Journal of Ethnopharmacology 102 (2005) 256–261

Anthelmintic activity ofCalotropis procera (Ait.) Ait. F.flowers in sheep

Zafar Iqbala,∗, Muhammad Lateefa, Abdul Jabbara,Ghulam Muhammadb, Muhammad Nisar Khana

a Department of Veterinary Parasitology, University of Agriculture, Faisalabad 38040, Pakistanb Department of Clinical Medicine and Surgery University of Agriculture, Faisalabad 38040, Pakistan

Received 4 November 2004; received in revised form 20 May 2005; accepted 16 June 2005Available online 8 August 2005

Abstract

The anthelmintic activity ofCalotropis (C.) procera flowers in comparison with levamisole was evaluated through in vitro and in vivostudies. In vitro studies revealed anthelmintic effects (P < 0.05) of crude aqueous (CAE) and crude methanolic extracts (CME) ofCalotropispp ointestinaln to as foundt vamisole( r than thoseu .©

K

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pe(wcpalarLh

htar

nicine

lax-naloci-,ry

theartsdadi-6astioneredut to

0d

rocera flowers on liveHaemonchus (H.) contortus as evident from their mortality or temporary paralysis. For in vivo studies,Calotropisrocera flowers were administered as crude powder (CP), CAE and CME to sheep naturally infected with mixed species of gastrematodes. Egg count percent reduction (ECR) was recorded as 88.4 and 77.8% in sheep treated with CAE and CP at 3 g kg−1 body weighn day 7 and 10 post-treatment (PT), respectively. CME was least effective resulting in 20.9% reduction in ECR on day 7 PT. It w

hat Calotropis procera flowers possess good anthelmintic activity against nematodes, yet it was lower than that exhibited by le97.8–100%). It is suggested that further research on large scale be carried out involving a large number of animals, doses highesed in the current study, identification of active principles, and standardization of dose and toxicity studies for drug development2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Calotropis procera; Flowers; Anthelmintic; Nematodes; Sheep

. Introduction

Helminthiasis is among the most important animal healthroblems, which inflicts heavy production losses. The dis-ase is highly prevalent particularly in developing countriesDhar et al., 1982). Chemical control of helminths coupledith improved management has been an important wormontrol strategy throughout the World. However, increasingroblems of development of resistance in helminths (Geertsnd Dorny, 1995; Coles, 1997) against anthelmintics have

ed to the proposal of screening medicinal plants for theirnthelmintic activities. The plants are known to provide aich source of botanical anthelmintics (Satyavati et al., 1976;ewis and Elvin Lewis, 1977). A number of medicinal plantsave been used to treat parasitic infections in man and animals

∗ Corresponding author. Tel.: +92 41 9201106; fax: +92 41 9200725.E-mail address: zafaruaf@yahoo.com (Z. Iqbal).

(Nadkarni, 1954; Chopra et al., 1956; Said, 1969; Aket al., 2000; Iqbal et al., 2004).

Calotropis procera (Ait.) Ait. F. (Asclepiadaceae) knowas Aak in Pakistan, is used in ethnoveterinary medsystem of Pakistan as an expectorant, anthelmintic,ative, purgative, anti-inflammatory and diuretic (persocommunication). Despite some reports of toxicity assated withCalotropis feeding to animals (Mahmoud et al.1979a,b; Badwi et al., 1998), its use in ethnoveterinamedicine is increasing based on empirical evidence insuccessful treatment of different ailments. Different pas well as latex ofCalotropis procera have been reporteto have emetic, purgative and anthelmintic effects in trtional medicine (Kirtikar and Basu, 1935; Jain et al., 199).In Pakistan,Calotropis procera flowers are mostly usedan anthelmintic in small ruminants in the form of decocand/or crude powder mixed with jaggery and administas physic drench/balls. The present study was carried o

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2005.06.022

Z. Iqbal et al. / Journal of Ethnopharmacology 102 (2005) 256–261 257

scientifically validate the use ofCalotropis procera flowersas an anthelmintic in the light of claims of the traditionalhealers and small ruminant farmers.

