In vitro efficacy of Elephantorrhiza elephantina root extracts against adult Paramphistomum cervi in goats.

Elephantorrhiza elephantina is a medicinally important plant whose roots are used to control gastrointestinal parasites in goats. The use of plant-based anthelmintics as potential alternatives to synthetic anthelmintics in controlling gastrointestinal worms in ruminants is a promising area of research. The study was carried out to ascertain the efficacy of E. elephantina in controlling goat trematodes. Standard procedures were used to quantify the phytochemicals in the water, ethanol and methanol root extracts of E. elephantina. The quantitative phytochemical analysis revealed that alkaloids (93.24 ± 1.68%), condensed tannins (450.52 ± 0.15 mg CE/g), flavonoids (803.93 ± 0.13 mg QE/g), phenols (476.11 ± 0.37 mg GAE/g) and saponins (83.28 ± 1.72%) were present in E. elephantina root extracts. Paramphistomum cervi adult worms recovered from the rumen of freshly slaughtered goats were used. In vitro screening of ethanol, methanol and water extracts of E. elephantina for potential anthelmintic activity against adult P. cervi worm motility showed time and dose-dependent significant effects. Ethanol extracts showed a highly significant (P < 0.05) inhibition of motility (86.67 and 96.67%) at concentrations 7.5 and 15 mg/mL respectively, after 12 h of treatment, while methanol and water extracts exhibited a significant (P < 0.05) inhibition of motility (96.67 and 66.67% respectively) at concentration of 15 mg/mL 16 h post-exposure. Our findings confirm the efficacy of E. elephantina in controlling goat trematodes.

Introduction

Gastrointestinal parasites account for huge losses experienced by the livestock industry worldwide (Veríssimo et al., 2012). While presently the major way of control and treatment of these gastrointestinal parasites depends profoundly on the utilization of conventional anthelmintics, there are challenges associated with the use of these synthetic anthelmintics (Behnke et al., 2008; Keiser and Utzinger, 2008). The challenges include unavailability and inconsistent supply; high cost to resource-limited farmers, environmental pollution and the build-up of residues in animal products (Kamaraj and Abdul Rahuman, 2010). The rapidly increasing resistance to anthelmintics may perhaps be as a result of the increased rate of dosing, under-dosing, prophylactic mass treatments and repeated chronic use of the same anthelmintic drugs (Shalaby, 2013). Regular use of these anthelmintics has also led to poor development of natural immunity against gastrointestinal parasites (Ketzis et al., 2006).

One practical way of developing cheaper and effective anthelmintics is to explore potential indigenous herbal remedies used as anthelmintics (Soetan et al., 2011). Plant-derived anthelmintics is a promising area of research in trying to mitigate the challenges around the use of synthetic anthelmintic drugs in controlling gastrointestinal parasites. Alternative ethnoveterinary medicines, for example, plant extracts with anthelmintic properties are considered to be of immense potential in overcoming anthelmintic resistance (Lans et al., 2007). There have been many reports, mainly from Africa and Asia have indicated the effectiveness of medicinal plants against helminth infections in livestock (Alawa et al., 2003; Carvalho et al., 2012). Hence, there is a need to investigate the potential active compounds from these plants.

Worldwide research has shown that some plants can be utilized to diminish the level of parasitism in livestock; thus are considered alternative options to the conventional chemical anthelmintics (Githiori et al., 2006). Elephantorrhiza elephantina is a medicinal plant commonly utilized in ethnoveterinary medicine. Root decoctions infusions of E. elephantina are used to control gastrointestinal worms (Van der Merwe et al., 2001; Barros et al., 2006; McGaw and Eloff, 2008). The anthelmintic effects of this plant are due to the activity of secondary metabolites, such as tannins, alkaloids, saponins and glycosides (Salem et al., 2011; Carvalho et al., 2012; Hernandez et al., 2014). Phytochemicals are beneficial in the treatment of diseases and control of parasites that nourish on the walls of the gastrointestinal tract (Nkohla et al., 2015). Plants produce them as a defense mechanism. Research demonstrates the ability of these phytochemicals to protect humans and animals against infections (Kumar et al., 2009).

