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Journal of Ethnopharmacology 114 (2007) 86–91

Anthelmintic activity of Chenopodium album (L.) and Caesalpiniacrista (L.) against trichostrongylid nematodes of sheep

Abdul Jabbar a,∗, Muhammad Arfan Zaman a, Zafar Iqbal a,Muhammad Yaseen b, Asim Shamim a

a Chemotherapy Laboratory, Department of Veterinary Parasitology, University of Agriculture, Faisalabad 38040, Pakistanb Department of Mathematics and Statistics, University of Agriculture, Faisalabad 38040, Pakistan

Received 3 October 2006; received in revised form 5 July 2007; accepted 29 July 2007Available online 2 August 2007

bstract

im of the study: The present study was carried out to determine the anthelmintic activity of Caesalpinia crista (L.) (Fabaceae) seed kernel andhenopodium album (L.) (Chenopodiaceae) whole plant in order to justify their traditional use in veterinary medicine.aterials and methods: In vitro anthelmintic activity of crude aqueous methanolic extract (AME) of both the plants was determined usingature Haemonchus contortus and their eggs in adult motility assay and egg hatch test, respectively. In vivo anthelmintic activity was evaluated

n sheep naturally infected with mixed species of gastrointestinal nematodes by administering crude powder (CP) and AME in increasing doses1.0–3.0 g/kg).esults: Both plants exhibited dose- and time-dependent anthelmintic effects by causing mortality of worms and inhibition of egg hatching.aesalpinia crista (LC50 = 0.134 mg/mL) was found to be more potent than Chenopodium album (LC50 = 0.449 mg/mL) in egg hatch test. In vivo,aximum reduction in eggs per gram (EPG) of faeces was recorded as 93.9 and 82.2% with Caesalpinia crista and Chenopodium album AME

t 3.0 g/kg on day 13 and 5 post-treatment, respectively. Levamisole (7.5 mg/kg), a standard anthelmintic agent, showed 95.1–95.6% reduction in

PG.onclusions: These data show that both Caesalpinia crista and Chenopodium album possess anthelmintic activity in vitro and in vivo, thus,

ustifying their use in the traditional medicine system of Pakistan.2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Caesalpinia crista; Chenopodium album; Anthelmintic activity; Ne

. Introduction

Gastrointestinal parasites pose a serious threat to the pro-uctivity of livestock in developing nations. Despite the fact ofevelopment of anthelmintic resistance (e.g., Praslicka, 1995;aller et al., 1996; Jabbar et al., 2006a; Saddiqi et al., 2006;

aeed et al., 2007) in parasites of high economic significance,hemotherapy is still the most widely used option for the con-rol of helminthes. However, many farmers in the developing

ountries are unable to afford synthetic anthelmintics for theirivestock. In this scenario, the farmers depend on time-honored,enturies-old, affordable and accessible treatments for parasites.

∗ Corresponding author. Tel.: +92 41 9201106; fax: +92 41 9200725.E-mail address: jabbaruaf@yahoo.co.uk (A. Jabbar).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.07.027

es; Sheep

his concept has led to an interest of scientists in ethnoveteri-ary medicine (EVM), i.e., the identification of the traditionalethods of managing livestock and treating livestock diseases

IIRR, 1994; McCorkle et al., 1996). In EVM, medicinal plantsre used to treat parasite infections. A number of plants haveeen listed to treat parasitic infections (Jabbar et al., 2006b) andfew of them have been scientifically validated (Hammond et

l., 1997; Akhtar et al., 2000; Iqbal et al., 2003, 2004, 2005,006a,b,c,d, 2007; Jabbar et al., 2006c; Lateef et al., 2006).

In Indo-Pak subcontinent, Ayurvedic and Unani medicinalystems are very popular and people have been using plantsot only for the treatment of their own ailments but also for

heir domesticated animals. Chenopodium album L. (Chenopo-iaceae) and Caesalpinia crista L. (Fabaceae) commonly knowns “Bathu” and “Kranjwa”, respectively, are valuable medici-al plants in Pakistan and their different parts are utilized in

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he traditional system of medicine (Said, 1969). Traditionally,henopodium album has been used for its diuretic, laxative,

edative, hepatoprotective and antiparasitic properties from cen-uries (Said, 1969; Fournier, 1999). Similarly, in folk medicineaesalpinia crista seeds are used to treat asthma, chronic fever,ough, headache, stomach or bowel upset and as an anthelminticNadkarni and Nadkarni, 1976; Satiyavati et al., 1976).