2. Materials and methods

FreshCalotropis procera flowers collected from Cholis-tan Rangeland, District Bahawalpur (Pakistan), identified andauthenticated by a botanist by comparing with specimensstored in the herbarium of Department of Botany, Univer-sity of Agriculture, Faisalabad, Pakistan. The voucher spec-imen (No. 03/2002;Calotropis procera flowers; Cholistan)is stored in the Ethnoveterinary Research and DevelopmentCentre (EVRDC), Faculty of Veterinary Science, Universityof Agriculture, Faisalabad, Pakistan. Flowers were dried inan oven at 40◦C, ground to a fine powder and stored in poly-thene bags at 4◦C until used.

2.1. Preparation of aqueous extract

Crude aqueous extract (CAE) of the powderedCalotropisprocera flowers was prepared according to the standardmethod (Fenado et al., 1989). Briefly, 100 g of the pow-dered flowers was mixed with 500 mL of distilled waterin a 1 L flask and boiled for 1.5 h. Following cooling to4 fil-t vac-ur

2

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2

dCtB thea attoirT phateb te toe es atr

1234

ss riteri

for anthelmintic activity. The motility was recorded after 0,1, 2, 3 and 6 h intervals. Finally, the treated worms were keptfor 30 min in the lukewarm fresh PBS to observe the revivalof motility.

2.4. In vivo anthelmintic activity

The in vivo trials were conducted at Livestock Experi-ment Station, Rakh Kherewala (Punjab, Pakistan). A total of44 Thalli sheep of both sexes (≤1 year), weighing 18–24 kgwas randomly selected for in vivo trials. Before the startof experiment, the animals were confirmed to be naturallyinfected with mixed species of gastrointestinal nematodesby fecal examination using standard qualitative and quanti-tative parasitological procedures (Soulsby, 1982). Identifica-tion of nematode eggs in the faeces was done using standarddescription ofMAFF (1979)andThienpont et al. (1979). Theanimals selected were suffering from mixed gastrointestinalnematodes species including mainlyHaemonchus contor-tus, Trichostrongylus colubriformis, Trichostrongylus axei,Strongyloides papillosus andTrichuris ovis. The experimen-tal sheep (n = 44) were randomly divided into 11 groups offour animals each and assigned different per os treatments assingle dose as given below:

Group 1: Untreated control.-

e rate

dose

ning,and

ce of

3

0◦C, the ‘brew’ was filtered using Whatman No.1er paper. The filtrate was then concentrated in aum rotary evaporator and the extract stored at 4◦C untilequired.

.2. Preparation of methanolic extract

PowderedCalotropis procera flowers were exhaustivextracted with methanol in a Soxhlet’s apparatus (Asuzu andnu, 1994). The crude methanolic extract (CME) was evrated to dryness and stored at 4◦C until used.

.3. In vitro anthelmintic activity

The in vitro trials for anthelmintic activity of CAE anME were conducted on mature liveHaemonchus contor-

us of sheep as described previously (Sharma et al., 1971).riefly, the female mature worms were collected frombomasums of freshly slaughtered sheep in the local abhe worms were washed and finally suspended in phosuffer saline (PBS). Ten worms were exposed in triplicaach of the following treatments in separate Petri dishoom temperature (25–30◦C):

. Levamisole, 0.55 mg mL−1;

. CAE ofCalotropis procera flowers at 25 mg mL−1;

. CME ofCalotropis procera flowers at 25 mg mL−1;

. PBS (control).

The inhibition of motility and/or mortality of the wormubjected to the above treatments were used as the c

.

a

Group 2: Levamisole HCl (Nilverm® 1.5%, w/v; ICI Pakistan Limited, Animal Health Division) at 7.5 mg kg−1 bodyweight (b.w.).Group 3: Crude powder (CP) at 1 g kg−1 b.w.Group 4: CP at 2 g kg−1 b.w.Group 5: CP at 3 g kg−1 b.w.Group 6: Crude aqueous extract at the equivalent dos1 g kg−1 b.w. of CP.Group 7: CAE at the equivalent dose rate 2 g kg−1 b.w. ofCP.Group 8: CAE at the equivalent dose rate 3 g kg−1 b.w. ofCP.Group 9: crude methanolic extract at the equivalentrate 1 g kg−1 b.w. of CP.Group 10: CME at the equivalent dose rate 2 g kg−1 b.w. ofCP.Group 11: CME at the equivalent dose rate 3 g kg−1 b.w. ofCP.

