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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

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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)

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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

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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

95

RECOMMENDATIONS

7

REFERENCES

98

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LIST OF TABLES

Table

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Title

Plants evaluated/used for anthelmintics activity In vitro assays of plant preparations evaluated against different species of nematodes Scientifically evaluated ethnobotanicals used for their in vitro anthelmintic activity in animals in Pakistan In vivo evaluation of plant preparations against Haemonchus contortus in sheep and goats In vivo evaluation of plant preparations against mixed gastrointestinal (GI) nematode infections in ruminant hosts In vivo evaluation of plant preparations against cestodes and trematode parasites in different host species Scientifically evaluated ethnobotanicals for their in vivo anthelmintic activity in animals in Pakistan Globally identified ethnobotanicals with their potential anthelmintic activity Plants screened to evaluate anthelmintic activity Frequency of use of medicinal plants for the treatment and/or management of helminthes of animals in district Sahiwal, Pakistan Ethnoveterinary practices for the treatment and/or management of heminthosis in animals in district Sahiwal, Pakistan In vitro effect of different indigenous plants on survival of Haemonchus contortus (Mean±SEM) of sheep in comparison with Levamisole Ranking of 10 plants according to their effects on adult Haemonchus contortus Per cent egg hatch and LC50 of different plants Regression values and correlation of regression of the effect of different plants on egg hatching

Ranking of 10 plants based on LC50 values and regression correlation values in egg hatch Summary of in vitro results

Page

8 20 21 22 23 24 25 29 34 42 44 49 53 54 55 56 57 59 63

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 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

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LIST OF FIGURES Figure

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10

Title

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 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 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 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 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 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 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 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 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 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

Page

64 65 66 67 68 69 70 71 72 73

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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 gastrointestinal nematodes, which may help veterinarians and stock raisers in future. · Scientifically validate some widely used ethnobotanicals for their anthelmintic activity.

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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.

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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.,

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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

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(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

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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; MathiusMundy 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 Allium sativum Annona senegalensis Acacia albida Adhatoda vesica Agati gratifola Ageratum conyzoides Aglaia odorattissima Agrimonia eupatori Agrimonia pilosa Alangium lamarckii Alangium larmarckii Albizia anthelmintica Part (s) used Bulb Leaf, bark, root Seeds Roots Not reported Leaves, flowers Root bark Not reported Agrimophol Root bark Root bark Bark Root Bark Bark Bark Bulb Bulb Bulb Leaves Parasite (s) Roundworms Nippostrongyllus braziliensis Worm infestation Mixed GI nematodes Ascaris lumbricoides Tapeworms Earthworms Anthelmintic Tapeworms Ascarids Hookworms, ascarids Anthelmintic Fasciolosis Lungwomms Fasciolosis, lungworms Ascaris lumbricoides Roundworms Ascaridia galli Ascaris lumbricoides Nippostrongyllus spp. 8 Target Cattle, goat, sheep Rat Sheep, goat Sheep Humans Not reported Not reported Humans Not reported Poultry Dogs, poultry Cattle, goat, sheep Cattle, goat,sheep Camel Cattle, goat, sheep In vitro Cattle, goat, sheep Chicken In vitro Rat Reference (s) Iqbal et al., 2001b Ibrahim et al., 1984 Nwude and lbrahim, 1980 Lateef et al., 2003 Kalesaraj, 1974 Sharma et al., 1979 Nanda et al., 1987 Farnsworth et al., 1985 Xiao and Lin, 1986 Dubey and Gupta, 1969 Dubey and Gupta, 1968 Minja, 1989; ITDG and IIRR, 1996

Albizia coriavera Albizia lebbeck Allium sativum

ITDG and IIRR, 1996 Kalesaraj, 1975 ITDG and IIRR, 1996; Iqbal et al., 2001b Das and Thakuria, 1974 Kalesaraj, 1975 Ibrahim et al., 1984

Aloe barteri

Name of plant Alpinia calcarata Cucuruma aramatica Ammora wallichii Calamintha umberosa Picus religiosa Sentia myrtina Sumplocos crataegoides Amomum aromaticum Anacardium occidentale Ananas comosus Ananas sativus Ananas sativus Annona cherimolia Annona muricata Annona braziliensis Molinema dessetae Anogeissus leiocarpus Anogeissus leiocarpus Securinega virosa Khaya senegalansis Nauclea latifolia Anthocephalus

Part (s) used Rhizomes Stem Whole plant Stem, bark Whole plant Leaves Roots and Rhizomes Not reported Fruit Not reported Not reported

Parasite (s) Ascaris 1umbricoides

Target In vitro

Reference (s) Kalesaraj,1975 Kaushik et al.,1981

Ascaridia galli

In vitro

Ascaridia galli Earthworms, tapeworms Ascaridia galli Taenia species and Paramphistomum cervi Earthworms Nippostroongylus sp.

In vitro Not reported Chicken in vitro in vitro Rat

Kaushik et al., 1981 Garg and Kasera, 1982a, b Fernandez, 199I Neogi et al, 1964 Chakraborty et al., 1976 Bories et al,, 1991

Bark, seeds Bark Leaves, stem Bark Roots Stem, Bark

Nippostrongyllus braziliensis Anthelmintic

Rat In vivo

Ibrahim et al., 1984 Bizimana, 1994

Ascaridia galli 9

In vitro

Kaushik et al., 1981

Name of plant indices Areca catechu Areca catechu Artabotrys odoratissimus Artemisia abrotanum Artemisia absinthium Artemisia annua Artemisia brevifolia Artemisia herbaalba Artemisia inforescence Artemisia maritima

Part (s) used Nuts Dried ripe seeds Leaves Not reported Not reported Not reported Not reported Shoots Leaves Whole plant Whole plant Flavonoids and sesquiterpene lactones Not reported Not reported Not reported

Parasite (s) Taenicidal Tape worms Pheretima posthuma (earthworms), Taenia solium and Ascaris lumbricoides Anthelmintic Anthelmintic Schistosoma mansoni Haemonchus conrortus Haemonchus contortus Ascaris suum Anthelmintic

Target Cattle, goat, dog Dogs, poultry In vitro Not reported Not reported Hamster, mice Sheep Goat Pig (in vitro) Not reported Buffalo calves Not reported Not reported Not reported Not reported 10

Reference (s) Roepke, I996 British Veterinary Codex, 1953 Siddiqui and Garg, 1990 Krause, 1993 Bara et al., 1999; Guarrera, 1999; Francois, 1974 Shuhua et al., 2000 Iqbal et al., 2004 Idris et al., 1982 Slepnev, 1970 Krantz and Carr, 1967; Narayana et al., 1976; Akhtar, 1984; Sharma, 1993; Hammond et al., 1997 Akhtar et al., 1982; Farnsworth et al., 1985; Sherif et al., 1987; Fernandez, 1991 Holeman et al., 1991 Abu-Niaaj et al., 1996 Anonymous, 1956; Nakhare and Garg, 1991 Naqvi et al., 1991

Neoascaris vitulorum Anthelmintic Anthelmintic Anthelmintic Anthelmintic

Artemisia mesatlantica Artemisia monosperma. Artemisia pallens Artemisia scoparia

Name of plant Artemisia senna Azadirachta indica Azadirachta indica Melia azedarach Ananas comosus Vernonia anthelmmtica Embelia ribes Fumarla parviflora Caesalpinia crista Bixa orellana Boswellia dalzelii Boswellia serrata Buddlea asiatica Butea frondosa

Part (s) used Not reported Cake and leaves Seeds Seeds Leaves Seeds Fruit Whole plant Seeds Seeds Bark Not reported Not reported Seeds

Parasite (s) Anthelmintic, Cestodes Anthelmintic Haemonchus contortus Trichostrongylus colubriformis

Target Canine Small ruminants Lambs

Reference (s) Francois, 1974; Narayana et al., 1976 Gowda, 1997; Mostofa et al., 1996 Hördegen et al., 2003

Ascaridia galli, Ascaris suum Anthelmintic Earthworms, tapeworms Earthworms, tapewonns, Hookworms Anthelmintic, Ascaridia galli, Ascaris lumbricoides Oxyurids Ascaridia galli Anthelmintic, G1 nematodes

Chicken, Pig Sheep, goat In vitro Not repoted Chicken (In vitro), canine, human

Fernandez, 1991 Nwude and 1brahim,1980 Girgune et al., 1978 Dengre, 1982

Butea frondosa Butea frondosa Butea monosperma

Not reported Seeds Seeds

Butea superba Caesalpina crista

Not reported Seeds Seeds

Anthelmintic Toxocara vitulorum, Ascaridia galli Haemonchus contortus

Kalesaraj and Kurup, 1962, 1968; Joshi, 1970; Narayana et al., 1976; Lal et al., 1976, 1978; Shilaskar and Parashar, 1989 Mice , Mehta and Parashar, 1966 Lal et al.,1976 In vitro Sheep and Kalesaraj and Kurup, 1968; Chandra others and Sabir, 1978; Lal et al., 1978; Prashanth et al., 2001; Iqbal et al., 2006b Not reported Charka, 1948; Chopra et al., 1958 Buffalo calves, Akhtar et al., 1985; Javed et al., 1994 Chicken Sheep, goats Sharma et al., 1971 (In vitro)

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Name of plant Calliandra calothyrsus Calliandra portoricensis Calotropis procera Capillipedium Foetidum Cymbopogon martini Carica papaya

Part (s) used Legume Roots, leaves, flowers Oil, grass

Parasite (s) Haemonchus contortus, Trichostrongylus, Strongyloides papillosus Toxocara canis, Gastrointestinal nematodes, Haemonchus contortus Pheretima posthuma (earthworms), Taenia solium and Ascaris lumbricoides Ascaris lumbricoides, Ascaridia galli Ascaridia galli, Ascaris suum, Heligmosomoides polygyrus Roundworms Ascaris lumbricoides Ascaridia galli Nippostrongylus braziliensis Roundworms Anthelmintic activity

Target Sheep Dog, Sheep

Reference (s) Parker and Palmer, l991 Adewunmi and Akubue,1981; Garg and Atal, 1963; Jain et al., 1996; A1-Qarawi et al., 2001; Iqbal et al., 2005 Siddiqui and Garg, 1990

In vitro

Seeds Latex from fruit

Carissa edulis Carum copticum Cassia alata Cassia accidentalis Cassia spectalis

Roots Seeds Seeds Leaves Roots

Human, Chicken Chicken, Pig, Mice Cattle, goats, sheep Human Chicken Rat Cattle, goat, sheep Not reported

