Pakistan Veterinary Journal

ISSN: 0253-8318 (PRINT), 2074-7764 (ONLINE)

Accessible at: www.pvj.com.pk
RESEARCH ARTICLE
Anthelmintic Activity of a Herbal Formulation Against Gastrointestinal Nematodes of Sheep
Muhammad Arfan Zaman*§, Zafar Iqbal, Muhammad Nisar Khan and Ghulam Muhammad1

Department of Parasitology; 1Department of Clinical Medicine & Surgery, University of Agriculture, Faisalabad;
§
Present Address: College of Veterinary and Animal Sciences, Jhang, Sub-Campus University of Veterinary and Animal
Sciences, Lahore, Pakistan
*Corresponding Author: [email protected]

ARTICLE HISTORY ABSTRACT

Received: July 09, 2011 This study was carried out to evaluate the anthelmintic activity of a herbal
Revised: September 05, 2011 formulation (HF) based on aqueous extracts of leaves of Azadirachta indica and
Accepted: September 06, 2011 Nicotiana tabacum, flowers of Calotropis procera and seeds of Trachyspermum
Key words: ammi. In vitro, eggs and adult Haemonchus contortus were exposed to different
Anthelmintic concentrations of HF following the standard procedures of egg hatch test (EHT; 50
Gastrointestinal nematodes to 0.024414 mg ml-1) and adult motility assay (AMA; 200-0.1953125mg ml-1),
Herbal extracts respectively. The reference drugs used in the study were oxfendazole (0.0056704 to
Sheep 0.0000027 mg ml-1) and levamisole (1.50 mg ml-1) for EHT and AMA, respectively.
In vivo, pre and post-treatment (4 mg, 2 mg and 500 µg kg-1 body weight) fecal egg
counts were determined following standard fecal egg count reduction test in sheep
naturally parasitized with mixed species of gastrointestinal nematodes. In EHT,
LC50 values of HF and oxfendazole (reference drug) were 275.1 and 0.016 µg ml-1,
respectively. In AMA, 100% mortality of H. contortus was observed 6 hr post-
exposure to 3.125-200 mg ml-1 concentrations of HF and 2 hr post-exposure to
levamisole. In vivo, maximum (96.2%) fecal egg count (EPG) reduction was
recorded in sheep treated with HF @ 4 mg kg-1 body weight; whereas, 89.3%
reduction in EPG was recorded in sheep treated with levamisole @ 7.5 mg kg-1 body
weight. A graded dose response was noted in all the tests used in the present study
to evaluate the anthelmintic activity of HF. Therefore, HF seems to be promising as
an anthelmintic for animals. Large scale trials on efficacy and safety, however, are
recommended before the HF is considered for commercialization in crude form.

©2011 PVJ. All rights reserved

To Cite This Article: Zaman MA, Z Iqbal, MN Khan and G Muhammad, 2012. Anthelmintic activity of a herbal
formulation against gastrointestinal nematodes of sheep. Pak Vet J, 32(1): 117-121.

INTRODUCTION and/or their products have been used for treatment of

different diseases for centuries. Anti-parasitic efficacy of
Gastrointestinal nematodes (GIN) impair animal different plants using standard parasitological procedures
productivity through reduction in voluntary food intake has been reported earlier (Dolan et al., 2007). In the
and/or inefficient use of nutrients. Disturbance in protein present study, a combination of the aqueous extracts of
metabolism and reduced absorption and/or retention of leaves of Azadirachta (A.) indica (locally known as
minerals are significant during parasite infection (Waller, Neem) and Nicotiana (N.) tabacum (locally known as
1987). The control of GINs predominantly depends upon Tambaku), flowers of Calotropis (C.) procera (locally
chemotherapy despite advancements in genetical, known as Aak) and seeds of Trachyspermum (T.) ammi
immunological and biotechnological methods (Lloyd- (locally known as Ajwain) have been tested for their
Evans, 1991). Development of drug resistance in the anthelmintic activity.
parasites, effect of drug residues on human (Bowman et
al., 1999) and high costs of the synthetic drugs, however, MATERIALS AND METHODS
have led to attention of the workers to find the alternatives
to the existing chemicals used for the treatment and/or Extraction of plant materials: Leaves of A. indica and
control of parasites (Al-Shaibani et al., 2009; Bachaya et N. tabacum, flowers of C. procera and seeds of T. ammi
al., 2009; Deeba et al., 2009; Sindhu et al., 2010). Plants were obtained from field and/or local market (Faisalabad-

