EVALUATION OF ANTHELMINTIC ACTIVITY OF SOME …prr.hec.gov.pk/jspui/bitstream/123456789/598/1/110S.pdf · anthelmintic activities of different plants reported in literature have been

i

EVALUATION OF ANTHELMINTIC ACTIVITY OF

SOME ETHNOBOTANICALS

BY

ALTAF HUSSAIN

A Thesis Submitted in Partial Fulfillment of the Requirement for the Degree

of

DOCTOR OF PHILOSOPHY

IN

PARASITOLOGY

DEPARTMENT OF PARASITOLOGY

FACULTY OF VETERINARY SCIENCE, UNIVERSITY OF AGRICULTURE,

FAISALABAD, PAKISTAN

2008

ii

To,

The Controller of Examinations, University of Agriculture, Faisalabad.

“We, the supervisory committee certify that contents and form of the thesis submitted by,

Mr. Altaf Hussain, Regd. No. 91-ag-774 have been found satisfactory and recommend that

it be processed for evaluation by the External Examiner(s) for the award of degree.

SUPERVISORY COMMITTEE

CHAIRMAN: ---------------------------------------------- (Prof. Dr. Muhammad Nisar Khan)

MEMBER: ---------------------------------------------- (Prof. Dr. Zafar Iqbal)

MEMBER: --------------------------------------------- (Prof. Dr. Muhammad Shoaib Akhtar)

iii

ACKNOWLEDGEMENT Thanks to Almighty ALLAH, the most compassionate, kind and merciful, Who blessed the mankind with

Holy Quran and the Prophet (PBUH) for their guidance.

The valuable guidance, constructive criticism and suggestions of my SUPERVISORY COMMITTEE

and a very kind and friendly guidance of DR. MUHAMMAD SOHAIL SAJID are highly appreciable for

the successful completion of this study. The services of all local farmers and veterinarians of district

Sahiwal are appreciable who contributed in the completion of the survey. Thanks to all my friends and

fellow students especially MR. MUHAMMAD KASIB KHAN for creating and maintaining an academic

atmosphere in the laboratories and hostel. Funds provided by the UNIVERSITY OF AGRICULTURE,

FAISALABAD, PAKISTAN for this project under the promotion of research scheme are gratefully

acknowledged.

Altaf Hussain

iv

CONTENTS

Chapter # Title Page #

1

INTRODUCTION

1

2 REVIEW OF LITERATURE 3

3 MATERIALS AND METHODS 31

4 RESULTS 40

5 DISCUSSION 74

6 SUMMARY, CONCLUSIONS AND

RECOMMENDATIONS

95

7 REFERENCES 98

v

LIST OF TABLES

Table Title Page

1 Plants evaluated/used for anthelmintics activity 8 2 In vitro assays of plant preparations evaluated against different species

of nematodes 20

3 Scientifically evaluated ethnobotanicals used for their in vitro anthelmintic activity in animals in Pakistan

21

4 In vivo evaluation of plant preparations against Haemonchus contortus in sheep and goats

22

5 In vivo evaluation of plant preparations against mixed gastrointestinal (GI) nematode infections in ruminant hosts

23

6 In vivo evaluation of plant preparations against cestodes and trematode parasites in different host species

24

7 Scientifically evaluated ethnobotanicals for their in vivo anthelmintic activity in animals in Pakistan

25

8 Globally identified ethnobotanicals with their potential anthelmintic activity

29

9 Plants screened to evaluate anthelmintic activity 34 10 Frequency of use of medicinal plants for the treatment and/or

management of helminthes of animals in district Sahiwal, Pakistan 42

11 Ethnoveterinary practices for the treatment and/or management of heminthosis in animals in district Sahiwal, Pakistan

44

12 In vitro effect of different indigenous plants on survival of Haemonchus contortus (Mean±SEM) of sheep in comparison with Levamisole

49

13 Ranking of 10 plants according to their effects on adult Haemonchus contortus

53

14 Per cent egg hatch and LC50 of different plants 54 15 Regression values and correlation of regression of the effect of different

plants on egg hatching 55

16 Ranking of 10 plants based on LC50 values and regression correlation values in egg hatch

56

17 Summary of in vitro results 57 18 Effect of different forms and doses of 10 selected plants on egg per

gram (Mean±SEM) of feces in sheep naturally infected with mixed species of gastrointestinal nematodes

59

19 Fecal egg count reduction (%) with crude aqueous methanolic extract at the dose rate of 8 g kg-1 body weight at day 15 post treatment

63

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LIST OF FIGURES

Figure Title Page

4.1 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Trianthema portulacastrum L. whole plant compared with control groups

64

4.2 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Lagenaria siceraria (Molina) Standl. leaves compared with control groups

65

4.3 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Tribulus terrestris L. whole plant compared with control groups

66

4.4 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Musa paradisiaca L. leaves compared with control groups

67

4.5 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Albizia lebbeck (L.) Benth. leaves compared with control groups

68

4.6 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Syzygium cumini (L.) Skeels leaves compared with control groups

69

4.7 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Bambusa arundinacea (Retz.) Willd. leaves compared with control groups

70

4.8 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Digera muricata L. whole plant compared with control groups

71

4.9 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Mangifera indica L. leaves compared with control groups

72

4.10 Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of Ziziphus mauritiana Lam. leaves compared with control groups

73

1

Chapter # 1

INTRODUCTION

Helminths are recognized as a major constraint to livestock production throughout the tropics

and elsewhere (Ibrahim et al., 1984; Waller, 1999; Githiori et al., 2004). Among different

types of helminths, nematodes are the most important as far as their prevalence and adverse

effects are concerned. They cause retarded growth (Ashraf, 1985; Kochapakdee et al., 1995),

lowered productivity (Perry and Randolph, 1999), mortality (FAO, 1974; Sykes, 1994) and

high economic losses (Irfan, 1984; Iqbal et al., 1993). The prevalence of nematodes in

different species of animals has been reported very high (25.1 to 92% ) in Pakistan (Durrani

et al., 1981; Mohiuddin et al., 1984; Khan, 1985; Iqbal et al., 1993; Qayyum, 1996).

Most of the parasite control programs are based upon a combination of chemotherapeutic

control, grazing management, dietary management, biological control, vaccination and

ethnoveterinary medicine (EVM) treatment (Waller, 1999; FAO, 2002). Various problems

have been evolved with chemotherapeutic control practices such as parasites are developing

resistance to several families of chemical anthelmintics (McKenna et al., 1995; Vermunt et

al., 1995; Chandrathani et al., 1999; Chartier et al., 2001; Leathwick et al., 2001), chemical

residues and toxicity problems (Kaemmerer and Buttenkotter, 1973; Muhammad et al.,

2004), un-economical, non-adaptability and non-availability of drugs in remote areas.

The concept of organic farming has stimulated a renewed interest in ethnoveterinary

medicine since last decade. McCorkle invented the term ethnoveterinary in 1986 and defined

it in 1996 as, the holistic interdisciplinary study of the local knowledge and socio-cultural

structures and environment associated with animal health care and husbandry. Historically,

both human and animal medicine has relied heavily on traditional treatments and plant

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materials. Even now in human healthcare 80 to 90% of the planet’s inhabitants still rely

mainly on traditional treatments and practitioners (Plotkin, 1992). Similar figures appear to

hold for animal health care (Mathias et al, 1996; Mc Corkle et al., 1996).

Ethnobotanical studies reveal that the indigenous knowledge of a community is a key player

in the identification of medicinal plants and such plants have been tested by generations of

indigenous people (Ole-Miaron, 1997; Makhubu, 1998; Tabrah, 1999; Cox, 2000). This

indigenous knowledge is passed on orally from one generation to the next and occasionally

within a family constitutes the basis for traditional bioprospecting. Traditional bioprospecting

forms the foundation for ethnomedicine (Sindiga et al., 1993) and ethnoveterinary practice

(Ole-Miaron, 1997). Traditional bioprospecting is often leaded to new herbal product

development. Ethnopharmacological surveys provide the rationale for selection and scientific

investigation of medicinal plants, since some of these indigenous remedies are already used

by significant numbers of people over extended periods of time (Lans, 2001). Most

pharmaceutical companies have some form of research programs investigating plants with

the aim of creating allelochemicals (bioactive secondary compounds) and new marketable

drugs. Their findings are often based on well funded research. It is estimated that it costs

$320 million to develop a new drug over 10-15 years (Anzuino, 1999).

In contrast to lot of research in many countries (Hooft, 1999; Mathias et al., 1999; Swaleh,

1999), written records on ethnoveterinary medicine are lacking in Pakistan. The present

project was therefore designed to:

• Document the indigenous knowledge of ethnoveterinary practices against gastro-

intestinal nematodes, which may help veterinarians and stock raisers in future.

• Scientifically validate some widely used ethnobotanicals for their anthelmintic activity.

3

Chapter # 2

REVIEW OF LITERATURE

Helminthiasis is one of the most important animal diseases worldwide that can cause heavy

production losses in grazing animals. The disease is prevalent all over the world especially in

developing countries (Dhar et al., 1982) and is always associated with poor management

practices and inadequate and inappropriate control strategies. An integrated approach is

required for the effective control of helminths which includes strategic and tactical use of

anthelmintics which remains the corner stone to this end and careful management of grazing

lands including control of stocking rates and appropriate rotation strategies. Role of

vaccinations is also vital for the control of various parasitic diseases as in the case of

lungworms. However, various problems have emerged with the use of anthelmintics and

among them; resistance against various species of helminthes is of utmost importance

(Waller and Prichard, 1985) to different anthelmintic compounds and classes, as well as

chemical residue and toxicity problems (Kaemmerer and Butenkotter, 1973). In addition,

recognition of the antigenic complexity of parasites has slowed vaccine development. For

these various reasons, interest in the screening of medicinal plants for their anthelmintic

activity remains of great scientific significance despite extensive use of synthetic chemicals

in modern clinical practices all over the world. The plant kingdom is known to provide a rich

source of botanical anthelmintics, antibacterials and insecticides (Satyavati et al., 1976;

Lewis and Elvin-Lewis, 1977). A number of medicinal plants have been used to treat

parasitic infections in man and animals (Nadkarni, 1954; Chopra et al., 1956; Said, 1969).

However, their scientific evaluation as compared to commercial anthelmintics is limited.

4

2.1. Plants used as anthelmintics

Plants with anthelmintic activity have been reviewed by Akhtar et al. (2000). Anthelmintic

activity of some plants has also been reported akin to that of sorghum (Iqbal et al., 2001a),

Aliium sativum, Zingiber officinale, Cucurbita mexicana and Ficus religiosa (Iqbal et al.,

2001b), Artemisia brevifolia (Iqbal et al., 2004), Calotropis procera (Iqbal et al., 2005),

Nicotiana tabacum (Iqbal et al., 2006a) and Butea monosperma (Iqbal et al., 2006b). The

anthelmintic activities of different plants reported in literature have been tabulated/reviewed

in Table 1.

2.2. In vitro anthelmintic activity

In the beginning, most of the in vitro researches regarding anthelmintic activity of plants,

their different extracts or oils have been based on their toxic effects on earthworm,

Pheritima posthuma (Gaind and Budhiraja, 1967; Ali and Mehta, 1970; Kokate and Varma,

1971; Dixit and Varma, 1975; Banerjee and Nigam, 1978; Girgune et al., 1978; Agarwal et

al., 1979; Girgune et al., 1979; Mishra et al., 1979; Mehta et al., 1981; Garg and Kasera,

1982a, b; Dengre, 1982; Nanda et al., 1987; Siddiqui and Garg, 1990; Garg and Siddiqui,

1992). Most of these substances which are toxic to earthworms produce a primary irritation

or agitation that results in the withdrawal of the worm from the neighborhood of the poison.

By asset of this effect, anthelmintics doubtless often drive out the parasite when the

concentration does not get sufficiently higher to kill the worm (Sollmann, 1918). Some

workers have also used hookworms, Haemonchus contortus, and tapeworms and/or Ascaris

lumbricoides for the evaluation of in vitro anthelmintic tivity of different plant materials

(Dubey and Gupta, 1968; Sharma et al., 1971; Kalesaraj, 1974, 1975; Dixit and Varma,

1975; Banerjee and Nigam, 1978; Girgune et al., 1978; Agarwal et al., 1979; Girgune et al.,

5

1979; Mishra et al., 1979; Sharma et al., 1979; Shrivastava, 1979; D’Cruz et al., 1980;

Mehta et al., 1981; Garg and Kasera, 1982a, b; Dengre, 1982; Kakrani and Kalyani, 1984;

Kalyani et al., 1989; Siddiqui and Garg, 1990; Nakhare and Garg, 1991; Garg and Siddiqui,

1992; Garg and Jain, 1992). A modified egg hatch assay (Coles et al., 1992) is often used

to evaluate the effect of plant products against eggs of Haemonchus contortus or other

trichostrongylids. Some other researchers conducting in vitro studies have used an

alteration of the larval development assay (LDA) or larval motility tests which are

commonly used for testing of resistance of parasites to anthelmintics (Menezes et al., 1992;

Nirmal et al, 1998; Al- Qarawi et al., 2001; Alawa et al., 2003; Assis et al., 2003; Lateef et

al., 2003). The anthelmintic activities of different plants reported in literature for their in

vitro anthelmintic activity have been tabulated/reviewed in Table 2 (world wide) and Table

3 (Pakistan).

2.3. In vivo anthelmintic activity

In vivo trials have also been conducted for the evaluation of anthelmintic activity of various

plant materials. The parameters for such an activity included expulsion of worms from their

hosts (Kalesaraj and Kurup, 1968; Lawrence, 1990; Philips, 1990; Pradhan et al., 1992;

Asuzu and Onu, 1994; Desta, 1995) or reduction in the number of eggs per gram of faeces

(EPG) passed by the infected hosts compared with commercial anthelmintic treated animals

(Akhtar, 1988). For example, in pigs experimentally infected with Ascaris suum, oral

administration of papaya (Carica papaya) latex, from Indonesia reduced parasitic burden

up to 100%, 7 days after treatment (Satrija et al., 1994). Similarly, some other plant

extracts identified from ethnoveterinary sources for their anthelmintic properties were

tested in experimentally infected sheep for their activity against gastrointestinal nematodes

6

(Hördegen et al., 2003). A 100% reduction was observed in faecal egg counts and a 72 and

88% mortality of adult Haemonchus contortus and Trichostrongylus colubriformis was

observed in sheep offered an ethanol extract of Fumaria parviflora, but no effect was

observed in sheep offered other plant extracts. Chakraborty et al. (1979), tested the

anthelmintic activity of alcoholic extracts of Tribulus terrestris, a perennial plant in India,

in an in vivo study. They reported a dose-related expulsion of Ascaridia galli worms, in

experimentally infected poultry. Recently, the anthelmintic activity of Khaya senegalensis,

a plant well known for its ethnoveterinary use, has been demonstrated anthelmintic activity

both in vitro and in vivo (Ademola et al., 2004). A few of the in vivo trial have been carried

out in sheep and goats infected with Haemonchus contortus (Table 4) or with mixed

nematode infections in ruminants (Table 5) or cestode and trematode infections in different

host species (Table 6) round the globe. Ample sum of work has been done as for as in vivo

anthelmintic trials are concerned (Table 7).

2.4. Survey of ethnoanthelmintic

Ethnobotanical studies reveal that the indigenous knowledge of a community is a key player

in the identification of medicinal plants and such plants have been often tested by generations

of indigenous people (Cox, 2000; Tabrah, 1999; Makhubu, 1998; Ole-Miaron, 1997). This

indigenous knowledge is passed on orally from one generation to the next and occasionally

within a family constitutes the basis for traditional bioprospecting. Traditional bioprospecting

form the foundation for ethnomedicine (Sindiga et al., 1993) and ethnoveterinary practice

(Ole-Miaron, 1997). Traditional bioprospecting often leads to new herbal product

development. For a very long time modern bioprospecting, which depends on scientific

7

analysis has preyed upon traditional bioprospecting to benefit the pharmaceutical industry

(Ole-Miaron, 2003).

In developing countries like Pakistan, the farmers and herdsmen do not have an easy access

to the professional veterinary personnel. In addition, despite availability of veterinarians,

farmers usually rely on their personal knowledge for prevention and treatment of

helminthiasis as reported elsewhere (Walzer et al., 1991). This situation has led to the fact

that ethnoveterinary systems are the only alternative to “Western” veterinary therapy.

Ethnoveterinary medicine (EVM) is a system of maintaining animal health and curing

diseases of animals that is based on folk beliefs and traditional knowledge (TK), skills,

methods and practices (Mathius-Mundy and McCorkle, 1989). EVM knowledge like all other

TK systems is transmitted orally from generation to generation (McCorkle, 1986; Mathius-

Mundy and McCorkle, 1989; McCorkle et al., 1996), and like the other TK systems, it is

disappearing because of rapid socioeconomic, environmental and technological changes. In

ethnomedicine, at least 80% of the worlds’ population in developing countries uses plant

materials as their source of primary health care (Farnsworth et al., 1985). To date there are

only few published research papers (Jabbar et al., 2006a) on documentation of

ethnoveterinary medicine in Pakistan in contrast to other countries where special attention

has been focused on this area (Anonymous, 1996). Documentation of indigenous knowledge

regarding ethnoanthelmintics has been tabulated/reviewed in Table 8.

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Table 1. Plants evaluated/used for anthelmintics activity Name of plant Part (s) used Parasite (s) Target Reference (s) Allium sativum Bulb Roundworms Cattle, goat,

sheep Iqbal et al., 2001b

Annona senegalensis

Leaf, bark, root Nippostrongyllus braziliensis Rat Ibrahim et al., 1984

Acacia albida Seeds Worm infestation Sheep, goat Nwude and lbrahim, 1980 Adhatoda vesica Roots Mixed GI nematodes Sheep Lateef et al., 2003 Agati gratifola Not reported Ascaris lumbricoides Humans Kalesaraj, 1974 Ageratum conyzoides

Leaves, flowers Tapeworms Not reported Sharma et al., 1979

Aglaia odorattissima

Root bark Earthworms Not reported Nanda et al., 1987

Agrimonia eupatori Not reported Anthelmintic Humans Farnsworth et al., 1985 Agrimonia pilosa Agrimophol Tapeworms Not reported Xiao and Lin, 1986 Alangium lamarckii

Root bark Ascarids Poultry Dubey and Gupta, 1969

Alangium larmarckii

Root bark Hookworms, ascarids Dogs, poultry Dubey and Gupta, 1968

Bark Anthelmintic Cattle, goat, sheep

Root Fasciolosis Cattle, goat,sheep

Albizia anthelmintica

Bark Lungwomms Camel

Minja, 1989; ITDG and IIRR, 1996

Albizia coriavera Bark Fasciolosis, lungworms Cattle, goat, sheep

ITDG and IIRR, 1996

Albizia lebbeck Bark Ascaris lumbricoides In vitro Kalesaraj, 1975 Bulb Roundworms Cattle, goat,

sheep ITDG and IIRR, 1996; Iqbal et al., 2001b

Allium sativum

Bulb Ascaridia galli Chicken Das and Thakuria, 1974 Bulb Ascaris lumbricoides In vitro Kalesaraj, 1975 Aloe barteri Leaves Nippostrongyllus spp. Rat Ibrahim et al., 1984

9

Name of plant Part (s) used Parasite (s) Target Reference (s) Alpinia calcarata Cucuruma aramatica

Rhizomes Ascaris 1umbricoides In vitro Kalesaraj,1975

Ammora wallichii Stem Calamintha umberosa

Whole plant

Picus religiosa Stem, bark Sentia myrtina Whole plant Sumplocos crataegoides

Leaves

Ascaridia galli In vitro Kaushik et al.,1981

Amomum aromaticum

Roots and Rhizomes

Ascaridia galli In vitro Kaushik et al., 1981

Anacardium occidentale

Not reported Earthworms, tapeworms Not reported Garg and Kasera, 1982a, b

Ananas comosus Fruit Ascaridia galli Chicken Fernandez, 199I Ananas sativus Not reported Taenia species and

Paramphistomum cervi in vitro Neogi et al, 1964

Ananas sativus Earthworms in vitro Chakraborty et al., 1976 Annona cherimolia Annona muricata Annona braziliensis Molinema dessetae

Not reported Nippostroongylus sp. Rat Bories et al„ 1991

Anogeissus leiocarpus

Bark, seeds Nippostrongyllus braziliensis Rat Ibrahim et al., 1984

Anogeissus leiocarpus

Bark

Securinega virosa Leaves, stem Khaya senegalansis

Bark

Nauclea latifolia Roots

Anthelmintic In vivo Bizimana, 1994

Anthocephalus Stem, Bark Ascaridia galli In vitro Kaushik et al., 1981

10

Name of plant Part (s) used Parasite (s) Target Reference (s) indices Areca catechu Nuts Taenicidal Cattle, goat,

dog Roepke, I996

Areca catechu Dried ripe seeds Tape worms Dogs, poultry British Veterinary Codex, 1953 Artabotrys odoratissimus

Leaves Pheretima posthuma (earthworms), Taenia solium and Ascaris lumbricoides

In vitro Siddiqui and Garg, 1990

Artemisia abrotanum

Not reported Anthelmintic Not reported Krause, 1993

Artemisia absinthium

Not reported Anthelmintic Not reported Bara et al., 1999; Guarrera, 1999; Francois, 1974

Artemisia annua Not reported Schistosoma mansoni Hamster, mice Shuhua et al., 2000 Artemisia brevifolia

Not reported Haemonchus conrortus Sheep Iqbal et al., 2004

Artemisia herba-alba

Shoots Haemonchus contortus Goat Idris et al., 1982

Artemisia inforescence

Leaves Ascaris suum Pig (in vitro) Slepnev, 1970

Whole plant Anthelmintic Not reported Krantz and Carr, 1967; Narayana et al., 1976; Akhtar, 1984; Sharma, 1993; Hammond et al., 1997

Artemisia maritima

Whole plant Neoascaris vitulorum Buffalo calves Akhtar et al., 1982; Farnsworth et al., 1985; Sherif et al., 1987; Fernandez, 1991

Artemisia mesatlantica

Flavonoids and sesquiterpene lactones

Anthelmintic Not reported Holeman et al., 1991

Artemisia monosperma.

