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

Journal of Ethnopharmacology 114 (2007) 86–91

Anthelmintic activity of Chenopodium album (L.) and Caesalpinia crista (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, Pakistan b 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 2007 Available online 2 August 2007

Abstract Aim of the study: The present study was carried out to determine the anthelmintic activity of Caesalpinia crista (L.) (Fabaceae) seed kernel and Chenopodium album (L.) (Chenopodiaceae) whole plant in order to justify their traditional use in veterinary medicine. Materials and methods: In vitro anthelmintic activity of crude aqueous methanolic extract (AME) of both the plants was determined using mature Haemonchus contortus and their eggs in adult motility assay and egg hatch test, respectively. In vivo anthelmintic activity was evaluated in sheep naturally infected with mixed species of gastrointestinal nematodes by administering crude powder (CP) and AME in increasing doses (1.0–3.0 g/kg). Results: Both plants exhibited dose- and time-dependent anthelmintic effects by causing mortality of worms and inhibition of egg hatching. Caesalpinia 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, maximum reduction in eggs per gram (EPG) of faeces was recorded as 93.9 and 82.2% with Caesalpinia crista and Chenopodium album AME at 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 EPG. Conclusions: These data show that both Caesalpinia crista and Chenopodium album possess anthelmintic activity in vitro and in vivo, thus, justifying their use in the traditional medicine system of Pakistan. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Caesalpinia crista; Chenopodium album; Anthelmintic activity; Nematodes; Sheep

1. Introduction Gastrointestinal parasites pose a serious threat to the productivity of livestock in developing nations. Despite the fact of development of anthelmintic resistance (e.g., Praslicka, 1995; Waller et al., 1996; Jabbar et al., 2006a; Saddiqi et al., 2006; Saeed et al., 2007) in parasites of high economic significance, chemotherapy is still the most widely used option for the control of helminthes. However, many farmers in the developing countries are unable to afford synthetic anthelmintics for their livestock. In this scenario, the farmers depend on time-honored, centuries-old, affordable and accessible treatments for parasites.

∗

Corresponding author. Tel.: +92 41 9201106; fax: +92 41 9200725. E-mail address: [email protected] (A. Jabbar).

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.07.027

This concept has led to an interest of scientists in ethnoveterinary medicine (EVM), i.e., the identification of the traditional methods of managing livestock and treating livestock diseases (IIRR, 1994; McCorkle et al., 1996). In EVM, medicinal plants are used to treat parasite infections. A number of plants have been listed to treat parasitic infections (Jabbar et al., 2006b) and a few of them have been scientifically validated (Hammond et al., 1997; Akhtar et al., 2000; Iqbal et al., 2003, 2004, 2005, 2006a,b,c,d, 2007; Jabbar et al., 2006c; Lateef et al., 2006). In Indo-Pak subcontinent, Ayurvedic and Unani medicinal systems are very popular and people have been using plants not only for the treatment of their own ailments but also for their domesticated animals. Chenopodium album L. (Chenopodiaceae) and Caesalpinia crista L. (Fabaceae) commonly known as “Bathu” and “Kranjwa”, respectively, are valuable medicinal plants in Pakistan and their different parts are utilized in

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the traditional system of medicine (Said, 1969). Traditionally, Chenopodium album has been used for its diuretic, laxative, sedative, hepatoprotective and antiparasitic properties from centuries (Said, 1969; Fournier, 1999). Similarly, in folk medicine Caesalpinia crista seeds are used to treat asthma, chronic fever, cough, headache, stomach or bowel upset and as an anthelmintic (Nadkarni and Nadkarni, 1976; Satiyavati et al., 1976). Both plants are being used separately or as a part of compound prescriptions in EVM as an anthelmintic and in anorexia in animals from centuries as crude powder mixed with jaggery or as water decoction (personal communication). The present study rationalizes the use of Chenopodium album and Caesalpinia crista as anthelmintics using standard parasitological procedures. 2. Materials and methods 2.1. Plant material Whole plant of Chenopodium album and seed kernels of Caesalpinia crista were procured from local market in Faisalabad (Pakistan), 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 specimens (no. 13/2001; Chenopodium album L. whole plant and no. 12/2000; Caesalapinia crista L. seed kernels) are stored in the Ethnoveterinary Research and Development Centre (EVRDC), Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan. Plant materials of both plants were dried in an oven at 40 ◦ C, ground to a fine powder and stored in polythene bags at 4 ◦ C until used. 2.2. Extraction The powdered material (450 g) of each plant was soaked in 3 L 70% aqueous methanol by cold maceration at room temperature. After 3 days, the filtrate was collected through a piece of porous cloth and filter paper, and the plant material was re-soaked twice and filtrate was collected. The combined filtrate was concentrated in a rotary evaporator at 40 ◦ C under reduced pressure, and dried in a vacuum oven. The w/w yield of Chenopodium album and Caesalpinia crista was 25.4 and 28.6%, respectively. These extracts were stored at 4 ◦ C until use. 2.3. In vitro anthelmintic activity The in vitro trials for anthelmintic activity of crude aqueous methanolic extract (AME) of both plants were carried out using the adult motility assay and egg hatch test. 2.3.1. Adult motility assay Adult motility assay was conducted on mature live Haemonchus (H.) contortus following Sharma et al. (1971). Briefly, the female mature worms were collected from the abomasums of freshly slaughtered sheep in the local abattoir. The worms were washed and finally suspended in phosphate buffer