Both plants are being used separately or as a part of com-ound prescriptions in EVM as an anthelmintic and in anorexian animals from centuries as crude powder mixed with jaggeryr as water decoction (personal communication). The presenttudy rationalizes the use of Chenopodium album and Cae-alpinia crista as anthelmintics using standard parasitologicalrocedures.

. Materials and methods

.1. Plant material

Whole plant of Chenopodium album and seed kernels of Cae-alpinia crista were procured from local market in FaisalabadPakistan), identified and authenticated by a botanist by compar-ng with the specimens stored in the herbarium of Departmentf Botany, University of Agriculture, Faisalabad, Pakistan. Theoucher specimens (no. 13/2001; Chenopodium album L. wholelant and no. 12/2000; Caesalapinia crista L. seed kernels) aretored in the Ethnoveterinary Research and Development CentreEVRDC), Faculty of Veterinary Science, University of Agricul-ure, Faisalabad, Pakistan. Plant materials of both plants wereried in an oven at 40 ◦C, ground to a fine powder and stored inolythene bags at 4 ◦C until used.

.2. Extraction

The powdered material (450 g) of each plant was soaked inL 70% aqueous methanol by cold maceration at room temper-ture. After 3 days, the filtrate was collected through a piecef porous cloth and filter paper, and the plant material wase-soaked twice and filtrate was collected. The combined fil-rate was concentrated in a rotary evaporator at 40 ◦C undereduced pressure, and dried in a vacuum oven. The w/w yieldf Chenopodium album and Caesalpinia crista was 25.4 and8.6%, respectively. These extracts were stored at 4 ◦C untilse.

.3. In vitro anthelmintic activity

The in vitro trials for anthelmintic activity of crude aqueousethanolic extract (AME) of both plants were carried out using

he adult motility assay and egg hatch test.

.3.1. Adult motility assayAdult motility assay was conducted on mature live

aemonchus (H.) contortus following Sharma et al. (1971).riefly, the female mature worms were collected from the abo-asums of freshly slaughtered sheep in the local abattoir. Theorms were washed and finally suspended in phosphate buffer

2

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armacology 114 (2007) 86–91 87

aline (PBS). Five worms were exposed in triplicate to eachf the following treatments in separate Petri dishes at roomemperature (25–30 ◦C):

. AME of Chenopodium album whole plant @ 6, 12, 24 and48 mg/mL.

. AME of Caesalpinia crista seed kernels @ 6, 12, 24 and48 mg/mL.

. Levamisole, 0.55 mg/mL.

. PBS (control).

The inhibition of motility and/or mortality of the wormsubjected to the above treatments were used as the criteria fornthelmintic activity. The motility was recorded after 0, 1, 2, 3nd 6 h intervals. Finally, the treated worms were kept for 30 minn the lukewarm fresh PBS to observe the revival of motility.

.3.2. Egg hatch test

.3.2.1. Egg recovery. Adult female Haemonchus contortusere collected after giving the longitudinal incision along thereater curvature of abomasums of naturally infected sheep. Theorms present in ingesta or attached to the surface of guts wereicked manually using forceps and placed in a bottle containingool (4 ◦C) PBS (pH 7.2) and later were triturated in pestle andortar. The suspension was filtered through sieves of different

izes based on the nematode species into a bowl. Filtrate wasentrifuged in Clayton Lane tubes for 2 min at about 300 × gnd supernatant was discarded. Tubes were agitated to loosenhe sediment and then saturated sodium chloride solution wasdded until a meniscus formed above the tube. A cover slip waslaced and sample re-centrifuged for 2 min at about 130 × g.overslip was plucked off carefully from tubes and eggs wereashed off into a conical glass centrifuge tube. Tube was filledith water and centrifuged for 2 min at about 300 × g. Super-atant was decanted and eggs were re-suspended in water. Theggs were then washed thrice in distilled water and adjusted to500 eggs/mL using the McMaster technique (Soulsby, 1982).