Dhar et al., 1965; Lal et al., 1976 Mursof and He, 1991; Satrija et al., 1994; Satrija et al., 1995 ITDG and IIRR, 1996 Krantz and Carr, 1967; Kalesaraj, 1974 Fernandez, 1991 Ibrahim et al., 1984 ITDG and IIRR, 1996 Gaind et al., 1964

Not reported Chebulic myrobalans Belleric myrobalans Emblic myrobalans Leaves Chenopodium album Chenopodium spp. Oil

Nematode Ascaris spp., Toxocara, Strongylus spp. 12

Sheep Horses, pigs, Dogs, Horses

Akhtar et al., 1999 British Veterinary Codex, 1953, 1965

Name of plant Chloroylon swientenia Chrysanthemum spp. Chrysophyllum cainito Cinnamomum tamala Cissampelos mucromata Citrus acida Citrus aromatica Citrus medico Combretum mucronatum Commiphora mukul Croton macrostachys Cucurbita rnexicana

Part (s) used Oil Not reported Stem Oil Roots Rind Roots Oleo-gum resin Leaves Seeds

Parasite (s) Earthworms, tapeworms, hookworms Haemonchus contortus Haemonchus contortus Earthworms, tapeworms Anthelmintic Ascaris lumbricoides Guinea worm Tapeworms, hookworms Anthelmintic Moniezia expansa, Fascialopsis buski, Ascaris lumbricoides, Hymenolepis diminuta Cestodes Haemonchus contortus (mature) Tapeworms, hookworms Earthworms

Target Not reported Chicken Cattle In vitro Not reported In vitro Humans Not reported Not reported Not reported

Reference (s) Dengre,1982 Rebrassier, 1934 Fernandez, 1991 Girgune et al., 1978 Minja, 1989 Kalesaraj, 1975 Sofowora, 1982 Kakrani and Kalyani, 1984 Minja, 1989 Shrivastava and Singh, 1967

Cucurbita moschata Cucurbita pepo Momordica charantia Cyathocline lyrata Cymbopogon nardus Cymbopogon

Seeds Not reported Essential oil Essential oil

Human Goats (in vitro) In vitro In vitro

Xiao and Lin, 1986 Sharma et al., 1971 Shrivastava, 1979 Kokate and Varma, 1971

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Name of plant citratus Cyperus rotendus Datura quercifolia Datura metal Diospyros mollis

Part (s) used Not reported Fruit Diospyrol Diospyrol Diospyrol

Parasite (s) Tapeworms, earthworms Ascaridia galli Necator americanus, Nematodirus dubius, Hymenolepis nana Necator americanus Nematodirus dubius, Hymenolepis nana Fasciolosis, lungworms Intestinal worms Moniezia, tapeworms, Dicrocoelium, Fasciola Anthelmintic Anthelmintic, Hynnenolepis diminuta Mixed nematode infection Taenia species, Paramphistomum cervi, GI nematodes Tapeworms Fasciolosis Ascaris lumbricoides and Taenia solium Ascarid nematodes, L4 of Ostertagia circumcincta 14

Target Not reported In vitro Golden, Hamster, Mice Golden hamster Mice Cattle, goat, sheep, camel Not reported Not reported Not reported Rat Ruminants Goats Poultry Ruminants Not reported Pig (in vitro) Sheep (in

Reference (s) Girgune et al., 1979 Kaushik et al.,1981 Sen et al., 1974 Sen et al.,1974 Sen et al.,1974 ITDG and 1IRR,1996 Sharma and Singh, 1989 British Veterinary Codex,1953 Minja,1989 Bøgh et al., 1996 Chopra et al., 1956; Ikram and Hussain, 1978 Neogi et al.,1964; Javed and Akhtar, 1990 Qureshi and Sabir, 1979 Nwude and Ibrahim, 1980 Garg and Nakhare, 1993 Perrett and Whitfield, 1995

Diospyros scabra Dodonea viscosa Dryopteris filixmas Embelia kilimandschiraca Embelia schimperi Embelia ribes

Seeds Leaves Male fern Roots Seeds, roots, fruit Not reported Fruit Seeds Bark Flowers Not reported

Embelia ribes Erythrina senegalensis Eupatorium triplinerve Evodia rutaecarpa

Name of plant Feruia foetidissima Ficus religiosa Flemingia vestita

Part (s) used Not reported Not reported Root-tuber peel

Parasite (s) Haemonchus, Bunostomum, Chabertia, Nematodirus Anthelmintic Raillietina echinobothrida Fasciolopsis buski Trichostrongylus, Haemonchus, Trichuris, Fasciola spp. Tapeworms, earthworms Roundworms Earthworms, tapeworms

Target vitro) Sheep In vitro Domestic fowl (in vitro) Pig (in vitro) Sheep, buffalo Not reported Cattle, goat, sheep Not reported

Reference (s) Pustovoi, 1968 Iqbal et al., 2001b Pal and Tandon, 1998 Kar et al., 2002 Akhtar and Javed, 1985; Kailani et al., 1995 Girgune et al.,1979 ITDG and IIRR, 1996 Dixit and Varma,1975

Root-tuber peel Flemingia vestita Fumaria parviflora Plant powder Essential oil Fruit Rhizomes

Gardenia lucida Hagenia abyssainicia Hedychium coronarium Hedychium spicatum Helleborus niger Heracleum sosnoskyi Hyoscyamus niger Inula racemosa Jugulans regia Musa paradisaca Scindapsus officinalis Khaya senegalansis Lagenaria siceraria Lansium domesticum

Stem Not reported Seeds Essential oil Not reported

Ascaris lumbricoides Strongylosis, GI nematodes Mixed nematode infection Earthworms, tapeworms Haemonchus contortus

Humans Sheep In vivo Not reported Goats (in vitro) Not reported Sheep Chicken Pig

Kalesaraj, 1974 Gadzhiev and Eminove, 1986a, b Akhtar and Ahmad, 1990 Mishra et al., 1979 Sharma et al., 1971

Bark Seeds Seeds

Fasciola spp. Cestodes, Moniezia, Avitelina spp. Ascaridia galli Ascaris suum 15

Bizimana, 1994 Akhtar and Riffat, 1987 Fernandez, 1991

Name of plant Lantana trifolia Lantana camara var. aculeata Lawsonia inermis Leucaena leucocephala Limnophila conferta Litsea chinensis Macuna prurita Mallotus philippinensis Mangifera indica Matteuccia orientalis Melia azedarach

Part (s) used Fruit Seeds Leaves Seeds Not reported Not reported Not reported Fruit powder Fruit Seeds Roots Fruit, leaves Fruit Fruit

Parasite (s) Haemonchus contortus Fasciolosis, lungworms Anthelmintic activity Fasciolosis Ascaridia galli Ascaris suum Haemonchus contortus Anthelmintic activity Earthworms, tapeworms Taenia species, Paramphistomum cervi Gastrointestinal cestodes Tapeworms Ascaris lumbricoides Fasciola sp. Taenia species, Paramphistomum cervi Haemonchus contortus Ascaridia galli Haemonchus, Trichostrongylus, Trichuris, Chabertia spp. Ascarids Haemonchus contortus Guinea worm Ascaridia galli 16

Target Goat Cattle, goat, sheep Not reported Sheep, goat Chicken Pig Goat Not reported Not reported Not reported Beetal goats Not reported Humans Cattle In vitro Chicken Goats Not reported Not reported Humans In vitro

Reference (s) 1TDG and IIRR, 1996 Avadhoot et al., 1980 Nwude and Ibrahim, 1980 Fernandez, 1991 Reddy et al., 1991 Mishra et al., 1979 Neogi et al., 1964 Akhtar and Ahmad, 1992 British Veterinary Codex, 1953 Kalesaraj, 1974 Shiramizu et al., 1993 Neogi et al., I964; Nirmal et al., 1998 Akhtar and Riffat, 1985a Akhtar and Riffat, 1984 Xiao and Lin, 1986 Fernandez, 1991 Sofowora, 1982 Lal et al., 1976

Melia toosendan Mimosa pudica Mitragyna stipulosa Momordica

Not reported Stem Roots Not reported

Name of plant charantia

Part (s) used Stem Seeds Roots Leaves Nicotine sulphate

Moringa olelfera

Myrsine africana Nicotiana tabacum

Parasite (s) Ascaris suum Haemonchus contortus Ascaridia galli Ascaris suum Haemonchus contortus Mixed nematode infection Roundworms Moneizia, Ascaridia, Cooperia, Haemonchus, Nematodirus, Ostertagia, Trichoslrogylus spp. Antifasciolic Mixed GI infection, cestode infection Gastrointestinal cestodes Earthworms Earthworms, tapeworms Gastrointestinal nematodes Gastrointestinal nematodes, cestodes Ascaris lumbricoides Haemonchus contortus Ascaris suum, Haemonchus contortus Ascaris suum Ascaridia galli Haemonchus contortus Ascaris spp. Earthworms, tapeworms Roundworms

Target Pigs Goats Chicken Pig Goats Sheep Cattle , goats, sheep Not reported

Reference (s) Fernandez ,1991; Farnsworth et al., 1985 Fernandez, 1991 Akhtar and Ahmad, 1990 ITDG and IIRR, 1996 British Veterinary Codex, 1953, 1965

Nigella sativa Peganum harmala Peganum harmala Piper betle Psitacia integrrima Psoralea coylifolia Punica granatum

Seeds Seeds Seeds Not reported Seeds Seed powder Fruit rind Not reported Not reported Stem

Buffalo Goats Goat In vitro Not reported Sheep Sheep In vitro In vitro Goats Pigs Chicken Goats Not reported Not reported Cattle, Goats,

Kailani et al., 1995 Akhtar and Ahmed, 1991 Akhtar and Riffat, 1986 Ali and Mehta, 1970 Mishra et al., 1979 laved and Akhtar, 1986 Akhtar and Riffat, 1985b Kalesaraj, 1975 Prakash et al., 1980 Farnsworth et al., 1985 Fernandez, 1991; Farnsworth et al., 1985 Xiao and Lin, 1986 Mishra et al., 1979 ITDG and IIRR,1996

Quisqualis indica

Quisqualis indica Randia dumetorum Rapanea

Seeds Seeds Seeds

17

Name of plant melanoploeos Rhamnus principides Rhus vulgaris Sapindus trifoliatum Saussurea lappa Semecarpus anacardium Senecio lyratiparitus Solanum nodiflorum Spigelia anthelmia Linn. Swertia chirata Tamarindus indica Terminalia avicennoides Tiinospora rumphii Tribulus terrestris Trichilia emetica Uvaria hookeri Uvaria narum Vernonia amygdalina Annona senegalensis

Part (s) used Leaves Roots Not reported Roots Nuts Seeds Leaves Fruit Aerial parts Whole plant Roots Leaves, roots Stem Whole plant Bark Root bark Stem bark Leaves

