Anthelmintic activity of Trianthema portulacastrum L. and Musa paradisiaca L. against gastrointestinal nematodes of sheep

Veterinary Parasitology 179 (2011) 92–99

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Anthelmintic activity of Trianthema portulacastrum L. and Musa paradisiaca L. against gastrointestinal nematodes of sheep Altaf Hussain ∗ , Muhammad Nisar Khan, Zafar Iqbal, Muhammad Sohail Sajid, Muhammad Kasib Khan Department of Parasitology, University of Agriculture, Faisalabad 38040, Pakistan

a r t i c l e

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Article history: Received 24 September 2010 Received in revised form 17 February 2011 Accepted 28 February 2011 Keywords: Anthelmintic activity Haemonchus contortus Trianthema portulacastrum Musa paradisiaca Pakistan Adult motility assay Fecal egg count reduction test

a b s t r a c t Evaluation of anthelmintic effects of Trianthema (T.) portulacastrum L. (Aizoaceae) whole plant and Musa (M.) paradisiaca L. (Musaceae) leaves against prevalent gastrointestinal worms of sheep was done that may justify their traditional use in veterinary clinical medicine. In vitro anthelmintic activity of the crude aqueous methanolic extract (CAME) of both the plants was determined using mature female Haemonchus (H.) contortus and their eggs in adult motility assay (AMA) and egg hatch test (EHT), respectively. In vivo anthelmintic activity of crude powder (CP) and CAME in increasing doses (1.0–8.0 g kg−1 ) was determined in sheep naturally infected with mixed species of nematodes using fecal egg count reduction test (FECRT) and larval counts. The study design also included untreated as well as treated controls. Fecal egg count reduction and larval counts from coprocultures were performed pre- and post-treatments to assess the anthelmintic activity of the plants. CAME of T. portulacastrum and M. paradisiaca showed a strong in vitro anthelmintic activity and pronounced inhibitory effects on H. contortus egg hatching as observed through AMA and EHT, respectively. Both plants exhibited dose and time dependent anthelmintic effects on live worms as well as egg hatching. M. paradisiaca (LC50 = 2.13 ␮g mL−1 ) was found to be more potent than T. portulacastrum (LC50 = 2.41 ␮g mL−1 ) in EHT. However, in vivo, maximum reduction in eggs per gram (EPG) of faeces was recorded as 85.6% and 80.7% with CAME of T. portulacastrum and M. paradisiaca at 8.0 g kg−1 on 15th day post-treatment, respectively as compared to that of Levamisole (7.5 mg kg−1 ) that caused 97.0% reduction in EPG. All the species of gastrointestinal nematodes (GINs), i.e. Haemonchus contortus, Trichostronglyus spp., Oesophagostomum columbianum and Trichuris ovis which were prevalent, found susceptible (P < 0.01) to the different doses of CP and CAME of both plants. The data showed that both T. portulacastrum and M. paradisiaca possess strong anthelmintic activity in vitro and in vivo, thus, justifying their use in the traditional medicine system of Pakistan. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Helminthiasis is considered as a major constraint in livestock productivity round the globe (Githiori et al., 2004). Chemotherapeutics remain the corner stone for treating helminthiasis even though the development of problems, e.g. resistance (Saeed et al., 2007), chemical

∗ Corresponding author. Tel.: +92 323 7673631. E-mail address: [email protected] (A. Hussain). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.02.022

residues and toxicity (Gasbarre et al., 2001), increased cost, non-adaptability of drugs and non-availability in remote areas (Jabbar et al., 2007). However, in developing countries like Pakistan, a huge proportion of farmers rely on alternative control strategies like ethnoveterinary medicine (EVM) as evident from the latest survey conducted by Hussain et al. (2008). A variety of plants have been scientifically validated for their anthelmintic properties in vitro and in vivo (Akhtar et al., 2000; Iqbal et al., 2003, 2004, 2005, 2006a,b,c,d, 2007).