117
 118 Pak Vet J, 2012, 32(1): 117-121.

Pakistan). The plant materials were identified and In vivo anthelmintic activity
authenticated by a botanist in the Department of Botany, Fecal egg count reduction test: For each dose of HF (4
University of Agriculture, Faisalabad (UAF), Pakistan, and mg, 2 mg and 500 µg kg-1 body weights) of extract, and
dried under shade. Extraction was carried out in the two control groups; 25, 4–8-months-old sheep (either sex)
Department of Parasitology, UAF. Five kilograms of each naturally infected with mixed gastrointestinal nematodes
plant material was soaked together in 50 liters of distilled species were selected from the private farms around
water at room temperature in large sized buckets covered district Jhang. The animals having ≥ 750 eggs per gram of
with a lid. The suspension was shaken vigorously every feces (EPG) were included in the study. The experimental
72 hrs. After 30 days, the suspension was concentrated at animals were stall fed on a uniform ration for all the
25-30°C till the volume reduced to 20 liters. Then, it was groups during the days of experimentation. Water was
further reduced to the weight of 10 kg in a vacuum oven. offered ad libitum.
The total w/w yield of extract was 20%. The extract was
stored at 4°C, and used for different biological assays Test Procedure: Before starting treatment, EPG of each
after dilution with distilled deionized water as desired. animal was calculated from the fecal samples obtained
directly from rectum, at least three times within the period
In vitro anthelmintic activity of three days. Coprocultures were done for identification
Egg hatch test (EHT): The EHT was performed of the nematode species composition following MAFF
following Coles et al. (1992). Adult H. contortus worms (1979). Experimental sheep were distributed into five
were obtained from the abomasal contents of slaughtered groups (A-E) through complete randomized design under
sheep. The female worms were separated and crushed in consideration of their live weight and EPG on day 0.
mortar and pestle to liberate the eggs. The concentration Group A was negative control while group B served as a
of eggs were estimated in 50 µl samples and adjusted to positive control. Levamisole HCl @ 7.5 mg kg-1 (ICI
100–150 eggs ml-1. Pakistan Limited, Animal Health Division) was drenched
once to positive control group.
Test procedure: One ml suspension containing Groups C to E were drenched with single dose of the
approximately 100-150 freshly collected H. contortus HF @ 4 mg, 2 mg and 500 µg kg-1 body weights. Fecal
eggs was added to each well of a 24-well flat-bottomed examination of each animal was carried out by the salt
microtitration plate. The eggs were exposed to different floatation technique (MAFF, 1979) in the morning on day
concentrations (50 to 0.024414 mg ml-1 in distilled 0, 3, 6, 9, 12 and 15 days post-treatment and EPG were
deionized water) of herbal formulation (HF) except in the calculated by the McMaster method (Soulsby, 1982). The
positive and negative control wells having egg suspension following formula was used to calculate the percent egg
with oxfendazole (0.0056704 to 0.0000027 mg ml-1) and count reduction (ECR):
distilled deionized water, respectively. The microtitration
plates were incubated for 48 hrs at room temperature ECR (%) = Pre-treatment egg count per gram–Post-treatment egg count per gram × 100
followed by addition of Lugol’s iodine solution in the Pre-treatment egg count per gram
wells. Un-hatched eggs and first-stage larvae (L1) were
counted in each well in all the plates. The experiment was Statistical analysis: LC50 of HF and oxfendazole were
carried out in triplicate for both control and extract. The calculated following Finney (1971) and probit was
LC50 of herbal extract and oxfendazole were calculated analyzed by Hubert and Kerboeuf (1992) method. In adult
following Finney (1971). motility assay, the comparison between means of dead
worms was assessed through DMR Test. For FECRT, the
Adult motility assay: Mature live H. contortus (females) results were expressed as eggs per gram (Mean±SEM) of
was obtained from abomasums of sheep freshly feces and means were compared by using DMR Test
slaughtered in the local abattoir. After washing, the (SAS, 1998).
collected worms (n=10) were suspended in phosphate
buffer saline (PBS). Afterward, they were exposed to each RESULTS
of the following treatments in separate petri-dishes, in
triplicate, at room temperature (25-30°C). PBS was added In vitro anthelmintic activity
in all the petri-dishes. The negative control dishes Egg hatch test: Exposure of H. contortus eggs to HF
contained more PBS so that all the dishes possessed resulted in a dose dependent inhibition of egg hatching.
uniformed volume. There was, however, a large difference between the LC50
1. The HF @ 200, 100, 50, 25, 12.5, 6.25, 3.125, of HF (275.1 µg ml-1; Table 1) and that of oxfendazole
1.5625, 0.78125, 0.390625 and 0.1953125 mg ml-1 (0.016 µg ml-1; Table 2). The 95% confidence intervals
2. Levamisole HCl @ 1.50 mg ml-1 were 231.3 to 327.1, and 0.013 to 0.020; and the fitness of
3. PBS (control) the model based on chi square analysis were P 0.263 and
The worms were observed after every two hours up P 0.192 for HF and oxfendazole, respectively.
to 8 hrs. Finally, the treated and negative control worms
were kept in the lukewarm fresh PBS for 5 min to assess Adult motility assay: HF exhibited anthelmintic effects
the restoration of motility and immotile worms were in a dose dependent manner and all the worms were found
supposed as dead. The number of live and dead worms dead at 6 hr post-exposure at 3.125 mg ml-1 and above
was recorded in all the Petri-dishes. (Table 3). Mortality of worms was comparable to that
 119 Pak Vet J, 2012, 32(1): 117-121.