However, reports on the phytochemical analysis of crude extracts from E. elephantina are limited. This study sought to quantify the phytochemical constituents of E. elephantina in ethanol, methanol and water extracts of the root. Furthermore, the study evaluated the in vitro anthelmintic potential of E. elephantina root extracts against adult Paramphistomum cervi in goats.

Materials and methodology

Plant collection

Roots of E. elephantina were collected from a characteristic populace in Mt. Frere, Alfred Nzo District Municipality, Eastern Cape, South Africa. Mt. Frere lies along latitude 30°55′0″ S and longitude 28°58′60″ E. Immediately after harvesting, the roots were rinsed in distilled water and air-dried in the shade for 10 weeks and after that were ground into powder using a grinder with 1 mm pore size sieve (IKA- Universal Mill M20, Laboratory and Scientific Equipment Co. Pty. Ltd). The root powder was kept in airtight containers at 4 °C until further analyses.

Extraction procedure

Three samples of 20 g of E. elephantina root powder were soaked in 200 mL each of ethanol, methanol, and distilled water. The mixtures were left on an orbital shaker for 24 h and then filtered under pressure using a Buchner funnel and Whatman filter paper (12.5 cm; 111v). Then the ethanol and methanol extracts were condensed in a rotary evaporator (Laborator 4000-efficient, Heidolph, Germany) while the water extract was freeze-dried (Vir Tis benchtop K, Vir Tis Co, Gardiner, NY). The dried extracts were used for the quantitative analysis of phytochemicals. The percentage yield was calculated for each extract using the formula:

Quantitative determination of phytochemicals

Alkaloids

The gravimetric analysis for total alkaloid content was used to determine alkaloids with a few modifications (Obadoni and Ochuko, 2001). A 0.5 mL of plant extract (1 mg/mL) was combined with 100 mL of 10% acetic acid in ethanol. The mixture was covered and left standing for 4 h. Afterwards, the mixture was filtered, and the filtrate was concentrated in a water bath at 60 °C to a quarter of its initial volume. Concentrated ammonium hydroxide (25%) was added to the mixture drop-wise until the formation of a precipitate was completed. The entire mixture was left to stand, then the accumulated precipitates were rinsed with 20 mL of 0.1 M dilute ammonium hydroxide and then filtered. The collected residue was oven-dried at 80 °C and weighed. The alkaloid content was calculated using the formula:

Condensed tannins

Determination of total condensed tannin was performed by applying the technique described by Sun et al. (1998) with a few modifications. A 0.5 mL of plant extract (I m/mL) was added to 3 mL of 4% vanillin-methanol. A 1.5 mL of 37% hydrochloric acid was added to the mixture, thoroughly mixed and left standing for 15 min at room temperature. The absorbance of catechin standard (0.02 to I mg/mL) solution and plant extracts were recorded at 500 nm using the UV 3000 PC Spectrophotometer. Distilled water was used as the blank. Readings were taken in triplicates. The calibration curve was plotted using standard catechin. The readings were expressed as mg of catechin equivalent per g dry weight extract (mg CE/g).

Flavonoids

Flavonoid content was determined as described by Kamtekar et al. (2014) using the aluminium chloride colorimetric assay technique with some modifications. Briefly, 2 mL of distilled water and 0.15 mL of 5% sodium nitrite were put into a test-tube with 0.5 mL of the plant extract (1 mg/mL). The mixture was left standing for about 5–6 min at room temperature. After 6 min 0.15 mL of 10% aluminium chloride was added to the mixture and then was left standing for an additional 6 min. Afterwards, 1 mL of 4% sodium hydroxide was added to the mixture. A 1.2 mL of distilled water was poured to make up the volume of the mixture to 5 mL. The mixture was incubated for 15 min to develop colour. The absorbance was taken at 420 nm using a UV 3000 PC Spectrophotometer. Each assay was done in triplicates. The calibration curve was plotted using standard quercetin (0.2 to 1 mg/mL). The results were calculated as mg of quercetin equivalent for each g dry weight (mg QE/g).