Not reported Anthelmintic Not reported Abu-Niaaj et al., 1996

Artemisia pallens Not reported Anthelmintic Not reported Anonymous, 1956; Nakhare and Garg, 1991

Artemisia scoparia Not reported Anthelmintic Not reported Naqvi et al., 1991

11

Name of plant Part (s) used Parasite (s) Target Reference (s) Artemisia senna Not reported Anthelmintic, Cestodes Canine Francois, 1974; Narayana et al., 1976 Azadirachta indica Cake and leaves Anthelmintic Small

ruminants Gowda, 1997; Mostofa et al., 1996

Azadirachta indica Seeds Melia azedarach Seeds Ananas comosus Leaves Vernonia anthelmmtica

Seeds

Embelia ribes Fruit Fumarla parviflora Whole plant Caesalpinia crista Seeds

Haemonchus contortus Trichostrongylus colubriformis

Lambs Hördegen et al., 2003

Bixa orellana Seeds Ascaridia galli, Ascaris suum

Chicken, Pig

Fernandez, 1991

Boswellia dalzelii Bark Anthelmintic Sheep, goat Nwude and 1brahim,1980 Boswellia serrata Not reported Earthworms, tapeworms In vitro Girgune et al., 1978 Buddlea asiatica Not reported Earthworms, tapewonns,

Hookworms Not repoted Dengre, 1982

Butea frondosa Seeds Anthelmintic, Ascaridia galli, Ascaris lumbricoides

Chicken (In vitro), canine, human

Kalesaraj and Kurup, 1962, 1968; Joshi, 1970; Narayana et al., 1976; Lal et al., 1976, 1978; Shilaskar and Parashar, 1989

Butea frondosa Not reported Oxyurids Mice , Mehta and Parashar, 1966 Butea frondosa Seeds Ascaridia galli In vitro Lal et al.,1976 Butea monosperma Seeds Anthelmintic, G1 nematodes Sheep and

others Kalesaraj and Kurup, 1968; Chandra and Sabir, 1978; Lal et al., 1978; Prashanth et al., 2001; Iqbal et al., 2006b

Butea superba Not reported Anthelmintic Not reported Charka, 1948; Chopra et al., 1958 Seeds Toxocara vitulorum, Ascaridia

galli Buffalo calves, Chicken

Akhtar et al., 1985; Javed et al., 1994 Caesalpina crista

Seeds Haemonchus contortus Sheep, goats (In vitro)

Sharma et al., 1971

12

Name of plant Part (s) used Parasite (s) Target Reference (s) Calliandra calothyrsus

Legume Haemonchus contortus, Trichostrongylus, Strongyloides papillosus

Sheep Parker and Palmer, l991

Calliandra portoricensis Calotropis procera

Roots, leaves, flowers

Toxocara canis, Gastrointestinal nematodes, Haemonchus contortus

Dog, Sheep Adewunmi and Akubue,1981; Garg and Atal, 1963; Jain et al., 1996; A1-Qarawi et al., 2001; Iqbal et al., 2005

Capillipedium Foetidum Cymbopogon martini

Oil, grass Pheretima posthuma (earthworms), Taenia solium and Ascaris lumbricoides

In vitro Siddiqui and Garg, 1990

Seeds Ascaris lumbricoides, Ascaridia galli

Human, Chicken

Dhar et al., 1965; Lal et al., 1976 Carica papaya

Latex from fruit Ascaridia galli, Ascaris suum, Heligmosomoides polygyrus

Chicken, Pig, Mice

Mursof and He, 1991; Satrija et al., 1994; Satrija et al., 1995

Carissa edulis Roots Roundworms Cattle, goats, sheep

ITDG and IIRR, 1996

Carum copticum Seeds Ascaris lumbricoides Human Krantz and Carr, 1967; Kalesaraj, 1974

Cassia alata Seeds Ascaridia galli Chicken Fernandez, 1991 Cassia accidentalis Leaves Nippostrongylus braziliensis Rat Ibrahim et al., 1984 Cassia spectalis Roots Roundworms Cattle, goat,

sheep ITDG and IIRR, 1996

Chebulic myrobalans Belleric myrobalans Emblic myrobalans

Not reported Anthelmintic activity Not reported Gaind et al., 1964

Chenopodium album

Leaves Nematode Sheep Akhtar et al., 1999

Chenopodium spp. Oil Ascaris spp., Toxocara, Strongylus spp.

Horses, pigs, Dogs, Horses

British Veterinary Codex, 1953, 1965

13

Name of plant Part (s) used Parasite (s) Target Reference (s) Chloroylon swientenia

Oil Earthworms, tapeworms, hookworms

Not reported Dengre,1982

Chrysanthemum spp.

Not reported Haemonchus contortus Chicken Rebrassier, 1934

Chrysophyllum cainito

Stem Haemonchus contortus Cattle Fernandez, 1991

Cinnamomum tamala

Oil Earthworms, tapeworms In vitro Girgune et al., 1978

Cissampelos mucromata

Roots Anthelmintic Not reported Minja, 1989

Citrus acida Citrus aromatica Citrus medico

Rind Ascaris lumbricoides In vitro Kalesaraj, 1975

Combretum mucronatum

Roots Guinea worm Humans Sofowora, 1982

Commiphora mukul

Oleo-gum resin Tapeworms, hookworms Not reported Kakrani and Kalyani, 1984

Croton macrostachys

Leaves Anthelmintic Not reported Minja, 1989

Cucurbita rnexicana

Seeds Moniezia expansa, Fascialopsis buski, Ascaris lumbricoides, Hymenolepis diminuta

Not reported Shrivastava and Singh, 1967

Cucurbita moschata

Seeds Cestodes Human Xiao and Lin, 1986

Cucurbita pepo Momordica charantia

Not reported Haemonchus contortus (mature)

Goats (in vitro)

Sharma et al., 1971

Cyathocline lyrata Essential oil Tapeworms, hookworms In vitro Shrivastava, 1979 Cymbopogon nardus Cymbopogon

Essential oil Earthworms In vitro Kokate and Varma, 1971

14

Name of plant Part (s) used Parasite (s) Target Reference (s) citratus Cyperus rotendus Not reported Tapeworms, earthworms Not reported Girgune et al., 1979 Datura quercifolia Datura metal

Fruit Ascaridia galli In vitro Kaushik et al.,1981

Diospyrol Necator americanus, Nematodirus dubius, Hymenolepis nana

Golden, Hamster, Mice

Sen et al., 1974

Diospyrol Necator americanus Golden hamster

Sen et al.,1974

Diospyros mollis

Diospyrol Nematodirus dubius, Hymenolepis nana

Mice Sen et al.,1974

Diospyros scabra Seeds Fasciolosis, lungworms Cattle, goat, sheep, camel

ITDG and 1IRR,1996

Dodonea viscosa Leaves Intestinal worms Not reported Sharma and Singh, 1989 Dryopteris filixmas Male fern Moniezia, tapeworms,

Dicrocoelium, Fasciola Not reported British Veterinary Codex,1953

Embelia kilimandschiraca

Roots Anthelmintic Not reported Minja,1989

Embelia schimperi Seeds, roots, fruit Anthelmintic, Hynnenolepis diminuta

Rat Bøgh et al., 1996

Not reported Mixed nematode infection Ruminants Chopra et al., 1956; Ikram and Hussain, 1978

Embelia ribes

Fruit Taenia species, Paramphistomum cervi, GI nematodes

Goats Neogi et al.,1964; Javed and Akhtar, 1990

Embelia ribes Seeds Tapeworms Poultry Qureshi and Sabir, 1979 Erythrina senegalensis

Bark Fasciolosis Ruminants Nwude and Ibrahim, 1980

Eupatorium triplinerve

Flowers Ascaris lumbricoides and Taenia solium

Not reported Garg and Nakhare, 1993

Evodia rutaecarpa Not reported Ascarid nematodes, L4 of Ostertagia circumcincta

Pig (in vitro) Sheep (in

Perrett and Whitfield, 1995

15

Name of plant Part (s) used Parasite (s) Target Reference (s) vitro)

Feruia foetidissima Not reported Haemonchus, Bunostomum, Chabertia, Nematodirus

Sheep Pustovoi, 1968

Ficus religiosa Not reported Anthelmintic In vitro Iqbal et al., 2001b Flemingia vestita Root-tuber peel Raillietina echinobothrida Domestic fowl

(in vitro) Pal and Tandon, 1998

Flemingia vestita Root-tuber peel Fasciolopsis buski Pig (in vitro) Kar et al., 2002 Fumaria parviflora Plant powder Trichostrongylus,

Haemonchus, Trichuris, Fasciola spp.

Sheep, buffalo Akhtar and Javed, 1985; Kailani et al., 1995

Gardenia lucida Essential oil Tapeworms, earthworms Not reported Girgune et al.,1979 Hagenia abyssainicia

Fruit Roundworms Cattle, goat, sheep

ITDG and IIRR, 1996

Hedychium coronarium Hedychium spicatum

Rhizomes Earthworms, tapeworms Not reported Dixit and Varma,1975

Helleborus niger Stem Ascaris lumbricoides Humans Kalesaraj, 1974 Heracleum sosnoskyi

Not reported Strongylosis, GI nematodes Sheep Gadzhiev and Eminove, 1986a, b

Hyoscyamus niger Seeds Mixed nematode infection In vivo Akhtar and Ahmad, 1990 Inula racemosa Essential oil Earthworms, tapeworms Not reported Mishra et al., 1979 Jugulans regia Musa paradisaca Scindapsus officinalis

Not reported Haemonchus contortus Goats (in vitro)

Sharma et al., 1971

Khaya senegalansis

Bark Fasciola spp. Not reported Bizimana, 1994

Lagenaria siceraria

Seeds Cestodes, Moniezia, Avitelina spp.

Sheep Akhtar and Riffat, 1987

Ascaridia galli Chicken Lansium domesticum

Seeds Ascaris suum Pig

Fernandez, 1991

16

Name of plant Part (s) used Parasite (s) Target Reference (s) Haemonchus contortus Goat

Lantana trifolia Fruit Fasciolosis, lungworms Cattle, goat, sheep

1TDG and IIRR, 1996

Lantana camara var. aculeata

Seeds Anthelmintic activity Not reported Avadhoot et al., 1980

Lawsonia inermis Leaves Fasciolosis Sheep, goat Nwude and Ibrahim, 1980 Ascaridia galli Chicken Ascaris suum Pig

Leucaena leucocephala

Seeds

Haemonchus contortus Goat

Fernandez, 1991

Limnophila conferta

Not reported Anthelmintic activity Not reported Reddy et al., 1991

Litsea chinensis Not reported Earthworms, tapeworms

Not reported Mishra et al., 1979

Macuna prurita Not reported Taenia species, Paramphistomum cervi

Not reported Neogi et al., 1964

Fruit powder Gastrointestinal cestodes Beetal goats Akhtar and Ahmad, 1992 Mallotus philippinensis Fruit Tapeworms Not reported British Veterinary Codex, 1953 Mangifera indica Seeds Ascaris lumbricoides Humans Kalesaraj, 1974 Matteuccia orientalis

Roots Fasciola sp. Cattle Shiramizu et al., 1993

Fruit, leaves Taenia species, Paramphistomum cervi

Fruit Haemonchus contortus

In vitro Neogi et al., I964; Nirmal et al., 1998

Fruit Ascaridia galli Chicken Akhtar and Riffat, 1985a

Melia azedarach

Haemonchus, Trichostrongylus, Trichuris, Chabertia spp.

Goats Akhtar and Riffat, 1984

Melia toosendan Not reported Ascarids Not reported Xiao and Lin, 1986 Mimosa pudica Stem Haemonchus contortus Not reported Fernandez, 1991 Mitragyna stipulosa

Roots Guinea worm Humans Sofowora, 1982

Momordica Not reported Ascaridia galli In vitro Lal et al., 1976

17

Name of plant Part (s) used Parasite (s) Target Reference (s) Ascaris suum Pigs Haemonchus contortus Goats

charantia Stem

Ascaridia galli Chicken

Fernandez ,1991; Farnsworth et al., 1985

Ascaris suum Pig Seeds Haemonchus contortus Goats

Fernandez, 1991 Moringa olelfera

Roots Mixed nematode infection Sheep Akhtar and Ahmad, 1990 Myrsine africana Leaves Roundworms Cattle , goats,


Nicotiana tabacum Nicotine sulphate Moneizia, Ascaridia, Cooperia, Haemonchus, Nematodirus, Ostertagia, Trichoslrogylus spp.

Not reported British Veterinary Codex, 1953, 1965

Nigella sativa Seeds Antifasciolic Buffalo Kailani et al., 1995 Peganum harmala Seeds Mixed GI infection, cestode

infection Goats Akhtar and Ahmed, 1991

Peganum harmala Seeds Gastrointestinal cestodes Goat Akhtar and Riffat, 1986 Piper betle Not reported Earthworms In vitro Ali and Mehta, 1970 Psitacia integrrima Seeds Earthworms, tapeworms Not reported Mishra et al., 1979 Psoralea coylifolia Seed powder Gastrointestinal nematodes Sheep laved and Akhtar, 1986

Fruit rind Gastrointestinal nematodes, cestodes

Sheep Akhtar and Riffat, 1985b

Not reported Ascaris lumbricoides In vitro Kalesaraj, 1975

Punica granatum

Not reported Haemonchus contortus In vitro Prakash et al., 1980 Ascaris suum, Haemonchus contortus

Goats Farnsworth et al., 1985

Ascaris suum Pigs Ascaridia galli Chicken

Quisqualis indica Stem

Haemonchus contortus Goats

Fernandez, 1991; Farnsworth et al., 1985

Quisqualis indica Seeds Ascaris spp. Not reported Xiao and Lin, 1986 Randia dumetorum Seeds Earthworms, tapeworms Not reported Mishra et al., 1979 Rapanea Seeds Roundworms Cattle, Goats, ITDG and IIRR,1996

18

Name of plant Part (s) used Parasite (s) Target Reference (s) melanoploeos Sheep Rhamnus principides


Rhus vulgaris Roots Roundworms Cattle, goats, sheep

ITDG and IIRR, 1996

Sapindus trifoliatum

Not reported Ascaridia galli In vitro Lal et al., 1976

Sheep Akhtar and Hassan, 1985 Saussurea lappa Roots Mixed species of nematodes Buffalo-calves Akhtar and Makhdoom, 1988

Nuts Anthelmintic Not reported Chattopadhyaya and Khare, 1969 Semecarpus anacardium Seeds GI cestodes Goats Akhtar, 1988 Senecio lyratiparitus


Solanum nodiflorum

Fruit Worm infestation Not reported Nwude and Ibrahim, 1980

Spigelia anthelmia Linn.

Aerial parts Haemonchus contortus In vitro Assis et al., 2003

Swertia chirata Whole plant Ascaridia galli Not reported Shilaskar and Parashar, 1989 Tamarindus indica Roots Roundworms Cattle, goats,


Terminalia avicennoides

Leaves, roots Nippostrongylus braziliensis Rats Ibrahim et al., 1984

Tiinospora rumphii Stem Haemonchus contortus Goats Fernandez, 1991 Tribulus terrestris Whole plant Ascaridia galli Poultry Chakraborty et al., 1979 Trichilia emetica Bark Fasciolosis, lungworms Cattle , goats,

sheep, camels ITDG and IIRR, 1996

Uvaria hookeri Uvaria narum

Root bark Haemonchus contortus Not reported Padmaja et al., 1993

Vernonia amygdalina

Stem bark

Annona senegalensis

Leaves

Haemonchus contortus In vitro Alawa et al., 2003

19

Name of plant Part (s) used Parasite (s) Target Reference (s) Seeds GI nematodes Ruminants Nadkarni, 1954; Awan, 1981; Ikram

and Hussain, 1978 GI nematodes, cestodes Sheep, Goats Oxyurids Not reported Ascaridia galli Chicken

Nadkarni, 1954; Chopra et al., 1956; Said, 1969; Awan, I981; Singh et al., 1985; Shilaskar and Parashar, 1989; Javed and Akhtar, 1990

Vernonia anthelmintica

Fruit/seeds

Oxyurids Mice Mehta and Parashar, 1966 Withania coagulans

Not reported Earthworms In vitro Gaind and Budhiraja, 1967

Essential oil Anthelmintic activity, earthworms, roundworms

Not reported Kokate and Varma, 1971; Mehta et al., 1981

Bark Ascaris lurnbricoides, Fasciolopsis buski, Hymenolepis nana

In vitro Singh et al., 1982

Zanthoxylum alatum

Not reported Earthworms, tapeworms, hookworms

Not reported Kalyani et al., 1989

G1 nematodes Sheep Iqbal et al., 2006c Ascaris Iumbricoides Human Kalesaraj, 1974, 1975 Anisakis larvae In vitro Goto et al., 1990 Dirofilaria immitis Canine Datta and Sukul, 1987; Chakraborty

et al., 1994

Zingiber officinale Rhizomes

Schistosoma mansoni Not reported Adewunmi et al., 1990

20

Table 2. In vitro assays of plant preparations evaluated against different species of nematodes Name of parasite/plant species Active principles Parts used Target Reference (s) 1. Against Caenorhabditis elegans Butea monosperma Sterols, palasonin S Not reported Prashanth et al., 2001 Combretum spp. Phenantherenes L Not reported McGaw et al., 2001 Cymbogon martini Geraniol W Not reported McGaw et al., 2000 Evodia ruteacarpa Atanine Fr Not reported Perrett and Whitfield, 1995 Ocimum sanctum Eugenol L Not reported Asha et al., 2001 Taverniera abyssinica Phytoalexins R Not reported Stadler et al., 1994 Terminalia macroptera Triterpenes W Not reported Conrad et al., 1998 2. Against Ascaris lumbricoides Acacia auriculiformis Not reported F Not reported El Garhy and Mahmoud, 2002 Albizia lebbek Not reported B E El Garhy and Mahmoud, 2002 Apium graveolens Not reported Sh E El Garhy andMahmoud, 2002 Artemesia santonica Not reported Sh E El Garhy and Mahmoud, 2002 Cassia obtusifolia Santonin Sh E El Garhy and Mahmoud, 2002 Inula helenium Alantalactone Sh E El Garhy and Mahmoud, 2002 3. Against Ascaridia galli Carica papaya Benzyl isothiocyanate S A Singh and Nagaich, 1999 4. Against Heligmosomoides polygyrus Albizia anthelmintica Not reported B E Gakuya, 2001 Embelia schimperi Embelin NR A Bøgh et al., 1996 Alstonia boonei Not reported B L3 Fakae et al., 2000 Nauclea latifolia Alkaloids saponin L L3 Fakae et al., 2000 Ocimum gratissimum Oleanolic acid L L3 Njoku and Asuzu, 1998 Piliostigma thonningii Tannins, alkaloids B L3 Fakae et al., 2000 5. Against Trichostrongylus colubriformis Peltophorum africanum Not reported L, Stem B, Root B E, L3 Bizimenyera et al., 2006 6. Against Haemonchus contortus Annona senegalensis Not reported B E, L3 Alawa et al., 2003 Spigelia anthelmia Not reported Aerial parts E, L3 Assis et al., 2003

21

Name of parasite/plant species Active principles Parts used Target Reference (s) Vernonia amygdalina Not reported L E, L3 Alawa et al., 2003 Parts used: B = Bark, F = funicle, Fr = fruits, L = leaves, R = root, S = seeds, Sh = Shoots, W = whole plant. Target: A = adult parasites, E = eggs, L3 = infective larvae. Table 3. Scientifically evaluated ethnobotanicals used for their in vitro anthelmintic activity in animals in Pakistan Botanical name of plant

Parts used Animal Parasite species Anthelmintic activity evaluated Reference (s)

Allium sativum Bulb Sheep H. contortus 100% at 6 hrs post exposure (PE) Iqbal et al., 2001 Artemisia brevifolia Whole

plant Sheep H. contortus 30% at 6 hrs PE with AE, 80% with CME

(at 25 mg mL-1) Iqbal et al., 2004

Calotropis procera Flowers Sheep H. contortus 50% with CAE, 57% with CAME (at 25 mg mL-1)

Iqbal et al., 2005

Chenopodium album Whole plant

Sheep H. contortus (eggs) LC50 = 0.449 mg mL-1 Jabbar et al., 2007

Caesalpinia crista

Seed kernel

Sheep H. contortus (eggs) LC50 = 0.134 mg mL-1 Jabbar et al., 2007

Cucurbita mexicana Whole fruit Sheep H. contortus 83.4% at 6 hrs PE Iqbal et al., 2001 Ficus religiosa Bark Sheep H. contortus 100% at 6 hrs PE Iqbal et al., 2001 Nicotiana tabacum Leaves Sheep H. contortus ≥75% at 6 hrs PT with CAE and CAME

at 25 mg mL-1 Iqbal et al., 2006a

Swertia chirata Whole plant

Sheep H. contortus 30% and 90% at 6 hrs PT with CAE and CME at 25 mg mL-1

Iqbal et al., 2006d

Trachyspermum ammi Seeds Sheep H. contortus (eggs) LC50 = 0.1698 and 0.1828 mg mL-1 of CAE and CME

Jabbar et al., 2006b,

Vernonia anthelmintica Seeds Goat H. contortus 50% at 6 hr PT with CME at 25 mg mL-1 Iqbal et al., 2006eZingiber officinale Rhizomes Sheep H. contortus 100% at 6 hrs PE Iqbal et al., 2001bAE=Aqueous extract; CAE=Crude aqueous extract; CAME=Crude aqueous methanolic extract; CME=Crude methanolic extract; PE=Post exposure

22

Table 4. In vivo evaluation of plant preparations against Haemonchus contortus in sheep and goats hosts Plant species Parts used Active principles Host Reference (s)

Allium sativum Bb Allicin G Vieira et al., 1999

Annona squamosa L Anthraquinone terpenoids G Vieira et al., 1999

Artemisia herba-alba Sh Santonin G Idris et al., 1982

Calotropis procera L Triterpenoids, anthocyanins, alkaloids S Al-Qarawi et al., 2001

Canavalia braziliensis S Not reported G Vieira et al., 1999

Carica papaya S Not reported G Vieira et al., 1999

Chenopodium ambrosioides L Benzyl isothiocyanate G Vieira et al., 1999

Chrysophyllum cainito St Ascaridole B Fernandez, 1991

Hymenaea courbaril B Not reported G Vieira et al., 1999

Menta spp. L Not reported G Vieira et al., 1999

Momordica charantia St Not reported G Vieira et al., 1999

Musa acuminate L Not reported G Vieira et al., 1999

Tinospora rumphii St Not reported G Fernandez, 1999 Parts used: B=bark, Bb=bulbs, L=leaves, S=seeds, Sh=shoots, St=stem. Host: B=bovids, G=goats, S=sheep.