Table 1: LC50 and percentage of eggs hatching at various concentrations of the herbal formulation
Dose (mg ml-1) Dose(µg ml-1) hatch % Log dose Probit Regression
0.024414 24.41406 70 1.38764 5.524 5.667744
0.048828 48.82813 65 1.68867 5.385 5.37073
0.097656 97.65625 63 1.9897 5.33 5.073716
0.195313 195.3125 45 2.29073 4.87 4.776702
0.390625 390.625 33 2.59176 4.56 4.479688
0.78125 781.25 18 2.89279 4.085 4.182674
1.5625 1562.5 9 3.19382 3.659 3.88566
3.125 3125 7 3.49485 3.524 3.588646
6.25 6250 6 3.79588 3.445 3.291632
12.5 12500 1 4.09691 2.676 2.994618
25 25000 1 4.39794 2.676 2.697604
50 50000 1 4.69897 2.676 2.40059
LC50 = 275.1 µg ml-1

Table 2: LC50 and percentage of eggs hatching at various concentrations of oxfendazole

Dose (mg ml-1) Dose(µg ml-1) hatch % Log dose Probit Regression
0.0000027 0.0027 98 2.56864 7.054 7.347628
0.0000055 0.0055 97 2.25964 6.881 7.125581
0.000011 0.011 96 1.95861 6.751 6.90926
0.0000221 0.0221 95 1.65561 6.645 6.691524
0.0000443 0.0443 96 1.3536 6.751 6.474498
0.0000886 0.0886 95 1.05257 6.645 6.258178
0.0001772 0.1772 95 0.75154 6.645 6.041857
0.0003544 0.3544 80 0.45051 5.842 5.825536
0.0007088 0.7088 75 0.14948 5.674 5.609215
0.0014176 1.4176 69 0.151554 5.496 5.392895
0.0028352 2.8352 47 0.452584 4.925 5.176574
0.0056704 5.6704 31 0.753614 4.504 4.960253
-1
LC50 = 0.016 µgml

