Amoebiasis is caused by a protozoan parasite, Entamoeba histolytica with or without clinical symptoms and it is the third leading cause of death from parasitic diseases after malaria and schistosomiasis (WHO, 1997). This infection remains a major health problem in developing countries and its prevalence varies between countries and between regions with different socio-economic conditions (Jackson, 2000). Sometimes, it may reach 50% of the population in regions with poor sanitary conditions (Caballero et al., 1994). The most effective and com-monly used drug for treatment of this intestinal protozoan infection is metronidazole (MTZ). However, this drug has been reported to have unpleasant side effects such as metallic taste, headache, dry mouth, and to a lesser extent nausea, glossitis, urticaria, pruritus, and dark colored urine. Carcinogenic, teratogenic, and embryogenic effects have also been documented (Upcroft et al., 1999; Upcroft and Upcroft, 2001) in addition to the fact that it may lower both cell mediated and humoral immune responses in drugs recipients (Saxena et al., 1985). Therefore, immunocom-promised recipients of MTZ including those with AIDS (full form) would constitute a high risk. According to these observations above, the search of alternative anti-amoebic compounds with high activity, and low toxicity is still necessary. Segment of the world’s population relies on traditional remedies to treat a plethora of diseases. Medicinal herbs constitute an indispensable part of traditional medicine practised all over the world due to the cost, easy access, and ancestral experiences (Martini-Bertolo, 1980).Euphorbia hirta belongs to the genus Euphorbia and the family of Euphorbiaceae. It is a common herb in the pan-tropic and partly subtropic areas worldwide, including China, India, Philippines, Australia, Africa, Malaysia, and so on(Huang et al., 2012). E. hirta is an important medicinal herb with various pharma-cological behaviours. The flavonol glycosides afzelin, quercitin and myricitrin, isolated from E. hirta showed inhibition of the proliferation of Plasmodium falcifarum at different concentrations (Jackson, 2000). The leaves, flowers, stems, and root extract of the plant exhibited antimicrobial activity against E. coli, C. albicans, S. aureus, and P. mirabilis (Mohammad et al., 2010). The antidiarrheal effect of the E. hirta herb decoction was studied in mice. It demonstrated an activity in models of diarrhea induced by castor oil, arachidonic acid, and prostaglandin E 2. Quercitrin, a flavonoid isolated from this crude drug contributed to the antidiarrheal activity at a dose of 50 mg/kg, against castor oil and prostaglandin E2-induced diarrhea in mice (Galvez et al., 1993).The in vitro and in vivo immunomodulatory properties of E. hirta is reported elsewhere (Ramesh and Vijaya, 2010). The finding has been proven through macrophage activity testing, carbon clearance test, and mast cell de-granu-lation assay. The aqueous extract of the leaves of E. hirta Linn could serve as an immunostimulant on the experi-ment of thepathogen-infected Cyprinuscarpio Linn. (Cyprinidae). The antiinflammatory activity of the chemicals in E. hirta showed that the flavonoids quercitrin (con-verted to quercetin in the alimentary canal) and myricitrin, as well as the sterols 24-methylenecycloartenol and sitosterol, exert noteworthy and dose dependent anti-inflammatory activity. Triterpene beta-amyrin also seems to exert a similar anti-inflammatory activity (Ekpo and Pretorius, 2007).The crude and polyphenolic extracts of E. hirta exhibited antiamoebic potential (Tona et al., 2000). 

However, the activity of the fractions obtained from this plant have not yet been tested against E. histolytica.  Therefore, the present study was planned to investigate the in vitro susceptibilities of the clinical isolates of E. histolytica to E. hirta (euphorbiaceae) aqueous extract and fractions.

Biological materials

Plant material

The aerial part of E. hirta (Euphorbiaceae) collected in Yaounde (Cameroon) on April 2011 during morning time was used in the present study.

Microorganisms

Clinical isolates of E. histolytica trophozoites from stool sample of Indian patients suffering from amoebiasis collected at the Department of Medical Parasitology of the Postgraduate Institute of Medical Education and Research (PGIMER) of Chandigarh, India were used for polyxenic cultivation.

