Extracts of Euphorbia hirta Linn. (Euphorbiaceae) and Rauvolfia vomitoria Afzel (Apocynaceae) demonstrate activities against Onchocerca volvulus Microfilariae in vitro

Background Onchocerciasis transmitted by Onchocerca volvulus is the second major cause of blindness in the world and it impacts negatively on the socio-economic development of the communities affected. Currently, ivermectin, a microfilaricidal drug is the only drug recommended for treating this disease. There have been speculations, of late, concerning O. volvulus resistance to ivermectin. Owing to this, it has become imperative to search for new drugs. World-wide, ethnomedicines including extracts of Euphorbia hirta and Rauvolfia vomitoria are used for treating various diseases, both infectious and non-infectious. Method In this study extracts of the two plants were evaluated in vitro in order to determine their effect against O. volvulus microfilariae. The toxicity of the E. hirta extracts on monkey kidney cell (LLCMK2) lines was also determined. Results The investigations showed that extracts of both plants immobilised microfilariae at different levels in vitro and, therefore, possess antifilarial properties. It was found that all the E. hirta extracts with the exception of the hexane extracts were more effective than those of R. vomitoria. Among the extracts of E. hirta the ethyl acetate fraction was most effective, and comparable to that of dimethanesulphonate salt but higher than that of Melarsoprol (Mel B). However, the crude ethanolic extract of E. hirta was found to be the least toxic to the LLCMK2 compared to the fractionated forms. Conclusions Extracts from both plants possess antifilarial properties; however, the crude extract of E. hirta was found to be least toxic to LLCMK2.


Background
Onchocerciasis, transmitted by Onchocerca volvulus is among the major causes of blindness in the world [1][2][3][4] and has a negative impact on the socio-economic development of the communities affected [5][6][7][8][9][10][11][12]. The pathology of the disease involves mainly the microfilariae (mff ) of the parasite. Ivermectin (Mectizan W ), the drug recommended for treatment is microfilaricidal only and has to be administered once within six to 12 months and continuously for several years [13][14][15][16][17][18]. Of late, there have been reports of emerging ivermectin resistance in the adult O. volvulus [19][20][21][22][23][24]. Should this be confirmed, the control of the disease will seriously be in jeopardy. In view of the limited candidate drugs available for testing against the parasite, it has become imperative to search for new drug(s) preferably a macrofilaricide(s) to treat the disease.
Presently, one area that is gaining grounds and acceptability world-wide is the evaluation and development of compounds from medicinal plants for the treatment of diseases. World-wide, ethnomedicines are used for treating various diseases, both infectious and non-infectious. In Ghana, a number of plant based products are claimed to be effective for the treatment of onchocerciasis. Among the medicinal plants involved Euphorbia hirta and Rauvolfia vomitoria reported to have antimicrobial properties [25][26][27][28][29][30][31][32] are used for this purpose. These have however, not been subjected to systematic evaluation to ascertain their effectiveness against O. volvulus. Owing to this, extracts from these two plants were selected for in vitro evaluation against the microfilariae (mff) of O. volvulus. The toxicity of the E. hirta extracts on monkey kidney cell (LLCMK2) lines was also determined following satisfactory results by testing its extracts.

Plant collection, identification and extract preparation
Whole plants of E. hirta and the leaves and roots of R. vomitoria were collected and identified at the herbarium of the Botany Department of the University of Ghana in Accra, Ghana. Voucher specimens of the plants: E. hirta (GC47751) and R. vomitoria (GC47752) were deposited at the herbarium. Whole plant specimens of E. hirta and root specimens of R. vomitoria were air dried for a week and pulverized into a fine powder. About 300 g of the pulverized E. hirta and 270 g of the R. vomitoria materials were macerated separately in three litres of 80% ethanol. After 24 hours the slurry of each plant was filtered with Whatman No.1 filter paper and concentrated under reduced pressure in a rotavapor (BUCHI Rotavapor R-114, Switzerland) at 50°C to recover the ethanol.
A portion of the concentrate was kept as crude extract. The remaining concentrate was partitioned with hexane (3 × 250 ml), chloroform (3 × 250 ml) and ethyl acetate (3 × 250 ml) sequentially (i.e. in an increasing order of polarity). Each fraction was then concentrated in vacuo at 50°C with the rotavapor to yield semi-solid masses. The residual (aqueous) solutions as well as the crude extracts were freeze-dried. All the extracts were stored at −20°C until they were used for testing. Dimethylsulfoxide (DMSO) was used to dissolve the plant extracts prior to diluting them into stock concentrations of 1000 μg/ml in MEM. Prior to testing against the mff, concentrations ranging from 6.25 to 800 μg/ml of each of the extracts were prepared from the working concentration such that the concentration of DMSO in each of the test solution was less than 0.1%. Melarsoprol (Mel B) and dimethanesulphonate (DMSPN) salt (Ash Stevens Inc. Detriot, Michigan 48202) used as reference drugs (Strote et al., 1990a and b, 1997 and 1998) were prepared similarly.
For testing against monkey kidney cell lines (LLCMK2) 1:10 serial dilutions of the E. hirta stock solutions were prepared down to 1 μg/ml. Potassium cyanide (KCN) (at 1 mg/ml of MEM) solution was used as a positive control and 1% DMSO in MEM as a solvent (or negative) control. All the solutions were sterile filtered with a 0.2 μm millipore filter before testing.