A. Hussain et al. / Veterinary Parasitology 179 (2011) 92–99

Trianthema (T.) portulacastrum L. (Aizoaceae), commonly called “It sit” is a prostrate, glabrous, succulent challenging global weed in cultivated and wastelands recognized by its high caliber of infestation in agricultural and vegetable crops (Balyan and Bhan, 1986). It has been reported for its traditional use as anthelmintic (Hussain et al., 2008) vermifuge and antirhematitis (Shastri, 1952), alexiteric, analgesic and stomachic, laxative (Kirtikar and Basu, 1975). Other medicinal uses of various parts of T. portulacastrum have been reported by Kumar et al. (2004, 2005), Shastri (1952), Bhattacharya and Chatterjee (1998), Mandal et al. (1998), Sarkar et al. (1999), Ahmad et al. (2000) and Kirtikar and Basu (1975). Musa (M.) paradisiaca L. (Musaceae), commonly called “Kaila” is traditionally used for the treatment of inflammation, rheumatism, gripe, diabetes, hypertension, cough and bronchitis. Unripe bananas are astringent and used to treat diarrhea. The roots can arrest hemoptysis and posses strongly astringent and anthelmintic properties (Morton, 1987). Plantain juice is used as an antidote for snake bite (Reid, 1961). Other medicinal uses of this plant have been reported by Morton (1987), Anjaria et al. (2002), Ojewole and Adewunmi (2003), Lewis et al. (1999), Orie (1997) and Pannangpetch et al. (2001). In EVM, M. paradisiaca (L.) are used in the problems of hooves and injuries while its green fruit is used for the treatment of diarrhea (Lans et al., 2006). The above mentioned reports of medicinal uses of M. paradisiaca and T. portulacastrum accompanied with the claims of traditional healers for their anthelmintic activity (Hussain et al., 2008) made the basis of designing this project for in vitro and in vivo scientific validation of these priceless novel herbs in Pakistan that may reveal their anthelmintic activity to contribute towards the help of the local livestock owners and veterinarians in combating nematode infections in sheep. 2. Methodology 2.1. Collection and processing of plant materials Whole plants of T. portulacastrum L. and leaves of M. paradisiaca L. were collected from the local fields of District Sahiwal (Punjab, Pakistan) during a survey (previous part of author’s research), identified and authenticated by a botanist by comparing with the specimens stored in the herbarium of Department of Botany, University of Agriculture, Faisalabad, Pakistan. The voucher specimen numbers 0110 (Trianthema portulacastrum L. whole plant) and 0135 (Musa paradisiaca L. leaves), described in previous report of Hussain et al. (2008), were stored in the Ethnoveterinary Research and Development Centre (EVRDC), Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan. Plant materials were processed through heat drying in an oven at 40 ◦ C, ground to a fine powder and stored in polythene bags at 4 ◦ C until use (Jabbar et al., 2007). 2.2. Extract preparation The plant extracts were prepared according to method described by Gilani et al. (2004). Briefly, powdered plant