Euphorbia hirta extract and fractions preparation

The aerial (leaves and stems) part of E. hirta was harvested, washed in chlorinated water and dried at room temperature (Moundipa et al., 2005). Dried materials were reduced in powder form and the extract was obtained by decoction of 200 g of the powder in 1000 ml of distilled water for 3 h. Decoction obtained was concentrated in the oven at 50°C for 72 h and the concentrated product constituted the aqueous extract. The extraction yield was calculated according to the following formula:

Fractionation of the aqueous extract was performed by using different polarity based solvent from hexane (nonpolar) to methanol (polar) (Zubair et al., 2011). To obtain the hexane fraction (HF), 200 g of the above aqueous extract was macerated in 1000 ml of pure hexane until exhaustion of the solvent. Resulting solution was concentrated using a BüchiRotavapor R-210/R-215 with the tem-perature of 40°C. The solid material obtained after a total evapo-ration of the solvent was conserved and constituted our hexane fraction. Then, methylenechloride fraction (CH₂Cl₂ F) was obtained by subjecting the residue to a second maceration in the same volume of pure methylene chloride followed by the concentration as described previously. The residue obtained from the second step was extracted with1000 ml of pure methanol as described above to obtain the methanol fraction (MF). Crude aqueous extract and fractions obtained were subjected to in vitro assays for the determination of theirantiamoebic potential.

E. histolytica cultivation

Polyxenic cultivation

Biphasic medium of Boeck and Drbohlav (Parija and Rao, 1995) that involves solid phase (ringer’s solution + egg) and liquid phase (lock’s solution containing nutrients) was used for E. histolytica poloyxenic cultivation. Before inoculation, complete media were pre incubated at 37°C for 30 min to 1 h and 1000 µl of diarrheal stool sample containing viable trophozoïtes of E. histolytica were introduced in each tube. The tubes were incubated at 37°C and the E. histolytica growth verified after every 48 or 72 h. Then, the tubes were removed from the incubator and shacked to detach parasites from the solid phase and left for 5 min then the supernatant was decanted to obtain the subculture. The pellet containing the para-sites was introduced in a tube containing pre incubated new medium as previously described (Moundipa et al., 2005).

Amoebicidal effect of the E. hirta extract and fractions assays

E. hirta aqueous extract (AE) and its fractions (AE, MF, CH₂Cl₂ F) were prepared using sterile DMSO (Sigma-Aldrich, and culture medium leading to concentrations of 200, 20, 2 and 0.2 mg/ml respectively). Each mixture was filtered with sterile syringe filters (Ø 22 µm) and aliquots were prepared from these stock solutions. Parasites grown were harvested at midlog phase at the concen-tration of 10⁷cells/ml of culture by counting using the haemo-cytometer (Neubauer,Hausser Scientific) and inoculated in tubes containing new 5 ml media in which 25 µl of plant materials were added. MTZ was used as a standard drug and was tested at 0.1, 1, 5 and 10 µg/ml. AE was tested at the concentration of 50, 100, 200 and 400 µg/ml; whereas all the different fractions were tested at 25, 50,100 and 200 µg/ml. One control tube was used in which para-sites were incubated on culture medium containing 0.5% DMSO without any drug. Each testing concentration was made in triplicate and the experiment was repeated three times for each compound. All the tested tubes were incubated at 37°C as described by Chitravanshi et al., 1992 and the viability was evaluated by trypan blue method after 24, 48 and 72 h.

Amoebicidal activity was evaluated using the method described by Bansal (1987). In 1.5 ml micro centrifuge tube, 25 µl of parasite suspension and 225 µl of 0.4% trypan blue solution prepared in 0.9% NaCl was introduced. The mixture was homogenized and 10 µl of this mixture was used for cells counting. The chamber was covered with cover slip and the viable (bright) cells as well as the dead (blue) cells were counted at 40X on a light microscope. The concentration of the cell has been calculated using the following formula:

N= (n×d)/v

Where, N= concentration of viable cells/ml; n= number of the viable cells counted in the chamber, d= dilution factor and v= the volume of the chamber (0.1 µl)

The percentages of inhibition were calculated also using the formula below and IC₅₀ were determined using the software Graphpad Prism 3.0

Percentage inhibition (%) = (N_C-N_T)/N_C ×100

N_C = Number of viable cells in the control tube and N_T= Number of viable cells in testing tube.