Faecal samples of each group collected in the morstarting from day 0 pre-treatment and at days 3, 5, 7, 1014 post-treatment (PT) were evaluated for the presenworm eggs by salt floatation technique (MAFF, 1979). Theeggs were counted by the McMaster method (Soulsby, 1982).Egg count percent reduction (ECR) (Pankavich et al., 197)was calculated using the following formula:

ECR (%)=

pre-treatment egg count/g

−post-treatment egg count/g

pre-treatment egg count/g× 100

258 Z. Iqbal et al. / Journal of Ethnopharmacology 102 (2005) 256–261

Table 1In vitro effect of crude aqueous and methanolic extracts ofCalotropis procera on Haemonchus contortus of sheep in comparison with positive control(levamisole)

Treatments Mean number of worms showing motility at different hours post-exposure

0 h 1 h 2 h 3 h 6 h Fresh PBS for 30 mina

Levamisole at 0.55 mg mL−1 10.0 a 3.6 b 1.6 c 0.3 d 0 d 0 dCalotropis procera CAE at 25 mg mL−1 10.0 a 7.0 b 5.3 c 3.3 d 3.0 d 5.0 bCalotropis procera CME at 25 mg mL−1 10.0 a 8.6 b 7.6 c 6.6 d 4.3 e 4.3 ePBS 10.0 a 10.0 a 10.0 a 10.0 a 9.6 a 9.6 a

(a–e) Means marked with the similar alphabets in a row do not differ significantly atP ≥ 0.05.a Indicates that worms were placed in PBS after exposure of 6 h to the treatments to confirm their mortality.

The observations were statistically analyzed using SASsoftware (SAS, 1998). Test of significance between the meanparameters were performed by analysis of variance.

3. Results and discussion

In vitro trials demonstrated time-dependent anthelminticactivity both of CAE and CME ofCalotropis procera flowersagainstHaemonchus contortus (Table 1). In vivo, maximumECR (88.4%) was recorded in sheep treated withCalotropisprocera CAE at 3 g on day 7 PT closely followed by CP at 3 g(77.8%) on day 10 PT (Tables 2 and 3). CME of Calotropisprocera flowers was least effective, i.e. 20.9% ECR on day 7PT (Table 4). The present investigation is the first scientific

validation of the anthelmintic activity ofCalotropis proceraflowers in view of its usage in ethnoveterinary practice inPakistan.

Earlier, however,Garg and Atal (1963)reported in vitroparalysis ofOesophagostomum columbianum and Bunos-tomum trigonocephalum in 4 and 6 h, respectively, post-exposure to 1% calotropain isolated from latex ofCalotropisprocera. In another study (Shivkar and Kumar, 2003), freshas well as aqueous extracts of dried latex ofCalotropis pro-cera exhibited a dose-dependent inhibition of spontaneousmotility (paralysis) of earthworms, the effects of higher doses(100 mg mL−1 of aqueous extract of dry latex and 100%fresh latex) were comparable with that of 3% piperazine.Al-Qarawi et al. (2001)showed a concentration-dependentlarvicidal activity againstHaemonchus contortus in vitro

Table 2Effect of per os administration of crude aqueous extract ofCalotropis procera flowers on egg count percent reduction (mean± S.E.M.) of faeces in sheepnaturally infected with gastrointestinal nematodes

Days PT Dose and ECR

1.0 g kg−1 body weight 2.0 g kg−1 body weight 3.0 g kg−1 body weight

0 997.5± 107.0 a 1005.0± 164.5 a 1035.0± 147.2 a3 922.5± 85.1 (7.5) a 765.0± 199.1 (23.9) ab 195.0± 60.6 (81.1) b5 982.5± 76.8 (1.5) a 525.0± 112.5 (47.8) b 135.0± 25.9 (86.9) b7 1027.5± 100.5 (−3.0) a 630.0± 138.5 (37.3) b 120.0± 0 (88.4) b