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material (1.5 kg) of each plant was soaked in 6 L of 70% aqueous (aq.) methanol by cold maceration at room temperature for 3 days. The filtrate collected through muslin cloth by repeated soaking of plant materials was concentrated in a rotary evaporator at 40 ◦ C under reduced pressure and dried in a vacuum oven. The w/w yield of T. portulacastrum and M. paradisiaca was 14.2% and 10.8%, respectively. The extracts were stored at 4 ◦ C until use. 2.3. In vitro anthelmintic activity 2.3.1. Collection of worms and recovery of eggs Collection of worms and recovery of eggs was done according to the method described by Jabbar et al. (2007). Briefly, mature live H. contortus female worms were collected from the abomasum of freshly slaughtered sheep at a local abattoir, washed and finally suspended in a bottle containing cool (4 ◦ C) phosphate buffer saline (PBS) (pH 7.2) for further use in adult worm motility assays. For egg hatch test (EHT), some of collected worms were triturated in pestle and mortar and the suspension was filtered through sieve of a 100-mesh (150 ␮m pore size). The suspension that passed through the sieve was collected and washed through another size 400 mesh sieve (38 ␮m pore size) with warm water. The material left on the sieve was back washed and transferred into Clayton Lane tubes. Filtrate was centrifuged in Clayton Lane tubes for 2 minutes at about 300 × g and supernatant was discarded. Tubes were agitated to loosen the sediment and then saturated sodium chloride solution was added until a meniscus formed above the tube. A cover slip was placed and sample re-centrifuged for 2 min at about 130 × g. Cover slip was plucked off carefully from tubes and eggs were washed off into a conical glass centrifuge tube. Tubes were filled with water and centrifuged for 2 min at about 300 × g. Supernatant was decanted and eggs were re-suspended in water. The eggs were then washed thrice in distilled water and adjusted to a 500 eggs mL–1 using Whitlock Universal egg counting technique (Whitlock, 1960) for further use in EHT. 2.3.2. Adult motility assay (AMA) The effect of crude aq. methanolic extract (CAME) of study plants was determined as per method described by Iqbal et al. (2004). Briefly, a minimum of 10 female H. contortus worms were exposed in 3 replicates to each of the following treatments in separate petri dishes at room temperature (25–30 ◦ C): 1. Twofold dilutions of CAME (100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39 and 0.19 mg mL−1 ). 2. Levamisole 0.5 mg mL−1 . 3. Phosphate buffer saline (PBS). The inhibition of motility and/or mortality of the worms kept in the above treatments were used as a criterion for anthelmintic activity. The motility was observed after 0, 2, 4, 6, 8 and 12 h intervals and post-treatment revival of motility (if any) was observed by keeping the treated worms in the lukewarm fresh PBS for 30 min.

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2.3.3. Egg hatch test (EHT) Egg hatch test was conducted by the method described by Coles et al. (1992) with slight modifications (Jabbar et al., 2007). Briefly, egg suspension of (0.2 ml; 100 eggs) was distributed in a 24-well flat-bottomed microtitre plate and mixed with the same volume of different concentrations (0.25–8 mg mL–1 ) of CAME of each plant. The positive control wells received different concentrations (0.09–3.0 ␮g mL–1 ) of oxfendazole (Systamex® —ICI Pakistan, Ltd.; 2.265%, w/v) while negative control wells contained the diluent (as placebo) and the egg solution. The plates were incubated at 27 ◦ C for 48 h and then a drop of Lugol’s iodine solution was added to stop the eggs from hatching. All the eggs and first-stage larvae in three replicates of treatment and control plates were counted. 2.4. In vivo anthelmintic activity In vivo anthelmintic activity of the study plants was determined through fecal egg count reduction test (FECRT) as described by Iqbal et al. (2004) 2.4.1. Study animals Sixty four Lohi sheep of both sexes (≤1 year of age) weighing 18–25 kg naturally infected with mixed parasitic worms having eggs per gram (EPG) of >500, i.e. 1300–1600 were selected from the Allah Dad cattle farm, District Khanewal, Punjab, Pakistan. The selected animals were routinely vaccinated against different bacterial/viral diseases and dipped with ectoparasiticide. The selected sheep were segregated into 8 groups of 4 animals each using completely randomized block design (Petrie and Watson, 1999). The sheep were kept on wood shaving and fed with fresh green fodder, concentrate and water ad libitum.

Group 6: Treated orally with single dose of CAME at equivalent dose rate 1 g kg−1 b.w. of CP. Group 7: Treated orally with single dose of CAME at the equivalent dose rate 4 g kg−1 b.w. of CP. Group 8: Treated orally with single dose of CAME at the equivalent dose rate 8 g kg−1 b.w. of CP. 2.4.3. Measurements The study sheep were regularly observed for clinical signs and weekly record of body weight was maintained. Post-treatment (PT) fecal egg count reduction (FECR) was assessed through EPG on days 3, 6, 9, 12 and 15 using Whitlock universal egg counting chamber (Whitlock, 1960). Percentage FECR was calculated using the following formula as described by Jabbar et al. (2007):