23

Table 5. In vivo evaluation of plant preparations against mixed gastrointestinal (GI) nematode infections in ruminant hosts Plant species Parts

used Active principles Host Reference (s)

Albizia anthelmintica B, RB Sesquiterpene, kosotoxins

S Gakuya, 2001; Gathuma et al., 2004; Grade and Longok, 2000

Ananas comosus L Bromelain S, B Baldo, 2001; Hördegen et al., 2003; Jovellanos, 1997 Annona squamosa L Anthraquinone

terpenoids G, B Jovellanos, 1997; Vieira et al., 1999

Azadirachta indica S, L Azadirachtin S, B Chandrawathani et al., 2003; Hördegen et al., 2003; Pietrosemoli et al., 1999

Chenopodium ambrosioides

L, S, O Ascaridole S Ketzis et al., 2002

Chrysanthemum cinerariaefolium

Fl Pyrethrins S Mbaria et al., 1998

Caesalpinia crista S Not reported S Hördegen et al., 2003 Embelia ribes Fr Not reported S Hördegen et al., 2003 Fumaria parviflora W Not reported S Hördegen et al., 2003 Hagenia abyssinica Fr Not reported G Abebe et al., 2000 Hildebrandtia sepalosa RB Not reported S Gathuma et al., 2004 Khaya anthotheca B Kosotoxin B Nfi et al., 1999 Khaya senegalensis B Not reported S Ademola et al., 2004 Maerua edulis Tb Not reported S Gakuya, 2001 Myrsine africana Fr Benzoquinone S Gathuma et al., 2004 Nauclea latifolia B Not reported S Onyeyili et al., 2001 Solanum aculeastrum R Resin, tannins,

alkaloids B Nfi et al., 1999

Terminalia glaucescens B Not reported B Nfi et al., 1999 Vernonia anthelmintica S Anthraquinone S Hördegen et al., 2003 Vernonia amygdalina L Not reported B Nfi et al., 1999

Parts used: B=bark, Fl=flowers, Fr=fruits, L=leaves, R=root, RB=root bark, O=oil, S=seeds, Tb=Tuber, W=whole plant. Host: B=bovids, G=goats, S=sheep.

24

Table 6. In vivo evaluation of plant preparations against cestodes and trematode parasites in different host species Plant species Parts used Active principles Parasite Host Reference Tested against cestodes Albizia anthelmintica RB Kosotoxin

sesquiterpene C S Gathuma et al., 2004

Embelia Schimperi Fr, S, R Embelin Hd, Hm, Ts R, M, H Desta, 1995; Bøgh, et al., 1996 Ficus insipida, Ficus carica Lx Ficin C M de Amorin et al., 1999 Hagenia abyssinica Fr Kosotoxin C H Desta, 1995 Hildebrandtia sepalosa B Not reported C S Gathuma et al., 2004 Mallotus philippinensis Fr Rottlerin C G Akhtar and Ahmad, 1992 Myrsine Africana Fr Benzoquinone C S Gathuma et al., 2004 Peganum harmala S Tetra-hydroharmine C G Akhtar and Riffat, 1986 Albizia anthelmintica B, R Not reported Tested against trematodes Albizia anthelmintica B Not reported Fg G Koko et al., 2000 Embelia schimperi Fr Benzoquinone Ec M Bøgh, et al., 1996 Albizia anthelmintica Rb Not reported Fasciolosis Catt, G, S,

Cam ITDG and IIRR, 1996

Albizia coriavera, Allium sativum

B Not reported Fasciolosis Catt, G, S ITDG and IIRR, 1996

Diospyrus scabra S Not reported Fasciolosis Catt, G, S, Cam

ITDG and IIRR, 1996

Lantana trifolia Fr Not reported Fasciolosis Catt, G, S, Cam

ITDG and IIRR, 1996

Lawsonia inermis L Not reported Fasciolosis G, S Nwude and Ibrahim, 1980 Trichilia emetica B Not reported Fasciolosis Catt, G, S,

Cam ITDG and IIRR, 1996

Parts used: B = bark, Fr = fruits, Lx = latex, S = seeds, R = root, RB = root bark Parasite: C=unspecified cestodes, Ec=Echinostoma caproni, Fg=Fasciola gigantica, Hd=Hymenolepis diminuta, Hm=Hymenolepis microstoma,

Ts=Taenia saginata Host: G=goats, H=humans, M=mice, R=rats, S=sheep, Cam=camel, Catt=cattle

25

Table 7. Scientifically evaluated ethnobotanicals for their in vivo anthelmintic activity in animals in Pakistan Botanical name of plant

Parts used Animal Parasite type/species

Anthelmintic activity evaluated

Phytochemicals isolated

Reference (s)

Adhatoda vesica Aerial parts Goat GINs 62±5.4% used as PR@2 g/kg b.wt. >>99±1.2% morantel

AL and GL and saponins

Akhtar, 1988

Allium sativum Bulb Sheep H. contortus 100% at 6 hrs post exposure (PE)

Not reported Iqbal et al., 2001b

Artemisia brevifolia

Whole plant Sheep H. contortus 30% at 6 hrs PE with AE, 80% with ME (at 25 mg/mL)

Not reported Iqbal et al., 2004

Artemisia brevifolia

Whole plant Sheep GINs 67.2% at 3 gm/kg b.wt. with ME at 14 days PT


Butea monosperma

Seeds Sheep Trichostrongylids 78.4% on day 10 PT with CP at 3 gm/kg b.wt.

Not reported Iqbal et al., 2006b

Caesalpinia crista

Seeds Buffalo calves

Neoascaris vitulorum

100±0.1% used as PR or ME @ 2 g/kg b.wt. >>100% morantel

GL and saponins Akhtar, 1988; Akhtar and Aslam, 1989

Calotropis procera

Flowers Sheep H. contortus 50% with CAE, 57% with CAME (at 25 mg/mL)


Calotropis procera

Flowers Sheep GINs 88.4% with CAE, at 3 gm/kg b.wt., ≥97.8% levamisole


Chenopodium album

Aerial parts Sheep GINs 87±6% used as WE @ 2 g/kg b.wt. >>96±4% morantel

GL Akhtar, 1988

Chenopodium album

Whole plant Sheep H. contortus (eggs)

LC50 = 0.449 mg/mL, Not reported Jabbar et al., 2007

Caesalpinia crista

Seed kernel Sheep H. contortus (eggs)

LC50 = 0.134 mg/mL, Not reported Jabbar et al., 2007

26

Botanical name of plant




Reference (s)

Chenopodium album

Whole plant Sheep GINs 82.2% on day 5 PT with AME

Not reported Jabbar et al., 2007

Caesalpinia crista

Seed kernel Sheep GINs 93.9% on day 13 PT with AME

Not reported Jabbar et al., 2007

Cinnamommum tamala

Leaves Sheep GINs 97.6±1.8% used as GL @ 150 mg/kg b.wt. >>98±3% morantel

AL and GL Akhtar, 1988

Cucurbita mexicana

Whole fruit Sheep H. contortus 83.4% at 6 hrs PE Not reported Iqbal et al., 2001b

Cyperus scariosus Embellia ribes/robusta

Seeds Buffalo calves

Neoascaris vitulorum

10±3% used as PR @ 3 g/kg b.wt. >>100±0% morantel

GL and essential oils

Akhtar, 1988

Euphorbia prostrata

Aerial parts Sheep GINs 56±26% used as PR @ 2 g/kg b.wt. >>97±2% morantel

GL and flavonoid Akhtar, 1988

Euphorbia prostrata

Aerial parts Sheep GINs 98.6±1.6% used as ME @ 3 g/kg b.wt. >>98.8±1.3% oxfendazole

CGL and GL Akhtar, 1988

Ficus religiosa Bark Sheep H. contortus 100% at 6 hrs PE Not reported Iqbal et al., 2001b

Fumaria parviflora

Aerial parts Sheep GINs 99.8±0.1% used as EE @ 2 g/kg b.wt. >>99.8±0.3% morantel

AL and GL Akhtar, 1988

Hyoscyamus niger

Seeds Sheep GINs 95.8±5.6% used as PR @ 3 g/kg b.wt. >> 98.8±1.3% oxfendazole

AL, CGL and GL Akhtar, 1988

Lagenaria siceraria

Seeds/flower Sheep Cestodes 91.4±3.9% used as GL @ 100 mg/kg b.wt. >>92.0±8.0% morantel

CGL and GL from seeds

Akhtar, 1988

27





Reference (s)

Mallotus philipinensis

Fruits Goat Cestodes 91.3±5.3% used as GL @ 100 mg/kg b.wt. >>100.0±0% Nilzan

Flavonoids and GL Akhtar, 1988; Akhtar and Ahmad, 1992

Melia azedarach Seeds Goat GINs 99.4±1.2% used as PR @ 30 mg/kg b.wt. >>99.2±1.6% morantel

Anthraquinone and GL

Akhtar and Riffat, 1984, 1985; Akhtar, 1988

Momordica charantia

Fruits Sheep GINs 99.6±0.5% used as WE @ 3 g/kg b.wt. >> 98.8±1.3% oxfendazole

AL, CGL, flavonoid, GL and saponins

Akhtar, 1988

Moringa olifera Roots Sheep GINs 94.4±2.6% used as PR @ 3 g/kg b.wt. >> 98.8±1.3% oxfendazole

CGL and GL Akhtar, 1988

Morus alba Leaves/stem/ bark

Goat GINs 85.0±2.0% used as GL @ 500 mg/kg b.wt. >>99.0±0.04% morantel

GL in stem bark Riffat et al., 1986

Nicotiana tabacum

Leaves Sheep GINs 73.6% at 5 days PT with CME at 3 gm/kg b.wt.

Not reported Iqbal et al., 2006a

Nigella sativa Seeds Sheep Cestodes 99.0±0.3% used as PR @ 2.5 g/kg b.wt. >>100.0±0% Niclosamide used against GI cestodes of sheep

GL and AL and anthraquinone

Akhtar, 1988; Akhtar and Javed, 1991; Akhtar and Aslam, 1997

Peganum harmala

Seeds Goat Cestodes 100.0±% used as PR @ 3 g/kg b.wt. >>98.0±6.2% levamisole + oxyclozanide

Flavonoid, GL and AL

Akhtar and Riffat, 1986

Prunus persica Leaves Sheep GINs 99±5% used as WE @ 3 g/kg b.wt. >> 97±7%

CGL, flavonoid and GL i.e.,

Akhtar, 1988

28





Reference (s)

Morantel used against GI nematodes of sheep

persicon and naringenin

Psoralea corylifolia

Seeds Sheep GINs 99±0.09% used as WE @ 2 g/kg b.wt. >>99.9±0.01% morantel

AL and GL Javed and Akhtar, 1986

Punica granatum Fruit Sheep Cestodes 95±12% used as AL @ 225 mg/kg b.wt.>>100±0% levamisole + oxyclozanide used against GI cestodes of sheep

AL, CGL, flavonoid and GL

Akhtar, 1988

Saussurea lappa Roots Sheep GINs 100±0% used as ME @ 2 g/kg b.wt. >>100±0% Morantel used against GI nematodes of sheep

AL, CGL and GL Akhtar and Hassan, 1985; Akhtar and Makhdoom, 1988

Semecarpus anacardium

Seed Goat Cestodes 29±3.2% used as GL @150 mg/kg b.wt. >>98±6.2% levamisole +oxyclozanide

Anthraquinone, flavonoid and GL

Akhtar, 1988

Swertia chirata Whole plant Sheep H.contortus 30% and 90% at 6 hrs PT with CAE and CME at 25 mg/mL

Not reported Iqbal et al., 2006d

Swertia chirata Whole plant Sheep GINs 79.7% at 14 days PT with CAE at 3 gm/kg b.wt.

Not reported Iqbal et al., 2006d

Trachyspermum ammi

Seeds Sheep H.contortus (eggs)

LC50 0.1698 and 0.1828 mg/mL of CAE and CME

Not reported Jabbar et al., 2006b,

Trachyspermum Seeds Sheep GINs 78.1% on day 5 PT with Not reported Lateef et al.,

29





Reference (s)

ammi CP at 3 gm/kg b.wt. 2006 Vernonia anthelmintica

Seeds Goat H. contortus 50% at 6 hr PT with CME at 25 mg/mL

Essential oils and GL

Iqbal et al., 2006e

Vernonia anthelmintica

Fruits Goat GINs 73.9% at day 5 PT with CAE at 3 gm/kg b.wt.

Essential oils and GL

Iqbal et al., 2006e

Zingiber officinale

Rhizomes Sheep H. contortus 100% at 6 hrs PE Not reported Iqbal et al., 2001

Zingiber officinale

Rhizomes Sheep GINs 66.6% after 10 days PT at 3 gm/kg b.wt., 99.2% levamisole

Not reported Iqbal et al., 2006c

AE=Aqueous extract; AL=Alkaloid; CAE=Crude aqueous extract; CAME=Crude aqueous methanolic extract; CGL=Cardiac glycoside; CP=Crude powder; GL=Glycoside; GINs=Gastrointestinal nematodes; PE=Post exposure; PR=Powder; PT=Post treatment; >>=Compared with Table 8. Globally identified ethnobotanicals with their potential anthelmintic activity Origin of survey No of plants with

Anthelmintic activity

Anthelmintic activity Hosts Reference (s)

South East Asia 23 Roundworms, cestodes, trematodes Monogastrics, Ruminants

Anonymous, 1994

Kenya 19 Roundworms, cestodes, trematodes

Monogastrics, Ruminants

Anonymous, 1996

Eastern and Southern Africa

>100 Hookworms, cestodes, roundworms, trematodes

Humans, Ruminants Watt and Breyer-Brandwijk, 1962

East Africa >100 Hookworms, roundworms, cestodes

Humans, Ruminants Kokwaro, 1993

West Africa 18 Roundworms, cestodes Monogastric Ibrahim et al., 1984 Zaire 11 Roundworms Ruminants Kasonia et al., 1991

15 Roundworms, trematodes Ruminants, Monogastric

Nwude and Ibrahim, 1980

Nigeria

4 Helminths Ruminants Alawa et al., 2002

30

Africa >50 Roundworms, trematodes, cestodes Ruminants, Monogastric

Bizimana, 1994

51 Anthelmintic Humans Guarrera, 1999 Italy 5 Heltminths, parasites Livestock Pieroni et al., 2004 6 Anthelmintic Dogs Lans et al. (2000) Trinidad and Tobago 4 Helminths Ruminants Lans and Brown, 1998

Cameroon 10 Helminthiasis Livestock Nfi et al., 2001 Worldwide 100 Cestodes, trematodes, nematodes Animals Tagboto and Townson,

2001 Saudi Arabia 6 Vermifuge Camels Abbas et al., 2002 Indian subcontinent 6 Helminths Monogastric Nadkarni, 1954 Pakistan (Southern Punjab)

29 Helminths Ruminants Jabbar et al., 2006a

31

Chapter # 3

MATERIALS AND METHODS

3.1. Study area

Sahiwal received its name with respect to the name of local tribesmen “Sahu”. It has the

distinction of being an important seat of one of the oldest urban civilizations in the history of

mankind, the Indus Valley Civilization, which flourished around 3,000 to 5000 B.C. Its

population is 1,843,194 (Population Census Organization, 1998). Sahiwal district (3201 km2)

lies between 29-59° and 30-57° north latitude and 72-25° and 73-21° east longitudes. It

roughly forms a parallelogram lying generally NE-SW along the Ravi River

(http://www.sahiwal.gov.pk/, accessed on April 2, 2008). The temperature rises as high as

52°C in summer and falls to –5°C in winter and average rainfall is 2000mm. It comprises two

Tehsils namely Sahiwal and Chichawatni comprising of 531 villages. Sahiwal is an agro

based district with a very fertile soil and wheat, cotton, sugarcane, maize and rice are major

cash crops in the district (http://en.wikipedia.org/wiki/Sahiwal_District, accessed on April 2,

2008). According to the Economic Survey of Pakistan (2006), total number of livestock

population in the district is 2,086,174with 238,437 cattle, 670,554 buffalos, 50,488 sheep,

477,782 goats, 1574 camels, 4624 horses, 1301 mules, 66,339 asses and 575,075 poultry.

Sahiwal is well known for its famous Sahiwal breed of cattle and Nili Ravi buffalos.

3.2. Ethnoveterinary medicine survey

Qualitative survey methodologies namely Rapid Rural Appraisal (RRA) and Participatory

Rural Appraisal (PRA) were used in this project. Both methodologies are widely used in

gathering information. RRA was first defined in 1985 by Grandstaff and Grandstaff, “It is a

process of learning about rural conditions in an iterative and expeditious manner. More often

32

than not, it is multi-disciplinary in nature and has an in-built flexibility in the process of

collecting information. It has been defined as ‘any systematic activity designed to draw

inferences, conclusions, hypotheses or assessments, including acquisition of new information

in a limited period of time” (Kashyap, 1992). Dunn, (1994) builds on Grandstaff &

Grandstaff’s definition and considers RRA to be a “qualitative survey methodology using a

multi-disciplinary team to formulate problems for agriculture and research development”.

Hence, RRA is a collection of cost effective ways to learn about research situations needed

and initiatives of rural people and collect relevant data for project planning (Waters-Bayer

and Bayer, 1994).

Participatory Rural Appraisal (PRA) goes further than RRA in actively involving rural

people in identifying their problems, seeking solutions and evaluating results (Dunn, 1994;

Chambers, 1992). It is an outgrowth of and often confused with RRA. PRA is an “approach

and method for learning about rural life and conditions from, with, and by rural people”

(Chambers, 1992). The key elements of RRA and PRA are quite similar, with the main

difference being that RRA generates information for planners and PRA shifts the

“presentation and analysis of information to community members”. Another key difference

between RRA and PRA is that in PRA “rushing is replaced by relaxation” and there is a

strong rapport with community members (Chambers, 1992). Tools used in both the

techniques include secondary data reviews, observations, semi-structured interviews,

analytical games, stories and portraits, diagrams and workshops; most of which were used

during the study.

33

3.2.1. Selection of respondents

Initially, an exploratory phase; a small-scale rapid rural appraisal (RRA) was conducted in

two tehsils viz; Sahiwal and Chichawatni. The exploratory phase of the study was intended to

provide primary data on traditional veterinary healers (TVHs) having the knowledge of

species of animals and ethnoveterinary practices used for the treatment and control of

helminths as a basis for selecting respondents for the second phase of the study. A total of

331 TVHs having good knowledge of EVM practices were selected for the second phase of

survey.

3.2.2. Surveillance and data collection

A 2-year field survey was conducted from August 2004 to September 2006. A well-

structured questionnaire (open-ended interviews and guided dialogue technique) was used to

collect the relevant information from 331 selected respondents as described previously (Iqbal

et al., 2007) which falls under the category of participatory rural appraisal (PRA). In

addition, the direct observation approach as described by Etkin (1993) was also used.

Interviews were also complemented by participant observations and field visits to identify

plants and collect ethnobotanical specimens as described by Cunningham (2000). The

informant consensus (Heinrich, 2000) on the documented plants was developed through

focused group discussions. Further information was recorded on the plants used as

anthelmintics, their mode of preparation and administration. The survey team comprised of a

veterinarian who worked both as the translator and a laboratory technologist. The trained

field assistant and a community leader were also recruited from the local community. Local

language of the interviewees was “Punjabi and Saraiki” in which the interviews were

conducted. The documented plants were collected and identified by Department of Botany,

34

University of Agriculture, Faisalabad, Pakistan. The voucher specimens of plants were

preserved in Ethnoveterinary Research and Development Centre, Faculty of Veterinary

Science, University of Agriculture, Faisalabad, Pakistan.

3.3. Collection of Plant Material

The plant material (Table 9) based on the information collected from ethno-medicinal survey

was selected and procured from the local market/field and got authenticated from an expert in

the Department of Botany, University of Agriculture, Faisalabad. The criteria for the

selection of plants was seasonal availability of plants and previous work done on them i.e. if

a plant is tested previously for anthelmintic activity in the Department of Parasitology,

University of Agriculture, Faisalabad, was not included in the study. Voucher specimens

were kept in the Department of Parasitology, University of Agriculture, Faisalabad. The

selected plant material was screened for anthelmintic activity.

Table 9. Plants screened to evaluate anthelmintic activity Sr. no. Plant species Plant family Part/s used English name Vernacular name

1 Albizia lebbeck (L.) Benth. Fabaceae Leaves Woman's tongue Shareen

2 Bambusa arundinacea (Retz.) Willd. Poaceae Leaves Bamboo Bans

3 Digera muricata L. Amaranthaceae Whole plant False amaranth Tandla

4 Lagenaria siceraria (Molina) Standl. Cucurbitaceae Leaves Calabash Kaddoo

5 Mangifera indica L. Anacardiaceae Leaves Mango Aam

6 Musa paradisiaca L. Musaceae Leaves Banana Kaila

7 Syzygium cumini (L.) Skeels Myrtaceae Leaves Jambolan plum Jaman

8 Trianthema portulacastrum L. Aizoaceae Whole plant Desert horse-purslane It Sit

9 Tribulus terrestris L. Zygophyllaceae Whole plant Puncturevine Bhakhrra

10 Ziziphus mauritiana Lam. Rhamnaceae Leaves Ber, Indian Jujube Bairy

3.3.1. Extract preparation

Plant material (in varying amount depending upon availability of plant) was dried under

shade at a well ventilated place, cleaned of adulterants and ground to powdered form. The

35

plant material was soaked in sufficient amount of 70% aqueous-methanol by cold maceration

at room temperature for a total of 3 days. After that the filtrate was collected through a piece

of porous cloth and filter paper and the plant material re-soaked twice. The combined filtrate

was concentrated in a rotary evaporator at 40 °C under reduced pressure to yield a thick and

dark colored crude extract. This extract was stored at -4 °C until use and dissolved in distilled

water on the day of the experiments to prepare stock solution and different dilutions for the

purpose of evaluating pharmacological activity.


