presence of ten compounds, the most abundant of which is Methyl linoleate (35.77%). This study demonstrated that P. nivosus leaf possesses antimalarial potential and contains bioactive compounds that could be beneficial in the development of new antimalarial agents.

Introduction

Malaria is an overwhelming parasitic disease that has remained a major global health concern. About 584,000 persons, 90% of which are from sub-Saharan Africa and 78% of which are children less than 5 years of age, died from malaria in 2013 (WHO 2014). This represent 47% reduction in global malaria mortality rates between 2000 and 2013 which is a result of strategic prevention and control measures like the use of insecticide-treated bed nets and most especially treatment of patients with artemisinin-based combinations (ACTs) (WHO 2014). According to the World Malaria Report of 2018, about 219 million cases of malaria occurred in 87 countries in 2017, and 435,000 people-many of which were children in the African region-died of malaria that year. Although there has been a period of significant success in global malaria control, progress seems to have slowed down (WHO 2018) and no substantial improvement was made between 2015 and 2017 (CDC 2019).

Furthermore, there is an increased incidence of parasite resistance to current antimalarial drugs including ACTs, which are regarded as the most effective to date (Johnson 2015; Oluba et al. 2017; Noedl et al. 2008). According to the World Health Organization (WHO), the drug resistance could lead to dire public health consequences (Johnson 2015). Total eradication of the disease remains an important option in the global strategy for combating malaria and antimalarial agents are essential tools at all stages of the process (Johnson 2015; Halper 2015). Hence, there is an exigent need for further research leading to the identification of new highly effective antimalarial drug candidates. Medicinal plants have remained an important source of alternative intervention (Kaushik et al. 2013; Lombardino and Lowe 2004; Oluba 2019) and plants used traditionally in the treatment of malaria are very crucial in antimalarial drug research.

The genus Phyllanthus has been used in herbal medicine for many years and many species of the plant are used as natural cures for a range of diseases. About 600 species of Phyllanthus have been found in tropical and subtropical regions and many of them are used medicinally (Watt and Breyer-Brandwijk 1962). Phyllanthus nivosus W. Bull (Breynia nivosa) is an attractive foliage plant belonging to the family of Euphorbiaceae. It grows well in Nigeria and it is being used traditionally for the treatment of malaria, fever, headaches, toothaches and tooth infections (Okokon et al. 2015). Extracts of P. nivosus leaf are reported to possess antioxidant, anti-inflammatory, antimicrobial and analgesic activities, and it is reported to have beneficial roles in the management of several health problems (Okokon et al. 2015; Onyegbule et al. 2014). However, little work has been done to explore its therapeutic potentials in antimalarial drug development. The aim of this study was therefore, to evaluate the antiplasmodial activity of P. nivosus leaf as a possible source of antimalarial agent.

Methods

The plant sample

Fresh leaves of P. nivosus were collected at the University of Jos, Bauchi road Campus and authenticated at the Department of Plant Science and Technology, University of Jos, Nigeria. It was preserved with the voucher number UJ/PCG/HSP/13E01. The leaves were spread under shade until completely dried. Sixty grams of the powdered leaf was dissolved in 500 ml of 70% ethanol at room temperature for twenty-four (24) h. The mixture was filtered through Whatmann filter paper no 42(125 mn) and the extract was concentrated using a rotary evaporator. Phytochemical screening of the extract was carried out using standard qualitative procedures described by Sofowora (1993) and Trease and Evans (1986).