Phenols

Phenol content was quantified through Folin-Ciocalteu's technique with a few modifications (Skerget et al., 2005). A 2.5 mL of Folin-Ciocalteu reagent was added to 0.5 mL of each plant extract (I mg/mL) and vortexed for about 1 min. The mixture was held at room temperature for about 3–8 min. A 2.5 mL of 7.5% anhydrous sodium carbonate was then added and incubated in a water bath at 40 °C for 30 min to develop colour. Absorbance was then read at 765 nm using the UV 3000 PC Spectrophotometer. The blank was performed using distilled water. Each assay was done in triplicates. Standard gallic acid (0.02 to 1 mg/mL) was used to plot the calibration curve. The readings were expressed as mg of gallic acid equivalent for each g dry weight (mg GAE/g).

Saponins

The saponin content was determined following the technique of Obadoni and Ochuko (2001) with some modifications. A 20 mL of 20% ethanol was added to 0.5 mL (1 mg/mL) of the plant extract and put on a shaker for 30 min. The resulting mixture was then left in a water bath at 55 °C for 4 h. The mixture was filtered and extraction was repeated with 20 mL of 20% ethanol. The collected filtrates were concentrated to one-quarter of the original volume in a water bath at 90 °C. Afterwards, the concentrate was put into a 250 mL separating funnel and extraction was done twice with 20 mL diethyl ether. The ether layer was disposed of, while the aqueous layer was kept. A 20 mL n-butanol was poured to the aqueous layer, and then washing was done twice using 5 mL of 5% sodium chloride. The solution was placed in a water bath to evaporate. Then the samples were oven-dried at 40 °C. Calculation of saponin content was determined using:

Worm recovery

Worms were collected from freshly slaughtered goats at East London Abattoir in the Eastern Cape Province (latitude 32.9702° S and longitude 27.8872° E). Worm recovery was done following the method described by Hansen and Perry (1994). Immediately after slaughter, rumens were collected from the animals, ligated and quickly transported in a cooler box with ice to the Animal Science Laboratory at the University of Fort Hare. The contents of the rumens were washed, several times with phosphate-buffered saline (PBS) until the worms were free from debris. The species and description of the worms are as shown in Table 3. The worms were then recovered from the rumens and kept in PBS at 37 °C for 2 h.

Table 3

Picture showing adult P. cervi recovered from the rumen of goats.

Preparation of solutions

All test solutions and the standard drug solutions were freshly prepared before starting the experiments. Concentrations of 1.875, 3.75, 7.5 and 15 mg/mL were prepared for each extract by dissolving the water extract in PBS, while the ethanol and methanol extracts were dissolved in PBS mixed with 5% dimethyl sulfoxide (DMSO). A 5% DMSO/PBS mixture was used as the negative control after ascertaining that the addition of 5% DMSO did not affect the worms (Barrau et al., 2005). Valbantel® (Albendazole 1.9% m/v and Closantel sodium 3% m/v Pfizer, South Africa) was used as the positive control after it was prepared in PBS to a final concentration of 1.875 mg/mL.

Experimental design and treatment procedures

Adult motility inhibition assay was done following the method of Hounzangbe-Adote et al. (2005). Ethanol, methanol and water extracts of E. elephantina were tested at different concentrations (1.875, 3.75, 7.5 and 15 mg/mL). Ten P. cervi adult worms were placed in each petri dish containing 3 mL of each of the above test solutions and incubated at 37 °C. Every concentration was tested in triplicate. Worm motility was observed at 2-hour intervals for 16 h, and each observation period lasted for 5–6 s. The number of motile worms was recorded per treatment during the observation period and expressed as a percentage. Time for motility inhibition was noted when no movement of any sort was observed except when the worms were shaken vigorously.

Statistical analysis

The results were presented as Mean ± SD. Fisher's Least Significant Difference (LSD) was used to compare the difference between the ethanol, methanol and water extracts at a 5% level (P < 0.05) using the Minitab 17 Statistical Software (2003). The mean percentages of worm motility inhibition were assessed using PROC GLM of SAS (SAS, 2003). Turkey post-hoc testing (SPSS version 22, Armonk, NY, 2013) was used to compare differences between treatment means. Two-way ANOVA test was performed to test for anthelmintic efficacy between treatment means at a 5% significance level (P < 0.05).