FECR (%) =

(pre-treatment EPG − post-treatment EPG) pre-treatment EPG



× 100

2.5. Statistical analyses For egg hatch test, probit transformation was performed to transform a typical sigmoid dose–response curve to linear function (Hubert and Kerboeuf, 1992). The extract concentration required to prevent 50%, i.e., lethal concentration 50 (LC50 ) of hatching of eggs was calculated from this linear regression (for y = 0 on the probit scale). The data of AMA, fecal egg counts and larval counts was presented as mean ± standard error of mean (Iqbal et al., 2010). The fecal egg count reduction (FECR) was determined by the method described by Coles et al. (1992). The data from AMA, FECRT and larval counts from various treatments among different days were compared using the analysis of variance (ANOVA) with SAS software (SAS, 1998). 3. Results

2.4.2. Treatment protocol The pre-treatment quantitative worm burden was assessed through examination of fresh fecal samples, collected directly from the rectum, using salt floatation technique (MAFF, 1979). Taxonomic identification of the eggs was conducted through keys (Soulsby, 1982) confirmed through identification of morphological characteristics of larvae through coproculture as described by Thienpont et al. (1979). The eggs of Haemonchus contortus, Trichostronglyus spp., Oesophagostomum columbianum and Trichuris ovis were identified from the selected animals. The groups were subjected to various treatments as mentioned below: Group 1: Untreated control. Group 2: Treated orally with Levamisole HCl (Nilverm® 1.5%, w/v; ICI Pakistan Limited, Animal Health Division) at 7.5 mg kg−1 body weight (b.w.). Group 3: Treated orally with single dose of Crude powder (CP) at 1 g kg−1 b.w. Group 4: Treated orally with single dose of CP at 4 g kg−1 b.w. Group 5: Treated orally with single dose of CP at 8 g kg−1 b.w.

3.1. In vitro anthelmintic activity CAME of T. portulacastrum and M. paradisiaca showed a strong in vitro anthelmintic activity and pronounced inhibitory effects on egg hatching as observed through AMA and EHT, respectively. All the concentrations of both study plants gave the excellent results up to maximum of 6th hours PT except 0.39 and 1.56 mg mL−1 of M. paradisiaca where all the worms died at 12th hour PT as determined by AMA. There was a significant (P < 0.05) dose and time dependent response observed in all the concentrations of both the subjected plant extracts (Figs. 1 and 2). Moreover, onset of activity of plant extracts was directly proportional to their dose rates, i.e. higher the dose of plant extract, earlier the onset of activity and vice versa. None of the worms showed the revival of motility after they were placed in PBS for 30 min. The LC50 values of M. paradisiaca and T. portulacastrum and positive control (oxfendazole) calculated graphically from the regression equation after correcting from negative control were 2.13, 2.41 and 1.88 ␮g mL–1 , respectively. The regression values and correlation of regression of M. paradisiaca, T. portulacastrum and positive control were y = −0.0002x + 4.6324; R2 = 0.9689, y = −0.0006x + 4.4134;

A. Hussain et al. / Veterinary Parasitology 179 (2011) 92–99 4.8

11

4.6

9

4.4

*

7

*

*

3

*

*

1

*

5

Probit hatching

Meannumber ofworms

95

*

*

4 hr

6 hr

*

4.2

y = -0.0002x + 4.6324 R2 = 0.9689

4 3.8 3.6 3.4

-1

3.2

0 hr

2 hr

3

Hours post-exposure 25.00 mg

6.25 mg

1.56 mg

0.39 mg

0

1000

2000

3000

Fig. 1. Graph showing the time and dose-dependent in vitro anthelmintic activity of Trianthema portulacastrum L. crude aqueous methanol extracts at 100.0–0.39 mg/mL concentrations in comparison with positive control levamisole (0.5 mg/mL), on mature live Haemonchus contortus of sheep. The inhibition of motility and/or mortality of the worms were used as the criterion for anthelmintic activity. Values shown are means, asterisk (*) indicates significantly different from previous value at P < 0.05.

R2 = 0.9793 and y = −0.2159x + 6.2447, R2 = 0.775, respectively (Figs. 3–5). It was observed that M. paradisiaca CAME produced more pronounced inhibitory effect on egg hatching of H.contortus as compared to that of T. portulacastrum. However, ovicidal activities of both plants were lower than that of positive control.