In vitro antiplasmodial assay

The extract was assessed for anti-plasmodial activity in vitro by the parasite Lactate Dehydrogenase (pLDH) assay (Ngemenya et al. 2006; Desjardins et al. 1979). Plasmodium falciparum positive blood sample was obtained from Faith Alive Foundation Hospital, Jos, Nigeria. The infected erythrocytes (4% hematocrit and 5% parasitemia) were incubated at 37 °C, under 3% O₂, 6% CO₂ and 91% N₂, in RPMI 1640 culture media (Sigma-aldrich) and varying concentrations (50 µg/ml, 25 µg/ml and 12 µg/ml) of ethanol extract of Phyllantus nivosus leaf. Same concentration of Chloroquine was used as standard. The incubation was done for 72 h after which 20 μl of culture was added to 100 μl of Malstat reagent in a flat-bottomed 96-well plate. This was followed by the addition of 25 µl of NTB/PES to each well. The experiment was done in four replicates. Absorbance was read at 630 nm using a spectrophotometric microplate reader. The absorbance from control (infected untreated) sample was taken to represent 100% pLDH activity (Kindala et al. 2016). Percentage (%) activity was calculated by dividing the sample’s absorbance by the absorbance of control and multiplied by 100. Percentage activity of sample was subtracted from the percentage activity of control (100) to give the % inhibition of pLDH activity.

In vivo antiplasmodial assay

Plasmodium berghei

Chloroquine sensitive Plasmodium berghei strain—NK-65 was obtained from the Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Research and Development (NIPRD), Abuja. The parasite was maintained by repeated passages in experimental mice. Blood samples were taken from highly infected donor mice and diluted with normal saline such that each experimental mice received 0.2 ml of 1 × 10⁻⁷ infected erythrocytes.

Experimental animals

Twenty (20) Swiss Albino mice of both sexes, aged 6–8 weeks old, weighing between 28 and 30 g obtained from the Animal Holding Unit of the University of Jos, Nigeria, were used. The mice were housed in standard cages and maintained under standard laboratory conditions and had access to food and clean drinking water. They were randomly separated into five groups of four mice each. Groups 1 and 2 mice were infected and treated with 100 and 200 mg/kg body weight of ethanol extract of P. nivosus leaf respectively. The doses used were chosen based on previous study by Okokon et al. (2015). Group 3 mice were infected and treated with 10 mg/kg body weight of the standard drug chloroquine. Group 4 mice were infected but not treated. Group 5 mice were neither infected nor treated. Administration of drugs, which commenced when percentage parasitemia of infected mice was above 10%, was done once daily for 4 days through oral gavage.

Determination of % parasitemia and % suppression of parasitemia

Percentage parasitemias of experimental mice were obtained daily according to the method of Dacie and Lewis (2000). Geimsa stained blood smears were examined under light microscope at X 100  magnification. Percentage parasitemia was estimated using the formula:

$$\frac{\text{Number of infected Red Blood Cells}}{\text{Total number of Red Blood Cells}}\times 100$$

Percentage (%) suppression of the parasitemia was calculated using the formula:

$$\%\text{Suppression}\left(\text{A}\right)=\frac{\text{C}-\text{B}}{\text{C}}\times 100$$

where C = Average % parasitemia of infected untreated mice. B = Average % parasitemia of infected treated mice.

Collection of blood and liver samples

At the end of the 4 day treatment, mice were placed under diethyl ether anesthesia and blood samples were collected through the jugular vein into EDTA containing bottles for the determination of PCV and Hemoglobin concentration. The blood was kept at 4 °C until required for hematological studies. Liver samples were removed, weighed, homogenized in ice cold sucrose solution (0.25 M), centrifuged at 5000 rpm for 5 min, and the supernatant kept in a refrigerator until required for the determination of malondialdehyde (MDA) concentration.

Determination of hematological parameters

Packed cell volume (PCV) was determined using capillary tube on a microhematocrit centrifuge at 10,000g for 5 min and read on a microhematocrit reader. Hemoglobin (Hb) concentration was determined using cyanmethemoglobin method.