Results

Methanol root extract exhibited higher yield (18.00%), followed by water (12.95%) and ethanol (9.91%) as shown in Table 1. The results of the quantitative phytochemical screening showed that E. elephantina root contains high amounts of alkaloids, flavonoids, phenols, saponins and condensed tannins (Fig. 1, Table 2). Higher alkaloid content was present in the water extract (93.24 ± 1.68%), followed by ethanol and methanol extracts (52.33 ± 1.38 and 34.77 ± 0.07%, respectively). There were significant differences in the saponin contents among the ethanol, methanol and water extracts at a 5% level (P < 0.05). Saponin content was highest in ethanol extract (83.28 ± 1.72%), followed by methanol extract (63.74 ± 0.75%) and water extract (50.83 ± 0.43%). Total condensed tannins in the roots were found to be (450.52 ± 0.15 mg CE/g) in ethanol extract, (354.53 ± 1.03 mg CE/g) in methanol extract and (41.31 ± 0.33 mg CE/g) in the water extract. A positive correlation was shown between standard catechin concentration and the absorbance values (R2 = 0.9986). Fisher's LSD analysis showed a significant difference in phytochemical quantities among the three solvents, which showed that ethanol had higher condensed tannin content than methanol and water extracts. The content of flavonoids was found to be high in ethanol (789.76 ± 0.21 mg QE/g) and methanol extracts (803.93 ± 0.13 mg QE/g) and there was no significant difference (P > 0.05) between them. However, the flavonoid content in ethanol and methanol extracts were significantly different (P < 0.05) from the water extract which contained lower content of flavonoids (403.44 ± 0.19 mg QE/g). A significant positive correlation was observed between standard quercetin concentration and the absorbance values (R2 = 0.998). The ethanol extract had the highest phenol content (476.11 ± 0.37 mg GAE/g), followed by methanol extract (462.86 ± 0.31 mg GAE/g) and lastly the water extract (359.91 ± 0.24 mg GAE/g). There was a significant difference (P < 0.05) between phenol contents in ethanol, methanol and water extracts. A positive correlation was observed between standard gallic acid concentration and the absorbance values (R2 = 0.9981).

Table 1

Percentage yield of extracts in ethanol, methanol and water solvents.

Solvent	Initial weight of the powder (g)	Final weight of the powder (g)	Weight of the crude extract (g)	Yield of crude extract (%)
Ethanol	20	18.019	1.981	9.91
Methanol	20	16.401	3.599	18.00
Water	20	17.41	2.59	12.95

Fig. 1

Phytochemical constituents in the ethanol, methanol and water extracts of E. elephantina roots. Results are expressed as means ± SD of three replicates. Bar graphs with dissimilar letter superscript in the same constituent are significantly different (P < 0.05).

Table 2

Content of alkaloids and saponins in extracts of E. elephantina.

Extract	Alkaloids (%)	Saponins (%)
Ethanol	52.33b ± 1.38	83.28a ± 1.72
Methanol	34.77c ± 0.07	63.74b ± 0.75
Water	93.24a ± 1.68	50.83c ± 0.43

Results are expressed as means ± SD of three replicates. Different superscripts within a column represent significant differences at (P < 0.05).

The extracts of E. elephantina showed time and dose-dependent anthelmintic activity on the adult P. cervi. Generally, all E. elephantina extracts showed anthelmintic effects against the adult worms (Table 4; Fig. 2, Fig. 3, Fig. 4, Fig. 5, respectively). The extracts of E. elephantina roots significantly inhibited adult worm motility over the 16 h treatment period (P < 0.05). The positive control (Valbantel) showed 100% motility inhibition within 10 h while the negative control (PBS) showed 100% motility. Ethanol extracts showed a highly significant (P < 0.05) inhibition of motility (86.67 and 96.67%) at concentrations 7.5 and 15 mg/mL respectively, after 12 h of treatment. However, there was no significant difference in the inhibition of motility between ethanol (15 mg/mL) and the positive control; while ethanol (7.5 mg/mL) was significantly different from the positive control at 12 h post-exposure. Methanol and water extracts exhibited significantly different (P < 0.05) motility inhibition (96.67 and 66.67%, respectively) at concentration of 15 mg/mL 16 h post-exposure. There was no significant difference in the inhibition of motility by methanol (15 mg/mL) and positive control at 16 h post-exposure (P > 0.05).