.3.2.2. Test procedure. Egg hatch test was conducted fol-owing Coles et al. (1992). Eggs suspension of (0.2 mL; 100ggs) was distributed in a 24-flat-bottomed microtitre platend mixed with the same volume of different concentrations0.15–5 mg/mL) of each plant extract. The positive controlells received different concentrations (3.0–0.0058 �g/mL) ofxfendazole (Systamex—ICI Pakistan, Ltd.; 2.265%, w/v) inlace of plant extracts while negative control plate containedhe diluent and the egg solution. The eggs were incubated inhis mixture at 27 ◦C. After 48 h, a drop of Lugol’s iodine solu-ion was added to stop the eggs from hatching. All the eggsnd first-stage larvae (L1) in each plate were counted. Thereas three replicates for each treatment and control for both thelants separately.

.4. In vivo anthelmintic activity

A total of 60 sheep (local Thalli breed) ≤1 year, havinglmost homogeneous characteristics viz., weight, eggs per gram

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f faeces (EPG) and composition of infecting nematode speciesor each plant were selected from a herd of more than 1000nimals being maintained at Livestock Experiment Station,akh Kherewala (Punjab, Pakistan). The animals were vac-inated against enterotoxemia and pleuropneumonia vaccines,upplied by the Veterinary Research Institute, Lahore (Punjab,akistan). The sheep had naturally acquired mixed parasitic

nfection of gastrointestinal nematodes. Infections were con-rmed before the beginning of study by collecting faecal samplesrom the animals, by rectum and the number of nematode eggherein determined by the floatation method (Soulsby, 1982). Forematode species composition, coproculture was done for iden-ification of larvae using standard description of MAFF (1979)nd Thienpont et al. (1979). The nematode eggs recovered fromhe experimental sheep were identified by larval cultures asaemonchus contortus, Trichostronglyus spp., Oesophagosto-um columbianum and Trichuris ovis. The experimental animalsere housed for one month before study initiation for acclimati-

ation. After treatment they were penned singly by treatmentntil the end of the study. No physical contact was possi-le between sheep from different treatment groups. The sheepere kept on plastered floor and fed with grass and water ad

ibitum.The experimental sheep (n = 30 for each plant) were ran-

omly divided into six groups of five animals each and assignedifferent per os treatments as single dose as given below:roup 1 served as negative control and received no treat-ent while group 2 was a positive control which was givensingle dose of levamisole HCl 7.5 mg/kg (ICI Pakistan Lim-

ted, Animal Health Division). Groups 3 and 4 received singleoses of CP 1.0 and 3.0 g/kg, respectively, while the groupsand 6 were given single doses of AME 1.0 and 3.0 g/kg,respectively.

Faecal samples from each animal were collected in the morn-ng, starting from day 0 pre-treatment and at days 5 and 13ost-treatment and were evaluated for the presence of wormggs by salt floatation technique (MAFF, 1979). The eggs wereounted by the McMaster method (Soulsby, 1982). Egg countercent reduction (ECR) was calculated using the followingormula:

CR (%) = Pre-treatment egg count per gram–Post-treatment e

Pre-treatment egg count per gram

.5. Statistical analysis

For egg hatch test, probit transformation was performedo transform a typical sigmoid dose-response curve to linearunction (Hubert and Kerboeuf, 1992). The extract concen-ration required to prevent 50%, i.e., lethal concentration0 (LC50) of hatching of eggs was calculated from thisinear regression (for y = 0 on the probit scale). The data

rom adult motility assay and in vivo experiments were sta-istically analysed using SAS software (SAS, 1998). Theesults were expressed as mean ± standard error of meanS.E.M.).

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armacology 114 (2007) 86–91

ount per gram × 100

. Results and discussion

In adult motility assay, all the worms were found deadt 6-h post-exposure to different concentrations of AMEf Chenopodium album and Caesalpinia crista except at.0 mg/mL in case of AME of Chenopodium album where oneorm was still alive (Fig. 1). The higher doses of both thelants resulted in an early onset of activity and higher num-er of dead worms compared with lower doses (Fig. 1). Crudequeous methanolic extracts of Chenopodium album and Cae-alpinia crista exhibited inhibitory effects on eggs hatching andC50 was determined graphically from the regression equa-

ion after correcting from negative control. The calculated LC50alues of Chenopodium album, Caesalpinia crista and posi-ive control (oxfendazole) were 0.449, 0.134 and 1.9 �g/mL,espectively. The regression values and correlation of regres-ion of the Chenopodium album were y = −0.112x + 5.051;2 = 0.869 and those of Caesalpinia crista and positive controlere y = −0.199x + 5.360; R2 = 0.600 and y = −1.263x + 7.485;2 = 0.786, respectively. It was observed that AME of Cae-alpinia crista was more effective (p ≥ 0.05) against egg hatchs compared to Chenopodium album and the ovicidal activityf both the plants was low (p ≤ 0.05) as compared to positiveontrol.