Determination of Lipid Peroxidation levels

The concentration of malondialdehyde (MDA)—a marker of Lipid peroxidation was determined using the method of Reilly and Aust (1999). Briefly, 0.4 ml of liver homogenate was mixed with 1.6 ml of Tris-KCl buffer containing 0.5 mL of 30% trichloroacetic acid (TCA). 0.5 ml of thiobarbituric acid (TBA) was then added and the mixture was incubated in a water bath at 80 °C for 45 min. The reaction mixture was cooled in ice and centrifuged at 14,000g for 15 min. The absorbance of the clear pink supernatant was then read at 532 nm. MDA concentration was calculated using the formula:

$$\text{MDA}\left(\text{units/mg}\text{protein}\right)=\frac{\text{Absorbance}\text{X}\text{Volume}\text{of}\text{mixture}}{\mathrm{\epsilon }532\text{X}\text{Volume}\text{of}\text{sample}\text{X}\text{mg}\text{protein}}$$

where ε532 (molar extinction coefficient of MDA-TBA at 532 nm) = 1.56 × 10⁵ M⁻¹ cm⁻¹

Gas chromatography–mass spectrometry (GC–MS) analysis of plant sample

GC–MS analysis of the ethanol extract of P. nivosus leaf was performed using Shimadzu GC-MS - QP2010 PLUS. Injector temperature was set at 250 °C. The oven temperature was programmed at 60 °C, the pressure was set at 100.2 kPa. The ion source and interface temperature were fixed at 200 and 250 °C respectively. The column and purge flow was 1.61 and 5.6 ml/min respectively, while the total flow time was 39.4 ml/min. Sample was manually injected with a split ratio of 20:0. Total running time of GC–MS was 18 min. The database of National Institute of Standard and Technology (NIST) library with more than 62,000 spectral patterns was used in the interpretation of the mass spectrum.

Statistical analysis

Data were presented as mean of four determinations ± SD. Statistical analysis was carried out using one-way analysis of variance (ANOVA). Differences were considered statistically significant at p < 0.05

Results

In vitro antiplasmodial assay

The absorbance value of the infected untreated sample was taken to represent 100% pLDH activity. Based on that, the pLDH activities of samples treated with 12, 25 and 50 µg/ml of P. nivosus leaf extract were calculated to be 60.48 ± 9.05, 57.93 ± 5.61 and 56.13 ± 2.57% respectively (Table 1). Chloroquine treated samples showed 51.55 ± 6.72, 47.94 ± 4.94 and 45.64 ± 1.82% pLDH activities. Hence, the extract reduced the activity of pLDH by 39.52, 42.07 and 43.87% and chloroquine by 48.45, 52.06 and 54.36% respectively (Fig. 1).

Table 1.

Percentage (%) pLDH activity of infected erythrocytes treated with Phyllanthus nivosus leaf extract and the standard drug chloroquine (CQ)

Concentration (µg/ml)	Percentage (%) pLDH activity
	Ethanol	Chloroquine
12	60.48 ± 9.05	51.55 ± 6.72
25	57.93 ± 5.61	47.94 ± 4.94
50	56.13 ± 2.57	45.64 ± 1.82

Values are expressed as mean ± SD, n = 4 for each group

Fig. 1.

Percentage inhibition of pLDH activity by Phyllantus nivosus leaf extract and the standard drug. Values are expressed as mean ± SD, n = 4 for each group

In vivo antiplasmodial assay

Figure 2 shows the effects of ethanol extract of P. nivosus leaf on % parasitemia of P. berghei infected mice. Parasitemias of infected mice were 11.25 ± 1.25, 15.0 ± 2.16 and 18.63 ± 2.43% before treatment. At the end of the 4 day treatment with 100 mg/kg extract, 200 mg/kg extract and 10 mg/kg chloroquine, parasitemias were 5.5 ± 0.57, 6.75 ± 0.95 and 4.0 ± 0.15 respectively. 100 and 200 mg/kg P. nivosus leaf extract suppressed parasitemia of infected mice by 82.76 and 81.11% respectively, and chloroquine by 86.87% (Fig. 3).