Selection of Subjects
Twenty-four onchocerciasis subjects between the ages of 18 and 60 from Kpedze-Anoe and Honuta-Gbogame, who had not taken any antifilarial drugs before were admitted to the Onchocerciasis Chemotherapy Research Centre (OCRC) at Hohoe in Ghana after a written consent had been sought from each of them. The study was carried out between May 5 and August 25,2005 following approval from the Ethical Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana.

Harvesting of microfilariae
Skin snips were taken from both iliac crests after sterilizing these sites with 70% alcohol. The snips were placed in a sterile petri-dish containing five millilitres of Eagle's Minimum Essential Medium (MEM) and left on the bench at 22°C. After three hours of incubation, the snips were removed, medium transferred into sterile test tubes and the mff recovered by centrifugation at 1,500 rpm.

Measurement of microfilaricidal action of extracts
One millilitre of each extract and drug preparations was delivered in triplicates into a 24-well plastic plate. About 30 mff were delivered into each well. The number of motile mff was determined at 2, 4, 6, 12, 24 and 48 hours of exposure to the extracts. The same amount of mff was exposed to concentrations between 0.01 and 1.0 per cent of DMSO (solvent control) and in plain MEM (i.e. without DMSO, drug or herbal preparation). The assays were carried out in triplicates. Microfilariae were observed under an inverted microscope and the proportion of motile/live ones determined.
Inhibition determination of Euphorbia hirta extracts on monkey kidney cells Three hundred microlitres of MEM solution containing about 1000 monkey kidney cells were placed in each well of 96-well plates. The cells were incubated at 35°C in a humidified atmosphere of 5% CO 2 for a day. At the beginning of day two the culture medium was removed from the wells and replaced with 300 μl of the different diluted extract to give a concentration of 0-1000 μg/ml and left to incubate for five days. The same volume of potassium cyanide (KCN) solution at 1 mg/ml concentration and DMSO in MEM were each delivered into three wells of the plate. The cells in three of the wells were killed with hot water and incubated with plain MEM culture medium (i.e., without any extract). In three other wells the cells were maintained with the plain culture medium. In other experiments, the cells were incubated for four days before the plant extracts were added and incubated for another two days.
At the end of the incubation period the plant extracts were removed from the wells and replaced with 300 μl of tetrazolium salt, 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution at 0.5 mg/ml and incubated at 35°C. After 30 minutes the MTT solution was removed and replaced with 100 μl DMSO and the plate placed in the dark at a temperature of 22°C for 1 hour. The formazan produced in each of the wells was transferred into corresponding wells of another microtitre plate in duplicates and the optical density (OD) determined spectrophotometrically at a wavelength of 510 nm.

Data management and statistical analysis
Data were analyzed using Excel 2000 and Epi-info 6 programme version 6.04d, January 2001. The 50% inhibition concentrations (IC 50s ) of the extracts were determined by log probit analysis, and the percentage inhibitions of the extracts on LLCMK2 were calculated using the means of the OD values. For comparison of results the chi-square test was used.

Effect of DMSO on microfilariae
All the mff maintained in the various concentrations of the solvent control retained their viability up to six hours of exposure. At 12 hours of exposure, there was a slight decrease in the number of live/motile mff in the 0.5% concentration (i.e. a decrease of 6.4%) and 1% concentration (i.e. a decrease of 1.8%). The mff maintained their survival above 90% at 24 hours and between 69% and 76% at 48 hours in all the concentrations. At 48 hours of exposure about 33% of the mff were immobilised in the 1% DMSO concentration and about 22% in the plain MEM. No significant difference was found between the proportions of mff immobilised in these two solutions.

