Efficacy of aqueous extracts of Genipa americana L. (Rubiaceae) in inhibiting larval development and eclosion of gastrointestinal nematodes of sheep

Abstract

The anthelminthic efficacy in vitro of the aqueous extract of Genipa americana L. leaves was evaluated at final concentrations of 20, 30, 50, 75 and 100 mg (dry matter)/mL through egg hatching inhibition (EHI) tests and quantitative cultures. The observed lethal concentration LC₉₀ values for hatching and L3 development inhibition were 79.8 and 28.7 mg/mL, respectively. The extract was more effective in larval development inhibition (LDI) than in hatching inhibition. Phytochemical analysis revealed tannins and flavonoids in the extract. The utilisation of G. americana as an anthelminthic may represent a viable alternative to synthetic products, although further studies are needed to verify its in vivo efficacy and to assess its toxicity in sheep.

Keywords:

• anthelminthic
• quantitative coproculture
• egg hatching inhibition
• phytotherapy
• small ruminants

1. Introduction

Demand for ovine meat has grown, mainly in large urban centres, and sheep farmers have endeavoured to optimise production. For success, it is important to ensure the health of animals. Haemonchosis is a major disease in sheep, affecting both sexes at all age levels, reducing weight gain and reproductive capacity, as well as milk, wool and hide production (Pires et al. 2000; Bizimenyera et al. 2006).

Gastrointestinal nematode (GIN) control has relied mainly on frequent use of commercial anthelminthic drugs. However, the emergence of drug resistant GIN populations (Jackson & Coop 2000; Melo & Bevilaqua 2002) has stimulated research into alternative approaches, including phytotherapy (Githiori et al. 2006).

The efficacy of anthelminthic compounds must be preserved through judicious use. Research into new anthelminthic molecules that may be used along with current drugs is laborious and costly, and the time span from trials to commercial availability is lengthy (Hennessy 1997). Research into alternatives for the control of GIN is essential.

Plant secondary compounds can be used as alternatives to control GINs in ruminant livestock (Kahn & Diaz-Hernandez 2000). However, scientific validation of the anthelminthic effects and investigation into possible side effects are necessary prior to the adoption of plant extract therapies (Githiori et al. 2006).

The genipap (Genipa americana L.) is a rubiaceous tree that has been used in folk medicine, foods and animal feed, leather tanning, forestry and logging industries. The species, native to South America, has ecological importance for feeding animals, and is suitable for planting in degraded areas and wetlands (Epistein 2001).

The use of medicinal plants for nematode control can reduce the presence of chemical residues in animal products and the development of nematodes with anthelminthic resistance (Hammond et al. 1997). The utilisation of these extracts for the reduction of anthelminthic-resistant populations of ovine nematodes may constitute a promising strategy in herds with high frequency of anthelminthic multiresistance (Nery et al. 2010). Although G. americana is not included as an anthelminthic in folk use, it is a plant rich in tannins (Hernes & Hedges 2004) and therefore, the goal of the present study was to evaluate in vitro the anthelminthic efficacy of G. americana on the inhibition of larval development and eclosion for GIN-infecting sheep.

2. Materials and methods

Leaves, flowers and fruits of G. americana were collected in a rural region of the Cerrado, Montes Claros City, northern Minas Gerais, Brazil (16°55′18″S 43°52′11″W), in January 2008, during the morning. Identification was based on Almeida et al. (1998) and Lorenzi (1998). Specimens were deposited at the herbarium of the Montes Claros State University in Brazil, number HMC 1490.

Healthy leaves were selected and dried to constant weight in a forced air circulating drier at 40 ± 5°C. Dried leaves were ground and stored at 5 ± 3°C until use. Extracts were obtained using the method of Nery et al. (2010) with modifications. Ground dried leaves were incubated in a distilled water bath at 60°C for 60 min and hot filtered through a gauze funnel. The aqueous extract was again dried in a forced air oven at 40°C.

