Ovicidal and larvicidal activity of Cassia alata leaf acetone extract and fractions on Haemonchus contortus: In vitro studies

Abstract

Context: The failure of modern anthelmintics to control nematode parasites of sheep and goats is a reality on many farms in the tropical/subtropical regions of the world. This necessitates chemotherapeutic control alternatives and plant secondary metabolite with activity is one of those potential solutions.

Objective: This study was design to evaluate the efficacy of solvent: solvent fractions of Cassia alata Gelenggang Besar (Leguminosae) leaf acetone extract against Heamonchus contortus Rudolphi (Trichostrongylidae).

Materials and methods: C. alata leaf was extracted with 70% acetone and fractions were obtained by solvent: solvent group separation procedures. The acetone extract and the fractions were tested by egg hatch assay (EHA) and larval development and viability assay to assess relative bioactivity against H. contortus eggs and larvae.

Results: The extracts inhibited egg hatchability and killed infective larvae of H. contortus in a concentration-dependent manner. The best-fit LC₅₀ values were 0.562, 0.243, 0.490, 0.314, and 0.119 mg/mL for the acetone extract, chloroform, hexane, butanol and 35% water in methanol fractions, respectively, when tested against nematode eggs. The best-fit LC₅₀ values were 0.191, 0.505, 1.444, 0.306, and 0.040 mg/mL for acetone extract, chloroform, hexane, butanol and 35% water in methanol fractions, respectively, when tested against larvae. The 35% water in methanol fraction was the most active against the larvae and eggs of H. contortus demonstrating the lowest LC₅₀ values

Discussion and conclusion: This study demonstrates that the leaf extracts of C. alata have anthelmintic activity; therefore it could find application in the control of helminths in livestock.

Keywords::

• Cassia alata
• egg hatch assay
• Haemonchus contortus
• larval development assay
• solvent:solvent fractions

Introduction

Helminthosis in livestock are the major constraint of productivity, especially in small ruminants in the tropics and subtropics (Perry et al., 2002). Infections by gastrointestinal helminth parasites of livestock are among the most common and economically important diseases of grazing livestock (Perry et al., 2002). They are characterized by lower outputs of animal products (meat, milk, hides, and skins), manure and traction, which all impact on the livelihood of small holder farmers (Perry & Randolph, 1999). The greatest losses associated with nematode parasite infections are sub-clinical, and economic assessments show that financial costs of internal parasitism are enormous (Preston & Allonby, 1979; McLeod, 1995). However, the highly pathogenic nematode parasite of small ruminants, H. contortus, is capable of causing acute disease and high mortality in all classes of stock (Allonby & Urquhart, 1975).

Livestock producers have derived substantial benefits from the use of anthelmintics in controlling livestock parasitoses. Synthetic anthelmintics are the main way of controlling nematode parasites of livestock today. However, these drugs may not be readily available to smallholder farmers, or to remote pastoralist communities. The misuse of these synthetic anthelmintics has led to the development of anthelmintic resistance (Lans & Brown, 1998). Cassia alata leaf is credited for the treatment of hemorrhoids, constipation, inguinal hernia, intestinal parasitosis, blennorrhagia, syphilis, and diabetes in humans (Adjanahoun et al., 1991). This plant contains many diverse constituents such as alkaloids, lectins, saponins, cyanogenic glycosides, isoflavones, and phytoestrogens (Leeuwenberg, 1987). Many alkaloids are pharmacologically active substances, which possess various physiological activities in humans and animals. The use of alkaloid-containing plants as dyes, spices, drugs, or poisons can be traced back almost to the beginning of civilization. Two glycosides were isolated from the seeds of C. alata: chrysoeriol-7-0-(2″-O-β-d-mannopyranosyl)-β-d-allopyranoside and rhamnetin-3-O-(2″-O-β-d-mannopyranosyl)-β-d-allopyranoside (Gupta & Singh, 1991).