Effect of the plant extracts on microfilariae
The results from the investigations showed that motile mff were immobilised in various degrees by the E. hirta and R. vomitoria extracts in a concentration and time dependent fashion. At 2 hrs of mff exposure more mff were already immobilised in the 400-800 μg/ml of the crude extract, 800 μg/ml of the hexane, 400 μg/ml and 800 μg/ml of the chloroform, 25-800 μg/ml of the ethyl acetate and 400 μg/ml and 800 μg/ml of the aqueous extracts of the E. hirta plant than the 1% DMSO concentration (p < 0.001). Immobilisation of mff continued in all the concentrations of the E. hirta extracts (with the exception of the 6.25 μg/ml and 12.5 μg/ml concentrations of the hexane extracts) and the reference drugs so much so that by 48 hrs, a significant difference was found between the proportions of mff that were immobilised in those concentrations and in the 1% DMSO concentration (p < 0.001). For the R. vomitoria extracts more mff were found immobilised in the 400 μg/ml and the 800 μg/ml concentrations than in the 1% DMSO (p < 0.001) from 2 hrs to 24 hrs. Similarly, with the exception of the 6.25 μg/ml of the crude extract and the 6.25-50 μg/ml of the hexane and ethyl acetate extracts, more mff were found immobilised in the lower concentrations (6.25-200 μg/ml) of the extracts of this plant than in the 1% DMSO concentration at 48 hrs (p < 0.001). Table 1 shows the percentages of motile O. volvulus mff found in different concentrations of extracts of the two plants ranging between 6.25 μg/ml and 200 μg/ml at 2 and 4 hrs. The minimum concentrations of E. hirta found to have immobilised the mff by 2 hrs of exposure were: the 12.5 μg/ml concentration of the ethyl acetate, 100 μg/ml of the aqueous, 200 μg/ml of the crude, 400 μg/ml of the chloroform and 800 μg/ml of the hexane extracts. The least concentration that reduced motile mff numbers to less than 50% among the E. hirta extracts was the 200 μg/ml concentration of the ethyl acetate extract. Reduction of live mff below 50% also occurred in the 400 μg/ml of the aqueous extract and in the crude and chloroform extracts at 800 μg/ml concentrations of that plant ( Table 2). The various concentrations of the hexane fraction were the least active among the E. hirta extracts. No significant reduction of mff was seen in all the concentrations of the R. vomitoria extracts except in the 800 μg/ml chloroform and the ethyl acetate extracts (Table 2).
Among the 6.25 μg/ml concentrations of E. hirta immobilisation of mff occurred only in the ethyl acetate extract at 4 hours, and also occurred in the chloroform extract at 6 hrs and aqueous extracts at 12 hrs. At 4 hrs the least concentration of each of the E. hirta extract that reduced motile mff numbers to less than 50% was 50 μg/ml of the ethyl acetate extract, 200 μg/ml of the crude extract, the chloroform extract and the aqueous extract and 800 μg/ml of the hexane extract. At this time point, the proportion of motile mff had been reduced significantly to 2% in the 200 μg/ml concentration of the E. hirta ethyl acetate extract. There was no significant reduction of motile mff numbers in the concentrations of R. vomitoria extracts up to 200 μg/ml at this time point (Table 1). Table 3 shows the percentage of motile O. volvulus mff found in concentrations of extracts of the two plants ranging between 6.25 μg/ml and 200 μg/ml at 6 and 12 hrs. At 6 hours of exposure to concentrations of the hexane extracts of E. hirta ranging between 6.25 μg/ml and 200 μg/ml no mff was found immobilised. Immobilisation of mff was observed in the 12.5 μg/ml concentrations of the remaining extracts of this plant. The reduction of motile mff numbers occurred gradually in the crude, chloroform and aqueous extracts of E. hirta but the rate of reduction was faster in the ethyl acetate extract of this plant. The minimum concentration that immobilised more than 90% of the mff at this time point was the 100 μg/ml of the ethyl acetate, 400 μg/ml of the chloroform, aqueous and crude extracts and 800 μg/ml of the hexane extract (Table 2). No significant difference was found between the effect of the aqueous extract and that of the chloroform or the crude extracts. A significant difference was however found between the effectiveness of the aqueous extract and that of the hexane    Table 2).