3.4.1. Adult motility assay

Mature live Haemonchus contortus from sheep were used to determine the effect of crude

aqueous methanolic extract (CAME) by method described previously by Iqbal et al. (2006a).

Briefly, the female mature worms were collected from the abomasum of freshly slaughtered

sheep in the local abattoir. The worms were washed and finally suspended in phosphate

buffer saline (PBS). A minimum of ten worms were exposed in three replicates to each of the

following treatments in separate petri dishes at room temperature (25-30oC):

1. CAME at the rate of 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 the criterion for anthelmintic activity. The motility was observed after 0, 2, 4, 6, 8 and 12

hour intervals. Finally, the treated worms were kept for 30 minutes in the lukewarm fresh PBS

to observe the revival of motility. The number of dead and survived worms was recorded for

each treatment.

36

3.4.2. Egg hatch test (EHT)

3.4.2.1. 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 minutes at 300 x 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 minutes at 130 x g. Cover slip 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 minutes at 300 x 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 the McMaster technique (Soulsby, 1982).

3.4.2.2. Test Procedure

Egg hatch test was conducted by the method described by Coles et al., 1992. Egg suspension of

(0.2 ml; 100 eggs) was distributed in a 24 well multi-well plate (Flow Laboratories) and mixed

with the same volume of different concentrations (0.25 to 8 mg mL–1) of plant extract (i.e.,

CAME). The positive control wells received different concentrations (0.09 to 3.0 µg mL–1) of

oxfendazole (Systamex—ICI Pakistan, Ltd., 2.265%, w/v) in place of plant extracts while

negative control wells contained the diluent and the egg solution. The eggs were incubated in

this mixture at 27°C. After 48 hours, two drops of Lugol’s iodine solution was added to stop the

37

eggs from hatching. All the eggs (dead and embryonated) and hatched larvae in each well were

counted. There were three replicates for each treatment and control.


3.5.1. Fecal egg count reduction test (FECR)

3.5.1.1. Study animals

A total 320 Lohi sheep of both sexes (≤1 year of age) weighing 18–25 kg having naturally

acquired mixed parasitic infection of gastrointestinal nematodes were selected from the Allah

Dad cattle farm of Jahaniyan, Punjab (Pakistan). Infection was confirmed before the beginning

of study by faecal examination of the animals, by the standard parasitological procedures

(Soulsby, 1982). The animals having higher than 500 eggs per gram of faeces were included in

the experiment. After selection of the animals, they were washed with an appropriate

ectoparasiticide. The animals were vaccinated against different bacterial/viral disease according

to the routine. The sheep were kept on wood shaving and fed with fresh grass/fodder,

concentrate (Anmol wanda®) and water ad libitum.

3.5.1.2. Treatment and follow-up procedures

Prior to the treatment, faecal samples were obtained by rectum from each animal, at least three

times at an interval of three days. On each occasion the number of eggs in the faeces according

to the genus was determined by larval culture and identification was done by morphological

characteristics described by MAFF (1986) and Thienpont et al. (1979). The animals selected

were suffering from mixed gastrointestinal nematodes species including mainly Haemonchus

contortus, Trichostrongylus colubriformis, Trichostrongylus axei, Strongyloides papillosus and

Trichuris ovis. On day zero, the sheep were allocated to eight groups of 4 animals each,

according to the complete randomized design, taking into consideration their live weight. These

38

groups were assigned different per os treatments as single dose for each plant as given below:

Group 1: Untreated control.

Group 2: Levamisole HCl (Nilverm® 1.5%, w/v; ICI Pakistan Limited, Animal Health

Division) at 7.5 mg kg−1 body weight (b.w.), served as treated control.

Group 3: Crude powder (CP) at 1 g kg−1 b.w.

Group 4: CP at 4 g kg−1 b.w.

Group 5: CP at 8 g kg−1 b.w.

Group 6: CAME at equivalent dose rate 1 g kg−1 b.w. of CP.

Group 7: CAME at the equivalent dose rate 4 g kg−1 b.w. of CP.

Group 8: CAME at the equivalent dose rate 8 g kg−1 b.w. of CP.

3.5.1.3. Measurements

Observation of clinical signs and/or death was undertaken daily. The body weight of the sheep

was recorded weekly. Faecal egg counts per gram of feces (EPG) were performed on each

animal on days 0, 3, 6, 9, 12 and 15 post-treatment (PT) 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 (%) = {(pre-treatment EPG – post-treatment EPG)/pre-treatment EPG} × 100

3.6. 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 from adult motility assay and

39

in vivo experiments were statistically analysed using SAS software (SAS, 1998). The results

were expressed as mean±standard error of mean (SEM).

40

Chapter # 4

RESULTS

4.1. Survey

The survey resulted in documentation of 41 plant species used in 49 different traditional

recipes representing 39 genera and 27 families (Table 10 to 11) for treatment of

helminthiasis. Most frequently used plants (≥5 times) were Brassica campestris L. and

Mallotus philippinensis (Lam.) Muell.-Arg. which represented the families Brassicaceae and

Euphorbiaceae respectively. Most frequently used part of the plants was leaves (n=10)

followed in order by seeds (n=9), whole fruit (n=5), aerial parts and whole plant (n=4), fruit

(n=3), bulb (n=2) and bark, rhizome, stem, stem plus root and twigs (n=1). Five recipes out

of forty-nine (10.2%) were containing more than one plant species and rest 44 (89.8%) were

containing single plant species. The methods of preparation of these botanical anthelmintics

comprised of crushing, grinding, soaking in water, boiling and mixing to obtain solutions and

mixtures. All the recipes were administered per os.


4.2.1. Adult motility assay

The criteria for interpretation of results of adult motility assay in the present study were (i) hours

taken for motility and/or mortality of worms (Haemonchus contortus) and (ii) dose dependant

response of worms to CAME of different plants. All the plants included in this study exhibited

anthelmintic activity against Haemonchus contortus. A wide variation however was recorded in

the anthelmintic effects among different plants. The plants in descending order of their

anthelmintic activity have been listed in Table 12. It is evident form data that all the plants have

dose dependant anthelmintic activity despite dissimilar levels of effect. The top 3 most effective

41

plants included Musa paradisiaca L., Trianthema portulacastrum L. and Tribulus terrestris L.,

followed in order by Ziziphus mauritiana Lam., Albizia lebbeck (L.) Benth., Digera muricata

L., Bambusa arundinacea (Retz.) Willd., Syzygium cumini (L.) Skeels, Lagenaria siceraria

(Molina) Standl. and Mangifera indica L (Table 13).

42

Table 10. Frequency of use of medicinal plants for the treatment and/or management of helminthes of animals in district Sahiwal, Pakistan

Sr. no. Plant family Plant speciesa (voucher specimen number) English name Vernacular name Frequency (n=331),

n (%)

1 Aizoaceae Trianthema portulacastrum L. (# 0110) Desert horse-

purslane

It Sit 29 (8.76)

2 Alliaceae Allium cepa L. (# 0111) Onion Piyaz 25 (7.55)

3 Amaranthaceae Digera muricata L. (# 0112) False amaranth Tandla 11 (3.32)

4 Anacardiaceae Mangifera indica L. (# 0113) Mango Aam 7 (2.11)

5 Apiaceae Coriandrum sativum L. (# 0114) Coriander Dhania 28 (8.45)

6 Apiaceae Foeniculum vulgare Mill. (# 0115) Fennel Sounf 4 (1.2)

7 Apiaceae Ferula assafoetida L. (# 0116) Stinking gum Hing 6 (1.81)

8 Apiaceae Cuminum cyminum L. (# 0117) Cumin Zeera 1 (0.3)

9 Arecaceae Cocos nucifera L. (# 0118) Coconut Garee/Khopa 3 (0.9)

10 Asteraceae Vernonia anthelmintica (L.) Willd. (# 0119) Ironweed Kali zeeri 47 (14.19)

11 Brassicaceae Brassica campestris L. (# 0120) Mustard Saron 67 (20.24)

12 Brassicaceae Eruca sativa Miller (# 0121) Garden Rocket Tarameera/Kusson 9 (2.71)

13 Capparaceae Capparis decidua (Forssk.) Edgew. (# 0122) Caper Kari 21 (6.34)

14 Convolvulaceae Convolvulus arvensis L. (# 0123) Field bindweed Laily 65 (19.63)

15 Cucurbitaceae Cucumis melo L. var. flexuosus (L.) Naud. (# 0124) Snake melon Chibbarr 7 (2.11)

16 Cucurbitaceae Lagenaria siceraria (Molina) Standl. (# 0125) Calabash Kaddoo 42 (12.68)

17 Cucurbitaceae Citrullus colocynthis (L.) Schrader (# 0126) Bitter apple Korr tumma 38 (11.48)

18 Cuscutaceae Cuscuta reflexa Roxb. (# 0127) Giant dodder Aakash bail 35 (10.57)

19 Euphorbiaceae Ricinus communis L. (# 0128) Castor bean Arind 79 (23.86)

20 Euphorbiaceae Mallotus philippinensis (Lam.) Muell.-Arg. (# 0129) Kamala tree Kameela 142 (42.9)

21 Fabaceae Cicer arietinum L. (# 0130) Chick pea Chana 1 (0.3)

43

Sr. no. Plant family Plant speciesa (voucher specimen number) English name Vernacular name Frequency (n=331),

n (%)

22 Fabaceae Medicago sativa L. (# 0131) Alfalfa Loosan 3 (0.9)

23 Fabaceae Albizia lebbeck (L.) Benth. (# 0132) Woman's tongue Shareen 2 (0.6)

24 Liliaceae Allium sativum L. (# 0133) Garlic Lassan 10 (3.02)

25 Meliaceae Azadirachta indica A. Juss. (# 0134) Neem Nim 30 (9.06)

26 Musaceae Musa paradisiaca L. (# 0135) Banana Kaila 13 (3.92)

27 Myrtaceae Syzygium cumini (L.) Skeels (# 0136) Jambolan plum Jaman 2 (0.6)

28 Poaceae Bambusa arundinacea (Retz.) Willd. (# 0137) Bamboo Bans 20 (6.04)

29 Poaceae Triticum aestivum L. (# 0138) Wheat Kanak 30 (9.06)

30 Ranunculaceae Helleborus niger L. (# 0139) Christmas Rose Karroo 26 (7.85)

31 Rhamnaceae Ziziphus mauritiana Lam. (# 0140) Ber, Indian Jujube Bairy 9 (2.71)

32 Rosaceae Prunus persica (L.) Batsch. (# 0141) Peach Aarroo 14 (4.22)

33 Scrophulariaceae Herpestis monniera L. (# 0142) Thyme leaved

gratiola

Jall booti 3 (0.9)

34 Solanaceae Nicotiana tabacum L. (# 0143) Tobacco Tamakoo 87 (26.28)

35 Solanaceae Withania coagulans Dunal. (# 0144) Indian rennet Paneer doda 26 (7.85)

36 Solanaceae Solanum xanthocarpum L. (# 0145) Yellow-Berried

Nightshade

Chamak namoly 2 (0.6)

37 Solanaceae Capsicum annuum L. (# 0146) Chili Mirch 12 (3.62)

38 Solanaceae Solanum tuberosum L. (# 0147) Potato Aaloo 1 (0.3)

39 Tamaricaceae Tamarix aphylla (L.) H.Karst. (# 0148) Tamarisk Okan di maieen/Maieen 16 (4.83)

40 Zingiberaceae Zingiber officinale Roscoe (# 0149) Ginger Adrak 10 (3.02)

41 Zygophyllaceae Tribulus terrestris L. (# 0150) Puncturevine Bhakhrra 13 (3.92) aScientific names of plants are according to the flora of Pakistan (Nasir and Ali, 1970–1988; Ali and Nasir, 1989–1991; Ali and Qaiser, 1992–to date); voucher specimens of the plants are kept in the Herbarium, Department of Parasitology, University of Agriculture, Faisalabad 38040, Pakistan.

44

Table 11. Ethnoveterinary practices for the treatment and/or management of heminthosis in animals in district Sahiwal, Pakistan

Sr.

no.

Name of plants/remediesb Parts used Dosage/administration Respondents

(n=331), n (%)

1 Albizia lebbeck (L.) Benth. L Crush ¼ to ½ kg leaves and administer per os or put leaves in front

of animal and allow the animal to eat ad libitum 2 (0.6)

2 Allium cepa L. Bulb Administer ½ kg jaggery per os, after half an hour administer ½ kg

crushed bulb per os 15 (4.53)

3

Allium sativum L.+Allium cepa

L.+Capsicum annum L.+Zingiber

officinale Roscoe

Bulb+Bulb+WF

(Green, raw

fruit)+Rhizomes

Grind 50 gm, 100 gm, 250 gm and 50 gm respectively, along with

25 gm sodium bicarbonate, mix them all and administer per os 10 (3.02)

4 Azadirachta indica A. Juss. L

Grind the leaves with pestle and mortar and sieve with muslin

cloth until ½ liter of extract is obtained, administer this extract per

os

25 (7.55)

5 Azadirachta indica A. Juss. L Boil one kg leaves in 3 liters of water, when water remains 1 liter

administer it per os 5 (1.51)

6 Bambusa arundinacea (Retz.) Willd. L Boil ½ kg leaves in 2 liters of water, when water remains 1 liter,

administer it per os 20 (6.04)

7 Brassica campestris L. S Mix ½ liter seed oil with ¼ kg curd and administer per os 50 (15.1)

8 Brassica campestris L. S Mix ½ liter seed oil with ½ liter of luke warm water and administer

per os 8 (2.41)

9 Brassica campestris L. S Boil ½ liter of oil, mix with ½ kg jaggery and administer per os 4 (1.2)

10 Capparis decidua (Forssk.) Edgew. Twigs Crush the twigs well, mix sufficient quantity of jaggery in it to

make the bolus and administer per os 9 (2.71)

45

Sr.

no.


(n=331), n (%)

11 Capparis decidua (Forssk.) Edgew. Aerial parts Mix 50 gm of coal (koila) of plant with butter 250 gm (Q.S. to

make the bolus) 12 (3.62)

12 Capsicum annuum L. WF

Make the syrup of jaggery and chilies by dissolving ¼ kg of them

each in ground form in water, drench the animal with syrup of

jaggery first then after 10 minutes drench the animal with syrup of

chilies

1 (0.3)

13

Capsicum annum L.+Cicer arietinum

L.+Cuminum cyminum

L.+Coriandrum sativum L.+Solanum

tuberosum L. (Pakoray+Chilies)

WF+S+S+S+St

tuber

Mix 150 gm to 200 gm of pkoray (the local recipe containing the

plants/plant material) with 60 gm of ground red chilies and

administer per os

1 (0.3)

14 Citrullus colocynthis (L.) Schrader WF Grind and give per os for 4 days 17 (5.13)

15

Citrullus colocynthis (L.)

Schrader+Veronica anthelmintica L.

Willd.

WF +S Grind 50 gm of both parts and administer per os 21 (6.34)

16 Cocos nucifera L. F Grind 125 gm of fruit and administer per os 3 (0.9)

17 Convolvulus arvensis L. Aerial parts Crush aerial parts, sieve with muslin cloth to give ½ to 1 liter of

extract and administer per os 33 (9.96)

18 Convolvulus arvensis L. Aerial parts Boil ½ to 1 kg of aerial parts in 1.5 to 2 liters of water, when water

remain only one liter administer it per os 32 (9.66)

19 Coriandrum sativum L. S Grind 50 gm seeds along with jaggery Q.S. to make bolus and

administer per os 27 (8.15)

20 Cucumis melo var. Flexuosus (L.)

naud. WF

Boil 1 kg of fruit in 2 liters of water for 1 to 2 hours then

administer the decoction per os 7 (2.11)

46

Sr.

no.


(n=331), n (%)

21 Cuscuta reflexa Roxb. WP Boil 1 kg the plant with 2 liters of water for 1 to 2 hours then

administer the decoction per os 35 (10.57)

22 Digera muricata L. WP Crush the plant and administer per os or animal is allowed to eat it

ad libitum 11 (3.32)

23 Eruca sativa Miller S Administer the oil per os 9 (2.71)

24 Ferula assafoetida L. St and R Grind 10 gm extracted gum (from stem and roots) along with

jaggery (Q.S to make bolus) and administer per os 6 (1.81)

25 Foeniculum vulgare Mill. S Grind 100 gm of seeds along with ¼ kg of jaggery and administer

per os 4 (1.2)

26 Incantation –

Some verses from Holy Quran are recited and air from mouth

blown on the animal or incantation done on a lump of doughed

flour and lump is administer per os or incantation is done on water

which is sprinkled on animal’s body (used usually for lumpy jaw,

typical symptom of fascioliasis)

30 (9.06)

27 Herpestis monniera L. Aerial parts Crush the aerial parts and administer per os 3 (0.9)

28 Lagenaria siceraria (Molina) Standl.

L

Crush leaves and administer per os or animal is allowed to eat the

leaves ad libitum 42 (12.68)

29 Mallotus philippinensis (Lam.)

Muell.-Arg. F

Mix the 10 gm fruit powder with ½ liters of milk and administer

per os 56 (16.91)


Muell.-Arg. F

Mix the 4 drama fruit powder with ½ kg of curd and administer per

os 41 (12.38)


Muell.-Arg. F

Mix the 4 dram fruit powder with ½ liter of milk whey and


47

Sr.

no.


(n=331), n (%)


Muell.-Arg. F

Mix the 4 dram fruit powder with ½ liter of mustard oil and



Muell.-Arg. F

Grind the 10 gm fruit powder jaggery (Q.S. to make the bolus) and



Muell.-Arg. F

Mix the 50 gm fruit powder with ½ liter of water and administer

per os 8 (2.41)

35

Mallotus philippinensis (Lam.)

Muell.-Arg.+Tamarix aphylla (L.)

H.Karst.+ Brassica campestris L.

F+F+S Mix ground fruit (10 gm each), ½ liter of mustard oil, ½ kg of

curd and administer per os 5 (1.51)

36 Mangifera indica L. L Crush ½ kg of leaves and administer per os or animal is allowed to

eat ad libitum 7 (2.11)

37 Medicago sativa L. Aerial part Crush ½ kg of leaves and administer per os or animal is allowed to


38 Musa paradisiaca L. L Crush leaves, sieve with muslin cloth to give ½ to 1 liter of extract

and administer per os 13 (3.92)

39 Nicotiana tabacum L. L Administer ½ to 1 liter of decoction type of water left (as a by

product) after smoking the Huqqa, per os 61 (18.42)

40

Nicotiana tabacum L.+Withania

coagulans Dunal.+Veronica

anthelmintica L. Willd.+Helleborus

niger L.

L+WF+S+Bark

Grind 100 gm each of fruit, seed, bark, mix with water of tobacco

leaves left after smoking huqqa, divide into 3 doses and administer

1 dose per os daily

26 (7.85)

41 Prunus persica L. Batsch. L Crush leaves, sieve with muslin cloth to give ½ to 1 liter of extract

and administer per os 14 (4.22)

48

Sr.

no.


(n=331), n (%)

42 Ricinus communis L. S Administer 125 ml of seed oil per os 6 (1.81)

43 Ricinus communis L. S Administer 125 ml of seed oil per os in ½ liter luke warm milk 73 (22.04)

44 Solanum xanthocarpum L. WF Crush 250 gm fruit and administer per os along with jaggery (Q.S.

to make bolus) 2 (0.6)

45 Syzygium cumini (L.) Skeels L Crush ½ kg leaves and administer per os or animal is allowed to


46 Tamarix aphylla (L.) H.Karst. F Grind 50 gm fruit and administer per os 11 (3.32)

47 Trianthema portulacastrum L. WP Crush ½ kg leaves and administer per os or animal is allowed to


48 Tribulus terrestris L. WP Crush ½ kg leaves and administer per os or animal is allowed to


49 Ziziphus mauritiana Lam. L Crush ½ kg leaves and administer per os or animal is allowed to


a1 dram=1.771845 grams, F = Fruit, L = Leaves, R = Roots, S = Seeds, St = Stem, WF = Whole fruit (fruit plus seeds), WP = Whole plant b Scientific names of plants are according to the flora of Pakistan (Nasir and Ali, 1970–1988; Ali and Nasir, 1989–1991; Ali and Qaiser, 1992–to date);

voucher specimens of the plants are kept in the Herbarium, Department of Parasitology, University of Agriculture, Faisalabad 38040, Pakistan.

49

Table 12. In vitro effect of different indigenous plants on survival of Haemonchus contortus (Mean±SEM) of sheep in comparison with Levamisole (Lev)

Mean number of dead worms at different hours Treatments mg mL-1

0 hr 2 hr 4 hr 6 hr 8 hr 10 hr 12 hr Fresh PBS for 30 min Lev 0.5 mg 10.00±0.0a 0.00±0.0h 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b

PBS 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a Musa paradisiaca L.