Fig. 2.

Effects of ethanol extract of P. nivosus leaf and standard drug on % parasitemia of P. berghei infected mice

Fig. 3.

Percentage (%) suppression of parasitemia of P. berghei infected mice treated with ethanol extract of P. nivosus leaf

Hematological parameters

The effects of P. nivosus leaf extract on packed cell volume (PCV) and hemoglobin concentration (Hb) of Plasmodium berghei infected mice are shown in Figs. 4 and 5. P. berghei infection significantly reduced PCV and Hb levels of experimental mice. PCV level reduced from 38.30 ± 6.19 to 28.30 ± 1.17% and Hb concentration reduced from 12.78 ± 2.02 to 9.43 ± 0.56 g/dl. In the infected groups treated with extract (100 mg/kg and 200 mg/kg) and standard drug (10 mg/kg chloroquine), PCV levels were restored to 38.50 ± 4.43, 39.25 ± 1.17 and 39.00 ± 2.58% respectively; and hemoglobin levels increased to 12.83 ± 1.50, 13.08 ± 0.56 and 13 ± 0.85 g/dl respectively.

Fig. 4.

Effects of ethanol extract of P. nivosus leaf on packed cell volume (PCV) of P. berghei infected mice. n = 4 ± SD; ⁰significantly lower compared with uninfected untreated, *significantly higher compared with infected untreated (p < 0.05)

Fig. 5.

Effects of ethanol extracts of P. nivosus leaf on hemoglobin concentration (Hb) of P. berghei infected mice. n = 4 ± SD; ⁰significantly lower compared with uninfected untreated, *significantly higher compared with infected untreated (p < 0.05)

Lipid peroxidation levels

Plasmodium berghei infection significantly increased liver MDA level of mice from 3.67 ± 1.5 (uninfected untreated) to 7.37 ± 1.5 µg/g (infected untreated). This elevated value was reduced to 6.53 ± 0.97, 5.83 ± 0.81 and 5.89 ± 0.62 µg/g in the 100 and 200 mg/kg extract and chloroquine treated groups respectively (Fig. 6).

Fig. 6.

- Effects of ethanol extracts of P. nivosus leaf on liver lipid peroxidation (MDA concentration) of P. berghei infected mice. n = 4 ± SD; ⁰significantly higher compared with uninfected untreated, *significantly lower compared with infected untreated (p < 0.05)

Qualitative phytochemical screening of ethanol extract of P. nivosus leaf

Phytochemical screening of ethanol extract of P. nivosus leaf revealed the presence of alkaloids, tannins, flavonoids, cardiac glycosides, anthraquinones, steroids and terpenes. The extract appeared to be relatively more abundant in flavonoids content, followed by alkaloids, tannins and cardiac glycosides. Saponins were not detected in the ethanol extract of P. nivosus in this study (Table 2).

Table 2.

Phytochemical constituents of ethanol extract of P. nivosus leaf

S. no	Constituent	Result
1	Alkaloids	++
2	Tannins	++
3	Saponins	–
4	Flavonoids	+++
5	Terpenes and Steroids	+
6	Anthraquinones	+
7	Cardiac glycosides	++

KEY: + = moderately present ++ = present +++ = richly present – = not detected

GC–MS analysis of the ethanol extract of P. nivosus leaf

GC-MS analysis of the extract showed the presence of 10 compounds, the most abundant of which is Linoleic acid methyl ester (Methyl linoleate). Others are palmitic acid methyl ester, undecanoic acid, 2-methyl–methyl ester, stearic acid methyl ester, oleic acid ethyl ester, heptanoic acid 2-methyl-methyl ester, methyl 2-hydroxydodecanoate, methyl 18-methylnonadecanoate, tetradecanoic acid 12-methyl-methyl ester, phthalic acid dibutyl ester (Fig. 7 and Table 3).

Fig. 7.