Parasite (s) Anthelmintic Roundworms Ascaridia galli Mixed species of nematodes Anthelmintic GI cestodes Anthelmintic Worm infestation Haemonchus contortus Ascaridia galli Roundworms Nippostrongylus braziliensis Haemonchus contortus Ascaridia galli Fasciolosis, lungworms Haemonchus contortus Haemonchus contortus

Target Sheep Not reported Cattle, goats, sheep In vitro Sheep Buffalo-calves Not reported Goats Not reported Not reported In vitro Not reported Cattle, goats, sheep Rats Goats Poultry Cattle , goats, sheep, camels Not reported In vitro

Reference (s) Minja, 1989 ITDG and IIRR, 1996 Lal et al., 1976 Akhtar and Hassan, 1985 Akhtar and Makhdoom, 1988 Chattopadhyaya and Khare, 1969 Akhtar, 1988 Minja, 1989 Nwude and Ibrahim, 1980 Assis et al., 2003 Shilaskar and Parashar, 1989 ITDG and IIRR, 1996 Ibrahim et al., 1984 Fernandez, 1991 Chakraborty et al., 1979 ITDG and IIRR, 1996 Padmaja et al., 1993 Alawa et al., 2003

18

Name of plant Vernonia anthelmintica

Part (s) used Seeds Fruit/seeds

Parasite (s) GI nematodes GI nematodes, cestodes Oxyurids Ascaridia galli Oxyurids Earthworms Anthelmintic activity, earthworms, roundworms Ascaris lurnbricoides, Fasciolopsis buski, Hymenolepis nana Earthworms, tapeworms, hookworms G1 nematodes Ascaris Iumbricoides Anisakis larvae Dirofilaria immitis Schistosoma mansoni

Target Ruminants Sheep, Goats Not reported Chicken Mice In vitro Not reported In vitro Not reported Sheep Human In vitro Canine Not reported

Withania coagulans Zanthoxylum alatum

Not reported Essential oil Bark Not reported

Reference (s) Nadkarni, 1954; Awan, 1981; Ikram and Hussain, 1978 Nadkarni, 1954; Chopra et al., 1956; Said, 1969; Awan, I981; Singh et al., 1985; Shilaskar and Parashar, 1989; Javed and Akhtar, 1990 Mehta and Parashar, 1966 Gaind and Budhiraja, 1967 Kokate and Varma, 1971; Mehta et al., 1981 Singh et al., 1982 Kalyani et al., 1989 Iqbal et al., 2006c Kalesaraj, 1974, 1975 Goto et al., 1990 Datta and Sukul, 1987; Chakraborty et al., 1994 Adewunmi et al., 1990

Zingiber officinale

Rhizomes

19

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

20

Name of parasite/plant species Vernonia amygdalina

Active principles Not reported

Parts used L

Target E, L3

Reference (s) 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 Allium sativum Artemisia brevifolia Calotropis procera Chenopodium album Caesalpinia crista Cucurbita mexicana Ficus religiosa Nicotiana tabacum Swertia chirata Trachyspermum ammi Parts used Animal Parasite species Bulb Whole plant Flowers Sheep Sheep Sheep H. contortus H. contortus Anthelmintic activity evaluated Reference (s) Iqbal et al., 2001 Iqbal et al., 2004 Iqbal et al., 2005 Jabbar et al., 2007 Jabbar et al., 2007 Iqbal et al., 2001 Iqbal et al., 2001 Iqbal et al., 2006a Iqbal et al., 2006d Jabbar et al., 2006b, Iqbal et al., 2006e Iqbal et al., 2001b

Whole Sheep plant Seed Sheep kernel Whole fruit Sheep Bark Sheep Leaves Sheep Whole plant Seeds Sheep Sheep

100% at 6 hrs post exposure (PE) 30% at 6 hrs PE with AE, 80% with CME (at 25 mg mL-1) 50% with CAE, 57% with CAME (at 25 H. contortus mg mL-1) H. contortus (eggs) LC50 = 0.449 mg mL-1 H. contortus (eggs) LC50 = 0.134 mg mL-1 83.4% at 6 hrs PE 100% at 6 hrs PE 75% at 6 hrs PT with CAE and CAME at 25 mg mL-1 30% and 90% at 6 hrs PT with CAE and H. contortus CME at 25 mg mL-1 H. contortus (eggs) LC50 = 0.1698 and 0.1828 mg mL-1 of CAE and CME 50% at 6 hr PT with CME at 25 mg mL-1 H. contortus 100% at 6 hrs PE H. contortus H. contortus H. contortus H. contortus

Goat Vernonia anthelmintica Seeds Rhizomes Sheep Zingiber officinale

AE=Aqueous extract; CAE=Crude aqueous extract; CAME=Crude aqueous methanolic extract; CME=Crude methanolic extract; PE=Post exposure

21

Table 4. In vivo evaluation of plant preparations against Haemonchus contortus in sheep and goats hosts Plant species Allium sativum Annona squamosa Artemisia herba-alba Calotropis procera Canavalia braziliensis Carica papaya Chenopodium ambrosioides Chrysophyllum cainito Hymenaea courbaril Menta spp. Momordica charantia Musa acuminate Tinospora rumphii Parts used Bb L Sh L S S L St B L St L St Active principles Allicin Anthraquinone terpenoids Santonin Triterpenoids, anthocyanins, alkaloids Not reported Not reported Benzyl isothiocyanate Ascaridole Not reported Not reported Not reported Not reported Not reported Host G G G S G G G B G G G G G Reference (s) Vieira et al., 1999 Vieira et al., 1999 Idris et al., 1982 Al-Qarawi et al., 2001 Vieira et al., 1999 Vieira et al., 1999 Vieira et al., 1999 Fernandez, 1991 Vieira et al., 1999 Vieira et al., 1999 Vieira et al., 1999 Vieira et al., 1999 Fernandez, 1999

Parts used: B=bark, Bb=bulbs, L=leaves, S=seeds, Sh=shoots, St=stem. Host: B=bovids, G=goats, S=sheep.

22

Table 5. In vivo evaluation of plant preparations against mixed gastrointestinal (GI) nematode infections in ruminant hosts Plant species Albizia anthelmintica Ananas comosus Annona squamosa Azadirachta indica Chenopodium ambrosioides Chrysanthemum cinerariaefolium Caesalpinia crista Embelia ribes Fumaria parviflora Hagenia abyssinica Hildebrandtia sepalosa Khaya anthotheca Khaya senegalensis Maerua edulis Myrsine africana Nauclea latifolia Solanum aculeastrum Terminalia glaucescens Vernonia anthelmintica Vernonia amygdalina Parts used B, RB L L S, L L, S, O Fl S Fr W Fr RB B B Tb Fr B R B S L Active principles Sesquiterpene, kosotoxins Bromelain Anthraquinone terpenoids Azadirachtin Ascaridole Pyrethrins Not reported Not reported Not reported Not reported Not reported Kosotoxin Not reported Not reported Benzoquinone Not reported Resin, tannins, alkaloids Not reported Anthraquinone Not reported Host S S, B G, B S, B S S S S S G S B S S S S B B S B Reference (s) Gakuya, 2001; Gathuma et al., 2004; Grade and Longok, 2000 Baldo, 2001; Hördegen et al., 2003; Jovellanos, 1997 Jovellanos, 1997; Vieira et al., 1999 Chandrawathani et al., 2003; Hördegen et al., 2003; Pietrosemoli et al., 1999 Ketzis et al., 2002 Mbaria et al., 1998 Hördegen et al., 2003 Hördegen et al., 2003 Hördegen et al., 2003 Abebe et al., 2000 Gathuma et al., 2004 Nfi et al., 1999 Ademola et al., 2004 Gakuya, 2001 Gathuma et al., 2004 Onyeyili et al., 2001 Nfi et al., 1999 Nfi et al., 1999 Hördegen et al., 2003 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.

23

Table 6. In vivo evaluation of plant preparations against cestodes and trematode parasites in different host species Plant species Tested against cestodes Albizia anthelmintica Embelia Schimperi Ficus insipida, Ficus carica Hagenia abyssinica Hildebrandtia sepalosa Mallotus philippinensis Myrsine Africana Peganum harmala Albizia anthelmintica Tested against trematodes Albizia anthelmintica Embelia schimperi Albizia anthelmintica Albizia coriavera, Allium sativum Diospyrus scabra Lantana trifolia Lawsonia inermis Trichilia emetica Parts used Active principles RB Fr, S, R Lx Fr B Fr Fr S B, R B Fr Rb B S Fr L B Kosotoxin sesquiterpene Embelin Ficin Kosotoxin Not reported Rottlerin Benzoquinone Tetra-hydroharmine Not reported Not reported Benzoquinone Not reported Not reported Not reported Not reported Not reported Not reported Parasite C Hd, Hm, Ts C C C C C C Host S R, M, H M H S G S G Reference Gathuma et al., 2004 Desta, 1995; Bøgh, et al., 1996 de Amorin et al., 1999 Desta, 1995 Gathuma et al., 2004 Akhtar and Ahmad, 1992 Gathuma et al., 2004 Akhtar and Riffat, 1986

Fg G Koko et al., 2000 Ec M Bøgh, et al., 1996 Fasciolosis Catt, G, S, ITDG and IIRR, 1996 Cam Fasciolosis Catt, G, S ITDG and IIRR, 1996 Fasciolosis Catt, G, S, ITDG and IIRR, 1996 Cam Fasciolosis Catt, G, S, ITDG and IIRR, 1996 Cam Fasciolosis G, S Nwude and Ibrahim, 1980 Fasciolosis Catt, G, S, ITDG and IIRR, 1996 Cam

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

24

Table 7. Scientifically evaluated ethnobotanicals for their in vivo anthelmintic activity in animals in Pakistan Botanical name Parts used of plant Adhatoda vesica Aerial parts Bulb Whole plant Whole plant Seeds Seeds Flowers Flowers Aerial parts Whole plant Seed kernel Animal Goat Sheep Sheep Sheep Sheep Buffalo calves Sheep Sheep Sheep Sheep Sheep Anthelmintic activity evaluated 62±5.4% used as PR@2 g/kg b.wt. >>99±1.2% morantel 100% at 6 hrs post H. contortus exposure (PE) 30% at 6 hrs PE with H. contortus AE, 80% with ME (at 25 mg/mL) GINs 67.2% at 3 gm/kg b.wt. with ME at 14 days PT Trichostrongylids 78.4% on day 10 PT with CP at 3 gm/kg b.wt. 100±0.1% used as PR Neoascaris or ME @ 2 g/kg b.wt. vitulorum >>100% morantel 50% with CAE, 57% H. contortus with CAME (at 25 mg/mL) GINs 88.4% with CAE, at 3 gm/kg b.wt., 97.8% levamisole GINs 87±6% used as WE @ 2 g/kg b.wt. >>96±4% morantel H. contortus LC50 = 0.449 mg/mL, (eggs) H. contortus LC50 = 0.134 mg/mL, (eggs) 25 Parasite type/species GINs Phytochemicals Reference (s) isolated AL and GL and Akhtar, 1988 saponins Not reported Not reported Not reported Not reported GL and saponins Not reported Not reported GL Not reported Not reported Iqbal et 2001b Iqbal et 2004 Iqbal et 2004 Iqbal et 2006b al., al.,