100 mg 10.00±0.0a 1.00±0.6fg 0.00±0.0e 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 50 mg 10.00±0.0a 2.33±0.7efg 0.00±0.0e 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 25 mg 10.00±0.0a 2.67±0.3ef 0.00±0.0e 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b

12.5 mg 10.00±0.0a 4.67±1.5de 0.00±0.0e 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 6.25 mg 10.00±0.0a 6.67±0.7cd 2.00±0.6d 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 3.12 mg 10.00±0.0a 7.33±1.2bc 3.00±1.0cd 0.67±0.7d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 1.56 mg 10.00±0.0a 7.33±2.2bc 3.33±0.9cd 1.00±0.6d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.78 mg 10.00±0.0a 9.67±0.3ab 4.67±0.3bc 2.67±0.3c 0.67±0.3c 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.39 mg 10.00±0.0a 10.00±0.0a 6.00±0.6b 4.00±0.6bc 2.00±0.6b 0.33±0.3c 0.00±0.0b 0.00±0.0b 0.19 mg 10.00±0.0a 10.00±0.0a 6.33±1.2b 4.33±1.2b 2.33±1.2b 1.00±0.6b 0.00±0.0b 0.00±0.0b

Trianthema portulacastrum L. 100 mg 10.00±0.0a 0.00±0.0h 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 50 mg 10.00±0.0a 0.00±0.0h 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 25 mg 10.00±0.0a 0.30±0.0gh 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b

12.5 mg 10.00±0.0a 1.30±0.3g 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 6.25 mg 10.00±0.0a 2.70±0.3f 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 3.12 mg 10.00±0.0a 4.30±0.3e 0.67±0.3e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 1.56 mg 10.00±0.0a 5.30±0.3de 3.00±0.6d 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.78 mg 10.00±0.0a 6.30±0.3cd 4.30±0.3c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.39 mg 10.00±0.0a 7.30±0.3bc 5.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.19 mg 10.00±0.0a 8.00±1.0b 6.30±0.3b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b

Tribulus trrestris L. 100 mg 10.00±0.00a 0.00±0.0e 0.00±0.0f 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 50 mg 10.00±0.00a 0.00±0.0e 0.00±0.0f 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b

50

Mean number of dead worms at different hours Treatments mg mL-1 0 hr 2 hr 4 hr 6 hr 8 hr 10 hr 12 hr Fresh PBS for 30 min

25 mg 10.00±0.00a 5.33±0.8d 0.00±0.0f 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 12.5 mg 10.00±0.00a 7.00±1.0c 0.00±0.0f 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 6.25 mg 10.00±0.00a 8.33±0.3bc 2.00±0.0e 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 3.12 mg 10.00±0.00a 8.67±0.8ab 3.67±0.3d 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 1.56 mg 10.00±0.00a 9.00±0.6ab 5.33±0.3c 0.33±0.3c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.78 mg 10.00±0.00a 10.00±0.0a 7.67±0.3b 0.67±0.7c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.39 mg 10.00±0.00a 10.00±0.0a 8.00±0.6b 1.00±0.6c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.19 mg 10.00±0.00a 10.00±0.0a 7.33±0.9b 2.67±1.2b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b

Ziziphus mauritiana Lam. 100 mg 10.00±0.0a 6.67±0.7b 2.00±1.5d 0.00±0.0e 0.00±0.0e 0.00±0.0e 0.00±0.0c 0.00±0.0c 50 mg 10.00±0.0a 7.00±1.2b 5.33±0.3c 0.00±0.0e 0.00±0.0e 0.00±0.0e 0.00±0.0c 0.00±0.0c 25 mg 10.00±0.0a 7.33±0.9b 6.00±0.6bc 3.33±0.9d 0.00±0.0e 0.00±0.0e 0.00±0.0c 0.00±0.0c

12.5 mg 10.00±0.0a 7.33±0.7b 7.33±0.7b 3.67±0.7d 0.00±0.0e 0.00±0.0e 0.00±0.0c 0.00±0.0c 6.25 mg 10.00±0.0a 9.67±0.3a 9.67±0.3a 6.33±0.9c 1.67±0.7d 0.00±0.0e 0.00±0.0c 0.00±0.0c 3.12 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.00±0.6b 2.33±0.7d 0.00±0.0e 0.00±0.0c 0.00±0.0c 1.56 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.67±0.3a 5.67±1.5c 1.00±0.0d 0.00±0.0c 0.00±0.0c 0.78 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.67±0.3a 6.00±0.6c 2.67±0.7c 0.33±0.3c 0.33±0.3c 0.39 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.00±0.6b 8.00±0.6b 1.33±0.3b 1.33±0.3b 0.19 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.67±0.3a 8.33±0.7b 1.33±0.3b 1.33±0.3b

Albizia lebbeck (L.) Benth. 100 mg 10.00±0.0a 1.33±0.9e 0.00±0.0e 0.00±0.0g 0.00±0.0d 0.00±0.0f 0.00±0.0d 0.00±0.0d 50 mg 10.00±0.0a 1.67±0.3e 0.00±0.0e 0.00±0.0g 0.00±0.0d 0.00±0.0f 0.00±0.0d 0.00±0.0d 25 mg 10.00±0.0a 7.00±0.6d 5.00±0.6d 4.33±0.7f 1.00±1.0d 0.00±0.0f 0.00±0.0d 0.00±0.0d

12.5 mg 10.00±0.0a 7.67±1.3cd 6.33±0.9c 5.67±0.3e 1.33±0.3d 0.00±0.0f 0.00±0.0d 0.00±0.0d 6.25 mg 10.00±0.0a 8.00±0.6bcd 8.00±0.6b 7.00±0.6d 3.67±0.3c 0.00±0.0f 0.00±0.0d 0.00±0.0d 3.12 mg 10.00±0.0a 8.33±0.3abcd 8.33±0.3b 8.00±0.6cd 4.00±1.0c 0.33±0.3ef 0.00±0.0d 0.00±0.0d 1.56 mg 10.00±0.0a 9.00±1.0abc 9.00±1.0ab 8.67±0.9bc 4.33±0.9c 1.67±0.7de 1.00±0.6cd 1.00±0.6cd 0.78 mg 10.00±0.0a 9.67±0.3ab 9.67±0.3a 9.67±0.3ab 4.67±0.9c 2.67±0.7cd 1.67±0.3c 1.67±0.3c 0.39 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 5.00±0.0c 3.33±0.9c 1.67±0.9c 1.67±0.9c 0.19 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 7.33±0.3b 5.67±0.9b 3.33±1.2b 3.33±1.2b

51


Digera muricata L. 100 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.00±1.2b 3.33±0.9c 0.00±0.0e 0.00±0.0f 0.00±0.0f 50 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 6.33±0.9b 0.00±0.0e 0.00±0.0f 0.00±0.0f 25 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 6.67±1.5b 0.00±0.0e 0.00±0.0f 0.00±0.0f

12.5 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.00±0.0a 3.00±0.6d 0.00±0.0f 0.00±0.0f 6.25 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 3.33±0.9d 0.00±0.0f 0.00±0.0f 3.12 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 6.67±0.3c 0.00±0.0f 0.00±0.0f 1.56 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.00±0.6b 4.00±0.6e 4.00±0.6e 0.78 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.00±0.6ab 6.33±0.3d 6.33±0.3d 0.39 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.67±0.3a 7.33±0.7c 7.33±0.7c 0.19 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.00±0.6b 9.00±0.6b

Bambusa arundinacea (Retz.) Willd. 100 mg 10.00±0.0a 8.33±1.2b 1.33±0.9b 0.00±0.0e 0.00±0.0g 0.00±0.0f 0.00±0.0e 0.00±0.0e 50 mg 10.00±0.0a 9.00±0.6ab 1.67±0.3b 0.00±0.0e 0.00±0.0g 0.00±0.0f 0.00±0.0e 0.00±0.0e 25 mg 10.00±0.0a 10.00±0.0a 8.33±1.2a 6.00±0.6d 2.67±0.3f 1.33±0.9ef 0.00±0.0e 0.00±0.0e

12.5 mg 10.00±0.0a 10.00±0.0a 9.00±0.0a 7.33±1.2cd 3.00±0.0f 1.67±0.3de 0.00±0.0e 0.00±0.0e 6.25 mg 10.00±0.0a 10.00±0.0a 9.00±0.0a 7.33±0.9cd 4.33±0.3e 3.00±0.6cd 0.00±0.0e 0.00±0.0e 3.12 mg 10.00±0.0a 10.00±0.0a 9.00±0.0a 7.67±0.3bcd 4.67±0.3e 3.67±0.9c 1.67±0.3d 1.67±0.3d 1.56 mg 10.00±0.0a 10.00±0.0a 9.00±1.0a 8.33±0.7abc 5.33±0.3d 3.67±0.7c 2.33±0.3d 2.33±0.3d 0.78 mg 10.00±0.0a 10.00±0.0a 9.33±0.3a 8.67±0.3abc 6.33±0.3c 4.00±0.0c 3.33±0.3c 3.33±0.3c 0.39 mg 10.00±0.0a 10.00±0.0a 9.33±0.3a 8.67±0.9abc 6.67±0.3bc 4.33±0.9c 3.67±0.3c 3.67±0.3c 0.19 mg 10.00±0.0a 10.00±0.0a 9.67±0.3a 9.33±0.3ab 7.00±0.0b 6.67±0.3b 4.67±0.8b 4.67±0.8b

Syzygium cumini (L.) Skeels 100 mg 10.00±0.0a 9.00±0.6b 0.00±0.0b 0.00±0.0d 0.00±0.0e 0.00±0.0d 0.00±0.0f 0.00±0.0f 50 mg 10.00±0.0a 10.00±0.0a 1.33±1.3b 0.00±0.0d 0.00±0.0e 0.00±0.0d 0.00±0.0f 0.00±0.0f 25 mg 10.00±0.0a 10.00±0.0a 9.00±1.0a 6.00±0.6c 2.33±1.5d 0.00±0.0d 0.00±0.0f 0.00±0.0f

12.5 mg 10.00±0.0a 10.00±0.0a 9.67±0.3a 8.33±0.7b 6.33±0.3c 2.00±2.0d 0.00±0.0f 0.00±0.0f 6.25 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.33±0.3a 8.00±0.0b 5.33±0.7c 0.00±0.0f 0.00±0.0f 3.12 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.33±0.3aa 9.33±0.3ab 7.33±0.7bc 0.67±0.3ef 0.67±0.3ef 1.56 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.33±0.3a 9.33±0.3ab 7.33±0.9bc 1.00±0.0de 1.00±0.0de

52


0.78 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.67±0.3a 9.33±0.3ab 1.67±0.3d 1.67±0.3d 0.39 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.67±0.3a 8.67±0.9ab 2.67±0.3c 2.67±0.3c 0.19 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.33±0.3ab 3.67±0.9b 3.67±0.9b

Lagenaria siceraria (Molina) Standl. 100 mg 10.00±0.0a 0.00±0.0b 0.00±0.0c 0.00±0.0d 0.00±0.0f 0.00±0.0d 0.00±0.0f 0.00±0.0f 50 mg 10.00±0.0a 10.00±0.0a 7.67±1.2b 2.67±1.2c 0.00±0.0f 0.00±0.0d 0.00±0.0f 0.00±0.0f 25 mg 10.00±0.0a 10.00±0.0a 9.67±0.3a 7.33±1.5b 5.67±0.3e 0.00±0.0d 0.00±0.0f 0.00±0.0f

12.5 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 7.33±0.9b 6.33±0.3de 4.67±0.3c 1.67±0.7e 1.67±0.7e 6.25 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 7.33±0.9b 7.33±0.9cd 7.00±1.2b 2.67±0.3de 2.67±0.3de 3.12 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.9ab 8.33±0.7bc 8.33±1.2ab 4.00±0.0cd 4.00±0.0cd 1.56 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.7ab 8.67±0.7b 8.33±0.9ab 5.33±0.3c 5.33±0.3c 0.78 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.00±0.6ab 9.00±0.6ab 8.33±0.9ab 7.33±0.3b 7.33±0.3b 0.39 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.33±0.3a 9.00±0.6ab 8.33±0.9ab 7.67±1.5b 7.67±1.5b 0.19 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.3ab 8.67±0.3ab

Mangifera indica L. 100 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.3b 6.33±0.3c 0.00±0.0f 0.00±0.0e 0.00±0.0e 50 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.00±0.6b 2.00±0.6e 0.00±0.0e 0.00±0.0e 25 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 3.67±0.9d 0.00±0.0e 0.00±0.0e

12.5 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 6.33±1.3c 1.00±1.0e 1.00±1.0e 6.25 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 7.33±0.9bc 4.00±1.0d 4.00±1.0d 3.12 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.9ab 5.33±1.8cd 5.33±1.8cd 1.56 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.7ab 6.67±0.9bc 6.67±0.9bc 0.78 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.3ab 8.00±0.6ab 8.00±0.6ab 0.39 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.9ab 8.33±0.9ab 8.33±0.9ab 0.19 mg 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.67±0.3a 9.67±0.3a

Mean number of live worms at different hours marked with similar alphabets in a column do not differ significantly (p< 0.05)

53

Table 13. Ranking of 10 plants according to their effects on adult Haemonchus contortus

Sr. no. Plant species Order of ranking

Part/s used

English name Vernacular name

1 Trianthema portulacastrum L.

01 Whole plant

Desert horse-purslane

It Sit

2 Tribulus terrestris L. 01 Whole plant

Puncturevine Bhakhrra

3 Lagenaria siceraria (Molina) Standl.

01 Leaves Calabash Kaddoo

4 Musa paradisiaca L. 02 Leaves Banana Kaila 5 Albizia lebbeck (L.)

Benth. 03 Leaves Woman's

tongue Shareen

6 Ziziphus mauritiana Lam.

04 Leaves Ber, Indian Jujube

Bairy

7 Bambusa arundinacea (Retz.) Willd.

05 Leaves Bamboo Bans

8 Syzygium cumini (L.) Skeels

05 Leaves Jambolan plum Jaman

9 Digera muricata L. 06 Whole plant

False amaranth Tandla

10 Mangifera indica L. 06 Leaves Mango Aam

4.2.2. Egg hatch test

The plant inhibiting egg hatching the most potently based on LC50 was Musa paradisiaca L.

(2.13 µg mL-1) followed in descending order of activity by Trianthema portulacastrum L.

(2.41 µg mL-1), Lagenaria siceraria (Molina) Standl. (2.53 µg mL-1), Albizia lebbeck (L.) Benth.

(2.75 µg mL-1), Tribulus terrestris L. (2.75 µg mL-1) Syzygium cumini (L.) Skeels (4.34 µg mL-1)

Mangifera indica L. (4.48 µg mL-1) Ziziphus mauritiana Lam. (4.69 µg mL-1) Bambusa

arundinacea (Retz.) Willd. (4.89 µg mL-1) and Digera muricata L. (5.36 µg mL-1) (Table 14).

The results suggest that all the 10 plats have potency to inhibit the egg hatch, indicating

ovicidal activity by all the plants.

54

Table 14. Per cent egg hatch and LC50 of different plants

CAME concentrations (µg mL-1) Plant

250 500 1000 2000 4000 8000

LC50

(µg mL-1)

Musa paradisiaca L. 36.00 32.00 27.00 22.50 19.00 4.00 2.13 Trianthema portulacastrum L. 33.00 20.00 10.00 2.00 0.50 0.00 2.41 Lagenaria siceraria (Molina) Standl.

51.00 28.00 10.00 4.00 1.33 0.00 2.53

Albizia lebbeck (L.) Benth. 58.00 44.00 39.50 35.50 20.00 0.00 2.75 Tribulus terrestris L. 66.00 55.00 37.00 25.00 10.00 2.50 2.75 Syzygium cumini (L.) Skeels 97.00 95.00 93.00 84.00 73.00 69.00 4.34 Mangifera indica L. 80.00 79.00 76.00 73.00 66.60 56.00 4.48 Ziziphus mauritiana Lam. 93.30 91.00 85.10 79.00 75.00 72.20 4.69 Bambusa arundinacea (Retz.) Willd.

95.00 92.00 88.80 83.00 80.00 75.00 4.89

Digera muricata L. 97.00 92.00 91.00 89.00 84.00 82.00 5.36

4.2.2.1. Regression values and correlation of regression of the effect of different plants on egg hatching

The data of correlation of regression (Table 15) revealed the best dose-dependant effects on egg

hatching with Trianthema portulacastrum L. (R2 = 0.9793) followed in descending order by

Albizia lebbeck (L.) Benth., Musa paradisiaca L. and Mangifera indica L. (R2 = 0.9689),

Lagenaria siceraria (Molina) Standl. (R2 = 0.9596), Tribulus terrestris L. (R2 = 0.9136),

Syzygium cumini (L.) Skeels and Bambusa arundinacea (Retz.) Willd. (R2 = 0.7454),

Ziziphus mauritiana Lam. (R2 = 0.6803) and Digera muricata L. (R2 = 0.6446). The results

reveal that all the plants have potent ovicidal compounds, which are responsible for the high

ovicidal activity.

55

Table 15. Regression values and correlation of regression of the effect of different plants on egg hatching

Plant LC50 Regression values and correlation of regression

Musa paradisiaca L. 2.13 y = -0.0002x + 4.6324, R2 = 0.9689 Trianthema portulacastrum L. 2.41 y = -0.0006x + 4.4134, R2 = 0.9793 Lagenaria siceraria (Molina) Standl. 2.53 y = -0.0006x + 4.7245, R2 = 0.9596 Albizia lebbeck (L.) Benth. 2.75 y = -0.0002x + 4.6324, R2 = 0.9689 Tribulus terrestris L. 2.75 y = -0.0003x + 5.1332, R2 = 0.9136 Syzygium cumini (L.) Skeels 4.34 y = -0.0001x + 6.4026, R2 = 0.7454 Mangifera indica L. 4.48 y = -0.0002x + 4.6324, R2 = 0.9689 Ziziphus mauritiana Lam. 4.69 y = -0.0001x + 6.2603, R2 = 0.6803 Bambusa arundinacea (Retz.) Willd. 4.89 y = -0.0001x + 6.4026, R2 = 0.7454 Digera muricata L. 5.36 y = -9E–05x + 6.5395, R2 = 0.6446 Oxfendazole 1.88 y = -0.2159x + 6.2447, R2 = 0.775

4.2.2.2. Salient findings of EHT

The data (Table 16) indicate ranking of 10 plants based on LC50 and correlation regression

values (egg hatch test), which indicate the potency and dose dependant effects, respectively. The

most potent plant inhibiting egg hatching based on LC50 was Musa paradisiaca L. (2.13 µg

mL-1) followed in descending order of activity by Trianthema portulacastrum L. (2.41 µg mL-

1), Lagenaria siceraria (Molina) Standl. (2.53 µg mL-1), Albizia lebbeck (L.) Benth. (2.75 µg

mL-1), Tribulus terrestris L. (2.75 µg mL-1) Syzygium cumini (L.) Skeels (4.34 µg mL-1)

Mangifera indica L. (4.48 µg mL-1) Ziziphus mauritiana Lam. (4.69 µg mL-1) Bambusa

arundinacea (Retz.) Willd. (4.89 µg mL-1) and Digera muricata L. (5.36 µg mL-1). The order

of ranking of these plants was different as far as their dose dependant effect is concerned.

The best dose-dependant effects on egg hatching was with Trianthema portulacastrum L. (R2 =

0.9793) followed in descending order by Albizia lebbeck (L.) Benth., Musa paradisiaca L. and

Mangifera indica L. (R2 = 0.9689), Lagenaria siceraria (Molina) Standl. (R2 = 0.9596),

Tribulus terrestris L. (R2 = 0.9136), Syzygium cumini (L.) Skeels and Bambusa arundinacea

56

(Retz.) Willd. (R2 = 0.7454), Ziziphus mauritiana Lam. (R2 = 0.6803) and Digera muricata L.

(R2 = 0.6446). Comparing and contrasting the LC50 (Table 14) and correlation (Table 15) and

variation in ranking based on preceding criteria (Table 16), it may be concluded that ovicidal

effect of different plants can not be attributed to the ovicidal compounds present in CAME of

platns.

Table 16. Ranking of 10 plants based on LC50 values and regression correlation values in egg hatch

Plant Ranking of

potency based on LC50

Ranking of potency based on dose dependant effect (R2 values)

Musa paradisiaca L. 01 02 Trianthema portulacastrum L. 02 01 Lagenaria siceraria (Molina) Standl.

03 03

Albizia lebbeck (L.) Benth. 04 02 Tribulus terrestris L. 04 04 Syzygium cumini (L.) Skeels 05 05 Mangifera indica L. 06 02 Ziziphus mauritiana Lam. 07 06 Bambusa arundinacea (Retz.) Willd.

08 05

Digera muricata L. 09 07

4.2.3. Summary of in vitro results

In vitro evaluation for anthelmintic activity of CAME of different plants was carried out using

egg hatch test (EHT) and adult motility assay (AMA). All the plants included in this study

exhibited anthelmintic activity against Haemonchus contortus as evident from inhibited egg

hatching and adult motility assay of the worms. A wide variation, however, was recorded in the

anthelmintic effects among different plants as far as the intensity and dose dependent effects

were concerned. A summary of the top most effect plants based on both in vitro tests is given in

Table 17. Trianthema portulacastrum L., Musa paradisiaca L., Lagenaria siceraria (Molina)

57

Standl., Albizia lebbeck (L.) Benth. and Tribulus terrestris L. were among top 5 plants in egg

hatch test as well as in adult motiltity assay. Musa paradisiaca L. showed best activity in egg

hatch test while Trianthema portulacastrum L. showed best anthelmintic activity in adult

motility assay.

In vivo, a graded dose response in fecal egg count reduction (range 35.20 to 70.18%; Table 18)

was recorded for all plants and CAME was found more effective than CP in all the experimental

groups. The best fecal egg count reduction was recorded with CAME of Trianthema

portulacastrum L. followed in order by Legnaria sisrarria, Tribulus tresstris, Musa

paradisiacal, Albezia lebbeck, Syzygium cumini, Bambusa arrundinacea, Digra muricata,

Mangifera indica and Ziziphus mauritiana at the dose rate of 8 g kg-1 body weight.