In this study, we investigated the ovicidal and larvicidal activity of an acetone extract of C. alata leaf, and the relative activity of the solvent: solvent fractions against the eggs and larvae of H. contortus; gastrointestinal nematode of sheep.

Materials and methods

Preparation of plant extracts

The leaf of C. alata was collected between February and March 2007 in Zaria, Nigeria. The voucher specimens (No: 2421) were identified by Mallam Musa Abdull and deposited by the Herbarium Section of the Biological Sciences Department, Ahmadu Bello University, Zaria. The leaves of the plant (120 g) were air-dried and ground to powder using a Macsalab Model 200 LAB grinder. The powdered plant material was extracted by maceration with shaking (Labotec Model 20.2 shaker) for 24 h in 70% acetone with a 10:1 solvent to dry weight ratio (Eloff, 1998). The extract was then filtered through Whatman No. 1 filter paper using a Buchner funnel, and the acetone removed under stream of air.

The solvent: solvent group separation procedure used by the USA National Cancer Institute as described by Suffness and Douros (1979) was adopted to fractionate the acetone extract with a slight modification. The acetone extract was dried in a rotary evaporator under reduced pressure and temperature of 40°C. The extract was then dissolved in a 1:1 mixture of chloroform and water. The water fraction was extracted with an equal volume of butanol in a separating funnel to yield the water and butanol fractions. The chloroform fraction was dried in a rotary evaporator under reduced pressure and subsequently extracted with a 1:1 mixture of hexane and 10% water in methanol. The hexane fraction was recovered with a separating funnel. The 10% water in methanol extract was diluted to 35% water in methanol. The 35% water in methanol was then extracted with chloroform to yield the chloroform and the 35% water in methanol fractions.

H. contortus egg recovery

Eggs of H. contortus were recovered according to Hubert and Kerboeuf (1992). Fecal sample (10–15 g) from a sheep experimentally infected with mono-specific larval suspensions of fresh H. contortus was collected. The fecal sample was mixed with distilled water and cleared of organic debris by filtration through 1 mm and 150 μm sieves. H. contortus eggs were collected on a 25 μm sieve. The eggs were further cleared of organic debris by centrifugation in magnesium sulfate (density 1.10) for 5 min at 1000g. The supernatant was filtered through 100 μm and 63 μm sieves and the eggs were washed in water and collected on a 25 μm sieve. The number of eggs in 200 μL samples was estimated and adjusted to 500 eggs/mL. Amphotericin B (Sigma, Germany) was added to the egg suspension (5 μg/mL) to avoid fungal development.

EHA

The in vitro egg hatch assay (EHA) was conducted according to the method described by Coles et al. (1992). Egg suspension (0.2 mL) was distributed in a 48-flat-bottomed microtitre plate containing approximately 100 fresh eggs/well and mixed with the same volume of plant extract dissolved in acetone at concentrations of 10 mg/mL in eight serial dilutions. Albendazole (Sigma, USA) (99.8% pure standard reference) was used as a positive control. The albendazole was dissolved in dimethyl sulfoxide (0.3% DMSO) and diluted at the concentrations of between 1 and 0.0075 μg/mL. The control plates contained the diluents, water and acetone or 0.3% DMSO and the egg solution. The experiment was replicated three times for each concentration. The egg suspensions were incubated for 48 h at 27°C and 70% relative humidity. After this time a drop of Lugol’s iodine solution (Reidel de Hae, Germany) was added to stop the eggs from hatching. All the eggs and first-stage larvae (L₁) in each plate were counted under an inverted microscope.

Larval development assay

The procedures used were a modification of the method described by Hubert and Kerboeuf (1992). Aliquots of 150 μL egg suspension which contained approximately 100 eggs and 20 μL of filtrate obtained by fecal washing during egg recovery were distributed to wells of a 48-well flat-bottomed microtiter plate. The egg suspension was supplemented with 30 μL of the nutritive medium described by Hubert and Kerboeuf (1984) and it comprised of Earle’s balanced salt solution (Sigma, Germany) and yeast extract (Sigma, Germany) in saline solution (1 g of yeast extract/90 mL of saline solution) at a ratio of 1:9 v/v. The plates were incubated at 27°C and 70% relative humidity. After 48 h, 200 μL of the extract, albendazole (Sigma, USA) or diluents (control) were added. The plates were incubated for a further 6 days. At this time the parasites were counted under an inverted microscope. The larvae were separated into two classes, third-stage larvae (L₃) and other developmental stages larvae (L₁ and L₂). Each concentration and control was replicated three times.