Tests to determine the main secondary metabolites in the extracts were conducted using the colorimetric method described by Matos (1997) and Maciel et al. (2006). Aliquots of the aqueous extract were diluted in distilled water. Tannins were identified with lead acetate, copper acetate and glacial acetic acid reactions. Phenols were identified by the ferric chloride test and flavonoids by the Shinoda method, ferric chloride and sodium hydroxide tests. Steroids and terpenoids were confirmed by the Lieberman-Burchard reaction; alkaloids using Dragendorff, Mayer and Bourchard reagents; and saponins using the foam test (Matos 1997; Maciel et al. 2006).

2.1. In vitro egg hatching inhibition test

The dry extract was dissolved 1:3 in distilled water at 40°C for 30 min. The mixture was filtered through gauze and cotton and stored at 4°C until use. Five aliquots of this solution were performed to dry matter determination at 105°C. The stored leaf aqueous extract solution was standardised at 200 mg of dry matter/mL and diluted in sterile distilled water to final concentrations of 40, 60, 100 and 150 mg/mL. Extract solutions were used immediately in the egg hatching inhibition (EHI) tests with four replicates (Coles et al. 1992).

Sedimentation in water, filtration and flotation in saturated saline were used to obtain nematode eggs from faeces of naturally infected Santa Inês ewes with an average faecal egg count of >300/g, determined by the modified McMaster technique (Gordon & Whitlock 1939).

Experimental mixtures comprised 300 µL faecal suspension with an average of 600 fresh eggs combined with either 300 µL of the extract concentrations, 300 µL sterile distilled water for the negative control or 300 µL of ivermectin suspension (16 µg/mL) as positive control. All experimental mixtures were homogenised and incubated in a BOD incubator at 28°C for 48 h. Subsequently, 15 µL Lugol's solution was added to each tube, which were stored at 4°C for counting of unembryonated and embryonated eggs and first stage larvae (L1; Coles et al. 1992).

The number of L1 relative to the total number of eggs plus L1 was determined for each repetition and subjected to variance analysis. The means were compared using the Scott–Knott test at 5% significance. Probit regression was employed to determine the concentrations sufficient to inhibit 50% (lethal concentration, LC₅₀) and 90% (lethal concentration, LC₉₀) of egg hatching using the statistical package, Saeg 9.1 (2007). The formula of Coles et al. (1992) was used to determine the EHI effectiveness:

2.2. In vitro larval development inhibition

Faeces of five naturally infected Santa Inês crossbred lambs, age six to eight months and producing >500 eggs/g of faeces were used. The leaf extract solution was prepared as described, standardised at 200 mg/mL, and diluted in sterile distilled water to 40, 60, 100 and 150 mg/mL. The extracts were used immediately in a larval development inhibition (LDI) test in quantitative culture (Borges 2003; Nery et al. 2010).

Unpreserved faeces collected directly from the rectal ampulla of each animal were pooled and divided into 2 g samples distributed among clean disposable cups. Two mL of each extract, 2 mL of ivermectin solution (Ranger LA, Vallée, Montes Claros, MG, Brazil) equivalent to 16 µg/mL (positive control), or 2 mL sterile distilled water (negative control) were added to the faeces. Each concentration and the controls were evaluated in four replications

On day 7 of the culture, the nematode larvae were collected in a test tube and held at ∼4°C until counting. To identify the genus, slides were prepared with Lugol's iodine, and identified using the key by Keith (1953). The number of L3 was divided by two to give the number of L3 per gram of faeces (LPGF). The following formula, adapted from Borges (2003), was used to determine the percent reduction in larva numbers per gramme of faeces:

The data were transformed, log (x + 1), and submitted to variance analysis. The means were compared by the Scott–Knott test and 5% probability calculated. The LC₅₀ and LC₉₀ were determined by probit analysis using the statistical package Saeg 9.1 (2007).