Table 17. Summary of in vitro results

Plant

Ranking of potency based on LC50

Ranking of potency based on dose

dependant effect (R2 values)

Ranking of potency based on adult motility assay (%

motility of worms after 2nd hour of treatment)

Musa paradisiaca L. 01 02 02

Trianthema portulacastrum L. 02 01 01

Lagenaria siceraria (Molina) Standl. 03 03 01

Albizia lebbeck (L.) Benth. 04 02 03

Tribulus terrestris L. 04 04 01

Syzygium cumini (L.) Skeels 05 05 05

Mangifera indica L. 06 02 06 Ziziphus mauritiana Lam. 07 06 04

Bambusa arundinacea (Retz.) Willd.

08 05 05

Digera muricata L. 09 07 06

58


All the 10 plants which were selected out 41 plants of survey, were subjected to evaluation for

their in vivo anthelmintic activity in sheep naturally parasitized with gastrointestinal helminthes.

The animals were drenched at different levels (1, 4 and 8 g kg-1 body weight) as crude powder

and CAME (at equivalent dose rate of 1, 4 and 8 g kg-1 body weight of CP) as single dose. Fecal

examination of the animals was carried out at 0, 3, 6, 9, 12 and 15 days post-treatment for egg

per gram of feces (EPG).

A graded dose response in EPG reduction was recorded for all the plants and crude aqueous

extracts were found more effective than CP in all the experiment groups except couple of plants

(Table 18).

59

Table 18. Effect of different forms and doses of 10 selected plants on egg per gram (Mean±SEM) of feces in sheep naturally infected with mixed species of gastrointestinal nematodes

Days PT Untreated control Levamisole 7.5 mg kg-1 CP 1 g kg-1 CP 4 g kg-1 CP 8 g kg-1 CAME 1 g kg-1 CAME 4 g kg-1 CAME 8 g kg-1

Trianthema partulacastrum L.

0 1450.00±20.4a (0%)

1425.00±32.3a (0%)

1475.00±14.4a

(0%) 1462.50±71.8a

(0%) 1450.00±28.9a

(0%) 1475.00±14.4a

(0%) 1450.00±28.9a

(0%) 1475.00±9.4a

(0%)

3 1440.00±5.8a (1%)

0.00±0.0d (100%)

1425.50±42.8a

(3%) 1387.50±12.5ab

(5%) 1300.00±20.4bc

(10%) 1390.00±29.2ab

(6%) 1312.50±23.9bc

(10%) 1275.50±59.6c

(13.5%)

6 1430.00±11.5a (1%)

0.00±0.0d

(100%) 1363.50±65.4ab

(8%) 1312.50±23.9abc

(10) 1225.00±43.3c

(16%) 1300.00±20.4bc

(12%) 1220.50±51.5c

(16%) 1200.00±57.7c

(18.6%)

9 1420.00±11.5a (2%)

0.00±0.0f

(100%) 1326.50±58.5a

(10%) 925.00±59.5c

(37%) 850.00±50.0cd

(41%) 1200.00±20.4b

(19%) 800.00±20.4d

(45%) 550.00±20.4e

(63%)

12 1410.00±5.8a (3%)

0.00±0.0e

(100%) 1239.50±44.5b

(16%) 800.00±67.7c

(45%) 637.50±42.7d

(56%) 1160.50±58.1b

(21%) 600.00±20.4d

(59%) 500.00±42.7e

(66%)

15 1400.00±20.4a (3%)

0.00±0.0e

(100%) 1190.00±76.5b

(19%) 712.50±65.7c

(51%) 550.00±35.4d

(62%) 1114.95±42.5b

(24%) 491.55±32.0d

(66%) 439.85±28.9e

(70%) Lagenaria siceraria (Molina) Standl.

0 1450.00±28.9a

(0%) 1425.00±14.4a

(0%) 1413.00±12.5a

(0%) 1425.00±14.4a

(0%) 1450.00±14.4a

(0%) 1438.00±12.5a

(0%) 1438.00±23.9a

(0%) 1450.00±35.4a

(0%)

3 1445.00±26.0a

(1%) 0.00±0.0d

(100%) 1384.00±11.8b

(2%) 1350.00±0.0b

(5%) 1296.00±2.3c

(11%) 1370.00±17.0b

(5%) 1368.00±19.7b

(5%) 1368.00±10.7b

(6%)

6 1435.00±20.2a

(1%) 0.00±0.0f

(100.%) 1295.00±5.0cd

(8%) 1275.00±14.4d

(11%) 1215.00±8.7e

(16%) 1325.00±14.4bc

(8%) 1344.00±25.9b

(7%) 1350.00±20.4b

(7%)

9 1422.00±12.7a

(2%) 0.00±0.0e

(100.%) 1192.00±7.7b

(16%) 950.00±20.4c

(33%) 870.00±17.3d

(40%) 1179.00±12.1b

(18%) 990.00±10.0c

(31%) 900.00±20.4d

(38%)

12 1400.00±0.0a

(3%) 0.00±0.0f

(100.%) 1075.00±9.6b

(24%) 930.00±17.3d

(35%) 812.00±6.9e

(44%) 1100.00±20.4b

(24) 987.00±7.2c

(31%) 842.00±21.7e

(42%)

15 1392.00±4.6a

(4.%) 0.00±0.0f

(100.%) 1015.00±10.0c

(28%) 895.00±2.9d

(37%) 773.50±11.0e

(47) 1056.00±15.1b

(27%) 923.40±13.5d

(36%) 783.60±16.6e

(46%) Tribulus terrestris L.

0 1562.50±7.2a

(0%) 1548.50±0.9a

(0%) 1552.00±4.6a

(0%) 1537.50±7.2a

(0%) 1550.00±14.4a

(0%) 1587.00±7.5a

(0%) 1537.50±23.9a

(0%) 1550.00±14.4a

(0%)

3 1545.00±2.9a

(1%) 0.00±0.0e

(100%) 1499.50±0.3b

(3%) 1437.00±7.5c

(7%) 1379.00±12.1d

(11%) 1525.00±14.4ab

(4%) 1462.50±23.9c

(9%) 1350.00±14.4d

(13%)

60


6 1533.50±3.8a

(2%) 0.00±0.0e

(100%) 1460.00±4.1ab

(6%) 1350.50±14.2c

(12%) 1200.00±64.5d

(19%) 1475.00±52.0ab

(7%) 1437.50±23.9bc

(14%) 1212.50±4.3d

(22%)

9 1522.50±4.3a

(2.6%) 0.00±0.0f

(100%) 1381.00±11.0b

(11%) 1250.00±14.4c

(19%) 1012.50±31.5de

(29%) 1312.50±37.5bc

(17%) 1050.00±67.7d

(29%) 937.50±1.4e

(40%)

12 1515.50±2.6a

(3%) 0.00±0.0f

(100%) 1289.00±6.4b

(17%) 1178.00±12.7c

(23%) 900.00±20.4e

(39%) 1225.00±32.3bc

(23%) 1025.00±59.5d

(33%) 850.00±14.4e

(45%)

15 1500.00±0.0a

(4.%) 0.00±0.0g

(100%) 1236.40±7.9b

(20%) 924.00±3.5e

(40%) 750.00±10.2f

(46%) 1165.38±11.8c

(27%) 987.50±42.7d

(36%) 750.00±14.4f

(52 %) Ziziphus mauritiana Lam.

0 1287.00±7.5a

(0%) 1313.00±7.2a

(0%) 1300.00±0.0a

(0%) 1287.00±7.5a

(0%) 1325.00±52.0a

(0%) 1330.00±17.3a

(0%) 1300.00±14.4a

(0%) 1297.00±2.0a

(0%)

3 1280.00±11.5a (1%)

0.00±0.0c (100%)

1287.00±7.8a (1%)

1256.00±2.6a (2%)

1263.00±42.7a

(5%) 1256.00±29.1a

(6%) 1229.00±0.6ab

(6%) 1193.00±4.3b

(8%)

6 1270.00±17.3ab

(1%) 0.00±0.0f (100%)

1274.00±3.8a (2%)

1193.00±4.0cd (7%)

1163.00±47.3d

(12%) 1219.00±0.6bc

(8%) 1172.00±4.9cd

(10%) 1099.00±0.6e

(15%)

9 1260.00±11.5a (1%)

0.00±0.0g (100%)

1169.00±0.9b (10%)

1067.00±1.7d (17%)

1038.00±23.9e

(22%) 1096.00±2.3c

(18%) 1072.00±4.9cd

(18%) 953.00±4.0f

(27%)

12 1250.00±5.8a (2%)

0.00±0.0f (100%)

1116.00±2.3b (14%)

1005.00±2.9d (22%)

987.50±23.9d (26%)

1047.00±2.0c (21%)

1000.00±0.0d (23%)

902.00±1.2e (30%)

15 1236.00±2.6a (4%)

0.00±0.0f (100%)

1090.00±5.8b (16%)

825.90±2.4d (28%)

900.00±54.0d (32%)

1022.00±4.6c (23%)

942.80±4.2d (28%)

840.10±5.7e (35%)

Bambusa arundinacea (Retz.) Willd.

0 1575.00±14.4a (0%)

1550.00±14.4a (0%)

1563.00±16.1a

(0%) 1563.00±7.2a

(0%) 1600.00±20.4a

(0%) 1550.00±10.2a

(0%) 1550.00±17.7a

(0%) 1575.00±22.8a

(0%)

3 1570.00±5.8a (0%)

0.00±0.0d (100%)

1514.00±6.8ab

(3%) 1447.00±16.4b

(7%) 1332.00±105.0c

(17%) 1498.00±7.5ab

(3%) 1452.00±10.2b

(6%) 1419.00±4.9bc

(10%)

6 1566.00±2.6a (1%)

0.00±0.0d (100%)

1453.00±35.5b

(7%) 1253.00±18.4c

(20%) 1270.00±99.5c

(21%) 1420.00±10.7b

(8%) 1355.00±7.0bc

(13%) 1354.00±7.1bc

(14%)

9 1550.00±0.0a

(2%) 0.00±0.0f (100%)

1270.00±12.1bc

(19%) 1072.00±44.1de

(31%) 1014.00±102.0e

(37%) 1329.00±8.0b

(14%) 1159.00±5.9cd

(25%) 1080.00±12.2de

(31%)

12 1526.00±2.6a (3%)

0.00±0.0g (100%)

1178.00±44.8bc

(25%) 1046.00±26.7de

(33%) 916.00±88.5f

(43%) 1251.00±10.0b

(19%) 1131.00±6.4cd

(27%) 975.00±10.2ef

(38%)

15 1512.00±4.6a (4.%)

0.00±0.0g (100%)

1160.00±44.5bc

(26%) 981.50±11.8de

(37%) 842.00±99.7f

(47%) 1237.00±13.6b

(20%) 1061.00±16.5cd

(32%) 898.00±8.7ef

(43%)

61

Days PT Untreated control Levamisole 7.5 mg kg-1 CP 1 g kg-1 CP 4 g kg-1 CP 8 g kg-1 CAME 1 g kg-1 CAME 4 g kg-1 CAME 8 g kg-1 Syzygium cumini (L.)

0 1450.00±14.4a (0%)

1425.00±14.4a (0%)

1450.00±14.4a

(0%) 1400.00±0.0a

(0%) 1438.00±7.2a

(0%) 1450.00±14.4a

(0%) 1438.00±7.2a

(0%) 1413.00±7.2a

(0%)

3 1445.00±2.9a (0%)

0.00±0.0h (100%)

1419.00±0.6b (2%)

1344.00±3.8e (4%)

1356.00±3.5d (6%)

1402.00±0.9c (3%)

1292.00±0.9f (10%)

1238.00±7.2g (12%)

6 1435.00±2.9a (1%)

0.00±0.0e (100%)

1342.00±4.6ab

(7%) 1288.00±7.2bc

(8%) 1302.00±1.2bc

(9%) 1328.00±1.2bc

(8%) 1180.00±0.3d

(18%) 1238.00±94.4cd

(12%)

9 1422.00±4.6a (2%)

0.00±0.0f (100%)

1172.00±1.2b (19%)

1102.00±0.9bc (21%)

1058.00±4.3cd (26%)

1045.00±2.6cd (21%)

995.00±2.9d (34%)

875.00±92.4e (38%)

12 1400.00±14.4a (3%)

0.00±0.0f (100%)

1157.00±2.0b (20%)

989.50±6.1c (29%)

977.00±4.0cd (32%)

1060.00±5.8bc (26%)

876.00±3.5de (39%)

812.00±96.6e (42%)

15 1392.00±4.6a (4%)

0.00±0.0f (100%)

1111.00±0.4b (23%)

952.00±1.2cd (32%)

895.50±2.6d (38%)

1024.00±2.1bc (29%)

842.30±1.3d (41%)

618.60±121.0e (56%)

Musa paradisiaca L.

0 1250.00±14.4a (0%)

1225.00±14.4a (0%)

1250.00±14.4a

(0%) 1237.50±80.0a

(0%) 1212.50±4.3a

(0%) 1237.50±62.5a

(0%) 1250.00±14.4a

(0%) 1225.00±14.4a

(0%)

3 1240.00±5.8a (1%)

0.00±0.0b (100%)

1223.50±2.0a (2%)

1225.00±59.5a (1%)

1154.00±2.3a (5%)

1175.00±52.0a

(5%) 1187.50±65.7a

(6%) 1147.50±1.4a

(6%)

6 1230.00±5.8a (2%)

0.00±0.0d (100%)

1210.50±5.5ab

(3%) 1187.50±65.7abc

(4%) 1096.00±2.3c

(10%) 1137.50±62.5abc

(8%) 1131.00±0.6bc

(10%) 1092.50±4.3c

(11%)

9 1220.00±5.8a (2%)

0.00±0.0e (100%)

1090.50±5.5b (13%)

1037.50±65.7bc

(16%) 723.00±4.0d

(40%) 1025.00±47.9bc

(17%) 988.00±1.2c

(21%) 684.50±3.2d

(44%)

12 1210.00±5.8a (3%)

0.00±0.0e (100%)

1050.50±5.5b (16%)

1000.00±70.7bc

(19%) 676.00±2.3d

(44%) 987.50±42.7bc

(20%) 952.50±1.4c

(24%) 640.00±5.8d

(48%)

15 1200.00±0.0a (4%)

0.00±0.0d (100%)

997.50±1.4b (20%)

975.15±75.0b (21%)

629.20±0.5c (48%)

937.50±42.7b (24%)

928.70±0.8b (26%)

595.30±2.7c (51%)

Mangifera indica L.

0 1350.00±14.4a (0%)

1350.00±14.4a (0%)

1325.00±14.4a

(0%) 1337.50±136.0a

(0%) 1300.00±0.0a

(0%) 1350.00±17.3a

(0%) 1312.50±7.2a

(0%) 1330.00±11.5a

(0%)

3 1340.00±5.8a (1%)

0.00±0.0c (100%)

1252.00±1.2ab

(6%) 1262.50±143.0ab

(6%) 1177.00±1.7b

(10%) 1275.50±14.7ab

(6%) 1239.00±9.0ab

(6%) 1204.00±2.3ab

(10%)

6 1325.00±14.4a (2%)

0.00±0.0c (100%)

1200.00±5.8ab

(9%) 1187.50±138.0ab

(11%) 1095.00±118.0b

(16%) 1222.50±4.3ab

(9%) 1165.50±9.0ab

(11%) 1092.00±4.6b

(18%)

62


9 1312.50±7.2a (3%)

0.00±0.0d (100%)

1130.00±5.8b (15%)

1075.00±120.0bc

(20%) 944.50±93.7c

(27%) 1137.50±12.5b

(16%) 1055.00±2.9bc

(20%) 938.00±6.9c

(30%)

12 1300.00±14.4a (3.7%)

0.00±0.0d (100%)

1128.00±1.2b (15%)

1050.00±117.0b

(22%) 889.50±77.5c

(32%) 1095.00±2.9b

(19%) 1030.50±11.3b

(22%) 868.00±18.5c

(35%)

15 1296.00±2.3a (4%)

0.00±0.0d (100%)

1064.25±2.5b (20%)

1012.60±107.0b

(24%) 875.80±65.8c

(33%) 1063.13±3.1b

(21%) 969.15±11.1bc

(26%) 854.15±2.4c

(36%) Albizia lebbeck (L.) Benth.

0 1550.00±14.4ª (0%)

1575.00±14.4ª (0%)

1600.00±54.0a

(0%) 1562.50±7.2ª

(0%) 1550.00±73.6ª

(0%) 1550.00±20.4ª

(0%) 1562.50±7.2ª

(0%) 1587.50±7.2a

(0%)

3 1540.00±5.8ª (1%)

0.00±0.0c (100%)

1525.00±52.0ab

(5%) 1486.00±8.1ab

(5%) 1462.50±42.7b

(6%) 1506.50±3.8ab

(3%) 1485.50±8.4ab

(5%) 1471.00±16.7ab

(7%)

6 1525.00±14.4ª (2%)

0.00±0.0e (100%)

1475.00±52.0ab

(8%) 1460.50±6.1bc

(7%) 1450.00±35.4bc

(7%) 1419.50±11.3c

(8%) 1362.00±6.9d

(13%) 1310.50±6.1d

(17%)

9 1510.00±5.8ª (3%)

0.00±0.0f (100%)

1312.50±37.5b

(18%) 1067.00±9.8d

(32%) 1025.00±32.3d

(34%) 1204.50±2.6c

(22%) 1160.00±5.8c

(26%) 936.00±12.1e

(41%)

12 1500.00±0.0a (3%)

0.00±0.0f (100%)

1225.00±32.3b

(23%) 1042.00±4.6d

(33%) 1012.50±31.5d

(35%) 1144.50±25.7c

(26%) 1104.50±2.6c

(29%) 869.00±12.0e

(45%)

15 1488.00±6.9ª (4%)

0.00±0.0g (100%)

1176.00±15.0b

(27%) 1004.70±2.7d

(36%) 945.50±2.6e

(39%) 1144.30±25.6b

(26%) 1101.50±0.9c

(30%) 842.15±7.9f

(47%) Digera muricata L.

0 962.50±7.2ª (0%)

950.00±28.9ª (0%)

1000.00±40.8ª(0%)

975.00±14.4ª (0%)

987.50±42.7ª (0%)

1000.00±40.8ª(0%)

987.50±7.2ª (0%)

962.50±7.2ª (0%)

3 950.00±14.4ab (1%)

0.00±0.0c (100%)

964.00±3.5a (4%)

937.50±31.5ab (4%)

937.50±31.5ab (5%)

954.00±2.3ab (5%)

939.50±6.1ab (5%)

908.00±4.6b (6%)

6 940.00±5.8ª (2%)

0.00±0.0c (100%)

940.00±5.8ª (6%)

912.50±42.7ab (6%)

912.50±37.5ab (8%)

922.00±16.2ab (8%)

907.50±4.3ab (8%)

861.50±6.6b (11%)

9 935.00±2.9ª (3%)

0.00±0.0d (100%)

866.00±29.3ab

(13%) 812.50±55.4b

(17%) 800.00±54.0b

(19%) 821.00±16.7b

(18%) 674.00±15.0c

(32%) 636.50±7.8c

(34%)

12 930.00±5.8ª (3%)

0.00±0.0e (100%)

842.00±4.6b (16%)

787.50±47.3bc (19%)

775.00±47.9bc (22%)

766.00±9.2c (23%)

658.50±4.9d (33%)

629.00±12.1d (35%)

15 924.00±2.3ª (4%)

0.00±0.0e (100%)

829.30±12.0b (17%)

762.50±55.4bc (22%)

737.50±55.4c (25%)

734.40±9.0c (27%)

634.28±9.1d (36%)

582.20±10.3d (40%)

PT = Post-treatment; Means marked with the same letters in a row do not differ significantly (p< 0.05); values in parenthesis indicate percentage reduction in EPG

63

4.3.1. Summary of in vivo results

The best in vivo anthelmintic activity based on FECRT (Table 19; Fig. 1 to 10) was exhibited

by CAME of Trianthema portulacastrum (70%), followed in descending order by Legnaria

sisrarria (56%), Tribulus tresstris (52%), Musa paradisiacal (51%), Albezia lebbeck (47%),

Syzygium cumini (46%), Bambusa arrundinacea (43%), Digra muricata (40%), Mangifera

indica (36%) and Ziziphus mauritiana (35%) at day 15 PT. However, FECR of CP of

Bambusa arrundinacea and Syzygium cumini was 47% and 47%, which was better than that

of, CAME of the plants which was 43% and 46% respectively. The data indicates that all the

selected plants possess active biochemical entities that have anthelmintic activity and

Bambusa arrundinacea and Syzygium cumini have active ingredients against helminths,

which are less soluble in methanol.

Table 19. Fecal egg count reduction (%) with crude aqueous methanolic extract at the dose rate of 8 g kg-1 body weight at day 15 post treatment

Plants FECR with CP @ 8 g gk-1

body weight

FECR with CAME @ 8 g gk-1

body weight

Trianthema portulacastrum 62 70

Legnaria sisrarria 38 56

Tribulus tresstris 46 52

Musa paradisiaca 48 51

Albezia lebbeck 39 47

Syzygium cumini 47 46

Bambusa arrundinacea 47 43

Digra muricata 25 40

Mangifera indica 33 36

Ziziphus mauritiana 33 35

64

66

24

62

51

19

100

4

70

-50

150

350

550

750

950

1150

1350

1550

Doses and forms of Trianthema portulacastrum L. used

Egg

s pe

r gra

m (E

PG

) of f

aece

s

Day 0 EPGDay 15 EPG

Crude Powder Crude Aqueous Methanolic ExtractControl

Untreated Treated 1 g 4 g 8 g 8 g4 g1 g

Fig. 1. Reduction in eggs per gram (EPG) of faeces in sheep treated at different doses and forms of

Trianthema portulacastrum L. whole plant compared with control groups. *(Numarical values show the per cent reduction)

65

46

4

100

28

37

47

27

36

-50

150

350

550

750

950

1150

1350

1550

Doses and forms of Lagenaria siceraria (Molina) Standl. used

Egg

s pe

r gra

m (E

PG

) of f

aece

s Day 0 EPGDay 15 EPG




Lagenaria siceraria (Molina) Standl. leaves compared with control groups.