Statistical analysis

The LC₅₀ values were determined by computing the concentration of extract and fractions that gave a response halfway between the minimum and maximum responses in a concentration-response sigmoid curve. The relationship below was used to calculate the egg hatch and larval development parameters, respectively:

The LC₅₀ of a sigmoidal concentration response (variable slope) curve was determined using GraphPad Prism version 4.01 for Windows (GraphPad, San Diego, CA). Global curve-fitting model of nonlinear regression was used to analyse the family of data sets generated by the four solvent: solvent fractions with top and bottom shared among the data sets. The bottom of the curve was also constrained as >0 and the top was constrained as <1.0. A (global) best-fit value that applies to the family of data sets was computed for each of these shared parameters, while the best-fit LC₅₀ value (unshared parameter) was calculated with 95% confidence interval for each of the data sets (fractions). One-way ANOVA and Tukey’s multiple comparison test was used to assess the relative bioactivity of the fractions by comparing the best-fit LC₅₀ value of the various fractions. This was performed using GraphPad Prism version 4.01 for Windows according to Ademola et al. (2005).

Results

Yield of extract and fractions

The acetone extract gave a yield of 11.80 g (9.83% w/w), whereas the hexane, chloroform, butanol and 35% water in methanol fractions of the acetone extract gave a yield of 3.49 g (39.08%), 0.817 g (9.15%), 0.64 g (7.14%), and 0.65 g (7.27%), respectively.

EHA

Both the acetone extract and the fractions of C. alata demonstrated an inhibitory effect on the hatching of eggs in a concentration-dependent manner (Figure 1). The 35% water in methanol fraction produced the lowest LC₅₀ value (0.119 mg/mL), indicating that it is more active than the other fractions, although not to a significant level (p > 0.05). The solvent used in the control did not inhibit egg hatching. Albendazole produced an LC₅₀ at a low concentration (0.164 μg/mL), indicating that the strain of H. contortus used in the current study was resistant. Taylor et al. (2002) reported that when the concentration of drug require to inhibit hatching of 50% of eggs is > 0.1 μg/mL it is an indication of resistance.

Figure 1.  Egg hatch assay concentration-response curve of acetone extract and fractions of C. alata, and Albendazole against eggs of H. contortus using global sigmoidal model of curve fitting (C: chloroform; H: hexane; B: butanol; MW: 35% water in methanol).

Larval development and viability assay

The acetone extract and the solvent: solvent fractions killed the infective stage larvae (L₃) of H. contortus (Figure 2). The 35% water in methanol was the most active fractions with an LC₅₀ value of 0.0404 mg/mL. However, it was not significantly more active than butanol fraction with an LC₅₀ value of 0.088 mg/mL (p > 0.05). The solvent used in the control did not affect larval viability. Tukey’s multiple comparison (post ANOVA) test shows that the 35% water in methanol fraction (LC₅₀ = 0.040 mg/mL) is significantly more active than chloroform and hexane fractions (p < 0.01). Albendazole produced an LC₅₀ at low concentration (0.061 μg/mL), indicating that the larvae of the strain of H. contortus used in this study was susceptible.

Figure 2.  Larval development and viability assay concentration-response curve of acetone extract and fractions of C. alata, and Albendazole against larvae of H. contortus using global sigmoidal model of curve fitting (C: chloroform; H: hexane; B: butanol; MW: 35% water in methanol).