Effect of E. hirta extract and fractions on some enzymes of E. histolytica

The effect of extract, fractions and metronidazole on some enzyme activities of polyxenically cultivated clinical isolate of E. histolytica was studied as described (Sohni et al., 1995).

Amoeba from different tubes of each type of culture were pooled and washed five times with cold phosphate-buffered saline then centrifuged at  /min for 10 min. The sediment was suspended in cold phosphate-buffered saline to yield a concentration of 10⁶cells/ml following which the suspension was homogenized in cold 0.25 M sucrose solution for 3 h at 4°C. The homogenate was then centri-fuged in cold and opalescent supernatant was used as the crude enzyme extract. The amount of total protein in the crude enzyme extract was determined according to the protein colorimetric method assay described by Bradford (1976). All the assays compounds (extract, fractions and metronidazole) were tested at the concen-tration of 100, 200, 400, and 800 µg/ml. Each concentration was made in triplicate and the experiments were repeated three times.

RNase activity assay

A micro centrifuge tube with a capacity of 1.5 ml was used for preparing the reaction mixture of 1.25 ml containing 150 µM of phosphate buffer (pH 7.6), 1.25 mg of yeast RNA, 0.5 ml of enzyme extract and the testing drug (Sparh and Hollingworth, 1961). The mixture was incubated at 37°C for 1 h. The reaction was stopped by the addition of 0.25 ml of uranyl acetate reagent. The suspension was mixed and chilled for 30 min to precipitate undigested RNA. The precipitate was centrifuged and the absorbance was read at 260 nm against blanks. An enzyme unit was defined as the amount of enzyme which gives an increase in absorbance of 0.1.

Aldolase activity assay

Aldolase activity was assayed according to the method describe by Sibley and Lehninger (1949) with some modifications. In a 1.5 ml micro centrifuge tube, a reaction mixture of 1.25 ml containing 0.5 ml of Tris buffer (pH 8.6), 0.125 ml of 0.05 M fructose-1,6-diphosphate, 0.125 ml of  0.0035 M hydrazine sulphate solution in 100 µM EDTA (pH 7.5) 0.25 ml of enzyme extract and the testing drugs was introduced. The mixture was incubated at 37°C for 30 min. The reaction was stopped by addition of 10% trichloroacetic acid (TCA) and the absorbance of supernatant was read after   at 240 nm against blanks. An enzyme unit was defined as the amount of enzyme which gives an increase in absorbance of 1.00.

Acid and alkaline phosphatase (ACP and ALP) activity assay

Aldolase activity was assayed according to the method described by Ashrafi et al.(1969). For the ALP analysis,  a reaction mixture containing 50 µl  of enzyme extract, 100 µl of substrate p-nitrophenylphosphate (PNP) 1% in 0.1 M  glycine/NaOH buffer pH 9, as well as the  tested compounds were prepared in a micro plate of 96 wells and the final volumes were adjusted to 250 µl. The reaction mixtures for the ACP activity assay contained 50 µl of enzyme extract, 100 µl of 1% substrate PNP in citrate buffer pH 4, as well as the tested compounds were prepared in a micro plate of 96 wells and the final volumes were adjusted to 250 µl. Both ACP and ALP mixtures were incubated at 37°C for 30 min. The reaction was stopped by addition of 100 µl 0.1 N NaOH and the absorbance was read at 405 nm against blanks. An enzyme unit was defined as the amount of enzyme which catalyses the formation of 1µmol of p-nitrophenolate ion.

The specific activities were calculated for all the enzymes assayed according and the IC₅₀ of the inhibition were determined using the software Graphpad Prism 5.0.

Statistical analysis

The tests were performed in triplicate and all data are presented as mean ± SD (standard deviation) values. Statistical analysis was performed using GraphPadPrism and student’s t-test was used to determine P-values for the differences observed between test compounds and control. Results were considered significantly different when P ≤ 0.05.