Table 4

In vitro effect of E. elephantina extracts on adult P. cervi in comparison with Valbantel.

Treatment	Conc(mg/mL)	Motility inhibition (%) of adult P. cervi worms at different hours (Mean ± SD)
		0	2	4	6	8	10	12	14	16	18
Ethanol	1.875	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	90.0be ± 0.00	90.0bg ± 0.00	76.7ae ± 15.28	40.0efg ± 10.00	33.3bcd ± 15.28	3.3c ± 5.77
	3.75	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	90.0a ± 10.00	86.7ae ± 11.55	83.3agh ± 15.28	66.7be ± 5.77	26.7fgh ± 20.82	26.7bcd ± 15.28	0.0c ± 0.00
	7.5	100.0a ± 0.00	100.0a ± 0.00	93.3ac ± 5.77	93.3ad ± 5.77	80.0be ± 10.00	70.0ch ± 10.00	13.3c ± 5.77	6.7h ± 5.77	3.3d ± 5.77	0.0c ± 0.00
	15	100.0a ± 0.00	100.0a ± 0.00	86.7ac ± 5.77	70.0be ± 10.00	50.0c ± 10.00	30.0e ± 10.00	3.3cg ± 5.77	0.0i ± 0.00	0.0d ± 0.00	0.0c ± 0.00
Methanol	1.875	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	96.7a ± 5.77	96.7a ± 5.77	96.7a ± 5.77	80.0aj ± 10.00	40.0bc ± 10.00	13.3bc ± 5.77
	3.75	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	96.7a ± 5.77	96.7a ± 5.77	93.3ag ± 0.5.77	86.7ae ± 5.77	70.0bj ± 10.00	33.3bcd ± 15.28	6.7bc ± 5.77
	7.5	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	90.0a ± 10.00	86.7ae ± 5.77	76.7agh ± 20.82	70.0bfh ± 10.00	40.0efg ± 10.00	10.0d ± 10.00	3.3c ± 5.77
	15	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	83.3ade ± 15.28	80.0be ± 10.00	56.7dh ± 5.77	56.7be ± 11.55	33.3g ± 5.77	3.3d ± 5.77	0.0c ± 0.00
Water	1.875	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	96.7a ± 5.77	96.7a ± 5.77	93.3a ± 5.77	56.7b ± 15.28	20.0b ± 10.00
	3.75	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	96.7a ± 5.77	96.7a ± 5.77	96.7a ± 5.77	86.7a ± 5.77	43.3bc ± 5.77	16.7b ± 5.77
	7.5	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	96.7a ± 5.77	93.3ae ± 5.77	83.3cg ± 5.77	70.0bfh ± 10.00	56.7bef ± 15.28	40.0bc ± 0.00	6.7bc ± 5.77
	15	100.0a ± 0.00	100.0a ± 0.00	96.7a ± 5.77	93.3ad ± 5.77	86.7ae ± 5.77	70.0ch ± 10.00	60.0be ± 10.00	36.7cef ± 15.28	33.3c ± 5.77	0.0c ± 0.00
Positive control (Valbantel)	1.875	100.0a ± 0.00	100.0a ± 0.00	60.0b ± 10.00	20.0c ± 10.00	13.3d ± 5.77	0.0f ± 0.00	0.0dg ± 0.00	0.0i ± 0.00	0.0d ± 0.00	0.0c ± 0.00
Negative control (PBS)		100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	100.0a ± 0.00	96.7a ± 5.77

abcdefghijMeans with the same lowercase letters within the same column are not significantly different (P < 0.05).