Experimental procedures with sheep were carried out in accordance with the Experience Ethical Committee of Minas Gerais Federal University CETEA – UFMG and approved by this committee under protocol number 042-2008.

3. Results and discussion

The phytochemical analysis of aqueous extract confirmed the presence of flavonoids and tannins. The addition of glacial acetic acid to lead acetate indicated the predominance of soluble tannins, since this reaction is specific to hydrolysable tannins.

The tannins and flavonoids are secondary metabolites with therapeutic potential. A recent study showed that a flavonoid-containing aqueous extract of immature mango is active against Haemonchus contortus (Nery et al. 2012). Tannins can complex with proteins, reducing available amino acids and resulting in larval death by starvation, or can bind to the glycoprotein-rich cuticle of the larvae, causing death (Athanasiadou et al. 2001). Reduction in the number of nematode eggs in faeces of sheep fed tanniferous plants has been shown by Marley et al. (2003), Lange et al. (2006) and Minho et al. (2008). Other hypotheses may explain the in vivo anthelmintic action of tannins. These compounds can bind dietary proteins and protecting them from ruminal degradation, increasing protein flux and amino acid absorption in the small intestine, favouring the immune response against the worms (Hoste 2006). Tannin intakes can have negative or positive effects on animal productivity (Frutos et al 2004), depending on which and how much is consumed the compound's structure and molecular weight, and on the physiology of the consuming species (Hagerman & Butler 1991). Thus, studies of the tannin characterisation and toxic analysis of acute or chronic ingestion be conducted carefully for treatment of helminthiasis in ruminants.

In the EHI test, the aqueous extract at 100 mg/mL completely inhibited hatching. The relative mean number of embryonated eggs was significantly greater than unembryonated eggs at 75 and 100 mg/mL (P < 0.05). This suggests greater efficacy in inhibiting hatching than in interfering with embryo development (Table 1). Probit analysis showed the LC₅₀ and LC₉₀ of aqueous extract of G. americana leaves to be 34.3 and 79.8 mg/mL, respectively. Culture and identification showed 99.8% of the L3 larvae to be Haemonchus spp., and 0.2% were Trichostrongylus spp.

Table 1. Efficacy of aqueous extracts of G. americana leaves on EHI of nematodes from sheep.

Concentrations (mg/mL)	Unlarvaed egg (mean)	Larvaed egg (mean)	L1 (mean)	Total eggs + L1	Efficacy* (%)
100	162.5	662.5	0.0	825.0	100.0a
75	90.0	447.5	100.0	637.5	84.3b
50	72.5	85.0	308.0	465.5	40.1c
30	50.0	52.5	357.5	460.0	33.2c
20	112.5	110.0	292.5	515.0	43.2c
Water	0.0	0.0	360.0	360.0	0.0d
Ivermectin	450.0	0.0	157.5	607.5	74.1

Note: Different lower case letters indicate significant differences. (P < 0.05) in Scott Knott test. Coefficient of variation = 30.4%.

* Efficacy (%) = 100 × (1–mean of L1/ mean eggs + L1).

In the LDI test, concentrations ≥ 30 mg/mL showed anthelminthic efficacy above 94% (Table 2). Based on probit analysis, the LC₅₀ and LC₉₀ were 14.6 and 28.7 mg/mL, respectively. The L3 larvae were 84.7% Haemonchus, 13.3% Trichostrongylus and 2% Strongyloides. This suggests that the extracts were effective against several nematodes considered to be the most prevalent and pathogenic in sheep (Wood et al. 1995), showing a wide spectrum of action.

Table 2. Mean nematode L3 count/g of faeces (LPGF) in coprocultures treated with aqueous extract of leaves of G. americana and efficacy in larval development inhibition.