4000

5000

6000

Concentraon (μg/mL)

PBS

7000

8000

Fig. 3. Linear relationships between egg hatching % on the probit scale of Haemonchus contortus and Musa paradisiaca L. aqueous methanolic extract concentrations (␮g mL−1 ). 5 4.5 4 3.5

Probit hatching

100.00 mg

y = -0.0006x + 4.4134 R 2 = 0.9793

3 2.5 2 1.5 1 0.5

3.2. In vivo anthelmintic activity

0 0

Both CP and CAME of each of the M. paradisiacal and T. portulacastrum plants exhibited strong (P < 0.05) anthelmintic activity vis-à-vis untreated control. Maximum post-treatment FECR (%) was recorded at 15th day PT at the dose rate of 8 g kg–1 which was 85.6% for CAME of T. portulacastrum (Fig. 6) and 80.7% for that of M. paradisiaca (Fig. 7). However, levamisole showed 99.3% FECR on 3rd day PT which was maintained up till day 6 followed by an insignificant (P ≥ 0.05) increase from days 9 to 15

9.00

2000

3000

4000

5000

6000

7000

8000

Concentraon (μg/mL) Fig. 4. Linear relationships between egg hatching % on the probit scale of Haemonchus contortus and Trianthema portulacastrum L. aqueous methanolic extract concentrations (␮g mL−1 ).

PT. Figs. 8 and 9 show the distribution of different genera/species of gastrointestinal nematodes (GINs) present in the flock determined by coprocultures on days 0 and 15. The reduction in mean larval counts followed the pattern of FECR and maximum reduction in all the prevalent GINs was observed with CP and CAME (P < 0.01) at the dose of 8 g kg−1 of both plants on day 15 PT (Figs. 8 and 9).

*

7.00 *

6.5

*

5.00 *

1.00 -1.00

*

*

3.00

*

2 hr

* *

*

0 hr

6.3

*

4 hr

*

6 hr

*

*

8 hr

10 hr

*

12 hr

Hours post-exposure 100.00 mg

25.00 mg

6.25 mg

1.56 mg

0.39 mg

PBS

Hatching (probit)

Mean number of worms

11.00

1000

6.1 y = -0.2159x + 6.2447 R2 = 0.775

5.9

5.7

Fig. 2. Graph showing the time and dose-dependent in vitro anthelmintic activity of Musa paradisiaca L. crude aqueous methanol extracts at 100.0–0.39 mg/mL concentrations in comparison with positive control levamisole (0.5 mg/mL), on mature live Haemonchus contortus of sheep. The inhibition of motility and/or mortality of the worms were used as the criterion for anthelmintic activity. Values shown are means, asterisk (*) indicates significantly different from previous value at P < 0.05.

5.5 0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

Concentration ( gm/mL) Fig. 5. Linear relationships between egg hatching % on the probit scale of Haemonchus contortus and Oxfendazole (␮g mL−1 ).

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Days 0

3

6

9

12

15

Eggs per gram (EPG) offaeces

1550 1350

3.4

1150 950 750 46.6

49.2

550 350 71.8

74.1

75.9

150 85.6

97.0

-50 Untreated

Treated

1.0 g

8.0 g

4.0 g

1.0 g

8.0 g

Crude Aqueous Methanolic Extract

Crude Powder

Control

4.0 g

Doses and forms ofTrianthema portulacastrum L. used Fig. 6. Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Trianthema portulacastrum L. compared with control. Numerical values show the per cent reduction of different treatment groups at day 15 post treatment.

Days 0

3

6

9

12

15

Eggs per gram (EPG) offaeces

1550 1350

3.4

1150 950 40.4

750

44.6

550 64.3

350

66.6

70.7

150

80.7 97.0

-50 Untreated

Control

Treated

1.0 g

8.0 g

4.0 g

Crude Powder

1.0 g

4.0 g

8.0 g

Crude Aqueous Methanolic Extract

Doses and forms ofMusa paradisiaca L. used Fig. 7. Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Musa paradisiaca L. compared with control. Numerical values show the per cent reduction of different treatment groups at day 15 post treatment.