GC–MS chromatogram of ethanol extract of P. nivosus leaf

Table 3.

Compounds identified in the ethanol extract of P. nivosus leaf

Peak	Retention time	Percentage (%) area (A)	Percentage (%) height (H)	A/H	Name	Formula	Structure
1	14.697	15.62	26.64	2.63	Palmitic acid, methyl ester (Metholene)	C17H34O2
2	15.411	0.51	1.48	1.55	Undecanoic acid, 2-methyl-, methyl ester (Methyl ester of 2-methylundecanoic acid)	C13H26O2
3	16.644	69.42	35.77	8.72	Linoleic acid, methyl ester (Methyl linoleate)	C19H34O2
4	16.840	8.93	20.70	1.94	Stearic acid, methyl ester (Kemester 9718)	C19H38O2
5	17.236	3.18	8.34	1.71	Oleic acid, ethyl ester (Ethyl Oleate)	C20H38O2
6	17.455	0.25	0.76	1.46	Heptanoic acid, 2-methyl-, methyl ester (Methyl 2-methylheptanoate)	C9H18O2
7	18.485	1.05	1.05	1.30	Methyl 2-hydroxydodecanoate	C13H26O3
8	18.703	0.30	3.99	1.53	Methyl 18-methylnonadecanoate	C21H42O2
9	20.430	1.36	1.05	1.37	Tetradecanoic acid, 12-methyl-, methyl ester, (S)-(Methyl 12-methyltetradecanoate)	C16H32O2
10	20.646	0.11	0.22	2.21	Phthalic acid, dibutyl ester (Dibutyl phthalate)	C16H22O4

Discussion

The reduced pLDH activity observed in the P. falciparum infected erythrocytes treated with P. nivosus leaf extract and standard drug in the in vitro study is an indication of P. falciparum suppressive effect (Shoemark et al. 2007). Plasmodium lactate dehydrogenase (pLDH), a terminal enzyme in the glycolytic pathway is one of the most abundant enzymes expressed by both the asexual and sexual stages of P. falciparum (Vander Jagt et al. 1981) and it is used as a specific biomarker for the presence of viable Plasmodium in the blood of infected host. Metabolic differences have been observed between the host- and parasite-specific enzymes. pLDH preferably use 3-acetylpyridine adenine dinucleotide (APAD) as coenzyme whereas human LDH requires β-nicotinamide adenine dinucleotide (NAD) for its activity (Makler and Hinrichs 1993). Hence the use of pLDH in malaria diagnosis and in the study of novel antimalarial agents (Shoemark et al. 2007; Makler and Hinrichs 1993).

The in vitro antiplasmodial activity demonstrated by the extract is further supported by the data obtained from the in vivo study, which shows the suppressive ability of the extract on P. berghei infection in mice. P. nivosus leaf extract at 100 and 200 mg/kg showed 82.76 and 81.11% suppression respectively as compared with 86.87% for the standard drug chloroquine. These results justify the traditional use of the plant by some communities in the eastern part of Nigeria (Okokon et al. 2015).

The significantly low Hb concentrations and PCVs observed in the infected untreated mice in the in vivo study is indicative of anaemia which is one of the major pathologic consequences of malaria infection. This condition has been suggested to be as a result of decrease in erythrocyte number or a reduction in hemoglobin concentration in each erythrocyte (Lathia and Joshi 2004; Atamna and Ginsburg 1993; Oluba et al. 2014). Atamna and Ginsburg (1993) reported double production of hydroxyl radicals (OH·) radicals and H₂O₂ by P. falciparum infected erythrocytes when compared with normal erythrocytes. The host’s hemoglobin molecule is said to be the potential source of free radical production as the parasite digests this molecule to obtain amino acids for its own nutrition during the erythrocytic stage of the disease. This explains the reduced Hb and PCV levels of infected mice observed in this study. Malaria parasite also induces the generation of OH· in the host’s liver, which most probably induces oxidative stress and lipid peroxidation (Guha et al. 2006). This corroborates the high level of lipid peroxidation observed in the liver of the infected untreated mice. The ability of the extracts to restore the PCV, Hb and liver lipid peroxidation levels of infected mice to normal values is of great significance in the antimalarial activity of the plant and it might be due to the antioxidant property of the plant (Onyegbule et al. 2014).