Concentrations (mg/mL)	Mean viable (LPGF)	Efficacy* (%)
100	0 ± 0.0c	100.0
75	1 ± 0.5c	99.6
50	1 ± 0.5c	99.6
30	4 ± 0.53b	94.5
20	19 ± 28.3b	74.9
Water	78 ± 34.6a	–
Ivermectin	0 ± 00c	100.0

Note: Different lower case letters indicate significant differences. (P < 0.05) in Scott–Knott test. Coefficient of variation = 23.5%.

* Efficacy (%) = 100 × (1 LPGF of treated group/LPGF of negative control).

Both the EHI and LDI showed the G. americana extract to have anthelminthic activity, suggesting action on two life stages of nematodes. The presence of tannins and flavonoids was detected in the leaf extract. Simões et al. (1999) stated that possible interaction and synergism between the active vegetal metabolites should not be ignored.

The 90% lethal concentrations for inhibiting larval development and hatchability were 28.7 and 79.8 mg/mL, respectively. These data suggest higher efficacy of the aqueous extract for inhibition of larval development than for inhibition of eclosion. However, using the hydro-alcoholic extract of leaves of genipap, Krychak-Furtado (2006) found 100% efficacy for EHI at 50 mg/mL, suggesting that the metabolites extracted with alcohol could also show action against nematode eggs.

Other in vitro studies have shown promising results for vegetal extracts. In tests of anthelminthic activity of Melia azedarach leaves, the aqueous and hydro-alcoholic extracts at 12.5 mg/mL inhibited 99.4% and 100% of egg hatching, respectively (Kamaraj et al. 2010). Ethanolic and dichloromethane extracts of Phytolacca icosandra produced in vitro activity against the H. contortus greater than 90% in EHI tests when used at concentrations of 0.90 mg/mL or higher (Hernández-Villegas et al. 2011).

The present study used the modified coproculture test in which the extract was added to the faeces, the natural environment for incubation and development of larvae, increasing the accuracy and precision of the method. Results from applying the extract directly to the faeces cannot be accurately extrapolated to the treatment of animals via ingestion of the plants. However, as reported in previous studies, the modified coproculture test may better approximate treatment by ingestion than does the in vitro EHI test, which is conducted in distilled water (Nery et al. 2012; Nogueira et al. 2012).

Using the coproculture quantitative test, Nery et al. (2010) reported that the aqueous extract of Anacardium humile leaves at 150 mg/mL was 97.3% effective, and the ethanol extract at 80 mg/mL was 99.6% effective, in LDI. In similar research, the LC₉₀ for LDI of aqueous extract of Caryocar Brasiliense was 53.19 mg/ml. (Nogueira et al. 2012) and the fresh juice of immature mangos showed greater than 95% efficacy in LDI at 44.4 mg/mL (Nery et al. 2012), Using conventional in vitro test, aqueous extracts of Leonotis ocymifolia, Leucas martinicensis, Albizia schimperiana and Senna occidentalis induced more than 96% efficacy at 50 mg/ml (Egualea et al. 2011). Comparing these results, in this study the LC₉₀ for LDI was only 28.7 mg/mL, suggesting the extract of G. americana as more effective for inhibiting the development of larval nematodes of sheep than these other plants.

In addition, the production of an aqueous extract of genipap leaves is accessible by sheep farmers in tropical regions, which could lower costs and reduce risks posed by chemicals to animals, humans and the environment. These results, together with future studies evaluating the acute and chronic toxicity in sheep and in vivo efficacy, may contribute to the use of this plant species as an alternative to synthetic anthelminthics.

4. Conclusion

The aqueous extract of G. americana leaves is effective for both inhibition of larval development and hatchability of GIN in sheep. The LC₉₀ for inhibition of larval development is 28.7 mg/mL and the extract is more effective against larvae than eggs.

Acknowledgements

We thank the Banco do Nordeste of the Brazil for providing financial support. The authors are also grateful to Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior CAPES, Brasília, DF, Brazil, Pro-Reitoria de Pesquia da Universidade Federal de Minas Gerais and the Fundação de Amparo à Pesquisa de Minas Gerais FAPEMIG.