66

52

4

100

20

40

46

27

36

-50

150

350

550

750

950

1150

1350

1550

1750

Doses and forms of Tribulus terrestris L. used

Egg

s pe

r gra

m (E

PG

) of f

aece





Tribulus terrestris L. whole plant compared with control groups.

67

51

4

100

20 21

48

24 26

-50

150

350

550

750

950

1150

1350

1550

1750

Doses and forms of Musa paradisiaca L. used

Egg

s pe

r gra

m (E

PG

) of f

aece

s

Day 0 EPGDay 15 EPG




Musa paradisiaca L. leaves compared with control groups.

68

3026

3936

27

100

4

47

-50

150

350

550

750

950

1150

1350

1550

1750

Doses and forms of Albizia lebbeck (L.) Benth. used

Egg

s pe

r gra

m (E

PG

) of f

aece





Albizia lebbeck (L.) Benth. leaves compared with control groups.

69

41

293832

23

100

4

56

-50

150

350

550

750

950

1150

1350

1550

1750

Doses and forms of Syzygium cumini (L.) Skeels used

Egg

s pe

r gra

m (E

PG

) of f

aece

s

Day 0 EPGDay 15 EPG




Syzygium cumini (L.) Skeels leaves compared with control groups.

70

32

20

4737

26

100

4

43

-50

150

350

550

750

950

1150

1350

1550

1750

Doses and forms of Bambusa arundinacea (Retz.) Willd. used

Egg

s pe

r gra

m (E

PG

) of f

aece





Bambusa arundinacea (Retz.) Willd. leaves compared with control groups.

71

36

27252217

100

4

49

-50

150

350

550

750

950

1150

Doses and forms of Digera muricata L. used

Egg

s pe

r gra

m (E

PG

) of f

aece

s

Day 0 EPGDay 15 EPG




Digera muricata L. whole plant compared with control groups.

72

26

21

33

2420

100

4

36

-50

150

350

550

750

950

1150

1350

1550

1750

Doses and forms of Mangifera indica L. used

Egg

s pe

r gra

m (E

PG

) of f

aece

s

Day 0 EPGDay 15 EPG




Mangifera indica L. leaves compared with control groups.

73

2823

3228

16

100

4

35

-50

150

350

550

750

950

1150

1350

1550

1750

Doses and forms of Ziziphus mauritiana Lam. used

Egg

s pe

r gra

m (E

PG

) of f

aece

s

Day 0 EPGDay 15 EPG




Ziziphus mauritiana Lam. leaves compared with control groups.

74

Chapter # 5

DISCUSSION

The discussion has been arranged in the following order:

Subject Page #

5.1. Survey 75

5.2. Tests used for evaluation of anthelmintic activity 79

5.2.1. Egg Hatch Test (EHT) 80

5.2.2. Adult Motility Assay (AMA) 81

5.2.3. Fecal Egg Count Reduction Test (FECRT) 83

5.3. In vitro and in vivo anthelmintic activity 84

5.3.1. Plants demonstrated anthelmintic activity in EHT, AMA and FECRT 85

5.3.1.1. Albizia lebbeck (L.) Benth. 85

5.3.1.2. Bambusa arundinacea (Retz.) Willd. 86

5.3.1.3. Digera muricata L. 87

5.3.1.4. Lagenaria siceraria (Molina) Standl. 87

5.3.1.5. Mangifera indica L. 88

5.3.1.6. Musa paradisiaca L. 89

5.3.1.7. Syzygium cumini (L.) Skeels 90

5.3.1.8. Trianthema portulacastrum L. 91

5.3.1.9. Tribulus terrestris L. 92

5.3.1.10. Ziziphus mauritiana Lam. 93

5.4. Phytoanthelmintics 94

75

5.1. Survey

The data of the present survey shows that despite of availability of veterinarians, farmers

usually rely on their personal knowledge for prevention and treatment of helminthiasis as

reported elsewhere (Walzer et al., 1991). They acquired the knowledge of EVM practices

against helminths from their parents and grandparents (ancestors), neighbours,

contemporaneous practitioners or practical experience. They had been paid high regards in

the society, they provide their expertise as do the family doctors in western medicine and this

process is going on generations after generations. The plants have been evaluated by

generations of indigenous people (Cox, 2000). This traditional knowledge (TK) is passed on

orally from one generation to the next and some times within the family, constitute the basis

for traditional bio prospecting. Traditional bio prospecting forms the foundation for the

ethnomedicine (Sindiga et al., 1993) and ethnoveterinary medicine (Ole-Miaron, 1997).

A progressive decrease in the percentage of farmers using medicinal plants was reported

from majority of informants. The probable causes may include a continued deforestation,

acculturization and generation gap due to modernization that took place in the area over

several years causing loss of transfer of knowledge to next generations (Giday et al., 2003).

For example, the plants at risk of high deforestation for human interest in expansion of

agriculture and change in socio-cultural activities include Ziziphus mauritiana Lam., Albizia

lebbeck (L.) Benth., Mangifera indica L., Tamarix aphylla (L.) H. Karst., Capparis decidua

(Forssk.) Edgew., Ricinus communis L., Tumma, Solanum xanthocarpum L., Azadirachta

indiaca A. Juss., Musa paradisiaca L., Syzygium cumini L., Bambusa arundinacea (Retz.)

Willd., Herpestis monniera L., Citrullus colocynthis (L.) Schrader and Prunus persica L.

Batsch. The plants like Tribulus terrestris L., Digera muricata L., Trianthema

76

portulacastrum L. and Cuscuta reflexa Roxb. grown spontaneously as weeds of different

crops, are going to parish because of well organized and efficient weed control programs

(Aneja et al., 2000; Chauhan et al., 1995). All the documented plants of pesent study except

Cocos nucifera L., Ferula assafoetida L. and Mallotus philippinensis (Lam.) Muell.-Arg.

were native to the study area.

Variation in the doses of traditional recipes as well as vehicles (carrier) were found from one

TVH to the other as well as from one animal to the other which may be a possible

determinant of the variable efficacy of traditional medicine. However, the variability of

carrier dose is not as outstanding in allopathic medicine as in EVM. In most of these recipes,

the principle of use of a carrier mechanism for the medicine to be administered is quite

common. The principle of using a carrier mechanism in Western veterinary medicine is well

recognized. Most of the traditional healers use capricious quantities of the carrier in most of

the recipes, which may alter the efficacy of the drug or reduce its relative potency. Variation

in the quantity of the carrier material is much prominent in ethnoveterinary medicine while in

allopathic medicine the case is otherwise (Jabbar et al., 2006a). A number of plants have so

far been reported for the anthelmintic activity round the globe e.g. Italy (Guarrera, 1999),

Trinidad and Tobago (Lans and brown, 1998; Lans et al., 2000), Cameroon (Nfi et al., 2001),

sub-humid zone of northern Nigeria (Alawa et al., 2002), Qassim region of Saudi Arabia

(Abbas et al., 2002) and Maasai (Ole-Miaron, 2003). This survey contributes in the

formation of database on the ethno-anthelmintics of Pakistan in continuation with the

previous research (Akhtar et al., 2000; Iqbal et al., 2004; Iqbal et al., 2006a; Jabbar et al.,

2006a).

77

There were 10 plants, the leaves of which were used and they include Albizia lebbeck (L.)

Benth., Azadirachta indica A. Juss., Bambusa arundinacea (Retz.) Willd., Lagenaria

siceraria (Molina) Standl., Mangifera indica L., Musa paradisiaca L., Nicotiana tabacum

L., Prunus persica L. Batsch., Syzygium cumini (L.) Skeels and Ziziphus mauritiana Lam.

Seeds of 9 plants vis; Brassica campestris L., Cicer arietinum L., Cuminum cyminum L.,

Coriandrum sativum L., Eruca sativa Miller, Foeniculum vulgare Mill., Ricinus communis

L., Triticum aestivum L. and Veronica anthelmintica L. Willd. and whole fruit (fruit plus

seeds) of 5 plants was used and these plants were Capsicum annum L., Citrullus colocynthis

(L.) Schrader, Cucumis melo var. Flexuosus (L.) naud., Solanum xanthocarpum L. and

Withania coagulans Dunal. Aerial parts of 4 plants were used and these plants were Capparis

decidua (Forssk.) Edgew., Convolvulus arvensis L., Herpestis monniera L. and Medicago

sativa L. Four plants were used as whole plant and they include Cuscuta reflexa Roxb.,

Digera muricata L., Trianthema portulacastrum L. and Tribulus terrestris L. Fruit of 3

plants was used as ethnoanthelmintic and these plants were Cocos nucifera L., Mallotus

philippinensis (Lam.) Muell.-Arg. and Tamarix aphylla (L.) H.Karst. Bulb of 2 viz; Allium

cepa L. and Allium sativum L. was used and only stem tuber of Solanum tuberosum L., bark

of Helleborus niger L., rhizome of Zingiber officinale Roscoe and twigs of Capparis

decidua (Forssk.) Edgew. were used as ethnoanthelmintics. Capparis decidua (Forssk.)

Edgew. is the only plant whose aerial parts and twigs were both used as anthelmintic.

Fourteen out of 41 plants (34.15%) reported in the present survey have already been

scientifically validated for their anthelmintic activity. These plants include Albizia lebbeck

(L.) Benth. (El Garhy and Mehmoud, 2002), Allium sativum L. (Iqbal et al., 2001b),

Azadirachta indica A. Juss., (Hordegen et al., 2003), Helleborus niger L., (Kalesaraj, 1974),

78

Lagenaria siceraria (Molina) Standl. (Akhtar and Riffat, 1987), Mallotus philippinensis

(Lam.) Muell.-Arg. (Akhtar and Ahmad, 1992), Mangifera indica L. (Kalesaraj, 1974), Musa

paradisiaca L. (Sharma et al., 1971), Nicotiana tabacum L. (Iqbal et al., 2006a), Prunus

persica L. Batsch. (Akhtar, 1988) Tribulus terrestris L. (Deepak et al., 2002), Veronica

anthelmintica L. Willd. (Iqbal et al., 2006e) Withania coagulans Dunal. (Gaind and

Budhiraja, 1967) and Zingiber officinale Roscoe. (Iqbal et al., 2006c) Seven plants (out of

total 41; 17.07%) viz; Brassica campestris L., Citrullus colocynthis (L.) Schrader,

Convolvulus arvensis L., Cuscuta reflexa Roxb., Eruca sativa Miller, Ferula assafoetida L.

and Foeniculum vulgare Mill. of our survey have previously been reported in another study

conducted by Jabbar et al., (2006a) but not yet scientifically validated. The remaining 20 (of

total 41; 48.78%) are being reported for the first time and need to be screened through

standard scientific procedures for their anthelmintic activity (if any). These include Allium

cepa L., Bambusa arundinacea (Retz.) Willd., Capparis decidua (Forssk.) Edgew.,

Capsicum annum L., Cicer arietinum L., Cocos nucifera L., Coriandrum sativum L.,

Cucumis melo var. Flexuosus (L.) naud., Cuminum cyminum L., Digera muricata L.,

Herpestis monniera L., Medicago sativa L., Ricinus communis L., Solanum tuberosum L.,

Solanum xanthocarpum L., Syzygium cumini (L.) Skeels, Tamarix aphylla (L.) H.Karst.,

Trianthema portulacastrum L., Triticum aestivum L. and Ziziphus mauritiana Lam.

The variability in efficacy of ethnoveterinary practices in contrast to the farmer’s claims

(Minja, 1989; Costa et al., 2006) therefore, necessitates the researchers to standardize the

procedures with respect to the methodology of the plant collection, extract preparation,

dilution making, dosage and mode of administration. Ethnobotanical and

ethnopharmacological survey shows that the plants are still in use in ethnoveterinary

79

medicine in the District Sahiwal which is helpful in improving the animal health care. The

survey contributes towards the development of an inventory of ethnobotanicals used as

anthelmintics and hence ensuring a thorough documentation, which would conserve the

ethnoveterinary practices against helminthiasis in the area. An exclusive variation in dose of

documented plants and non-plant materials used in the present survey indicated scarcity of

knowledge in this arena and needs to be explored (Dilshad et al., 2008). The reported plants

may be promising candidates for their future use as anthelmintics. In addition, an extension

service to the small-holder dairy farmers about the traditional knowledge of plants and non-

plant materials around themselves specifically used for treatment of a wide variety of

diseases, will not only be beneficial for the developing world (Gesler, 1991) but also for the

other advanced countries with modern farming systems.

5.2. Tests used for evaluation of the anthelmintic activity

Screening of plants for their anthelmintic activity has multiple objectives. These include: (i)

validation of the claims of the farmers using different plants for anthelmintic purposes using

standard parasitological procedures (ii) exploring the possibilities of discovering new plants

with anthelmintic properties (Bachaya, 2007).

This thesis reports screening of 10 plants for their anthelmintic activity using in vitro and in

vivo tests. Results of in vitro tests with plant products against nematodes using methods such

as larval (Robinson et al., 1990; Perrett and Whitfield, 1995) and adult (Kaushik et al., 1981;

Parveen, 1991) paralysis tests, egg hatch assays (Ketzis et al., 2002; Pessoa et al., 2002;

Alawa et al., 2003), or motility and biochemical tests (Kumar et al., 1995; Khunkitti et al.,

2000) have been reported. In vitro screening for anthelmintic activity of CAME s of different

plants was carried out using egg hatch test (EHT) and adult motility assay (AMA). The

80

authenticity of the in vitro and in vivo tests used for evaluation of the anthelmintic activity in

the light of current results and available literature is discussed below:

5.2.1. Egg Hatch Test (EHT)

The egg hatch test (EHT) was originally developed for the detection of benzimidazole (BZ)

resistance in livestock helminths. It is based on the ovicidal activity of BZ. However, the test

has also been used for screening of plants and/or other compounds for their anthelmintic

activity (Molan et al., 1999; Molan et al., 2000a; Waghorn and Molan, 2001; Molan et al.,

2002; Min et al., 2004). The test was originally described by Le Jambre (1976). A

standardized protocol was adopted by the World Association for the Advancement of

Veterinary Parasitology (WAAVP) (Coles et al., 1992). The reliable data can be obtained by

freshly collected faecal samples (within 3 hours of being shed). This is because of a false

positive result due to development of eggs beyond the ventral indentation stage leading to

embryonation (Le Jambre, 1976; Weston et al., 1984; Riou et al., 2005). If fresh collection of

faeces is not possible, samples must be stored anaerobically. This storage does not influence

the outcome of the test at least for the major gastrointestinal (GI) helminths of small

ruminants (Hunt and Taylor, 1989).

In the present study, EHT was employed on Haemonchus contortus eggs using two fold

dilutions (8.00, 4.00, 2.00, 1.00, 0.50 and 0.mg mL-1) of crude aqueous methanolic extracts

of different plants (to be tested) and benzimidazole (control). Egg hatch test was found useful

in obtaining reliable data as evident from the varying efficacies (LC50) and dose-dependent

effects of different plants screened in this study. Therefore, reliability of EHT as a drug/plant

screening assay was in support of the earlier workers (Molan et al., 1999; Molan et al.,

2000b; Waghorn and Molan, 2001; Molan et al., 2002; Min et al., 2004).

81

5.2.2. Adult Motility Assay (AMA)

There are constraints to raise parasitic nematodes in continuous culture, though maturation of

larvae to egg-laying adults has been attained in many cases (e.g., Stringfellow, 1986). The

ability to raise parasites in the laboratory outside of a host would be of enormous benefit to

study the basic biology of these organisms and the effects of drugs on them; the absence of a

suitable culture system is a major impediment to such research. Since the adult stage is a

primary target for chemotherapy, it would be most desirable to be able to determine the intrinsic

potency of anthelmintics against them. Available systems for maintaining adult stages in

culture, following isolation from the host, seems to be inevitably plagued by a continuous drop

in viability, complicating the interpretation of drug toxicity tests (Bachaya, 2007). Some species,

such as Haemonchus contortus, are less robust in culture (Geary et al., 1993) than others, e.g.

Trichostrongylus colubriformis (Rapson et al., 1985; Jenkins et al., 1986) and Nippostrongylus

braziliensis (Rapson et al., 1987).

Adult motility assay (AMA) is, however, the most convenient test used for assaying the

anthelmintic activity of drugs/plants/plant-products. In AMA, worms are exposed to varying

concentrations of drugs and observed for their inhibited motility and/or mortality at different

intervals. Most of the in vitro research on anthelmintic activity of plants, their oils or extracts

have been based on their toxic effects on earthworm, Pheritima posthuma (Gaind and

Budhiraja, 1967; Ali and Mehta, 1970; Kokate and Varma, 1971; Dixit and Varma, 1975;

Banerjee and Nigam, 1978; Girgune et al., 1978; Agarwal et al., 1979: Girgune et al., 1979;

Mishra et al., 1979; Mehta et al., 1981; Dengre, 1982; Garg and Kasera, 1982a, b; Nanda et al.,

1987; Siddiqui and Garg, 1990; Garg and Siddiqui, 1992). Most of the substances which are

toxic to earthworms produce a primary irritation or agitation that results in the withdrawal of the

82

worm from the neighborhood of the poison. By virtue of this effect, anthelmintics doubtless

often expel the parasite when the concentration does not rise sufficiently high to kill the worm

(Sollmann, 1918). Some workers have also used roundworms, Haemonchus contortus, Ascaris

lumbricoides, Ostertagia cicumcincta and Trichostrongylus colubriformis for the evaluation of

in vitro anthelmintic activity of different plant materials (Dubey and Gupta, 1968; Sharma et al.,

1971; Kalesaraj, 1974, 1975; Dixit and Varma, 1975; Lal et al., 1976; Banerjee and Nigam,

1978; Girgune et al., 1978; Agarwal et al., 1979; Girgune et al., 1979; Mishra et al., 1979;

Sharma et al., 1979; Shrivastava, 1979; D’Cruz et al., 1980; Prakash et al., 1980; Mehta et al.,

1981; Dengre, 1982; Garg and Kasera, 1982, 1982a; Kakrani and Kalyani, 1984; Singh et al.,

1985; Kalyani et al., 1989; Siddiqui and Garg, 1990; Nakhare and Garg, 1991; Garg and

Siddiqui, 1992; Garg and Jain, 1992; Asuzu and Njoku, 1996; Amorium et al., 1998; Nirmal et

al, 1998; Paolini et al., 2003; Hounzangbe-Adote et al., 2005).

In this study, Haemonchus contortus proved to be good test worm because of its longer survival

in PBS. By high merit of its longer survival, more number of observations was recorded on the

motility of worms. Adult motility assay used in present study is simple and economical. Worms

from few animals are sufficient to test many drugs and their concentrations and only a little

amount of chemical compound/plant extract is required. Moreover, no previous toxicity tests are

necessary. Although, in vitro tests upon parasites in the blood or tissues are not justified,

theoretically this method can be used for screening compound/plant extracts against intestinal

worms. Since these live in the lumen of gut, the drugs which have been given by mouth reach

the parasite in the intestine without much opportunity for chemical modification. It is, however,

true that no single chemotherapeutic test can be reliable to detect 100% of the compounds/plant

83

extracts. But as conciliation between time, expense and labor, the test used in the current study

is good.

5.2.3. Faecal Egg Count Reduction Test (FECRT)

Faecal egg count reduction test is the most commonly used test to detect the problem of

anthelmintic resistance (AR). It was used in this study with an increased number of observations

on faecal egg count reduction. FECRT compares the egg counts before and after treatment with

an anthelmintic drug (Boersema, 1983; Presidente, 1985). The standard procedure used for egg

counting through McMaster chamber was employed following Urquhart et al. (2003). An

untreated and a treated group was also included to monitor any change that occurs in nematode

egg counts during the test period. One of the important limitations of FECRT is that the result of

test may not estimate anthelmintic efficacy accurately because nematode egg output does not

always correlate well with actual worm numbers, and the test only measures effects on egg

production by mature worms. Moreover, if the interval between treatments is less than 10 days,

egg production may be suppressed leading to an overestimation of anthelmintic efficacy

(Hotson et al., 1970; Martin et al., 1985). Therefore, observations on faecal egg counts were

extended up to the day 15 post-treatment as recommended earlier by Coles et al. (1992). Faecal

egg count reduction test can lead the worker to a false situation either false negative (Jackson,

1993) or false positive (Grimshaw et al., 1996) due to difference in developmental stages of the

parasite.

To stabilize the variances in FECRT data, egg counts are logarithmically transformed and

expressed as geometric means for the groups (Sangster et al., 1979; Martin et al., 1982). Dash et

al. (1988) which urged that the arithmetic mean may be more appropriate in FECRTs because

the geometric mean underestimates total egg output and transformation of data may vary

84

between laboratories, thus, making comparisons difficult. A modification of the FECRT has

been described in which no pre-treatment samples are taken (Vizard and Wallace, 1987). The

FECRT may not provide sufficient information on its own for correct interpretation. Larval

culture can be used to determine the species involved, but culture conditions may favour the

development of one species over another (Presidente, 1985). Parasites with a high biotic

potential, e.g. Haemonchus contortus, may exert a disproportionate influence on the results and,

therefore, correction factors have to be included (Webb et al., 1979).

In conclusion, application of FECRT may be useful for preliminary in vivo testing of drugs,

which can also be combined with copro-cultures to measure the efficacy against individual

worm species. The test was found useful as evident from the graded dose response recorded for

the plants used as crude powder or as an extract.

5.3. In vitro and in vivo anthelmintic activity

All the plants included in this study exhibited anthelmintic activity against Haemonchus

contortus as evident from inhibited egg hatching and mortality of worms. A wide variation,

however, was recorded in the anthelmintic effects among different plants as far as the intensity

and dose dependent effects were concerned. In this section, the anthelmintic efficacy of different

plants recorded in the present study and/or previous studies, traditional uses/pharmacological

activities (particularly antimicrobial) and phytochemicals of the considered plants are given

without much discussion on the mechanism of action except of those already reported. The main

route of acquisition of broad-spectrum anthelmintics by nematodes appears to be via trans-

cuticular diffusion as proposed to oral ingestion (Ho et al., 1994; Sims et al., 1996). This

concept is consistent with the hypothesis that anthelmintics must be in the compartment of

residence in order to exert broad spectrum activity. For this purpose, measurements of the

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physiochemical properties of an anthelmintic, coupled with an understanding of its

pharmacokinetic behavior in the gut, would enable one to predict the concentration versus time

profile attained within the parasite in any given section of the tract (Ho et al., 1994). Therefore,

the variation of in vitro effectiveness of different plants may also be due to differences in their

pharmacokinetic behavior besides other factors like chemical composition.