Table 3: In vitro effect of the herbal formulation(HF) on survival of Haemonchus contortus of sheep in comparison with levamisole
Treatments Mean (±SD) number of dead worms at different hrs post-exposure1
0 hr 2 hrs 4 hrs 6 hrs 8 hrs
Levamisole @ 1.50 mg ml-1 0.0±0.00 k 10±0.00a 10±0.00 a 10±0.00 a 10±0.00 a
k k k k
PBS 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00 k
HF (mg ml-1)
0.195 0.0±0.00 k 0.0±0.00 k 0.0±0.00 k 0.0±0.00 k 2.66±1.52 i
0.390 0.0±0.00 k 0.0±0.00 k 0.0±0.00 k 1.67±1.52 j 2.33±1.52 i
0.781 0.0±0.00 k 0.0±0.00 k 4.33±0.57 h 5.66±1.15 g 7.33±1.52 de
1.562 0.0±0.00 k 5.67±1.15g 6.33±1.15 ef 9.33±0.57 ab 10±0.00 a
3.125 0.0±0.00 k 6.00±1.00f 7.33±0.57 de 10±0.00 a 10±0.00 a
6.25 0.0±0.00 k 7.33±0.57 de 8.00±1.00 d 10±0.00 a 10±0.00 a
12.5 0.0±0.00 k 7.33±0.57de 8.00±1.00 d 10±0.00 a 10±0.00 a
25 0.0±0.00 k 8.33±0.57 cd 9.00±0.00 b 10±0.00 a 10±0.00 a
50 0.0±0.00 k 8.33±0.57cd 9.33±0.57ab 10±0.00 a 10±0.00 a
100 0.0±0.00 k 8.67±0.57bc 10±0.00 a 10±0.00 a 10±0.00 a
200 0.0±0.00 k 9.0±1.00b 10±0.00 a 10±0.00 a 10±0.00 a
1
Each treatment group had three replicates each having 10 worms; the values with same superscript in a row do not differ significantly at P≥0.05.

Table 4: In vivo anthelmintic activity of the herbal formulation (HF) based reduction in eggs per gram of feces (Mean+SE) in sheep naturally
parasitized with gastrointestinal nematodes
Days PT Doses of HF Control groups
500 µgkg-1 body weight 2 gkg-1 body weight 4 gkg-1 body weight Levamisole Negative Control
0 2526.7± 2 0 0 a 2214± 2 0 3 a 5053.3± 2 0 9 a 2487.6± 2 1 9 a 5335.5± 1 9 8 a
3 1972.6± 336.5b (21.9) 1370.9± 404.9b (38.1) 2285± 465.9b (54.8) 862.8± 382.9b (65.3) 5320.3± 1 0 1 . 1 a ( 0 . 3 )
6 1743.3± 307.1c (31.0) 591.6± 169.6c (73.3) 1099.7± 261.7c (78.3) 606.4± 306.6c (75.6) 5298.7± 1 2 0 . 2 a ( 0 . 7 )
9 1736.7± 308.3c (31.3) 338.3± 70.4d (84.7) 448.5± 74.4d (91.1) 453.9± 288.0d (81.8) 5307.1± 1 2 0 . 5 a ( 0 . 5 )
12 1780± 306.7c (29.6) 254± 47.8e (88.5) 317.5± 61.8e (93.7) 321.9± 279.0e (87.1) 5290.4± 1 6 6 . 8 a ( 0 . 8 )
15 1859.5± 312.6d (26.4) 186.5± 18.0f (91.6) 190.9± 39.0f (96.2) 265.9± 279.0f (89.3) 5300.9± 1 7 4 . 8 a ( 0 . 6 )
Each group had five sheep; PT= Post-treatment; Values with different superscripts in a column differ significantly (P≥0.05). Figures in parenthesis
indicate percentage.