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saline (PBS). Five worms were exposed in triplicate to each of the following treatments in separate Petri dishes at room temperature (25–30 ◦ C): 1. AME of Chenopodium album whole plant @ 6, 12, 24 and 48 mg/mL. 2. AME of Caesalpinia crista seed kernels @ 6, 12, 24 and 48 mg/mL. 3. Levamisole, 0.55 mg/mL. 4. PBS (control). The inhibition of motility and/or mortality of the worms subjected to the above treatments were used as the criteria for anthelmintic activity. The motility was recorded after 0, 1, 2, 3 and 6 h intervals. Finally, the treated worms were kept for 30 min in the lukewarm fresh PBS to observe the revival of motility. 2.3.2. Egg hatch test 2.3.2.1. Egg recovery. Adult female Haemonchus contortus were collected after giving the longitudinal incision along the greater curvature of abomasums of naturally infected sheep. The worms present in ingesta or attached to the surface of guts were picked manually using forceps and placed in a bottle containing cool (4 ◦ C) PBS (pH 7.2) and later were triturated in pestle and mortar. The suspension was filtered through sieves of different sizes based on the nematode species into a bowl. Filtrate was centrifuged in Clayton Lane tubes for 2 min 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. Coverslip was plucked off carefully from tubes and eggs were washed off into a conical glass centrifuge tube. Tube was 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 using the McMaster technique (Soulsby, 1982). 2.3.2.2. Test procedure. Egg hatch test was conducted following Coles et al. (1992). Eggs suspension of (0.2 mL; 100 eggs) was distributed in a 24-flat-bottomed microtitre plate and mixed with the same volume of different concentrations (0.15–5 mg/mL) of each plant extract. The positive control wells received different concentrations (3.0–0.0058 ␮g/mL) of oxfendazole (Systamex—ICI Pakistan, Ltd.; 2.265%, w/v) in place of plant extracts while negative control plate contained the diluent and the egg solution. The eggs were incubated in this mixture at 27 ◦ C. After 48 h, a drop of Lugol’s iodine solution was added to stop the eggs from hatching. All the eggs and first-stage larvae (L1) in each plate were counted. There was three replicates for each treatment and control for both the plants separately. 2.4. In vivo anthelmintic activity A total of 60 sheep (local Thalli breed) ≤1 year, having almost homogeneous characteristics viz., weight, eggs per gram

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of faeces (EPG) and composition of infecting nematode species for each plant were selected from a herd of more than 1000 animals being maintained at Livestock Experiment Station, Rakh Kherewala (Punjab, Pakistan). The animals were vaccinated against enterotoxemia and pleuropneumonia vaccines, supplied by the Veterinary Research Institute, Lahore (Punjab, Pakistan). The sheep had naturally acquired mixed parasitic infection of gastrointestinal nematodes. Infections were confirmed before the beginning of study by collecting faecal samples from the animals, by rectum and the number of nematode egg therein determined by the floatation method (Soulsby, 1982). For nematode species composition, coproculture was done for identification of larvae using standard description of MAFF (1979) and Thienpont et al. (1979). The nematode eggs recovered from the experimental sheep were identified by larval cultures as Haemonchus contortus, Trichostronglyus spp., Oesophagostomum columbianum and Trichuris ovis. The experimental animals were housed for one month before study initiation for acclimatization. After treatment they were penned singly by treatment until the end of the study. No physical contact was possible between sheep from different treatment groups. The sheep were kept on plastered floor and fed with grass and water ad libitum. The experimental sheep (n = 30 for each plant) were randomly divided into six groups of five animals each and assigned different per os treatments as single dose as given below: group 1 served as negative control and received no treatment while group 2 was a positive control which was given a single dose of levamisole HCl 7.5 mg/kg (ICI Pakistan Limited, Animal Health Division). Groups 3 and 4 received single doses of CP 1.0 and 3.0 g/kg, respectively, while the groups 5 and 6 were given single doses of AME 1.0 and 3.0 g/kg, ? respectively. Faecal samples from each animal were collected in the morning, starting from day 0 pre-treatment and at days 5 and 13 post-treatment and were evaluated for the presence of worm eggs by salt floatation technique (MAFF, 1979). The eggs were counted by the McMaster method (Soulsby, 1982). Egg count percent reduction (ECR) was calculated using the following formula: ECR (%) =