Discussion

The 35% water in methanol fraction gave the lowest LC₅₀ with the egg hatch inhibition and larval viability test (LC₅₀ = 0.119 and 0.0404 mg/mL, respectively). However the LC₅₀ is high compared with albendazole (LC₅₀ = 0.164 and 0.061 μg/mL, respectively). This shows that the level of activity of the extract and fractions are significantly (p < 0.01) lower than the activity of albendazole. This significant difference can be explained by the presence of small concentrations of the active ingredient in the plant extracts, among these the compound with ovicidal and larvicidal activity, unlike synthetic anthelmintics, where the chemical compounds are isolated in pure forms (Rates, 2001).

The higher levels of activity observed in the 35% water in methanol fraction suggest that the anthelmintic component of C. alata is a relatively polar compound. Tukey’s multiple comparison (post ANOVA) test suggested a significant (p < 0.01) difference in the activity of 35% water in methanol compared with the chloroform and hexane fractions when tested on H. contortus larvae. However, while the 35% water in methanol fraction showed better activity against H. contortus eggs compared with other fractions, these differences were not significant.

The activity of the acetone extract of C. alata with LC₅₀ values of 0.562 and 0.191 mg/mL against nematode eggs and larvae, respectively, is comparable to that obtained for the ethanol extract of Spigelia anthelmia on the eggs (LC₅₀: 0.625 mg/mL) (Ademola et al., 2007). An in vitro test for anthelmintic activity against Ascaris suum showed that a chloroform alkaloidal extract of Leucaenia leucocephala gave a comparable result to mebendazole at a concentration of 5 mg/mL (Irene, 1998). The partially purified C. alata extracts exhibited a relatively high antifungal activity against mycelial growth with total suppression of sporulation for 4 days at a concentration of 2 mg/mL, while preventing fungal growth after the seventh day (Adedayo et al., 1999). The acetone and ethanol extract of C. alata also showed high activity against Staphylococcus aureus, S. aureus coagulase positive, Bacillus. subtilis, B. cereus, B. stearothermophillus, Escherichia coli, Vibrio cholerae, Salmonella typhi, Shigella dysenteriae, and Klebsiella pneumoniae (Sakharkar & Pati, 1998).

Some anthelmintics act by paralysing the helminth (such as cestodes), which then may have to be expelled by a purge; others destroy the parasites through lysis, because they contain proteolytic enzymes such as bromelain [Ananas comosus (L.) Merr.], calotropain [Calotropis procera (Aiton) W.T. Aiton] and pawpaw (Carica papaya L.) (Stepek et al., 2004). Many constituents with antibacterial activity also have anthelmintic properties and alkaloids are often the active constituents.

A large number of the biological effects of saponins have been ascribed to their action on membranes. In fact, their specific ability to form pores in membranes has contributed to their common use in physiological research (Menin et al., 2001; Plock et al., 2001). Saponins have long been known to have a lytic action on erythrocyte membranes and this property has been used for their detection. Saponins could induce a permeability change on the liposomal membrane without cholesterol when they are glycosylated at both the C3 and C28 (bidesmosidic) of the oleanolic aglycone (Hu et al., 1996). Therefore either of these constituents found in C. alata could be responsible for the activity or a synergistic effect of the constituents.

Although the use of plant extracts as phytomedicines is becoming increasingly popular as alternative to the use of synthetic single molecule drugs, accurate knowledge of the composition is still warranted.

Conclusions

The crude methanol extract of the powdered leaf of C. alata may be a useful and effective phtyomedicine for controlling nematodes in livestock production. Further in vitro and in vivo investigations into the isolated fractions should be conducted to investigate the potential therapeutic use of this plant. The active principles that induced the observed anthelmintic activity may be found in one or more classes of chemicals in C. alata.

Acknowledgement

The authors wish to express gratitude to the National Research Foundation South Africa for providing the funds for this study. Onderstepoort Veterinary research Institute, South Africa and Dr A.F Vatta are thanked for the provision of facility and technical assistance. The authors would like to thank Dr. F. van Schalkwyk, Biozetica Agri-source (Pty) Ltd., South Africa for supplying the H. contortus larvae.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.