In the 6.25 μg/ml concentration of the E. hirta extracts all the mff were motile in the crude and hexane extracts up to 12 hours of their exposure. At this time point, no live mff were found in concentrations higher than 25 μg/ml of the ethyl acetate extract. Live mff numbers fell below 20% in the 100 μg/ml and 200 μg/ml   concentrations, and fell to zero in the 400 μg/ml and 800 μg/ml concentrations of the crude, chloroform and aqueous extracts. In the R. vomitoria extracts all the mff were motile up to 12 hours when they were exposed to the 6.25 μg/ml concentration. No mff was found immobilised in the hexane extracts of E. hirta at concentrations lower than 400 μg/ml except in the 100 μg/ml and 200 μg/ml concentrations. Table 4 shows the percentage of motile mff exposed to the 6.25 μg/ml to 200 μg/ml concentrations of E. hirta and R. vomitoria extracts at 24 hrs and 48 hrs and Table 2 shows the percentage of motile mff exposed to the 400 μg/ml and 800 μg/ml concentrations of E. hirta and R. vomitoria extracts at 2-24 hrs. At 24 hrs of exposure to the E. hirta extracts the proportion of motile mff were found to have been reduced drastically. The lowest concentration that killed all the mff at this time point in the case of the ethyl acetate extract was 25 μg/ml; chloroform extract, 100 μg/ml (Table 4) and aqueous extract, 400 μg/ml; hexane extract, 800 μg/ml and the crude extract, 400 μg/ml ( Table 2). In all the plant extract concentrations, only in the 800 μg/ml concentration of the aqueous extract of R. vomitoria were motile mff seen at the same time point (Table 2). By 48 hrs, all the mff were immobilised in all the concentrations of the ethyl acetate extract of E. hirta and in the 800 μg/ml concentrations of both the E. hirta and R. vomitoria extracts (Tables 2 and 4). At this time point, with the E. hirta plant, motile mff were found in the 6.25 μg/ml and 12.5 μg/ml concentrations of the aqueous extract and the 6.25 μg/ml to 25 μg/ml concentrations of the chloroform and crude extracts and 6.25-200 μg/ml of the hexane extract.
Generally, the extracts of E. hirta were more effective in immobilising the mff and their IC 50s were lower than those of R. vomitoria. At 2-24 hrs the extracts of E. hirta were found to be more effective in immobilising mff than those of R. vomitoria and the difference was significant (P < 0.004). There was no significant difference between the ethyl acetate extract of E. hirta and DMSPN at any time point with regards to their effectiveness. The various concentrations of Mel B were less effective in immobilising motile mff than those of the ethyl acetate extract of E. hirta (P < 0.019) and those of DMSPN (P < 0.01) at 2-6 hrs (Tables 1 and 3). Table 5 shows the 50% inhibition concentrations (IC 50s ) of the plant extracts and the reference drugs on O. volvulus mff. The IC 50s of all the extracts and the reference drugs decreased consistently over time. The E. hirta extracts with the exception of that of the hexane had lower IC 50s than those of R. vomitoria at 24 hrs and Mel B at 12 hrs. Among the E. hirta extracts that of ethyl acetate had the least IC 50s which were comparable to those of DMSPN up to 24 hrs. With the exception of the hexane extract of E. hirta and the ethyl acetate extract of R. vomitoria, the IC 50s of all the extracts as well as DMSPN and Mel B fell below 6.25 μg/ml at 48 hrs. From the foregoing, it can be deduced that the E. hirta ethyl acetate extract (which had the least IC 50s ) was the most effective.
Effect of E. hirta extracts on monkey kidney cell lines Figure 1 shows the effect of E. hirta extracts on the viability of monkey kidney cells maintained for one day prior to the exposure of the extracts for five days. In this figure the crude extracts were seen to exhibit negative inhibition on the LLCMK2. All the extracts with the exception of that of chloroform at 1000 μg/ml concentration also inhibited the cells negatively (i.e. promoted cell proliferation) when they were maintained in culture for four days before being exposed to the extracts for two days (Figure 2).
Among these extracts only the crude extract inhibited the cells in a concentration dependent fashion with the lowest concentration having the highest effect and vice versa. At each concentration the crude extract exhibited the lowest inhibition on the cells. The 1000 μg/ml concentration of the hexane extract showed the highest inhibition and the 100 μg/ml the lowest in these two experiments. In the first experiment the highest inhibition on the cells was exhibited by the 10 μg/ml, the 1000 μg/ml and the 1 μg/ml concentrations of the chloroform, ethyl acetate and water respectively whilst the 100 μg/ml, 1 μg/ml, and 100 μg/ml concentrations in the same order had the lowest effect on the cells (Figure 1). In the second experiment the 1000 μg/ml concentration of the chloroform and ethyl acetate extract as well as the 1 μg/ml concentration of the aqueous extract had the highest whilst the 100 μg/ml concentrations of the chloroform, ethyl acetate and aqueous extracts respectively had the lowest  inhibitory effect on the cells (Figure 2).