3. Results and discussion In adult motility assay, all the worms were found dead at 6-h post-exposure to different concentrations of AME of Chenopodium album and Caesalpinia crista except at 6.0 mg/mL in case of AME of Chenopodium album where one worm was still alive (Fig. 1). The higher doses of both the plants resulted in an early onset of activity and higher number of dead worms compared with lower doses (Fig. 1). Crude aqueous methanolic extracts of Chenopodium album and Caesalpinia crista exhibited inhibitory effects on eggs hatching and LC50 was determined graphically from the regression equation after correcting from negative control. The calculated LC50 values of Chenopodium album, Caesalpinia crista and positive control (oxfendazole) were 0.449, 0.134 and 1.9 ␮g/mL, respectively. The regression values and correlation of regression of the Chenopodium album were y = −0.112x + 5.051; R2 = 0.869 and those of Caesalpinia crista and positive control were y = −0.199x + 5.360; R2 = 0.600 and y = −1.263x + 7.485; R2 = 0.786, respectively. It was observed that AME of Caesalpinia crista was more effective (p ≥ 0.05) against egg hatch as compared to Chenopodium album and the ovicidal activity of both the plants was low (p ≤ 0.05) as compared to positive control. In vivo, both CP and AME of Caesalpinia crista and Chenopodium album exhibited anthelmintic activity (p < 0.05) compared with negative control. In Caesalpinia crista treated sheep, the maximum reduction in eggs per gram of faeces was recorded as 93.9% with AME at 3.0 g/kg on day 13 PT (Fig. 2B), whereas, it was 82.2% with Chenopodium album AME at 3.0 g/kg on day 5 PT (Fig. 3A). Levamisole (7.5 mg/kg), a standard anthelmintic agent, showed maximum reduction (95.1–95.6%) in EPG on day 5 PT (Figs. 2 and 3) compared with negative control. The dose-dependent anthelmintic effect of Caesalpinia crista was recorded on day 13 PT, which was not evident on day 5 PT. The possible explanation of this difference may be due to the direct effect of Caesalpinia crista through paralysis and expulsion of worms leading to drop in EPG. The added adverse effects on the ovaries of surviving worms may have led to further

Pre-treatment egg count per gram–Post-treatment egg count per gram × 100 Pre-treatment egg count per gram

2.5. Statistical analysis 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 from adult motility assay and in vivo experiments were statistically analysed using SAS software (SAS, 1998). The results were expressed as mean ± standard error of mean (S.E.M.).

drop in EPG on day 13 PT making a dose-dependent difference. Levamisole seems to affect only through paralysis of worms as there was a little increase in EPG on day 13 PT compared with day 5 PT; whereas, there was further reduction in EPG with CP and AME of Caesalpinia crista suggesting its more than one actions, i.e., paralysis of worms and lowered fecundity due to possible effects on ovaries of the worms. Similarly, in case of Chenopodium album, there was no notable dose-dependent response on day 5 PT while on day 13 PT significant reduction in EPG was recorded with CP. Caesalpinia crista might have affected both adult worms through paralysis caused by its cholinergic effects as reported for Caesalpinia bonduc (Datt´e et al., 1998, 2004), which resulted

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Fig. 1. Graph showing the time- and dose-dependent in vitro anthelmintic activity of Caesalpinia crista (A) and Chenopodium album (B) crude aqueous methanol extracts at 6.0–48.0 mg/mL concentrations in comparison with positive control levamisole (0.55 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.