with levamisole (reference drug) at 200 mg ml-1 at 2 hr gram of feces (EPG) was recorded on day 15 post-
post-exposure. There was no mortality of worms in PBS treatment in sheep treated @ 4 g kg-1 body weight (Table
till 8 hrs. 4). The reduction in EPG (89.3%) with levamisole was
comparable to that with the HF @ 4 g kg-1 body weight.
In vivo anthelmintic activity: The experimental sheep
were found to have mixed infection with H. contortus, DISCUSSION
Chabertia ovina, Trichostrongylus spp., Telodorsagia
spp. and Oesophagostomum columbianum. HF exhibited Results revealed anthelmintic efficacy of the HF in all
anthelmintic activity in a time and dose-dependent the tests used in this study. The plants used in the HF have
manner. The maximum reduction (96.2%) in eggs per been reported for their anthelmintic activities individually
 120 Pak Vet J, 2012, 32(1): 117-121.

in numerous studies. For example, crude methanolic (gastrointestinal). Likewise, the aqueous extract of C.
extract of A. indica leaves have been reported for their procera exhibited spasmolytic effect on muscle chain
ovicidal (LC50=371.53 µg ml-1), larvicidal (LC50=199.53 (smooth) of trachea in guinea-pig (Iwalewa et al., 2005).
µg ml-1) and wormicidal (>500 µg ml-1) effects (Bachaya, A. indica contained triterpenol derivatives such as nimbin,
2007). Iqbal et al. (2010) have demonstrated mild azadirachtin, salannin, diacetyl-nimbin (Verma et al.,
anthelmintic activity of crude powder and methanolic 1995) and its anthelmintic properties are attributed to
extract of A. indica seeds as evident from 29.3 and 40.2% azadirachtin (Sharma et al., 2003). N. tabacum possesses
reduction in EPG, respectively in sheep naturally nicotine, a ganglion stimulant, which may be responsible
parasitized with mixed species of gastrointestinal for its anthelmintic properties through the activation of
nematodes (GINs). In contrast; however, Worku et al. neuromuscular junctions leading to spastic paralysis,
(2009) could not demonstrate anthelmintic effects of A. death and expulsion of worms from the body of host
indica leaves. Iqbal et al. (2006) have reported in vitro (Neal, 2002). Major chemical constituent of T. ammi is
and in vivo anthelmintic activity of crude aqueous and Thymol (Khajeh et al., 2004). Thymol may have exerted
methanol extracts of N. tabacum leaves. Aqueous extract anthelmintic actions (Sánchez et al., 2004) by (i)
was more effective in in vitro adult motility assay; increasing surface curvature and polarity of the
whereas, methanolic extract caused higher reduction membranes at the level of packing densities of natural
(73.6%) in EPG compared with aqueous extract (49.4%) membranes, (b) activating and stimulating the binding of
in sheep naturally parasitized with GINs. Bachaya (2007) GABAA-R to FNZ at the extent of concentration more
has also demonstrated ovicidal (LC50=13.96 µg ml-1) and than critical micellar concentration, and (c) effecting the
larvicidal (LC50=478.63 µg ml-1) effects of methanolic binding (allosteric) sites of GABA leading to disturbances
extract of N. tabacum leaves against H. contortus. Crude in the coupling of GABA with FNZ.
powder, and/or aqueous and methanolic extracts of C. In conclusion, HF tested in this study is conveniently
procera flowers have been reported for their in vitro and available, economical and effective against GINs of
in vivo anthelmintic activity (Iqbal et al., 2005). In vitro, sheep. Standardization of procedures for its preparation,
methanolic extract was more effective; whereas, aqueous doses, and large scale trials are recommended before the
extract (88.4%) and crude powder (77.8%) caused higher tested HF is considered for commercialization.
reduction in EPG compared with methanolic extract
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