Allium sativum Artemisia brevifolia Artemisia brevifolia Butea monosperma Caesalpinia crista Calotropis procera Calotropis procera Chenopodium album Chenopodium album Caesalpinia crista

al., al.,

Akhtar, 1988; Akhtar and Aslam, 1989 Iqbal et al., 2005 Iqbal 2005 et al.,

Akhtar, 1988 Jabbar et al., 2007 Jabbar et al., 2007

Botanical name of plant Chenopodium album Caesalpinia crista Cinnamommum tamala Cucurbita mexicana Cyperus scariosus Embellia ribes/robusta Euphorbia prostrata Euphorbia prostrata

Parts used Whole plant Seed kernel Leaves Whole fruit Seeds

Animal Sheep Sheep Sheep Sheep Buffalo calves Sheep Sheep

Parasite type/species GINs GINs GINs

H. contortus Neoascaris vitulorum GINs GINs

Anthelmintic activity evaluated 82.2% on day 5 PT with AME 93.9% on day 13 PT with AME 97.6±1.8% used as GL @ 150 mg/kg b.wt. >>98±3% morantel 83.4% at 6 hrs PE

Phytochemicals isolated Not reported Not reported AL and GL Not reported

Reference (s) Jabbar et al., 2007 Jabbar et al., 2007 Akhtar, 1988

Iqbal et al., 2001b 10±3% used as PR @ 3 GL and essential Akhtar, 1988 g/kg b.wt. >>100±0% oils morantel 56±26% used as PR @ GL and flavonoid 2 g/kg b.wt. >>97±2% morantel 98.6±1.6% used as ME CGL and GL @ 3 g/kg b.wt. >>98.8±1.3% oxfendazole 100% at 6 hrs PE Not reported 99.8±0.1% used as EE @ 2 g/kg b.wt. >>99.8±0.3% morantel 95.8±5.6% used as PR @ 3 g/kg b.wt. >> 98.8±1.3% oxfendazole 91.4±3.9% used as GL @ 100 mg/kg b.wt. >>92.0±8.0% morantel 26 AL and GL AL, CGL and GL Akhtar, 1988 Akhtar, 1988

Aerial parts Aerial parts

Ficus religiosa Fumaria parviflora Hyoscyamus niger Lagenaria siceraria

Bark Aerial parts Seeds

Sheep Sheep Sheep

H. contortus GINs GINs Cestodes

Iqbal et al., 2001b Akhtar, 1988 Akhtar, 1988

Seeds/flower Sheep

CGL and GL from Akhtar, 1988 seeds

Botanical name Parts used of plant Fruits Mallotus philipinensis Melia azedarach Seeds

Animal Goat Goat

Parasite type/species Cestodes GINs

Momordica charantia Moringa olifera

Fruits Roots

Sheep Sheep

GINs GINs GINs GINs Cestodes

Morus alba

Leaves/stem/ Goat bark Leaves Seeds Sheep Sheep

Nicotiana tabacum Nigella sativa

Peganum harmala

Seeds

Goat

Cestodes

Prunus persica

Leaves

Sheep

GINs

Phytochemicals Reference (s) isolated Flavonoids and GL Akhtar, 1988; Akhtar and Ahmad, 1992 Anthraquinone and Akhtar and GL Riffat, 1984, 1985; Akhtar, 1988 99.6±0.5% used as WE AL, CGL, Akhtar, 1988 @ 3 g/kg b.wt. >> flavonoid, GL and 98.8±1.3% oxfendazole saponins 94.4±2.6% used as PR CGL and GL Akhtar, 1988 @ 3 g/kg b.wt. >> 98.8±1.3% oxfendazole 85.0±2.0% used as GL GL in stem bark Riffat et al., @ 500 mg/kg b.wt. 1986 >>99.0±0.04% morantel 73.6% at 5 days PT with Not reported Iqbal et al., CME at 3 gm/kg b.wt. 2006a 99.0±0.3% used as PR GL and AL and Akhtar, 1988; @ 2.5 g/kg b.wt. anthraquinone Akhtar and >>100.0±0% Javed, 1991; Niclosamide used Akhtar and against GI cestodes of Aslam, 1997 sheep 100.0±% used as PR @ Flavonoid, GL and Akhtar and 3 g/kg b.wt. AL Riffat, 1986 >>98.0±6.2% levamisole + oxyclozanide 99±5% used as WE @ 3 CGL, flavonoid Akhtar, 1988 g/kg b.wt. >> 97±7% and GL i.e.,

Anthelmintic activity evaluated 91.3±5.3% used as GL @ 100 mg/kg b.wt. >>100.0±0% Nilzan 99.4±1.2% used as PR @ 30 mg/kg b.wt. >>99.2±1.6% morantel

27

Botanical name Parts used of plant Seeds Fruit

Animal

Parasite type/species GINs Cestodes

Psoralea corylifolia Punica granatum

Sheep Sheep

Saussurea lappa

Roots

Sheep

GINs

Anthelmintic activity evaluated Morantel used against GI nematodes of sheep 99±0.09% used as WE @ 2 g/kg b.wt. >>99.9±0.01% morantel 95±12% used as AL @ 225 mg/kg b.wt.>>100±0% levamisole + oxyclozanide used against GI cestodes of sheep 100±0% used as ME @ 2 g/kg b.wt. >>100±0% Morantel used against GI nematodes of sheep 29±3.2% used as GL @150 mg/kg b.wt. >>98±6.2% levamisole +oxyclozanide 30% and 90% at 6 hrs PT with CAE and CME at 25 mg/mL 79.7% at 14 days PT with CAE at 3 gm/kg b.wt. LC50 0.1698 and 0.1828 mg/mL of CAE and CME 78.1% on day 5 PT with

Phytochemicals Reference (s) isolated persicon and naringenin AL and GL Javed and Akhtar, 1986 AL, CGL, Akhtar, 1988 flavonoid and GL

AL, CGL and GL

Semecarpus anacardium

Seed

Goat

Cestodes

Anthraquinone, flavonoid and GL Not reported Not reported Not reported Not reported

Akhtar and Hassan, 1985; Akhtar and Makhdoom, 1988 Akhtar, 1988

Swertia chirata

Whole plant Whole plant Seeds Seeds

Sheep Sheep Sheep Sheep

H.contortus GINs

Iqbal et 2006d Iqbal et 2006d

al.,

Swertia chirata

al.,

Trachyspermum ammi Trachyspermum

H.contortus (eggs) GINs

Jabbar et al., 2006b, Lateef et al.,

28

Botanical name of plant ammi Vernonia anthelmintica Vernonia anthelmintica Zingiber officinale Zingiber officinale

Parts used Seeds Fruits Rhizomes Rhizomes

Animal Goat Goat Sheep Sheep

Parasite type/species H. contortus GINs H. contortus GINs

Anthelmintic activity evaluated CP at 3 gm/kg b.wt. 50% at 6 hr PT with CME at 25 mg/mL 73.9% at day 5 PT with CAE at 3 gm/kg b.wt. 100% at 6 hrs PE

Phytochemicals isolated

Reference (s)

2006 Essential oils and Iqbal GL 2006e Essential oils and Iqbal GL 2006e Not reported Iqbal 2001 66.6% after 10 days PT Not reported Iqbal at 3 gm/kg b.wt., 99.2% 2006c levamisole

et et et et

al., al., al., al.,

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 South East Asia Kenya Eastern and Southern Africa East Africa West Africa Zaire Nigeria No of plants with Anthelmintic activity 23 19 >100 >100 18 11 15 4 Anthelmintic activity Hosts Reference (s) Anonymous, 1994 Anonymous, 1996 Watt and BreyerBrandwijk, 1962 Kokwaro, 1993 Ibrahim et al., 1984 Kasonia et al., 1991 Nwude and Ibrahim, 1980 Alawa et al., 2002

Roundworms, cestodes, trematodes Monogastrics, Ruminants Roundworms, cestodes, Monogastrics, trematodes Ruminants Hookworms, cestodes, Humans, Ruminants roundworms, trematodes Hookworms, roundworms, Humans, Ruminants cestodes Roundworms, cestodes Monogastric Roundworms Ruminants Roundworms, trematodes Ruminants, Monogastric Helminths Ruminants

29

Africa Italy Trinidad and Tobago Cameroon Worldwide Saudi Arabia Indian subcontinent Pakistan (Southern Punjab)

>50 51 5 6 4 10 100 6 6 29

Roundworms, trematodes, cestodes Ruminants, Monogastric Anthelmintic Humans Heltminths, parasites Livestock Anthelmintic Dogs Helminths Ruminants Helminthiasis Livestock Cestodes, trematodes, nematodes Animals Vermifuge Helminths Helminths Camels Monogastric Ruminants

Bizimana, 1994 Guarrera, 1999 Pieroni et al., 2004 Lans et al. (2000) Lans and Brown, 1998 Nfi et al., 2001 Tagboto and Townson, 2001 Abbas et al., 2002 Nadkarni, 1954 Jabbar et al., 2006a

30

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

31

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.

32

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 wellstructured 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,

33

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. 1 2 3 4 5 6 7 8 9 10 Plant species Albizia lebbeck (L.) Benth. Digera muricata L. Mangifera indica L. Musa paradisiaca L. Syzygium cumini (L.) Skeels Trianthema portulacastrum L. Tribulus terrestris L. Ziziphus mauritiana Lam. Plant family Fabaceae Amaranthaceae Anacardiaceae Musaceae Myrtaceae Aizoaceae Zygophyllaceae Rhamnaceae Part/s used Leaves Leaves Leaves Leaves Leaves Leaves English name Woman's tongue Bamboo Calabash Mango Banana Jambolan plum Vernacular name Shareen Bans Tandla Kaddoo Aam Kaila Jaman Bhakhrra Bairy

Bambusa arundinacea (Retz.) Willd. Poaceae Lagenaria siceraria (Molina) Standl. Cucurbitaceae

Whole plant False amaranth

Whole plant Desert horse-purslane It Sit Whole plant Puncturevine Leaves Ber, Indian Jujube

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

34

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. In vitro anthelmintic 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.