5.3.1. Plants demonstrated anthelmintic activity in EHT, AMA and FECRT

Selected 10 plants, which exhibited broader range effectiveness in all the three tests, were

Albizia lebbeck (L.) Benth., Bambusa arundinacea (Retz.) Willd., Digera muricata L.,

Lagenaria siceraria (Molina) Standl., Mangifera indica L., Musa paradisiaca L., Syzygium

cumini (L.) Skeels, Trianthema portulacastrum L., Tribulus terrestris L. and Ziziphus

mauritiana Lam.

5.3.1.1. Albizia lebbeck (L.) Benth.

In vitro anthelmintic trials (AMA and EHT) of CAME of Albizia lebbeck (L.) Benth. leaves

exhibited time and dose dependant anthelmintic activity. CAME of the plant leaves proved

good anthelmintic at higher doses. In vivo trials (FECR) of CAME and CP of Albizia lebbeck

(L.) Benth. leaves also showed time and dose dependant anthelmintic activity. Albizia

lebbeck is a tree from leguminosea family originally

from Africa and wide spread in Asia and in the American continent as an ornamental tree. In

China, it has been used as a folk medicine for treating psychological disorders, insomnia and

warts (Kan, 1979). Other native medicine uses include insecticidal and anthelmintic (Allen

and Allen, 1981; Hussain et al., 2008). Albizia lebbeck (L.) Benth. has been reported to have

antiparasitic, anti-dysentric and anti-tubercular activities (Chadha, 1985). Saponins, tannins

and xanthones have been extracted from the bark and associated with the medicinal

86

properties (Chiu and Chang, 1992; Ma et al., 1997). Aqueous extract (5%) of Albizia lebbeck

(L.) Benth. has been evaluated against Ascaris lumbricoides and it was found effective in

killing both infective larvae and eggs in less than 40 and 20 days respectively. The results

showed that Albizia lebbeck (L.) Benth. proved promising anthelmintic against Ascaris

lumbricoides (El Garhy and Mahmoud, 2002). Phytochemical reports on Albizia species

have revealed the presence of saponins, tannins and xanthones (Chiu and chang, 1992; Pal,

1995; Ma et al., 1997), echinocystic acid glycosides (Carpani et al., 1989; Orsini et al.,

1991), flavonol glycosides (Souleman, 1991; Barkat et al., 1999), triterpenoid saponins and

sapogenin lactones (Debella et al., 2000), flavonoids (El-Mousallamy, 1998) and a novel

phenolic glycoside, “albizinin” and four known flavan-3-ols (Ma et al., 1997), tannins and a

proportion of aluminium and heavy metals (Anderson and Morrison, 1990). These

phytochemicals are known for their antimicrobial activity (Cowan, 1999) and may also have

their application as an anthelmintic (Bachaya, 2007).

5.3.1.2. Bambusa arundinacea (Retz.) Willd.

Crude aqueous methanolic extract of Bambusa arundinacea (Retz.) Willd. leaves exhibited time

and dose-dependent in vitro anthelmintic activity as well as in vivo anthelmintic activity with

CAME and CP of the leaves of the plant. Although the plant and its extracts have been used in

the folk medicine extensively, but no scientific evidence for such activities is available in

established scientific journals of repute. The leaves of Bambusa arundinacea (Retz.) Willd. are

useful for its inflammatory and antiulcer activities (Muniappan and Sundararaj, 2003) and

healing of wounds and they are also used in diarrhea in cattle (Kirtikar and Basu, 1990) and has

ethnoanthelmintic effect (Hussain et al., 2008). The leaves of the plant are also used in

Ayurvedic medicine in ptosis and paralytic complaints (Kirtikar and Basu, 1990). It has also

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been reported for its use in wounds, menstrual disorders, antifertility, cuts and abortifacient

activities (Adhikari et al., 2007). Further, the methanol extract of Bambusa arundinacea showed

the antihypersensitivity activity, immunosuppressive activity, wound healing property. The

antibacterial activity has also been proved experimentally (Muniappan, 1998).

5.3.1.3. Digera muricata L.

In vitro anthelmintic trials (AMA and EHA) of CAME of Digera muricata L. whole plant

exhibited time and dose dependant anthelmintic activity. CAME of the plant showed late onset

of anthelmintic activity even at higher doses. In vivo trials (FECR) of CAME and CP of Digera

muricata L. leaves also showed time and dose dependant anthelmintic activity. Digera muricata

L. has been reported for its use in folk medicine as anthelmintic (Hussain et al., 2008). As for as

it could be ascertained, there is not a single example of published data regarding anthelmintic

activity of Digera muricata L. in any reputed journal. However, the plant as a whole is laxative

while flowers and seeds are used for urinary discharge (Anjaria et al., 2002).

5.3.1.4. Lagenaria siceraria (Molina) Standl.

Crude aqueous methanolic extract of Lagenaria siceraria (Molina) Standl. leaves exhibited time


CAME and CP of leaves of the plant. Though the plant is previously reported to be used as

anthelmintic (Hussain et al., 2008) but as for as it could be ascertained, the leaves of this plant

have been evaluated for their anthelmintic activity for the first time. However, comparative

anthelmintic activity of seed powder of Lagenaria siceraria (Molina) Standl. at the dose rate of

3 g kg-1, its equivalent water extract, methanol extract with Niclosamide at the dose rate of 100

mg/kg caused 89±14, 67±15, 81±13 and 91±13% reduction in EPG, respectively in sheep

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infected with cestodes, predominantly being the Moniezia and Avitellina species (Akhtar and

Riffat, 1987).

5.3.1.5. Mangifera indica L.

In vitro anthelmintic trials (AMA and EHA) of CAME of Mangifera indica L. leaves exhibited

time and dose dependant anthelmintic activity. There was late onset of in vitro anthelmintic

activity during adult motility assay. In vivo trials (FECR) of CAME and CP of Mangifera indica

L. leaves also showed time and dose dependant anthelmintic activity. Traditionally the plant is

used as anthelmintic (Hussain et al., 2008). A study was conducted to investigated the

antiallergic and anthelmintic properties of vimang (an aqueous extract of Mangifera indica

family stem bark) and mangiferin (the major polyphenol present in vimang) administered orally

to mice experimentally infected with the nematode, Trichinella spiralis. Treatment with vimang

or mangiferin (500 or 50 mg kg-1 body weight per day, respectively) throughout the parasite life

cycle led to a significant decline in the number of parasite larvae encysted in the musculature.

However, no treatment was effective against adults in the gut. Treatment with vimang or

mangiferin likewise led to a significant decline in serum levels of specific anti-Trichinella IgE,

throughout the parasite life cycle. Finally, oral treatment of rats with vimang or mangiferin,

daily for 50 days, inhibited mast cell degranulation as evaluated by the passive cutaneous

anaphylaxis test (sensitization with infected mouse serum with a high IgE titre, then stimulation

with the cytosolic fraction of Trichinella spiralis muscle larvae). Since IgE plays a key role in

the pathogenesis of allergic diseases, these results suggest that vimang and mangiferin may be

useful in the treatment of diseases of this type (Garcia et al., 2003).

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5.3.1.6. Musa paradisiaca L.

In vitro anthelmintic trials (AMA and EHA) of CAME of Musa paradisiaca L. leaves exhibited

a very good time and dose dependant anthelmintic activity. In vivo trials (FECR) of CAME and

CP of Musa paradisiaca L. leaves exhibited very good anthelmintic activity. Higher levels of

anthelmintic activity of CAE of Musa paradisiaca revealed that active ingredient; responsible

for the anthelmintic activity is relatively a polar compound. As far as ascertained, only one

instance of anthelmintic activity of Musa paradisiaca against the eggs of gastrointestinal

nematodes of ovine has been reported (Krychak-Furtado et al., 2005). In this report, ethanolic

extract and pure latex of Musa paradisiacal have been found possessing only low anthelmintic

activity. In the present study, this plant has been tested for the first time for its anthelmintic

activity against gastrointestinal helminths in Pakistan.

Traditionally, the plant is reported to be used as anthelmintic (Hussain et al., 2008). Other

pharmacological uses of the plant have been reported from various countries e.g. Reid (1961)

used the plantain juice of the plant as an antidote for snake bite. The extract of Musa

paradisiaca green fruits reduced hyperglycemia in normal and diabetic mice (Ojewole and

Adewunmi, 2003) and protected the gastric mucosa from aspirin-induced erosion (Lewis et al.,

1999). It has direct vasodilation effect and nonspecific relaxing and inhibiting effect on aortic

and portal smooth muscles (Orie, 1997). The plant has also been tested for the anti-ulcerogenic

activity (Pannangpetch et al., 2001). Musa paradisiaca contains tannins, eugenol, tyramine.

Serotonin, levarterenol, norepinephrine and dopamine are available in the ripe fruit and peel.

Other chemical constituents are alkaloids, steroidal lactones, and iron (Morton, 1987). The

chemical constituent responsible for the anthelmintic activity of Musa paradisiaca has not yet

been explored but this speculation is supported due to the presence of phytochemicals like

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norepinephrine and alkaloids which have been reported to possess anthelmintic activity (Lateef

et al., 2003). Tannins, another constituent present in the plant also have anthelmintic activity

(Molan et al., 2000a). Further research in this area may be helpful to jot down the exact

mechanism of action of this plant.

5.3.1.7. Syzygium cumini (L.) Skeels

In vitro anthelmintic trials (AMA and EHT) of CAME of Syzygium cumini (L.) Skeels leaves

exhibited time and dose-dependant anthelmintic activity. In vivo trials (FECR) of CAME and

CP of Syzygium cumini (L.) Skeels leaves exhibited dose-dependant anthelmintic activity. The

plant is used in indigenous system of medicine as anthelmintic where leave of the plant are used

for this purpose (Hussain et al., 2008). The anthelmintic activity of the plant may be attributed

to condensed tannins which were found up to 8.65% of dry matter (DM) (Bachaya, 2007).

Condensed tannins (CT) have been reported to exert direct or indirect biological effects on the

control of gastrointestinal parasites. There are reports that direct effect of CT might be mediated

by CT nematode interaction and in this way affecting physiological functioning of parasites.

Condensed tannins may also react directly by interfering with parasite egg hatching and hence

interfering with development of infective stage larvae. Molan et al. (2000) demonstrated that the

CT extracted from Lotus pedunculatus, Lotus corniculatus, Hedysarum coronarium and

Onobrychus viciifolia forages reduced the rate of larval development (eggs to third stage larvae).

There are reports that CTs extracted from various forages markedly decrease the viability of the

larval stages of several nematodes in sheep and goats (Kahn and Diaz-Hernandez, 2000). Some

other compounds (ellagitannins, casuarictin and eugeniin) isolated form methanolic extracts of

clove from another species of Syzygium (Syzygium aromaticum) were found to be the rat

intestinal maltase inhibitor (Toda et al., 2000) indicating thereby some other biochemical effects

91

on parasites as well. As for as it could be ascertained the leaves of this plant have been tested for

the first time as anthelmintic. However, there are various reports of use of different parts of this

plant for different ailments e.g. bark juice is used for dysentery, the leaves are also used as

antibacterial agents and also used for strengthning the teeth and gums.. The fruit and seeds are

sweet, acrid, sour, tonic and cooling and are used in diabities, diarrhoea and ringworm infection.

The bark is astringent, sweet sour, diuretic, digestive and anthelmintic (www.info@heart-

intl.net, accessed on July 16, 2008).

5.3.1.8. Trianthema portulacastrum L.

In the present study, CAME extract of Trianthema (T) portulacastrum L. whole plant

exhibited a time and dose-dependent anthelmintic activity in EHT, AMA and FECRT and

caused 70.18% reduction in fecal egg counts in sheep naturally parasitized with

gastrointestinal nematodes.

The present investigation is the first scientific validation of the anthelmintic activity of

Trianthema portulacastrum L. In view of its usage in ethnoveterinary practice in Pakistan,

exhibited very much promising results as far as in vitro and in vivo results are concerned. The

results are comparable with results of other plants in Pakistan e.g. Allum sativum, Curcurbita

maxicana, Ficus religiosa, (Iqbal et al., 2001b), Artimisia bravifolia, (Iqbal et al., 2004),

Calotropis procera, Zingiber officinale (Iqbal et al., 2006c), Nictiana tabacum (Iqbal et al.,

2006a), Swetia chirata (Iqbal et al., 2006d), Vernonia anthelmintica (Iqbal et al., 2006e), Butea

monosperma (Iqbal et al., 2006b), Trachyspermum ammi (Jabbar et al., 2006b), Chinopodium

album and Caesalpinia crista (Jabbar et al., 2007). However, Trianthema portulacastrum L. has

been evaluated for its hepatoprotective activity against paracetamol and thioacetamide

intoxication (Kumar et al., 2004) and alcohol poison (Shastri, 1952). Some other compounds

92

e.g. diethylnitrosamine induced hepatocarcinogenesis (Bhattacharya and Chatterjee, 1998),

acute and chronic carbon tetrachloride-induced hepatocellular injury (Mandal et al., 1998;

Sarkar et al., 1999), edema of liver and spleen (Ahmad et al., 2000) and also has antioxidant

activity (Kumar et al., 2004). Trianthema portulacastrum L. has also been reported to be used as

anthelmintic in folk medicine (Hussain et al., 2008). The probable mechanism by which T.

portulacastrum L. exerts its potent anthelmintic activity may be due to the fact that it contains

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-7-methoxy-6,8-dimethylflavone, along

with 5,7-dihydroxy-6,8-dimethylchromone (leptorumol). Some of these compounds e.g.

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 proved as good antelmintics. So it can be concluded that

this wonderful plant justifies its traditional use by livestock holders as anthelmintic (Hussain et

al., 2008). However, its further biochemical analysis may lead to successful isolation of some

wonderful biochemical compounds with anthelmintic properties for further commercialization

after in vitro and in vivo trials.

5.3.1.9. Tribulus terrestris L.

Crude aqueous methanolic extract of Tribulus terrestris L. whole plant exhibited very time


CAME and CP of whole plant. Tribulus terrestris Linn. (Zygophyllaceae) is a herb

distributed throughout subcontinent and is known in Ayurveda for its anti-urolithiatic,

diuretic and aphrodisiac properties (Sivarajan and Balachandran, 1994). The plant is reported

93

to be used as anthelmintic in folk medicine (Hussain et al., 2008). Pharmacological studies

reported in the literature (Anand et al., 1994; Ross, 2001) have confirmed these properties.

The plant is reported to contain steroidal saponins (Fang et al., 1999), alkaloids (Wu et al.,

1999), lignanamides (Li et al., 1998) and flavonoids (Saleh et al., 1982). Recently a study

was conducted in India to detect the anthelmintic activity of Tribulus terrestris L. whole

plant against Caenorhabditis elegans and it was observed that the activity could be detected

only in 50% methanol extract which on further bioactivity guided fractionation and

chromatographic separation yielded a spirostanol type saponin, tribulosin and β-sitosterol-D-

glucoside. Both the compounds exhibited anthelmintic activity with ED50 of 76.25 and 82.50

µg mL-1 respectively (Deepak et al., 2002).

5.3.1.10. Ziziphus mauritiana Lam.

Crude aqueous methanolic extract of Ziziphus mauritiana Lam. leaves exhibited time and

dose-dependent in vitro anthelmintic activity as well as in vivo anthelmintic activity with

CAME and CP of the leaves of the plant. As for it could be ascertained, this plant has been

evaluated for anthelmintic activity for the first time in the world (no such example could be

found through literature search), however it is used in indigenous system of medicine as

anthelmintic (Hussain et al., 2008). The combined powder of Ziziphus mauritiana Lam.

(Rhamnaceae) with Vitellaria paradoxa C.F. Gaertn. (syn. Butyrospermum paradoxum) has

ability to produce a more effective insecticide (Cisse, 2004). Phytochemical reports on

Ziziphus species has revealed the presence of polysaccharides (Yamada et al., 1985; Zhao et

al., 2006a), a pectin composed of D-galacturonic acid, L-rhamnose, D-galacturonic acid as

methyl ester and O-acetyl groups (Shimizu and Tomoda, 1983), cyclopeptides (Barboni et

al., 1994; Gournelis et al., 1998; Singh et al., 2002), peptide alkaloids (Tschesche et al.,

94

1974), flavonoides (Nawar et al., 1984; Cheng et al., 2000), dodecaacetylprodelphinidin B3

(Weinges and Schick, 1995), Ziziphine N, O, P and Q (Suksamrarn et al, 2005), saponins and

fatty acids (Zhao et al., 2006b). These phytochemicals are known for their antimicrobial

activity (Cowan, 1999) and may have their application as an anthelmintic as well.

5.4. Phytoanthelmintic activity

All the 10 plants evaluated in the present study exhibited anthelmintic activity in one or the

other tests. The anthelmintic activity of the subjected plants, however, varied in different

tests. Some of the top ranked plants e.g. Musa paradisiaca L., Trianthema portulacastrum

L., Lagenaria siceraria (Molina) Standl., Albizia lebbeck (L.) Benth. and Tribulus terrestris

L. proved their anthelmintic activity in all the tests used whereas these plants showed

variable results. The probable reasons of variable anthelmintic activities of study plants

might be due to variable (i) chemistry of the plants and (ii) targets on the parasites to exert

anthelmintic effects.

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CHAPTER # 6

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

The present research was conducted to document the indigenous knowledge of

ethnoveterinary practices against gastro-intestinal nematodes and scientifically validate some

widely used ethnobotanicals being currently used in the ethno-veterinary medicinal system of

Pakistan, for their anthelmintic activity. For this purpose, documentation of 41 plant species

was done which were used in 49 different traditional recipes representing 39 genera and 27

families for the treatment of helminthiasis. Most frequently used plants (≥5 times) were

Brassica campestris L. and Mallotus philippinensis (Lam.) Muell.-Arg. which represented

the families Brassicaceae and Euphorbiaceae respectively. Most frequently used part of the

plant was leaves (n=10) followed in order by seeds (n=9), whole fruit (n=5), aerial parts and

whole plant (n=4), fruit (n=3), bulb (n=2) and bark, rhizome, stem, stem plus root and twigs

(n=1). Five recipes out of forty-nine (10.2%) were containing more than one plant species

and rest 44 (89.8%) were containing single plant species. Out of these 41 plants, a total of 10

plants were selected to be tested in vitro and in vivo studies. All the plant materials were

procured from local market and fields (Sahiwal, Pakistan), identified and authenticated by a

botanist in the Department of Botany, University of Agriculture, Faisalabad, Pakistan. The

materials were dried in shade, ground finally in powder in electric grinder, and stored in

cellophane bags at 4°C until use. In vitro screening for anthelmintic activity of crude aqueous

methanolic extracts of different plants was carried out using egg hatch test (EHT) and adult

motility assay (AMA). The methanolic extracts of the plants were used for in vitro studies on

Haemonchus contortus. In AMA the motility/survival of the worms was selected as the

criteria for the anthelmintic activity. All plants demonstrated anthelmintic activity in both

96

the tests EHT and AMA. These plants were; Trianthema portulacastrum L., Lagenaria

siceraria (Molina) Standl., Tribulus tresstris L., Musa paradisiaca L., Albizia lebbeck (L.)

Benth., Syzygium cumini (L.) Skeels, Bambusa arrundinacea (Retz.) Willd., Digra muricata

L., Mangifera indica L. and Ziziphus mauritiana Lam.

For in vivo studies same 10 plants were used. The experiment was conducted on

sheep naturally infected with mixed gastrointestinal nematode species including Haemonchus

contortus, Trichostrongylus colubriformis, Trichostrongylus axei, Strongyloides papillosus and

Trichuris ovis. All the plants were found to possess varying anthelmintic activity. This activity

also varied in the form of plants used, i.e., crude powder and methanol extract. The best in vivo

anthelmintic activity based on EPG reduction was exhibited by CAME of Trianthema

portulacastrum L. (70%), followed by Lagenaria siceraria (Molina) Standl. (56%), Tribulus

tresstris L. (52%), Musa paradisiaca L. (51%), Albizia lebbeck (L.) Benth. (47%), Syzygium

cumini (L.) Skeels (46%), Bambusa arrundinacea (Retz.) Willd. (43%), Digra muricata L.

(40%), Mangifera indica L. (36%) and Ziziphus mauritiana Lam. (35% PT) at dose rate of 8 g

kg-1 body weight at day 15 PT. The results indicate that biochemical contents responsible for

anthelmintic activity are present in plants. There were no observed untoward effects of any plant

or the form of drug used.

Conclusions and recommendations

1. These types of surveys provide a baseline data of ethno-anthelmintics which may

contribute to further investigations in relation to a professional ethnoveterinary

medicinal approach.

2. The plants considered in this study and used in ethnoveterinary system of Pakistan have

a potential to be used as anthelmintics.

97

3. Information gained from this study will be made available to all livestock farmers in the

region, through veterinarians, agricultural agents or some other means of dissemination.

The farmers can benefit greatly from seeing the information about the performance of

the various plants, according to standardized methods of formulation, dosage level and

treatment regimes. However, it is recommended that further research be carried out on

large number of animals, identification of active ingredients of plants with proven

anthelmintic activity, standardization of dose and toxicity studies for drug development.

In addition to this, large number of samples of the same plant from different geographic

areas should be subjected to experimentation keeping in view the possibility of

differences in chemical composition of the same plant having different soil origin.

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practice of traditional healers in Qassim Region, Saudi Arabia. J. Arid Env., 50: 367–

379.

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