Fig. 2

Time- and dose-dependent in vitro anthelmintic activity of ethanol, methanol and water extracts of E. elephantina (at a concentration of 1.875 mg/mL) in comparison with positive (Valbantel 1.875 mg/mL) and negative (PBS) controls on adult P. cervi.

Fig. 3

Time- and dose-dependent in vitro anthelmintic activity of ethanol, methanol and water extracts of E. elephantina (at a concentration of 3.75 mg/mL) in comparison with positive (Valbantel 1.875 mg/mL) and negative (PBS) controls on adult P. cervi.

Fig. 4

Time- and dose-dependent in vitro anthelmintic activity of ethanol, methanol and water extracts of E. elephantina (at a concentration of 7.5 mg/mL) in comparison with positive (Valbantel 1.875 mg/mL) and negative (PBS) controls on adult P. cervi.

Fig. 5

Time- and dose-dependent in vitro anthelmintic activity of ethanol, methanol and water extracts of E. elephantina (at a concentration of 15 mg/mL) in comparison with positive (Valbantel 1.875 mg/mL) and egative (PBS) controls on adult P. cervi.

Discussion

The findings of this work have shown that the root extracts of E. elephantina exhibited anthelmintic activity against the adult P. cervi motility that was significantly different (P < 0.05) from the positive control (Valbantel). These findings are similar to studies by Maphosa et al. (2010) and Sanhokwe et al. (2016) who reported anthelmintic activity of root fractions of E. elephantina against nematodes. The results of this study also exhibited time and dose-dependent anthelmintic activity which resulted in the inhibition of motility. Thus increasing the concentration of the extracts caused early motility inhibition. The time and dose-dependent anthelmintic effects could be due to more compounds with anthelmintic activity being present at higher concentrations of the extract (Maphosa and Masika, 2012). The ethanol extract was the most effective at the test concentration of 7.5 and 15 mg/mL compared to methanol and aqueous extracts. This could be attributed to other molecules present or the different mechanisms of action against the parasites. Methanolic and aqueous extracts exhibited significantly different (P < 0.05) motility inhibition (96.67 and 66.67%, respectively) at a concentration of 15 mg/mL, 16 h post-exposure. The effect of most worm expellers such as albendazole is to cause worm paralysis leading to expulsion of the worm via faecal matter of the host (Islam et al., 2019). Therefore, the methanolic extract at the test concentration of 15 mg/mL showed a significant anthelmintic activity as compared to the positive control (Valbantel).

In this present study, the anthelmintic effects of E. elephantina could be attributed to one or more of the phytochemicals present in its roots such as alkaloids, condensed tannins, flavonoids, phenols and saponins. However, the precise mechanism of action of these phytochemicals against gastrointestinal nematodes is not well understood as their effects might be additive, synergistic, or antagonistic and act at single or multiple target sites (Wynn and Fougere, 2007; Hernandez et al., 2014). Tannins are postulated to cause anthelmintic activity by either binding to the proteins found in the gastrointestinal tract of the host or to glycoprotein on the cuticle of the parasite resulting in the death of the worms (Hoste et al., 2012; Williams et al., 2014). Also, tannins are thought to interfere with energy generation by uncoupling oxidative phosphorylation (Roy et al., 2010; Weldemariam et al., 2015) which can lead to worm death. On the other hand, saponins are reported to disrupt the cell membrane of the parasites thereby changing the morphology of the cells in the cuticle (Hrckova and Velebny, 2013). Saponins may also affect feed intake and nourishment of the parasites, resulting in parasites dying (Hernandez et al., 2014). Alkaloids have been reported to act on the central nervous system and are thought to cause paralysis of worms (Roy et al., 2010; Weldemariam et al., 2015).

Conclusions and recommendations

This study showed that aqueous, ethanol and methanol extracts of E. elephantina root exhibited anthelmintic activity: they inhibited motility in adult P. cervi. Therefore, further research is needed to determine their bioactivities in vivo as E. elephantina could contribute to the development of novel anthelmintic agents. The findings thereof may be useful in developing phytotherapic products that are more cost-effective, safer, and accessible with reduced risk of resistance than the conventional drugs presently in use.