35

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 mL­1 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 mL­1) of plant extract (i.e., CAME). The positive control wells received different concentrations (0.09 to 3.0 µg mL­1) 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

36

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. In vivo anthelmintic activity 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 18­25 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

37

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 doseresponse 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

38

in vivo experiments were statistically analysed using SAS software (SAS, 1998). The results were expressed as mean±standard error of mean (SEM).

39

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. In vitro anthelmintic activity 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

40

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).

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

Sr. no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Plant family Aizoaceae Alliaceae Amaranthaceae Anacardiaceae Apiaceae Apiaceae Apiaceae Apiaceae Arecaceae Asteraceae Brassicaceae Brassicaceae Capparaceae Convolvulaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Cuscutaceae Euphorbiaceae Euphorbiaceae Fabaceae Plant speciesa (voucher specimen number) Trianthema portulacastrum L. (# 0110) Allium cepa L. (# 0111) Digera muricata L. (# 0112) Mangifera indica L. (# 0113) Coriandrum sativum L. (# 0114) Foeniculum vulgare Mill. (# 0115) Ferula assafoetida L. (# 0116) Cuminum cyminum L. (# 0117) Cocos nucifera L. (# 0118) Vernonia anthelmintica (L.) Willd. (# 0119) Brassica campestris L. (# 0120) Eruca sativa Miller (# 0121) Capparis decidua (Forssk.) Edgew. (# 0122) Convolvulus arvensis L. (# 0123) Cucumis melo L. var. flexuosus (L.) Naud. (# 0124) Lagenaria siceraria (Molina) Standl. (# 0125) Citrullus colocynthis (L.) Schrader (# 0126) Cuscuta reflexa Roxb. (# 0127) Ricinus communis L. (# 0128) Mallotus philippinensis (Lam.) Muell.-Arg. (# 0129) Cicer arietinum L. (# 0130) English name Desert horsepurslane Onion False amaranth Mango Coriander Fennel Stinking gum Cumin Coconut Ironweed Mustard Garden Rocket Caper Field bindweed Snake melon Calabash Bitter apple Giant dodder Castor bean Kamala tree Chick pea Piyaz Tandla Aam Dhania Sounf Hing Zeera Garee/Khopa Kali zeeri Saron Tarameera/Kusson Kari Laily Chibbarr Kaddoo Korr tumma Aakash bail Arind Kameela Chana 25 (7.55) 11 (3.32) 7 (2.11) 28 (8.45) 4 (1.2) 6 (1.81) 1 (0.3) 3 (0.9) 47 (14.19) 67 (20.24) 9 (2.71) 21 (6.34) 65 (19.63) 7 (2.11) 42 (12.68) 38 (11.48) 35 (10.57) 79 (23.86) 142 (42.9) 1 (0.3) Vernacular name It Sit Frequency (n=331), n (%) 29 (8.76)

42

Sr. no. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

a

Plant family Fabaceae Fabaceae Liliaceae Meliaceae Musaceae Myrtaceae Poaceae Poaceae Ranunculaceae Rhamnaceae Rosaceae Scrophulariaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Tamaricaceae Zingiberaceae Zygophyllaceae

Plant speciesa (voucher specimen number) Medicago sativa L. (# 0131) Albizia lebbeck (L.) Benth. (# 0132) Allium sativum L. (# 0133) Azadirachta indica A. Juss. (# 0134) Musa paradisiaca L. (# 0135) Syzygium cumini (L.) Skeels (# 0136) Bambusa arundinacea (Retz.) Willd. (# 0137) Triticum aestivum L. (# 0138) Helleborus niger L. (# 0139) Ziziphus mauritiana Lam. (# 0140) Prunus persica (L.) Batsch. (# 0141) Herpestis monniera L. (# 0142) Nicotiana tabacum L. (# 0143) Withania coagulans Dunal. (# 0144) Solanum xanthocarpum L. (# 0145) Capsicum annuum L. (# 0146) Solanum tuberosum L. (# 0147) Tamarix aphylla (L.) H.Karst. (# 0148) Zingiber officinale Roscoe (# 0149) Tribulus terrestris L. (# 0150)

English name Alfalfa Woman's tongue Garlic Neem Banana Jambolan plum Bamboo Wheat Christmas Rose Ber, Indian Jujube Peach Thyme leaved gratiola Tobacco Indian rennet Yellow-Berried Nightshade Chili Potato Tamarisk Ginger Puncturevine

Vernacular name Loosan Shareen Lassan Nim Kaila Jaman Bans Kanak Karroo Bairy Aarroo Jall booti Tamakoo Paneer doda Chamak namoly Mirch Aaloo Okan di maieen/Maieen Adrak Bhakhrra

Frequency (n=331), n (%) 3 (0.9) 2 (0.6) 10 (3.02) 30 (9.06) 13 (3.92) 2 (0.6) 20 (6.04) 30 (9.06) 26 (7.85) 9 (2.71) 14 (4.22) 3 (0.9) 87 (26.28) 26 (7.85) 2 (0.6) 12 (3.62) 1 (0.3) 16 (4.83) 10 (3.02) 13 (3.92)

Scientific names of plants are according to the flora of Pakistan (Nasir and Ali, 1970­1988; Ali and Nasir, 1989­1991; Ali and Qaiser, 1992­to date); voucher specimens of the plants are kept in the Herbarium, Department of Parasitology, University of Agriculture, Faisalabad 38040, Pakistan.

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

Sr. no. 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 Administer ½ kg jaggery per os, after half an hour administer ½ kg crushed bulb per os Grind 50 gm, 100 gm, 250 gm and 50 gm respectively, along with 25 gm sodium bicarbonate, mix them all and administer per os Grind the leaves with pestle and mortar and sieve with muslin 4 Azadirachta indica A. Juss. L cloth until ½ liter of extract is obtained, administer this extract per os 5 6 7 8 9 10 Azadirachta indica A. Juss. Bambusa arundinacea (Retz.) Willd. Brassica campestris L. Brassica campestris L. Brassica campestris L. Capparis decidua (Forssk.) Edgew. L L S S S Twigs Boil one kg leaves in 3 liters of water, when water remains 1 liter administer it per os Boil ½ kg leaves in 2 liters of water, when water remains 1 liter, administer it per os Mix ½ liter seed oil with ¼ kg curd and administer per os Mix ½ liter seed oil with ½ liter of luke warm water and administer per os Boil ½ liter of oil, mix with ½ kg jaggery and administer per os Crush the twigs well, mix sufficient quantity of jaggery in it to make the bolus and administer per os 5 (1.51) 20 (6.04) 50 (15.1) 8 (2.41) 4 (1.2) 9 (2.71) 25 (7.55) Name of plants/remediesb Parts used Dosage/administration Respondents (n=331), n (%)

1 2

Albizia lebbeck (L.) Benth. Allium cepa L. Allium sativum L.+Allium cepa

L Bulb Bulb+Bulb+WF (Green, raw fruit)+Rhizomes

2 (0.6) 15 (4.53)

3

L.+Capsicum annum L.+Zingiber officinale Roscoe

10 (3.02)

44

Sr. no.

Name of plants/remediesb

Parts used

Dosage/administration

Respondents (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) 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

12 (3.62)

12

Capsicum annuum L.

WF

1 (0.3)

Capsicum annum L.+Cicer arietinum 13 L.+Cuminum cyminum L.+Coriandrum sativum L.+Solanum tuberosum L. (Pakoray+Chilies) 14 15 16 17 18 19 20 Citrullus colocynthis (L.) Schrader Citrullus colocynthis (L.) Schrader+Veronica anthelmintica L. Willd. Cocos nucifera L. Convolvulus arvensis L. Convolvulus arvensis L. Coriandrum sativum L. Cucumis melo var. Flexuosus (L.) naud. F Aerial parts Aerial parts S WF WF +S WF 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 Grind and give per os for 4 days Grind 50 gm of both parts and administer per os Grind 125 gm of fruit and administer per os Crush aerial parts, sieve with muslin cloth to give ½ to 1 liter of extract and administer per os 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 Grind 50 gm seeds along with jaggery Q.S. to make bolus and administer per os Boil 1 kg of fruit in 2 liters of water for 1 to 2 hours then administer the decoction per os 17 (5.13) 21 (6.34) 3 (0.9) 33 (9.96) 32 (9.66) 27 (8.15) 7 (2.11) 1 (0.3)

45

Sr. no.

Name of plants/remediesb

Parts used

Dosage/administration

Respondents (n=331), n (%)

21 22 23 24 25

Cuscuta reflexa Roxb. Digera muricata L. Eruca sativa Miller Ferula assafoetida L. Foeniculum vulgare Mill.

WP WP S St and R S

Boil 1 kg the plant with 2 liters of water for 1 to 2 hours then administer the decoction per os Crush the plant and administer per os or animal is allowed to eat it ad libitum Administer the oil per os Grind 10 gm extracted gum (from stem and roots) along with jaggery (Q.S to make bolus) and administer per os Grind 100 gm of seeds along with ¼ kg of jaggery and administer per os Some verses from Holy Quran are recited and air from mouth blown on the animal or incantation done on a lump of doughed

35 (10.57) 11 (3.32) 9 (2.71) 6 (1.81) 4 (1.2)

26

Incantation

­

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 28

Herpestis monniera L. Lagenaria siceraria (Molina) Standl.

Aerial parts L

Crush the aerial parts and administer per os Crush leaves and administer per os or animal is allowed to eat the leaves ad libitum Mix the 10 gm fruit powder with ½ liters of milk and administer per os Mix the 4 drama fruit powder with ½ kg of curd and administer per os Mix the 4 dram fruit powder with ½ liter of milk whey and administer per os

3 (0.9) 42 (12.68)

29 30 31

Mallotus philippinensis (Lam.) Muell.-Arg. Mallotus philippinensis (Lam.) Muell.-Arg. Mallotus philippinensis (Lam.) Muell.-Arg.

F F F

56 (16.91) 41 (12.38) 17 (5.13)

46

Sr. no.

Name of plants/remediesb

Parts used

Dosage/administration

Respondents (n=331), n (%)

32 33 34

Mallotus philippinensis (Lam.) Muell.-Arg. Mallotus philippinensis (Lam.) Muell.-Arg. Mallotus philippinensis (Lam.) Muell.-Arg. Mallotus philippinensis (Lam.) Muell.-Arg.+Tamarix aphylla (L.) H.Karst.+ Brassica campestris L.

