Ovicidal effect of ethanolic extracts of selected plants on eggs of liver flukes in vitro

Twenty-nine plant samples (Table 1) were selected based on previous reports of their bioactivity against parasites (Ahmed et al., 2012; Fomum, 2018). Nine were collected from the University of KwaZulu-Natal (UKZN) Botanical Garden, Pietermaritzburg (KwaZulu-Natal Province), latitude 29.37 S, longitude 30.24 E, 655 meters above sea level. Eighteen plants were collected from a private garden in Pietermaritzburg, South Africa. Allium sativum bulbs and Syzygium aromaticum (clove) were purchased from a local market. All plants used were identified by the Department of Botany, UKZN. Voucher specimens of plants were deposited at the UKZN Herbarium, Pietermaritzburg and were assigned voucher numbers. Plant samples were washed and cut into small pieces, air dried under shade, and then dried in an oven (Model 5SOE1B, Labcon, Maraisburg, South Africa) at 50°C for 3 – 5 days. All plant samples were ground into a fine powder using an electric grinder (Retsch Haan, Germany). Crude powders fitted with a 1 mm diameter sieve, then preserved in airtight plastic containers, labeled and stored at 4°C until used in extract preparation. The same plant samples were used for all subsequent experiments.

Plant extraction techniques were carried out in the Department of Animal and Poultry Science, School of Agricultural, Earth, and Environmental Sciences (SAEES), UKZN. Extraction was carried out using ethanol as the solvent, which has been consistently proven to be the most effective solvent for extracting potentially bioactive compounds (McGaw & Eloff, 2008; Ahmed et al., 2012). Extraction was performed following (Ahmed et al., 2012). Ten grams of each plant sample were boiled in 100 mL of 80 % ethanol for 24 hours using a Soxhlet extractor. Each extract was transferred to a beaker and placed in a water bath (Model 101, Labotec, South Africa) at 60°C. Extracts were evaporated till dry. The dried extracts were stored in sealed glass vials in the dark at 4°C until tested for antihelmintic efficacy. The dried extracts were reconstituted in 80 % ethanol to bring up the concentration required for a specific assay.

Seventy-two fecal samples from randomly selected cattle from three different areas in Pietermaritzburg, KwaZulu-Natal Province, Impendle, (latitude 29.599696 S, longitude 29.867056 E, 6000 meters above sea level), the Cedara Research Institute (latitude 29.32 S, longitude 30.16 E, 1085 meters above sea level) and Mpumuza, (latitude 29.62 S, longitude 30.38 E, 596 meters above sea level). Twenty-four fecal samples were collected from naturally infected cattle (1 – 4 years old) in each area. All experimental animals were allowed to graze freely on planted Kikuyu pasture (Pennisetum clandestinum Hochst. ex Chiov.) under the same conditions as other animals. The experimental animals were also treated with an antiparasitic drug, following the deworming protocol used in each farm. Rectal fecal samples were taken by hand and stored in plastic bags. Samples were transported to the Department of Plant Pathology, UKZN, where egg samples were prepared and treated.

For each sample, 100 g of feces was weighed and placed in a 1000 ml beaker filled with 500 ml distilled water. The contents were mixed thoroughly using a glass rod and poured through a tea strainer to remove large debris. The solution was poured into a 1000 ml conical flask, and the suspension was sedimented overnight. The supernatant was then removed. To obtain pure eggs, samples were passed twice through a 100 mm diameter sieve (330 mm pore size).

Egg samples were stored in sterilized sealed bottles at 4°C for further use.

Ethanolic extracts of 29 plants were evaluated for their ovicidal efficacy against eggs of liver flukes at a 20 % concentration of the raw extract. Treatments were prepared by pipetting 125μl from the egg samples (containing approximately 10 eggs) into 96 wells of cell culture plate. From each plant extract, 125μl was added to the egg samples (3 replicates each). Two controls were undertaken: one treated with ethanol and one with distilled water. To determine the ovicidal activity of each plant extract, the treated eggs were incubated at 28°C for 15 days. Miracidial formation was observed under a light microscope using a 10-magnification.

The experiment was arranged in a randomized complete blocks design (RCBD).

The five plant extracts that caused the highest mortality levels in the primary bioassay were selected to evaluate the effect of concentrations on their efficacy. All samples were then used at three concentrations, 5, 10 and 20 % of the raw extract. Each concentration was used to treat fluke egg samples. As in the first part of the experiment, ethanol and distilled water are used as controls. The experiment was a 5 × 3 factorial arranged in a randomized complete block design (RCBD).

Control mortalities were zero in both experiments. Data was subjected to analysis of variance (ANOVA) using GenStat for Windows, 18th edition (Payne et al., 2014). Means were compared using Fisher’s Least Significant Difference at a 5 % significance level.

There were highly significant differences in the effects of plant extracts in their effects on eggs of liver flukes of cattle at a 20 % concentration (F= 320.08; P˂0.001) (Table 2). Mortality levels ranged from 0 to 100 % (Fig. 1). Extracts of two plants, M. oleifera and A. comosus, caused the highest mortality levels of 100 %. Extracts of three plants, F. vulgare, C. moorei and Plectranthus sp2, caused mortality levels higher than 80 %. Extracts of three plants, C. nardus, A. afra and W. salutaris, caused 50 to 62 % mortality levels. Extracts of the remaining 24 plants caused mortality levels of less than 50 % (Fig. 1).

Fig. 1.

The effects of ethanolic extracts of 29 plants on the mortality of eggs of liver flukes from cattle, observed for 15 days after treatment. Mortality was zero in the control treatment.

There were highly significant differences between the plant extracts (F=67.51; P˂0.001) and between the three concentrations (F=72.26; P˂0.001). The interaction between plant extracts and concentrations was also significant (F=3.26; P˂0.005) (Table 3). The five selected plant extracts were effective at the three concentrations of 5, 10 and 20 % against the eggs of liver flukes. Mortality levels ranged from 30 – 82.67 % due to the 5 % extracts, 42 – 90 % for the 10 % extracts and 60 – 100 % for the 20 % extracts. Extracts of M. oleifera and A. comosus caused more significant mortalities than the other plant extracts at the 5, 10 and 20 % concentrations (Table 4). The concentration of 20 % was consistently more effective than 5 or 10 %. A relatively constant performance ranking resulted in straight-line regressions for efficacy × concentrations (Fig. 2).

Fig. 2:

In vitro efficacy of three concentrations of five ethanolic plant extracts against eggs of liver flukes from cattle. 5% versus 10%: y = 13.8x + 27.2 R² = 0.9115; 5% versus 20%: y = 11.8x + 46.8 R² = 0.8808 ▼– five plants at 20% concentrations; – five plants at 10% concentrations; ● – five plants and bromelain at 5% concentrations