Fig. 2. Bar diagram showing anthelmintic activity of Caesalpinia crista crude powder (CP) and aqueous methanol extract (AME) in sheep naturally infected with 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) was used as the criterion for anthelmintic activity. Activity of CP and AME is compared with that of positive control, levamisole (7.5 mg/kg). Values shown are mean ± S.E.M., n = 5; Both CP and AME exhibited anthelmintic activity (p < 0.05) vs. negative control; dose-dependent effects, however, were recorded on day 13 PT.

in expulsion of the worms from the host. The cholinergic effect of Caesalpinia bonduc extracts (Datt´e et al., 1998, 2004) could be explained by three acting pathways, as reported by Sandow (1965). This action could be due to: (i) a direct effect on the muscle, (ii) an activation of the transmission process in the neuromuscular junction, or (iii) both. A similar study was performed by Clausen and Everts (1991) on isolated rat skeletal muscle. Tannins (1.56% of DM) in Caesalpinia crista seed kernels may have affected establishment or fecundity of the nematodes as demonstrated for tannin containing plants by Molan et al. (2000). The present results suggest that whole plant of Chenopodium album possess anthelmintic activity against sheep gastrointestinal nematodes. Previously, oil and infusion of leaves of Chenopodium ambrosioides have been reported for their anthelmintic activity (Gibson, 1965; Ketzis et al., 2002; MacDonald et al., 2004). As far as ascertained, this is the first report of anthelmintic activity of Chenopodium album.

The active principles for the anthelmintic effects of both Chenopodium album and Caesalpinia crista have not been exactly identified so far. Better efficacy of alcoholic fractions in case of Caesalpinia crista indicates that the active principle may be lipophilic while in case of Chenopodium album it may be lipophilic as well as hydrophilic. Earlier, ascaridole was considered to be the active constituent of Chenopodium ambrosioides oil for its anthelmintic activity but now titerpenoid saponins are also well thoughtout to be important constituents in this regard. For example, Kiuchi et al. (2002) isolated four p-menthane-type monoterpene hydroperoxides (2a–5a) together with ascaridole from AcOEt extract of aerial parts of Chenopodium ambrosioides L. var. antherminticum A. Gray, and tested for anti-trypanosomal activity. These constituents exhibited trypanocidal activity against the epimastigotes of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chaga’s disease). Similarly, the

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attributed to CH2 Cl2 extract of the seed kernels of Caesalpinia crista which was found to contain seven furanocassanetype diterpenes [caesalpinins C-G (1–5) and norcaesalpinins D and E (6, 7)] together with norcaesalpinins A-C (8–10) and 11 known compounds (norcaesalpinins A-C, 2-acetoxy3-deacetoxycaesaldekarin e, caesalmin B, caesaldekarin e, caesalpin F, 14(17)-dehydrocaesalpin F, 2-acetoxycaesaldekarin e, 7-acetoxybonducellpin C and caesalmin G) (Linn et al., 2005). The anthelmintic activity of Caesalpinia crista observed in the present study might be due to the presence of cassane- and noncassane-type diterpenes in the seed kernels. 4. Conclusion This study shows that Caesalpinia crista and Chenopodium album possess in vitro and in vivo anthelmintic activity, thus, justifying the traditional use of these plants against helminths. However, further controlled experiments on these plants and/or or isolated bioactive compounds using larger number of animals and experimental infections with individual nematode species are suggested. References

Fig. 3. Bar diagram showing anthelmintic activity of Chenopodium album crude powder (CP) and aqueous methanol extract (AME) in sheep naturally infected with 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) was used as the criterion for anthelmintic activity. Activity of CP and AME is compared with that of positive control, levamisole (7.5 mg/kg). Values shown are mean ± S.E.M.; n = 5. Both CP and AME exhibited anthelmintic activity (p < 0.05) vs. negative control; dose-dependent effects, however, were recorded on day 13 PT with CP.

compounds like betalain alkaloids, phenolic acids, betain, oxalic acid, oleanolic acid, sitosterol, furanocoumarins and saponins (Nicholas et al., 1955; Hegnauer, 1964, 1989) may be responsible for anthelmintic activity of Chenopodium album. Earlier, Akhtar and Aslam (1989) have reported 94.5% anthelmintic activity of glycosides isolated from Caesalpinia crista seeds. In contrast, however, H¨ordegen et al. (2003) could not demonstrate reduction in EPG with 28 mg/kg of an aqueous ethanol extract of the seeds of Caesalpinia crista. The discrepancy among the findings of the present study and those of Akhtar and Aslam (1989), and H¨ordegen et al. (2003) could be attributed to (i) different doses, parts and chemical composition of the Caesalpinia crista seeds used, (ii) different extraction procedures employed, (iii) variations in the susceptibility of the nematodes to various phytochemicals and (iv) food–drug interactions which can influence the absorption of bioactive plant compounds. Recently, Linn et al. (2005) have reported antimalarial activity of cassane- and norcassane-type diterpenes against the growth of Plasmodium falciparum. The antimalarial activity was

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