F F F

Mix the 4 dram fruit powder with ½ liter of mustard oil and administer per os Grind the 10 gm fruit powder jaggery (Q.S. to make the bolus) and administer per os Mix the 50 gm fruit powder with ½ liter of water and administer per os Mix ground fruit (10 gm each), ½ liter of mustard oil, ½ kg of curd and administer per os Crush ½ kg of leaves and administer per os or animal is allowed to eat ad libitum Crush ½ kg of leaves and administer per os or animal is allowed to eat ad libitum Crush leaves, sieve with muslin cloth to give ½ to 1 liter of extract and administer per os Administer ½ to 1 liter of decoction type of water left (as a by product) after smoking the Huqqa, per os Grind 100 gm each of fruit, seed, bark, mix with water of tobacco

12 (3.92) 3 (0.9) 8 (2.41)

35

F+F+S

5 (1.51)

36 37 38 39

Mangifera indica L. Medicago sativa L. Musa paradisiaca L. Nicotiana tabacum L. Nicotiana tabacum L.+Withania

L Aerial part L L

7 (2.11) 3 (0.9) 13 (3.92) 61 (18.42)

40

coagulans Dunal.+Veronica anthelmintica L. Willd.+Helleborus niger L.

L+WF+S+Bark

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)

47

Sr. no. 42 43 44 45 46 47 48 49

a b

Name of plants/remediesb

Parts used

Dosage/administration

Respondents (n=331), n (%)

Ricinus communis L. Ricinus communis L. Solanum xanthocarpum L. Syzygium cumini (L.) Skeels Tamarix aphylla (L.) H.Karst. Trianthema portulacastrum L. Tribulus terrestris L. Ziziphus mauritiana Lam.

S S WF L F WP WP L

Administer 125 ml of seed oil per os Administer 125 ml of seed oil per os in ½ liter luke warm milk Crush 250 gm fruit and administer per os along with jaggery (Q.S. to make bolus) Crush ½ kg leaves and administer per os or animal is allowed to eat ad libitum Grind 50 gm fruit and administer per os Crush ½ kg leaves and administer per os or animal is allowed to eat ad libitum Crush ½ kg leaves and administer per os or animal is allowed to eat ad libitum Crush ½ kg leaves and administer per os or animal is allowed to eat ad libitum

6 (1.81) 73 (22.04) 2 (0.6) 2 (0.6) 11 (3.32) 29 (8.76) 13 (3.92) 9 (2.71)

1 dram=1.771845 grams, F = Fruit, L = Leaves, R = Roots, S = Seeds, St = Stem, WF = Whole fruit (fruit plus seeds), WP = Whole plant Scientific names of plants are according to the flora of Pakistan (Nasir and Ali, 1970­1988; Ali and Nasir, 1989­1991; Ali and Qaiser, 1992­to date); voucher specimens of the plants are kept in the Herbarium, Department of Parasitology, University of Agriculture, Faisalabad 38040, Pakistan.

48

Table 12. In vitro effect of different indigenous plants on survival of Haemonchus contortus (Mean±SEM) of sheep in comparison with Levamisole (Lev) Treatments mg mL-1 Lev 0.5 mg PBS 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 0 hr 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 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 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±0.00a 10.00±0.00a 2 hr 0.00±0.0h 10.00±0.0a 1.00±0.6fg 2.33±0.7efg 2.67±0.3ef 4.67±1.5de 6.67±0.7cd 7.33±1.2bc 7.33±2.2bc 9.67±0.3ab 10.00±0.0a 10.00±0.0a 0.00±0.0h 0.00±0.0h 0.30±0.0gh 1.30±0.3g 2.70±0.3f 4.30±0.3e 5.30±0.3de 6.30±0.3cd 7.30±0.3bc 8.00±1.0b 0.00±0.0e 0.00±0.0e Mean number of dead worms at different hours 4 hr 6 hr 8 hr 10 hr 12 hr Fresh PBS for 30 min e b b b b 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b a a a a a 10.00±0.0 10.00±0.0 10.00±0.0 10.00±0.0 10.00±0.0 10.00±0.0a Musa paradisiaca L. 0.00±0.0e 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0e 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b e d c c b 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 0.00±0.0e 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b 2.00±0.6d 0.00±0.0d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b cd d c c b 3.00±1.0 0.67±0.7 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 3.33±0.9cd 1.00±0.6d 0.00±0.0c 0.00±0.0c 0.00±0.0b 0.00±0.0b bc c c c b 4.67±0.3 2.67±0.3 0.67±0.3 0.00±0.0 0.00±0.0 0.00±0.0b 6.00±0.6b 4.00±0.6bc 2.00±0.6b 0.33±0.3c 0.00±0.0b 0.00±0.0b b b b b b 6.33±1.2 4.33±1.2 2.33±1.2 1.00±0.6 0.00±0.0 0.00±0.0b Trianthema portulacastrum L. 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b e b b b b 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b e b b b b 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 0.00±0.0e 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b e b b b b 0.67±0.3 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 3.00±0.6d 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 4.30±0.3c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b c b b b b 5.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 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. 0.00±0.0f 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b f c b b b 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b

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Treatments mg mL-1 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 0 hr 10.00±0.00a 10.00±0.00a 10.00±0.00a 10.00±0.00a 10.00±0.00a 10.00±0.00a 10.00±0.00a 10.00±0.00a 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 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 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 2 hr 5.33±0.8d 7.00±1.0c 8.33±0.3bc 8.67±0.8ab 9.00±0.6ab 10.00±0.0a 10.00±0.0a 10.00±0.0a 6.67±0.7b 7.00±1.2b 7.33±0.9b 7.33±0.7b 9.67±0.3a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 1.33±0.9e 1.67±0.3e 7.00±0.6d 7.67±1.3cd 8.00±0.6bcd 8.33±0.3abcd 9.00±1.0abc 9.67±0.3ab 10.00±0.0a 10.00±0.0a

Mean number of dead worms at different hours 4 hr 6 hr 8 hr 10 hr 12 hr Fresh PBS for 30 min f c b b b 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 0.00±0.0f 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b e c b b b 2.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 3.67±0.3d 0.00±0.0c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b c c b b b 5.33±0.3 0.33±0.3 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b 7.67±0.3b 0.67±0.7c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b 8.00±0.6b 1.00±0.6c 0.00±0.0b 0.00±0.0b 0.00±0.0b 0.00±0.0b b b b b b 7.33±0.9 2.67±1.2 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0b Ziziphus mauritiana Lam. 2.00±1.5d 0.00±0.0e 0.00±0.0e 0.00±0.0e 0.00±0.0c 0.00±0.0c 5.33±0.3c 0.00±0.0e 0.00±0.0e 0.00±0.0e 0.00±0.0c 0.00±0.0c bc d e e c 6.00±0.6 3.33±0.9 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0c 7.33±0.7b 3.67±0.7d 0.00±0.0e 0.00±0.0e 0.00±0.0c 0.00±0.0c 9.67±0.3a 6.33±0.9c 1.67±0.7d 0.00±0.0e 0.00±0.0c 0.00±0.0c a b d e c 10.00±0.0 8.00±0.6 2.33±0.7 0.00±0.0 0.00±0.0 0.00±0.0c 10.00±0.0a 9.67±0.3a 5.67±1.5c 1.00±0.0d 0.00±0.0c 0.00±0.0c a a c c c 10.00±0.0 9.67±0.3 6.00±0.6 2.67±0.7 0.33±0.3 0.33±0.3c 10.00±0.0a 10.00±0.0a 8.00±0.6b 8.00±0.6b 1.33±0.3b 1.33±0.3b a a a b b 10.00±0.0 10.00±0.0 9.67±0.3 8.33±0.7 1.33±0.3 1.33±0.3b Albizia lebbeck (L.) Benth. 0.00±0.0e 0.00±0.0g 0.00±0.0d 0.00±0.0f 0.00±0.0d 0.00±0.0d e g d f d 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0d 5.00±0.6d 4.33±0.7f 1.00±1.0d 0.00±0.0f 0.00±0.0d 0.00±0.0d c e d f d 6.33±0.9 5.67±0.3 1.33±0.3 0.00±0.0 0.00±0.0 0.00±0.0d 8.00±0.6b 7.00±0.6d 3.67±0.3c 0.00±0.0f 0.00±0.0d 0.00±0.0d 8.33±0.3b 8.00±0.6cd 4.00±1.0c 0.33±0.3ef 0.00±0.0d 0.00±0.0d ab bc c de cd 9.00±1.0 8.67±0.9 4.33±0.9 1.67±0.7 1.00±0.6 1.00±0.6cd 9.67±0.3a 9.67±0.3ab 4.67±0.9c 2.67±0.7cd 1.67±0.3c 1.67±0.3c a a c c c 10.00±0.0 10.00±0.0 5.00±0.0 3.33±0.9 1.67±0.9 1.67±0.9c 10.00±0.0a 10.00±0.0a 7.33±0.3b 5.67±0.9b 3.33±1.2b 3.33±1.2b

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Treatments mg mL-1 0 hr 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 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 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 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 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 2 hr 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 10.00±0.0a 10.00±0.0a 8.33±1.2b 9.00±0.6ab 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 9.00±0.6b 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a 10.00±0.0a