Discussion
To date, not much work has been done to investigate the efficacy of plant extracts against human filarial parasites except those conducted by Kilian et al. (1990) using Cassia aubrevillei [33] and Titanji et al. (1990) using Polyalthia suaveolens [34]. Most of the work conducted so far involved the use of animal filarial parasites. Some of the plants used were Acacia auriculiformis [35], Sancio nudicaulis Buch. Ham. [36], Saxifraga stracheyion root [37], Centratherum anthelminticum seeds [38], Azadirachta indica and Ficus racemosa Linn. [39,40]. Comley et al. (1990) reported the macrofilaricidal effects in vitro of filaricid (a crude extract of the stem bark of Streblus asper, Lour) and filarin (a crude extract comprising five plants in different proportions) against Acanthocheilonema viteae females at 500 μg/ml [41]. They also found that oliverine derived from n-hexane extract of the stem bark of Pachypodanthium staudtii has similar effects on the Acanthocheilonema viteae worm at 120 hours down to 10 μg/ml. In spite of reports concerning E. hirta and R. vomitoria possessing antimicrobial properties, no systematic studies on their effectiveness against filarial parasites either in vivo or in vitro have been carried out. The results of this study has indicated that extracts of these two plants possess antifilarial properties. Among the E. hirta extracts, that of ethyl acetate was most effective, and this was comparable to that of DMSPN but more effective than Mel B. The hexane extract of E. hirta and the extracts of R. vomitoria showed least activity before 48hours of mff exposure. A lot of chemical constituents including diterpenes, flavonoids, tannins, aromatic acids, alkaloids, coumarins and anthocyanins, euphorbon, triterpenes and quercetol have been isolated from every part of E. hirta [28,29]. The root of R. vomitoria, on the other hand, contains flavonoids and over 100 alkaloids including reserpine, rescinnamine, deserpidine, yohimbine, aijmaline and alstonine which are therapeutically important [27][28][29].
Notwithstanding the efficacy of E. hirta extracts against these diseases, issues have been raised concerning the toxicity of the extracts. For instance, Adedapo et al. (2005) in conducting in vivo work showed that some chromatographic fractions of the plant have potentially deleterious effect on the serum chemistry of rats. They therefore cautioned against the use of this plant for medicinal purposes. Contrary to these findings, Hashemi et al. (2008) observed a significant decrease in Aspartate transaminase (AST), Alanine aminotransferase (ALT), and Alkaline phosphatase (ALP) after 14 days of oral administration of 2000 mg/kg of the crude aqueous extracts of E. hirta to chicks [42]. Ogueke et al. (2007) found the crude ethanolic extracts of the plant at concentrations ranging from 60.4 to 483.0 mg/kg body weight to be haematologically safe to albino rats. Biochemical analyses of the renal and hepato-biliary function following oral administration of up to 3000 mg/kg body weight of the crude extracts of the plant were also found to be haematologically safe and did not show any signs of nephrotoxicity and hepatotoxicity in rats, as confirmed by histopathological examination [43].
E. hirta has been used to treat various diseases by traditional herbal practitioners and its administration has mainly been through the oral route [25,[28][29][30]. In this study, the crude extract of E. hirta inhibited cell growth the least. Moreover, it was the only extract that was found to have exhibited its effect in a concentration dependent fashion with the highest concentration having the least inhibitory effect. This observation is in agreement with that of Brindha et al. (2010) who observed a dose-dependent increase in viability when antitubercular drug exposed human liver derived HepG2 cells were treated with different concentrations of extracts of the plant [44].
In the current study, when the cells were maintained for a longer period before being exposed to the extracts, the survival of the cells was enhanced more than when they were maintained for a shorter period before their exposure to the extracts. This observation was most pronounced when the cells were exposed to the crude extracts. This observation can be explained by the fact that once the cells form confluence they are inhibited less than when they have not. A previous investigation by Raja Sidambaram et al. (2011) using the methanol extract of the leaves of E. hirta on Hep--2 cells from human epithelioma of the larynx showed a dose dependent antitumor activity in vitro [45]. The extracts in the current investigation have also revealed antiproliferative effects on the cells that have not achieved complete attachment to the plates. This finding shows that extracts from the plant have the potential to serve as anticancer agents. The promotion of the vitality of the monkey kidney cells particularly by the crude extract of E. hirta implies that the administration of plant extracts in the crude form might be safer than the fractionated or purified forms. This work justifies the use of these plants particularly in their crude form for treating diseases by herbal practitioners.

Conclusions
It might be concluded from the foregoing that extracts of both E. hirta and R. vomitoria possess antifilarial properties. Of the E. hirta extracts, that of ethyl acetate was found to be the most effective whilst the crude extract was the least toxic to monkey kidney cell lines. It is recommended that further efficacy determination of the