In vivo, both CP and AME of Caesalpinia crista andhenopodium album exhibited anthelmintic activity (p < 0.05)ompared with negative control. In Caesalpinia crista treatedheep, the maximum reduction in eggs per gram of faecesas recorded as 93.9% with AME at 3.0 g/kg on day 13 PT

Fig. 2B), whereas, it was 82.2% with Chenopodium albumME at 3.0 g/kg on day 5 PT (Fig. 3A). Levamisole (7.5 mg/kg),standard anthelmintic agent, showed maximum reduction

95.1–95.6%) in EPG on day 5 PT (Figs. 2 and 3) comparedith negative control.The dose-dependent anthelmintic effect of Caesalpinia crista

as recorded on day 13 PT, which was not evident on dayPT. The possible explanation of this difference may be due

o the direct effect of Caesalpinia crista through paralysis andxpulsion of worms leading to drop in EPG. The added adverseffects on the ovaries of surviving worms may have led to further

rop in EPG on day 13 PT making a dose-dependent difference.evamisole seems to affect only through paralysis of worms as

here was a little increase in EPG on day 13 PT compared withay 5 PT; whereas, there was further reduction in EPG withP and AME of Caesalpinia crista suggesting its more thanne actions, i.e., paralysis of worms and lowered fecundity dueo possible effects on ovaries of the worms. Similarly, in casef Chenopodium album, there was no notable dose-dependentesponse on day 5 PT while on day 13 PT significant reduction

n EPG was recorded with CP.

Caesalpinia crista might have affected both adult wormshrough paralysis caused by its cholinergic effects as reported foraesalpinia bonduc (Datte et al., 1998, 2004), which resulted

A. Jabbar et al. / Journal of Ethnopharmacology 114 (2007) 86–91 89

Fig. 1. Graph showing the time- and dose-dependent in vitro anthelminticactivity of Caesalpinia crista (A) and Chenopodium album (B) crude aque-ous methanol extracts at 6.0–48.0 mg/mL concentrations in comparison withpositive control levamisole (0.55 mg/mL), on mature live Haemonchus contor-tai

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Fig. 2. Bar diagram showing anthelmintic activity of Caesalpinia crista crudepowder (CP) and aqueous methanol extract (AME) in sheep naturally infectedwith mixed species of gastrointestinal nematodes at (A) 5 days post-treatment(PT) and (B) 13 days PT. Reduction in eggs per gram of faeces (EPG) wasused as the criterion for anthelmintic activity. Activity of CP and AME iscompared with that of positive control, levamisole (7.5 mg/kg). Values shownare mean ± S.E.M., n = 5; Both CP and AME exhibited anthelmintic activity(o

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us of sheep. The inhibition of motility and/or mortality of the worms were useds the criterion for anthelmintic activity. Values shown are means, asterisk (*)ndicates significantly different from previous value at p < 0.05.

n expulsion of the worms from the host. The cholinergic effectf Caesalpinia bonduc extracts (Datte et al., 1998, 2004) coulde explained by three acting pathways, as reported by Sandow1965). This action could be due to: (i) a direct effect on theuscle, (ii) an activation of the transmission process in the neu-

omuscular junction, or (iii) both. A similar study was performedy Clausen and Everts (1991) on isolated rat skeletal muscle.annins (1.56% of DM) in Caesalpinia crista seed kernels mayave affected establishment or fecundity of the nematodes asemonstrated for tannin containing plants by Molan et al. (2000).

The present results suggest that whole plant of Chenopodiumlbum possess anthelmintic activity against sheep gastroin-estinal nematodes. Previously, oil and infusion of leaves

f Chenopodium ambrosioides have been reported for theirnthelmintic activity (Gibson, 1965; Ketzis et al., 2002;acDonald et al., 2004). As far as ascertained, this is the first

eport of anthelmintic activity of Chenopodium album.