Mean number of dead worms at different hours 4 hr 6 hr 8 hr 10 hr 12 hr Fresh PBS for 30 min Digera muricata L. 10.00±0.0a 8.00±1.2b 3.33±0.9c 0.00±0.0e 0.00±0.0f 0.00±0.0f a a b e f 10.00±0.0 10.00±0.0 6.33±0.9 0.00±0.0 0.00±0.0 0.00±0.0f 10.00±0.0a 10.00±0.0a 6.67±1.5b 0.00±0.0e 0.00±0.0f 0.00±0.0f a a a d f 10.00±0.0 10.00±0.0 9.00±0.0 3.00±0.6 0.00±0.0 0.00±0.0f 10.00±0.0a 10.00±0.0a 10.00±0.0a 3.33±0.9d 0.00±0.0f 0.00±0.0f 10.00±0.0a 10.00±0.0a 10.00±0.0a 6.67±0.3c 0.00±0.0f 0.00±0.0f a a a b e 10.00±0.0 10.00±0.0 10.00±0.0 8.00±0.6 4.00±0.6 4.00±0.6e 10.00±0.0a 10.00±0.0a 10.00±0.0a 9.00±0.6ab 6.33±0.3d 6.33±0.3d a a a a c 10.00±0.0 10.00±0.0 10.00±0.0 9.67±0.3 7.33±0.7 7.33±0.7c 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. 1.33±0.9b 0.00±0.0e 0.00±0.0g 0.00±0.0f 0.00±0.0e 0.00±0.0e 1.67±0.3b 0.00±0.0e 0.00±0.0g 0.00±0.0f 0.00±0.0e 0.00±0.0e a d f ef e 8.33±1.2 6.00±0.6 2.67±0.3 1.33±0.9 0.00±0.0 0.00±0.0e 9.00±0.0a 7.33±1.2cd 3.00±0.0f 1.67±0.3de 0.00±0.0e 0.00±0.0e a cd e cd e 9.00±0.0 7.33±0.9 4.33±0.3 3.00±0.6 0.00±0.0 0.00±0.0e 9.00±0.0a 7.67±0.3bcd 4.67±0.3e 3.67±0.9c 1.67±0.3d 1.67±0.3d a abc d c d 9.00±1.0 8.33±0.7 5.33±0.3 3.67±0.7 2.33±0.3 2.33±0.3d 9.33±0.3a 8.67±0.3abc 6.33±0.3c 4.00±0.0c 3.33±0.3c 3.33±0.3c 9.33±0.3a 8.67±0.9abc 6.67±0.3bc 4.33±0.9c 3.67±0.3c 3.67±0.3c a ab b b b 9.67±0.3 9.33±0.3 7.00±0.0 6.67±0.3 4.67±0.8 4.67±0.8b Syzygium cumini (L.) Skeels 0.00±0.0b 0.00±0.0d 0.00±0.0e 0.00±0.0d 0.00±0.0f 0.00±0.0f 1.33±1.3b 0.00±0.0d 0.00±0.0e 0.00±0.0d 0.00±0.0f 0.00±0.0f 9.00±1.0a 6.00±0.6c 2.33±1.5d 0.00±0.0d 0.00±0.0f 0.00±0.0f a b c d f 9.67±0.3 8.33±0.7 6.33±0.3 2.00±2.0 0.00±0.0 0.00±0.0f 10.00±0.0a 9.33±0.3a 8.00±0.0b 5.33±0.7c 0.00±0.0f 0.00±0.0f a aa ab bc ef 10.00±0.0 9.33±0.3 9.33±0.3 7.33±0.7 0.67±0.3 0.67±0.3ef 10.00±0.0a 9.33±0.3a 9.33±0.3ab 7.33±0.9bc 1.00±0.0de 1.00±0.0de

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Treatments mg mL-1 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 100 mg 50 mg 25 mg 12.5 mg 6.25 mg 3.12 mg 1.56 mg 0.78 mg 0.39 mg 0.19 mg 0 hr 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 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 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 2 hr 10.00±0.0a 10.00±0.0a 10.00±0.0a 0.00±0.0b 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 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 10.00±0.0a 10.00±0.0a 10.00±0.0a

Mean number of dead worms at different hours 4 hr 6 hr 8 hr 10 hr 12 hr Fresh PBS for 30 min a a a ab d 10.00±0.0 10.00±0.0 9.67±0.3 9.33±0.3 1.67±0.3 1.67±0.3d 10.00±0.0a 10.00±0.0a 9.67±0.3a 8.67±0.9ab 2.67±0.3c 2.67±0.3c a a a ab b 10.00±0.0 10.00±0.0 10.00±0.0 9.33±0.3 3.67±0.9 3.67±0.9b Lagenaria siceraria (Molina) Standl. 0.00±0.0c 0.00±0.0d 0.00±0.0f 0.00±0.0d 0.00±0.0f 0.00±0.0f 7.67±1.2b 2.67±1.2c 0.00±0.0f 0.00±0.0d 0.00±0.0f 0.00±0.0f 9.67±0.3a 7.33±1.5b 5.67±0.3e 0.00±0.0d 0.00±0.0f 0.00±0.0f a b de c e 10.00±0.0 7.33±0.9 6.33±0.3 4.67±0.3 1.67±0.7 1.67±0.7e 10.00±0.0a 7.33±0.9b 7.33±0.9cd 7.00±1.2b 2.67±0.3de 2.67±0.3de a ab bc ab cd 10.00±0.0 8.67±0.9 8.33±0.7 8.33±1.2 4.00±0.0 4.00±0.0cd 10.00±0.0a 8.67±0.7ab 8.67±0.7b 8.33±0.9ab 5.33±0.3c 5.33±0.3c a ab ab ab b 10.00±0.0 9.00±0.6 9.00±0.6 8.33±0.9 7.33±0.3 7.33±0.3b 10.00±0.0a 9.33±0.3a 9.00±0.6ab 8.33±0.9ab 7.67±1.5b 7.67±1.5b 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. 10.00±0.0a 8.67±0.3b 6.33±0.3c 0.00±0.0f 0.00±0.0e 0.00±0.0e a a b e e 10.00±0.0 10.00±0.0 9.00±0.6 2.00±0.6 0.00±0.0 0.00±0.0e 10.00±0.0a 10.00±0.0a 10.00±0.0a 3.67±0.9d 0.00±0.0e 0.00±0.0e a a a c e 10.00±0.0 10.00±0.0 10.00±0.0 6.33±1.3 1.00±1.0 1.00±1.0e 10.00±0.0a 10.00±0.0a 10.00±0.0a 7.33±0.9bc 4.00±1.0d 4.00±1.0d 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.9ab 5.33±1.8cd 5.33±1.8cd a a a ab bc 10.00±0.0 10.00±0.0 10.00±0.0 8.67±0.7 6.67±0.9 6.67±0.9bc 10.00±0.0a 10.00±0.0a 10.00±0.0a 8.67±0.3ab 8.00±0.6ab 8.00±0.6ab a a a ab ab 10.00±0.0 10.00±0.0 10.00±0.0 8.67±0.9 8.33±0.9 8.33±0.9ab 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)

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Table 13. Ranking of 10 plants according to their effects on adult Haemonchus contortus Sr. no. Plant species 1 2 3 4 5 6 7 8 9 10 Trianthema portulacastrum L. Tribulus terrestris L. Lagenaria siceraria (Molina) Standl. Musa paradisiaca L. Albizia lebbeck (L.) Benth. Ziziphus mauritiana Lam. Bambusa arundinacea (Retz.) Willd. Syzygium cumini (L.) Skeels Digera muricata L. Mangifera indica L. Order of ranking 01 01 01 02 03 04 05 05 06 06 Part/s used Whole plant Whole plant Leaves Leaves Leaves Leaves Leaves Leaves Whole plant Leaves English name Desert horsepurslane Puncturevine Calabash Banana Woman's tongue Ber, Indian Jujube Bamboo Vernacular name It Sit Bhakhrra Kaddoo Kaila Shareen Bairy Bans

Jambolan plum Jaman False amaranth Mango Tandla 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.

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Table 14. Per cent egg hatch and LC50 of different plants CAME concentrations (µg mL-1) Plant Musa paradisiaca L. Trianthema portulacastrum L. Lagenaria siceraria (Molina) Standl. Albizia lebbeck (L.) Benth. Tribulus terrestris L. Syzygium cumini (L.) Skeels Mangifera indica L. Ziziphus mauritiana Lam. Bambusa arundinacea (Retz.) Willd. Digera muricata L. 250 500 1000 2000 4000 36.00 32.00 27.00 22.50 19.00 33.00 20.00 10.00 2.00 0.50 51.00 28.00 10.00 4.00 1.33 58.00 66.00 97.00 80.00 93.30 95.00 44.00 55.00 95.00 79.00 91.00 92.00 39.50 37.00 93.00 76.00 85.10 88.80 35.50 25.00 84.00 73.00 79.00 83.00 20.00 10.00 73.00 66.60 75.00 80.00 8000 4.00 0.00 0.00 0.00 2.50 69.00 56.00 72.20 75.00 LC50 (µg mL-1) 2.13 2.41 2.53 2.75 2.75 4.34 4.48 4.69 4.89 5.36

97.00 92.00 91.00 89.00 84.00 82.00

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.

54

Table 15. Regression values and correlation of regression of the effect of different plants on egg hatching Plant Musa paradisiaca L. Trianthema portulacastrum L. Lagenaria siceraria (Molina) Standl. Albizia lebbeck (L.) Benth. Tribulus terrestris L. Syzygium cumini (L.) Skeels Mangifera indica L. Ziziphus mauritiana Lam. Bambusa arundinacea (Retz.) Willd. Digera muricata L. Oxfendazole LC50 2.13 2.41 2.53 2.75 2.75 4.34 4.48 4.69 4.89 5.36 1.88 Regression values and correlation of regression y = -0.0002x + 4.6324, R2 = 0.9689 y = -0.0006x + 4.4134, R2 = 0.9793 y = -0.0006x + 4.7245, R2 = 0.9596 y = -0.0002x + 4.6324, R2 = 0.9689 y = -0.0003x + 5.1332, R2 = 0.9136 y = -0.0001x + 6.4026, R2 = 0.7454 y = -0.0002x + 4.6324, R2 = 0.9689 y = -0.0001x + 6.2603, R2 = 0.6803 y = -0.0001x + 6.4026, R2 = 0.7454 y = -9E­05x + 6.5395, R2 = 0.6446 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 mL1

), 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

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(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 Ranking of potency based on LC50 01 02 03 04 04 05 06 07 08 09 Ranking of potency based on dose dependant effect (R2 values) 02 01 03 02 04 05 02 06 05 07

Plant Musa paradisiaca L. Trianthema portulacastrum L. Lagenaria siceraria (Molina) Standl. Albizia lebbeck (L.) Benth. Tribulus terrestris L. Syzygium cumini (L.) Skeels Mangifera indica L. Ziziphus mauritiana Lam. Bambusa arundinacea (Retz.) Willd. Digera muricata L.

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)

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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 Ranking of potency Ranking of potency based on Ranking of potency based based on dose adult motility assay (% dependant effect (R2 motility of worms after 2nd on LC50 values) hour of treatment) 01 02 03 04 04 05 06 07 08 09 02 01 03 02 04 05 02 06 05 07 02 01 01 03 01 05 06 04 05 06

Plant Musa paradisiaca L. Trianthema portulacastrum L. Lagenaria siceraria (Molina) Standl. Albizia lebbeck (L.) Benth. Tribulus terrestris L. Syzygium cumini (L.) Skeels Mangifera indica L. Ziziphus mauritiana Lam. Bambusa arundinacea (Retz.) Willd. Digera muricata L.

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4.3. In vivo anthelmintic activity 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).