4. Discussion According to the survey conducted by Hussain et al. (2008), T. portulacastrum and Musa paradisiaca are tradi-

tionally used anthelmintics in small and large animals of District Sahiwal (Punjab, Pakistan). Regarding T. portulacastrum, as far as could be ascertained, no scientific report on the anthelmintic activity is available so far.

A. Hussain et al. / Veterinary Parasitology 179 (2011) 92–99

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Fig. 8. Bar diagram showing the anthelmintic activity of Trianthema portulacastrum L. crude powder and crude aqueous methanolic extract on number of larvae of different nematodes before (day 0) and after treatment (day 15). Activity is compared with that of positive control levamisole and untreated control.

Fig. 9. Bar diagram showing the anthelmintic activity of Musa paradisiaca L. crude powder and crude aqueous methanolic extract on number of larvae of different nematodes before (day 0) and after treatment (day 15). Activity is compared with that of positive control levamisole and untreated control.

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The present investigation is the first in vitro and in vivo scientific validation of T. portulacastrum showing its promising anthelmintic activity as screened through in vitro (AMA and EHT) and in vivo (FECRT and Larval Counts) assays in view of its usage in ethnoveterinary practices in Pakistan. The probable reason for its potent anthelmintic activity may be due to the constituents including an alkaloid trianthemine, ecdysterone (a potent chemosterilant) (Shastri, 1952), saponin and punarnavine (Chopra et al., 1956). The extraction of T. portulacastrum with dichloromethane led to the isolation of a new flavonoid, 5,2-dihydroxy-7methoxy-6,8-dimethylflavone, along with 5,7-dihydroxy6,8-dimethylchromone (leptorumol) (Udom et al., 1997). The compounds including alkaloids (Akhtar, 1988; Asuzu and Onu, 1993; Roepke, 1996; Fakae et al., 2000) saponins (Akhtar, 1988; Akhtar and Aslam, 1989; Fakae et al., 2000) and flavonoids (Akhtar, 1988; Akhtar and Ahmad, 1992) have been proven as good anthelmintics. Previously, M. paradisiaca has been screened for its in vitro anthelmintic activity against the eggs of gastrointestinal nematodes of ovine has been reported by Krychak-Furtado et al. (2005) in which ethanolic extract and pure latex of M. paradisiaca were found possessing only low anthelmintic activity. The plant was also screened for its in vitro anthelmintic activity by the Hussain et al. (2010) through a pilot project in which aqueous and aqueous methanolic extracts of the plant showed a strong inhibitory effect on the hatching of eggs of mixed GINs demonstrating the significance of its selection for the present study. However, in the present study, the results were well marked as the CAME of M. paradisiaca showed in vitro as assayed through AMA and EHT, respectively. Moreover, CP of the plant also provided better efficacy as compared to that reported by Krychak-Furtado et al. (2005). The chemical constituents of M. paradisiaca include tannins, eugenol, tyramine (Morton, 1987), serotonin, levarterenol, norepinephrine and dopamine (Pari and Umamaheswari, 2000), alkaloids, steroidal lactones, and iron (Morton, 1987), however, those responsible for its anthelmintic activity have not yet been explored. Anyhow, anthelmintic activity of various phytochemicals including norepinephrine, alkaloids (Lateef et al., 2003) and tannins (Molan et al., 2000a,b; Iqbal et al., 2007) strongly support this speculation. Levamisole seems to affect only through paralysis of worms as there was a little increase in EPG on day 15 PT compared with day 6 PT; while, there was further reduction in EPG with CP and CAME of M. paradisiacal and T. portulacastrum suggesting their more than one actions, i.e., paralysis of worms and lowered fecundity due to possible effects on ovaries of the worms. 5. Conclusion In conclusions, the results of present study justify the traditional use of T. portulacastrum L. (Aizoaceae) whole plant and M. paradisiaca L. (Musaceae) leaves against prevalent gastrointestinal nematodes of sheep by livestock holders in Pakistan. However, their further biochemical analysis may result not only in isolation of some wonderful biochemical compounds with anthelmintic properties but

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