Phytochemical screening of the extracts showed high presence of flavonoids, followed by alkaloids, tannins and cardiac glycosides, with considerable levels of anthraquinones, steroids and terpenes. Most of this compounds have been reported to be associated with the antiplasmodial and/or antioxidant activity of many plants (Philipson and Wright 1991; Christensen and Kharazmi 2001). Various classes of alkaloids have been known to exhibit antimalarial activity. Quinine which is the first isolated antimalarial compound is an alkaloid (Johnson 2015). Flavonoids act as an inhibitor of the nucleic acid base pairing of the malaria parasite, as an antioxidant and as an iron chelator (malaria parasites require iron for proliferation and survival) (Lui et al. 1992). Some terpenes are protein inhibitors (Liao et al. 1976) and others like Artemisinin (a sesquiterpene trioxane lactone) produce toxic plasmocidal free radicals through the cleavage of their peroxide bridge (Johnson 2015).

The GC-MS analysis of P. nivosus leaf extract also revealed the presence of some bioactive compounds. Linoleic acid methyl ester (Methyl linoleate) which showed the highest peak in the GC-MS chromatogram is the methyl ester of linoleic acid. Linoleic acid is a polyunsaturated omega-6 fatty acid, an 18-carbon chain with two double bonds in cis configuration. The antimalarial activity of some fatty acids (including linoleic acid, stearic acid and oleic acid) and their methyl esters have been reported and polyunsaturation of the fatty acids as well as inhibition of fatty acid biosynthetic activity of the parasite are suggested to be responsible for the action of these compounds. This recent discovery is currently been considered as a likely approach to fight the parasite (Carballeira 2007). Kumaratilake et al. (1992) studied the antimalarial activities of omega-3 and omega-6 polyunsaturated fatty acids and they suggested a positive correlation between the degree of unsaturation and the antiplasmodial action of the fatty acids towards the parasite. Krugliak et al. (1995) later studied the antiplasmodial activity of some fatty acids against the FCR3 strain of P. falciparum, and they reported that these fatty acids exhibited some inhibitory activity against both the intact infected cells and the free parasites. Oleic acid (9–18:1) was found to exhibit the highest inhibitory activity followed by linoleic acid (9, 12–18:2). They suggested that inhibition of fatty acid biosynthetic pathway of P. falciparum is a possible mechanism for the antiplasmodial activity of the C18 fatty acids (Carballeira 2007).

Conclusions

This study demonstrated that Phyllanthus nivosus leaf possesses antimalarial activity thereby justifying the traditional use of the plant by the Ibibios of Niger Delta region of Nigeria. The plant was also found to contain bioactive compounds that could be beneficial in the development of new antimalarial agents.

Authors’ contributions

TO designed the work, carried out the in vivo study and the GCMS analysis, supervised and prepared the manuscript. IG conducted the in vitro study and contributed to the writing of the manuscript. RJ was involved in the design and co-supervision of the work as well as preparation of the manuscript. All authors read and approved the final manuscript.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical standard

The study was performed in accordance with the Declaration of Helsinki. An ethical approval with Reference Number FAFEC/08/34/5 was obtained from Faith Alive Foundation Hospital, Jos, Nigeria where the Plasmodium falciparum positive blood sample used for the in vitro study was obtained. The in vivo study was carried out in accordance with the National Institute of Health guide for care and use of Laboratory animals. An ethical approval with Reference Number UJ/FPS/F1700379 was obtained from the ethical committee of the University of Jos to carry out the study.