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p < 0.05) vs. negative control; dose-dependent effects, however, were recordedn day 13 PT.

The active principles for the anthelmintic effects of bothhenopodium album and Caesalpinia crista have not beenxactly identified so far. Better efficacy of alcoholic fractionsn case of Caesalpinia crista indicates that the active principleay be lipophilic while in case of Chenopodium album it may

e lipophilic as well as hydrophilic.Earlier, ascaridole was considered to be the active con-

tituent of Chenopodium ambrosioides oil for its anthelminticctivity but now titerpenoid saponins are also well thought-ut to be important constituents in this regard. For example,iuchi et al. (2002) isolated four p-menthane-type monoterpeneydroperoxides (2a–5a) together with ascaridole from AcOEtxtract of aerial parts of Chenopodium ambrosioides L. var.ntherminticum A. Gray, and tested for anti-trypanosomal activ-

ty. These constituents exhibited trypanocidal activity againsthe epimastigotes of Trypanosoma cruzi, the etiologic agentf American trypanosomiasis (Chaga’s disease). Similarly, the

90 A. Jabbar et al. / Journal of Ethnoph

Fig. 3. Bar diagram showing anthelmintic activity of Chenopodium album crudepowder (CP) and aqueous methanol extract (AME) in sheep naturally infectedwith mixed species of gastrointestinal nematodes at (A) 5 days post-treatment(PT) and (B) 10 days PT. Reduction in eggs per gram of faeces (EPG) wasused as the criterion for anthelmintic activity. Activity of CP and AME iscompared with that of positive control, levamisole (7.5 mg/kg). Values showna(o

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re mean ± S.E.M.; n = 5. Both CP and AME exhibited anthelmintic activityp < 0.05) vs. negative control; dose-dependent effects, however, were recordedn day 13 PT with CP.

ompounds like betalain alkaloids, phenolic acids, betain, oxaliccid, oleanolic acid, sitosterol, furanocoumarins and saponinsNicholas et al., 1955; Hegnauer, 1964, 1989) may be responsi-le for anthelmintic activity of Chenopodium album.

Earlier, Akhtar and Aslam (1989) have reported 94.5%nthelmintic activity of glycosides isolated from Caesalpiniarista seeds. In contrast, however, Hordegen et al. (2003) couldot demonstrate reduction in EPG with 28 mg/kg of an aqueousthanol extract of the seeds of Caesalpinia crista. The discrep-ncy among the findings of the present study and those of Akhtarnd Aslam (1989), and Hordegen et al. (2003) could be attributedo (i) different doses, parts and chemical composition of the Cae-alpinia crista seeds used, (ii) different extraction proceduresmployed, (iii) variations in the susceptibility of the nematodeso various phytochemicals and (iv) food–drug interactions which

an influence the absorption of bioactive plant compounds.

Recently, Linn et al. (2005) have reported antimalarial activ-ty of cassane- and norcassane-type diterpenes against therowth of Plasmodium falciparum. The antimalarial activity was

H

armacology 114 (2007) 86–91

ttributed to CH2Cl2 extract of the seed kernels of Caesalpiniarista which was found to contain seven furanocassane-ype diterpenes [caesalpinins C-G (1–5) and norcaesalpinins

and E (6, 7)] together with norcaesalpinins A-C (8–10)nd 11 known compounds (norcaesalpinins A-C, 2-acetoxy--deacetoxycaesaldekarin e, caesalmin B, caesaldekarin e,aesalpin F, 14(17)-dehydrocaesalpin F, 2-acetoxycaesaldekarin, 7-acetoxybonducellpin C and caesalmin G) (Linn et al., 2005).he anthelmintic activity of Caesalpinia crista observed in theresent study might be due to the presence of cassane- andoncassane-type diterpenes in the seed kernels.

. Conclusion

This study shows that Caesalpinia crista and Chenopodiumlbum possess in vitro and in vivo anthelmintic activity, thus,ustifying the traditional use of these plants against helminths.owever, further controlled experiments on these plants and/orr isolated bioactive compounds using larger number of animalsnd experimental infections with individual nematode speciesre suggested.

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