11

F PT, po o not diffes

TE ers on llyi

D

11

Fs

0 1020.0± 32.4 (−2.2) a4 982.5± 78.7 (1.5) a

igures in parenthesis indicate egg count percent reduction (ECR);ignificantly atP ≥ 0.05.

able 3ffect of per os administration of crude powder ofCalotropis procera flow

nfected with gastrointestinal nematodes

ays PT Dose and ECR

1.0 g kg−1 body weight

0 1140.0± 86.6 a3 1035.0± 77.9 (9.2) a5 1080.0± 69.2 (5.3) a7 1140.0± 103.9 (0) a0 930.0± 34.6 (18.4) b

4 1095.0± 95.2 (3.9) a

igures in parenthesis indicate egg count percent reduction (ECR); PT, poignificantly atP ≥ 0.05.

570.0± 207.8 (43.2) b 135.0± 8.6 (86.9) b495.0± 112.5 (50.7) b 180.0± 17.3 (82.6) b

st-treatment; ECR marked with the similar alphabets in a column dr

egg count percent reduction (mean± S.E.M.) of faeces in sheep natura

2.0 g kg−1 body weight 3.0 g kg−1 body weight

1000.0± 40.4 a 1080.0± 207.8 a885.0± 43.3 (11.5) b 810.0± 277.1 (15.7) ab885.0± 25.9 (11.5) b 630.0± 329.0 (41.7) ab675.0± 25.9 (32.5) c 360.0± 155.8 (66.7) b615.0± 43.3 (38.5) c 240.0± 103.9 (77.8) b

585.0± 8.6 (41.5) c 315.0± 129.9 (70.8) b

st-treatment; ECR marked with the similar alphabets in a column do not differ

Z. Iqbal et al. / Journal of Ethnopharmacology 102 (2005) 256–261 259

Table 4Effect of per os administration of crude methanolic extract ofCalotropis procera flowers on egg count percent reduction (mean± S.E.M.) of faeces in sheepnaturally infected with gastrointestinal nematodes

Days PT Dose and ECR

1.0 g kg−1 body weight 2.0 g kg−1 body weight 3.0 g kg−1 body weight

0 1035.0± 77.9 a 1005.0± 112.5 a 945.0± 43.3 a3 1005.0± 112.5 (2.9) a 975.0± 112.5 (2.9) ab 870.0± 17.3 (7.9) b5 1050.0± 138.5 (−1.4) a 1155.0± 77.9 (−14.9) a 975.0± 8.6 (−3.1) a7 900.0± 69.2 (13.0) a 795.0± 60.6 (20.9) b 1050.0± 155.8 (−11.1) a

10 1140.0± 155.8 (−10.1) a 930.0± 103.9 (7.5) ab 1080.0± 190.5 (−14.3) a14 930.0± 103.9 (9.1) a 870.0± 121.2 (13.4) ab 960.0± 138.5 (−1.5) ab

Figures in parenthesis indicate egg count percent reduction (ECR); PT, post-treatment; ECR marked with the similar alphabets in a column do not differsignificantly atP ≥ 0.05.

within 20 min of application ofCalotropis procera latex,and significant reduction in egg production and lesser wormburden in sheep experimentally infected withHaemonchuscontortus and treated with single oral doses of 0.01 mL or0.02 mL kg−1 body weight ofCalotropis procera latex.

It is evident from the results of present study that CAEhad higher activity as compared to CP and CME forms,which may be considered as an indication of the presenceof water soluble active anthelmintic principles inCalotropisprocera. The in vivo findings were consistent with thoserecorded under in vitro wherein CME exhibited relativelymilder effect than CAE. The difference in anthelmintic activ-ity of CAE and CME was, however, more pronounced in vivo.The anthelmintic activity ofCalotropis procera was also con-firmed by graded dose response in sheep treated with CAEand CP (Tables 2 and 3). The graded dose response was,however, not observed for CME ofCalotropis procera.