Amoebicidal effect of the E. hirta aqueous extract and fractions against polyxenic culture of clinical isolates of E. histolytica

The clinical isolates of E. histolytica grown maintained on biphasic medium of Boeck and Drbohlav (Figure 1) were incubated with different plant extract and fractions. MTZ and E. hirta aqueous extract, methanol fraction, methylene chloride fraction exhibited amoebicidal effects that were concentration dependant (Figure 2). 

In contrast no amoebicidal activity was found to be associated with HF. The IC₅₀ values of MTZ and E. hirta AE, MF, and CH₂Cl₂ F were respectively about 4.30, 145.95, 67.18, and 194.04 µg/ml after 72 h of incubation. E. hirta MF exhibited higher amoebicidal effect than AE and CH₂Cl₂ F but, remained lower as compared to reference drug MTZ activity (Table 1).

Effect of E. hirta extract and fractions on some enzymes of E. histolytica

The results of the experiments on the effect of metronidazole and E. hirta AE, MF, and CH₂Cl₂ F on enzymes of E. histolytica by mean of specific activities is presented in Figure 3. Specific activity was defined as the enzyme unit per milligram of protein. The specific activity units of compound containing reactions were converted into a percentage basis by correlating with those of controls, the inhibitory effect of MF is significantly higher than those of AE and CH₂Cl₂ F, and comparable to that of metronidazole for all the enzymes assayed. The higher inhibition percentage observed with MF was on ACP activity (73.0435 ± 2.30%) at the concentration of 800 µg/ml. The lower percentage observed with the same fraction was on ALP activity (52.2 ± 1.42 %) (Table 2). 

Several mechanisms for the pathogenesis of E. histolytica by which the parasite can engage in tissue damage are available. These mechanisms include secretion of enzymes and cell free cytotoxins, contact dependant cytolysis and phagocytosis (Sohni, et al., 1995). Some of the secreted enzymes which have been investigated in the present study are believed to play an important role in the virulence and survival of the para-site. The nucleopolymerases play an important role in the metabolism of all living cells. Amoebas which were fed with cholesterol have been shown to increase in lysosomal level of DNase and RNase activities (Narain, 1979). It is also well documented previously that cholesterol increases the virulence of E. histolytica (Meerovitch and Ghadirian, 1978). 

There is significantly higher level of ACP than ALP in trophozoites of E. histolytica and axenically growth amoeba exhibit increased level of ACP activity (Sohni et al., 1995). ACP may play an important role in the utilization of phagocytised food materials. It is also demon-strated that ACP gene expression increases during invasion and cells lesions by E. histolytica suggesting that this enzyme plays an important role during tissue invasion by the pathogenic amoeba (Fernandes et al., 2014).

In the present study, clinical isolates of amoeba grown in polyxenic medium exhibit also a higher level of ACP than ALP. It is apparent from the Figure 3 that MF of E. hirta has inhibitory effect non significantly different from that of MTZ for all the enzyme activities studied. CH₂Cl₂ F and AE have inhibitory effects lower than those of MF.

However further studies of MF of the aerial part of E. hirta are suggested, mainly to confirm the finding in axenic culture of E. histolytica and its effect on the activity and expression of the cysteine proteinase which is the main compound involved in virulence of E. histolytica. In future in vivo studies can be carried out to confirm the antiamoebic activity of E. hirta extracts, so that these can be used for therapeutics.

Methanol fraction of the aerial part of E. hirta exhibited higher antiamoebic activity than methylene chloride fractions and aqueous extract against clinical isolate of E. histolytica. The extract and fractions also had varying degree of inhibition on enzymes of E. histolytica which are thought to play a role in its survival and virulence. The present finding justifies the use of E. hirta aerial part in the traditional medicine for the treatment of dysentery.

The authors have not declared any conflict of interests.

The authors thank the World Academy of Science for the Advancement of science in Developing Countries (TWAS), the Department of Biotechnology (DBT) India, for providing financial assistances through the DBT-TWAS sandwich postgraduate fellowship FR number: 3240255096.