In the light of available information (Garg and Atal, 1963;Al-Qarawi et al., 2001; Shivkar and Kumar, 2003), oneor more constituents of latex in flowers, separately or incombination may be responsible for the anthelmintic activityrecorded in the present study. Latex is chemically com-posed of various compounds like cardenolides, proteolyticenzymes, alkaloids and carbohydrates (Dhar and Singh,1973; Seiber et al., 1982). It contains a number of car-dioactive glycosides, calactin, calotropain, calotropagenin,p nin,u nd/ort 82;M n-z .,1Mcea

op-ee ro-sw edn

wise, 830 mg kg−1 body weight oral dose of dried latexdid not produce toxic effects in mice and the LD50 wasfound to be 3 g kg−1 body weight (Dewan et al., 2000). Thedoses ofCalotropis procera flowers used in the present studywere based on their traditional use for small ruminants, andnone of the treatments revealed signs of toxicity. Ethano-lic extracts of flowers, buds and roots ofCalotropis procerahave been reported to contain alkaloids, carbohydrates, gly-cosides, phenolic compounds/tannins, proteins and aminoacids, flavonoids, saponins, sterols, acidic compounds andresins in (Sharma and Sharma, 1999). We, however, could notfind scientific evidence of quantitative differences in the com-pounds isolated from flowers and other parts ofCalotropisprocera. It is, therefore, not possible to ascertain the quantityof latex, known to be toxic on higher doses to small rumi-nants (Mahmoud et al., 1979b), contained in the doses ofvarious treatments given in the current study. Varying levelsof antiplasmodial and schizonticidal activity of different partsincluding flowers ofCalotropis procera againstPlasmod-ium falciparum (Sharma and Sharma, 2000, 2001), however,suggest qualitative and/or quantitative differences in theirchemical composition.

The active principles responsible for anthelmintic activityof Calotropis procera flowers or latex and the mechanismof action involved have not so far been identified/elucidated.Recently, however, in vitro and in vivo spasmogenic effectsoa use thm d.

e thef udy.Tc uldb fort mallr con-t ganica rch isn rtic-u ell as

roceroside, syriogenine, calotoxin, uscharin, uzarigescharidine, voruscharin, tannins, flavonoids, sterols a

riterpenes (Singh and Rastogi, 1972; Seiber et al., 19ossa et al., 1991). Other compounds found were be

oylisolineolon and benzoyllineolone (Chandler et al968), procesterol, a steroidal hydroxy ketone (Khan andalik, 1989), flavonol glycosides (Sen et al., 1992), organic

arbonate along with stigmasterol and�-sitosterol (Oleat al., 2002), and procerain, a stable cysteine protease (Dubeynd Jagannadham, 2003).

It is important to note that latex also exhibits toxic prrties like irritation, inflammation and iridocyclitis (Tomart al., 1970), cardiotoxicity, liver damage and testicular necis (Garg, 1979). Oral doses of 0.01 or 0.02 mL kg−1 bodyeight ofCalotropis procera latex were, however, reporton-toxic to sheep and goats (Mahmoud et al., 1979b). Like-

f latex on gastrointestinal smooth muscles of rats (Kumarnd Shivkar, 2004), and in vitro spasmolytic effect of aqueoxtract ofCalotropis procera on guinea-pig trachea smoouscle chain (Iwalewa et al., 2005) have been demonstrateIn conclusion, the use of driedCalotropis procera flow-

rs as an anthelmintic in the form of water decoction byarmers seems valid in the light of results of the current stherefore, quality controlled extracts ofCalotropis pro-era flowers or possibly isolated bioactive compounds coe promising alternatives to conventional anthelmintics

he treatment of gastrointestinal trichostrongylides of suminants in the future. Such a treatment could be used inrol strategies against gastrointestinal nematodes in ornd conventional production systems. However, reseaeeded for studies on artificially induced infections of palar species of parasites, the bioactive constituents, as w

260 Z. Iqbal et al. / Journal of Ethnopharmacology 102 (2005) 256–261

on the reproducibility, dosage, application regime, toxicity,and effectiveness ofCalotropis procera. Moreover, chem-ical constituents can vary considerably between individualplants due to genetic or environmental differences, develop-ment stages of the plant at harvesting, drying process andstorage technique (Croom, 1983). Thus, a quality control ofthe plant material, the extraction scheme and the extract itselfis strongly recommended for further studies.

Acknowledgements

This research was funded by the University of Agricul-ture, Faisalabad (Pakistan) under Promotion of ResearchProgramme.

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