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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 0 3 6 9 12 15 1450.00±20.4a (0%) 1440.00±5.8a (1%) 1430.00±11.5a (1%) 1420.00±11.5a (2%) 1410.00±5.8a (3%) 1400.00±20.4a (3%) 1450.00±28.9a (0%) 1445.00±26.0a (1%) 1435.00±20.2a (1%) 1422.00±12.7a (2%) 1400.00±0.0a (3%) 1392.00±4.6a (4.%) 1562.50±7.2a (0%) 1545.00±2.9a (1%) 1425.00±32.3a (0%) 0.00±0.0d (100%) 0.00±0.0d (100%) 0.00±0.0f (100%) 0.00±0.0e (100%) 0.00±0.0e (100%) 1425.00±14.4a (0%) 0.00±0.0d (100%) 0.00±0.0f (100.%) 0.00±0.0e (100.%) 0.00±0.0f (100.%) 0.00±0.0f (100.%) 1548.50±0.9a (0%) 0.00±0.0e (100%) CP 1 g kg-1 CP 4 g kg-1 CP 8 g kg-1 Trianthema partulacastrum L. 1475.00±14.4a 1462.50±71.8a 1450.00±28.9a (0%) (0%) (0%) 1425.50±42.8a 1387.50±12.5ab 1300.00±20.4bc (3%) (5%) (10%) 1363.50±65.4ab 1312.50±23.9abc 1225.00±43.3c (8%) (10) (16%) 1326.50±58.5a 925.00±59.5c 850.00±50.0cd (10%) (37%) (41%) 1239.50±44.5b 800.00±67.7c 637.50±42.7d (16%) (45%) (56%) b c 1190.00±76.5 712.50±65.7 550.00±35.4d (19%) (51%) (62%) Lagenaria siceraria (Molina) Standl. 1413.00±12.5a 1425.00±14.4a 1450.00±14.4a (0%) (0%) (0%) 1384.00±11.8b 1350.00±0.0b 1296.00±2.3c (2%) (5%) (11%) cd d 1295.00±5.0 1275.00±14.4 1215.00±8.7e (8%) (11%) (16%) 1192.00±7.7b 950.00±20.4c 870.00±17.3d (16%) (33%) (40%) b d 1075.00±9.6 930.00±17.3 812.00±6.9e (24%) (35%) (44%) 1015.00±10.0c 895.00±2.9d 773.50±11.0e (28%) (37%) (47) Tribulus terrestris L. 1552.00±4.6a 1537.50±7.2a 1550.00±14.4a (0%) (0%) (0%) 1499.50±0.3b 1437.00±7.5c 1379.00±12.1d (3%) (7%) (11%) CAME 1 g kg-1 CAME 4 g kg-1 CAME 8 g kg-1 1475.00±14.4a (0%) 1390.00±29.2ab (6%) 1300.00±20.4bc (12%) 1200.00±20.4b (19%) 1160.50±58.1b (21%) 1114.95±42.5b (24%) 1438.00±12.5a (0%) 1370.00±17.0b (5%) 1325.00±14.4bc (8%) 1179.00±12.1b (18%) 1100.00±20.4b (24) 1056.00±15.1b (27%) 1450.00±28.9a (0%) 1312.50±23.9bc (10%) 1220.50±51.5c (16%) 800.00±20.4d (45%) 600.00±20.4d (59%) 491.55±32.0d (66%) 1475.00±9.4a (0%) 1275.50±59.6c (13.5%) 1200.00±57.7c (18.6%) 550.00±20.4e (63%) 500.00±42.7e (66%) 439.85±28.9e (70%)

0 3 6 9 12 15

1438.00±23.9a 1450.00±35.4a (0%) (0%) 1368.00±19.7b 1368.00±10.7b (5%) (6%) b 1344.00±25.9 1350.00±20.4b (7%) (7%) 990.00±10.0c 900.00±20.4d (31%) (38%) c 987.00±7.2 842.00±21.7e (31%) (42%) 923.40±13.5d 783.60±16.6e (36%) (46%)

0 3

1587.00±7.5a 1537.50±23.9a 1550.00±14.4a (0%) (0%) (0%) 1525.00±14.4ab 1462.50±23.9c 1350.00±14.4d (4%) (9%) (13%)

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Days PT Untreated control Levamisole 7.5 mg kg-1 0.00±0.0e 1533.50±3.8a 6 (2%) (100%) 0.00±0.0f 1522.50±4.3a 9 (2.6%) (100%) a 0.00±0.0f 1515.50±2.6 12 (3%) (100%) 0.00±0.0g 1500.00±0.0a 15 (4.%) (100%) 0 3 6 9 12 15 1287.00±7.5a (0%) 1280.00±11.5a (1%) 1270.00±17.3ab (1%) 1260.00±11.5a (1%) 1250.00±5.8a (2%) 1236.00±2.6a (4%) 1575.00±14.4a (0%) 1570.00±5.8a (0%) 1566.00±2.6a (1%) 1550.00±0.0a (2%) 1526.00±2.6a (3%) 1512.00±4.6a (4.%) 1313.00±7.2a (0%) 0.00±0.0c (100%) 0.00±0.0f (100%) 0.00±0.0g (100%) 0.00±0.0f (100%) 0.00±0.0f (100%) 1550.00±14.4a (0%) 0.00±0.0d (100%) 0.00±0.0d (100%) 0.00±0.0f (100%) 0.00±0.0g (100%) 0.00±0.0g (100%)

0 3 6 9 12 15

CP 1 g kg-1 CP 4 g kg-1 CP 8 g kg-1 ab c 1460.00±4.1 1350.50±14.2 1200.00±64.5d (6%) (12%) (19%) 1381.00±11.0b 1250.00±14.4c 1012.50±31.5de (11%) (19%) (29%) b c 1289.00±6.4 1178.00±12.7 900.00±20.4e (17%) (23%) (39%) 1236.40±7.9b 924.00±3.5e 750.00±10.2f (20%) (40%) (46%) Ziziphus mauritiana Lam. 1300.00±0.0a 1287.00±7.5a 1325.00±52.0a (0%) (0%) (0%) 1287.00±7.8a 1256.00±2.6a 1263.00±42.7a (1%) (2%) (5%) 1274.00±3.8a 1193.00±4.0cd 1163.00±47.3d (2%) (7%) (12%) 1169.00±0.9b 1067.00±1.7d 1038.00±23.9e (10%) (17%) (22%) b d 1116.00±2.3 1005.00±2.9 987.50±23.9d (14%) (22%) (26%) 1090.00±5.8b 825.90±2.4d 900.00±54.0d (16%) (28%) (32%) Bambusa arundinacea (Retz.) Willd. 1563.00±16.1a 1563.00±7.2a 1600.00±20.4a (0%) (0%) (0%) ab b 1514.00±6.8 1447.00±16.4 1332.00±105.0c (7%) (3%) (17%) 1453.00±35.5b 1253.00±18.4c 1270.00±99.5c (20%) (7%) (21%) bc de 1270.00±12.1 1072.00±44.1 1014.00±102.0e (19%) (31%) (37%) 1178.00±44.8bc 1046.00±26.7de 916.00±88.5f (43%) (25%) (33%) 1160.00±44.5bc 981.50±11.8de 842.00±99.7f (37%) (47%) (26%)

CAME 1 g kg-1 CAME 4 g kg-1 CAME 8 g kg-1 1475.00±52.0ab 1437.50±23.9bc 1212.50±4.3d (7%) (14%) (22%) 1312.50±37.5bc 1050.00±67.7d 937.50±1.4e (17%) (29%) (40%) bc d 1225.00±32.3 1025.00±59.5 850.00±14.4e (23%) (33%) (45%) 1165.38±11.8c 987.50±42.7d 750.00±14.4f (27%) (36%) (52 %) 1330.00±17.3a (0%) 1256.00±29.1a (6%) 1219.00±0.6bc (8%) 1096.00±2.3c (18%) 1047.00±2.0c (21%) 1022.00±4.6c (23%) 1550.00±10.2a (0%) 1498.00±7.5ab (3%) 1420.00±10.7b (8%) 1329.00±8.0b (14%) 1251.00±10.0b (19%) 1237.00±13.6b (20%) 1300.00±14.4a (0%) 1229.00±0.6ab (6%) 1172.00±4.9cd (10%) 1072.00±4.9cd (18%) 1000.00±0.0d (23%) 942.80±4.2d (28%) 1550.00±17.7a (0%) 1452.00±10.2b (6%) 1355.00±7.0bc (13%) 1159.00±5.9cd (25%) 1131.00±6.4cd (27%) 1061.00±16.5cd (32%) 1297.00±2.0a (0%) 1193.00±4.3b (8%) 1099.00±0.6e (15%) 953.00±4.0f (27%) 902.00±1.2e (30%) 840.10±5.7e (35%) 1575.00±22.8a (0%) 1419.00±4.9bc (10%) 1354.00±7.1bc (14%) 1080.00±12.2de (31%) 975.00±10.2ef (38%) 898.00±8.7ef (43%)

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Days PT Untreated control Levamisole 7.5 mg kg-1 0 3 6 9 12 15 1450.00±14.4a (0%) 1445.00±2.9a (0%) 1435.00±2.9a (1%) 1422.00±4.6a (2%) 1400.00±14.4a (3%) 1392.00±4.6a (4%) 1250.00±14.4a (0%) 1240.00±5.8a (1%) 1230.00±5.8a (2%) 1220.00±5.8a (2%) 1210.00±5.8a (3%) 1200.00±0.0a (4%) 1350.00±14.4a (0%) 1340.00±5.8a (1%) 1325.00±14.4a (2%) 1425.00±14.4a (0%) 0.00±0.0h (100%) 0.00±0.0e (100%) 0.00±0.0f (100%) 0.00±0.0f (100%) 0.00±0.0f (100%) 1225.00±14.4a (0%) 0.00±0.0b (100%) 0.00±0.0d (100%) 0.00±0.0e (100%) 0.00±0.0e (100%) 0.00±0.0d (100%) 1350.00±14.4a (0%) 0.00±0.0c (100%) 0.00±0.0c (100%)

0 3 6 9 12 15

0 3 6

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.) 1450.00±14.4a 1400.00±0.0a 1438.00±7.2a 1450.00±14.4a 1438.00±7.2a 1413.00±7.2a (0%) (0%) (0%) (0%) (0%) (0%) b e d c f 1419.00±0.6 1344.00±3.8 1356.00±3.5 1402.00±0.9 1292.00±0.9 1238.00±7.2g (2%) (4%) (6%) (3%) (10%) (12%) 1342.00±4.6ab 1288.00±7.2bc 1302.00±1.2bc 1328.00±1.2bc 1180.00±0.3d 1238.00±94.4cd (8%) (9%) (8%) (18%) (12%) (7%) b bc cd cd d 1172.00±1.2 1102.00±0.9 1058.00±4.3 1045.00±2.6 995.00±2.9 875.00±92.4e (19%) (21%) (26%) (21%) (34%) (38%) 1157.00±2.0b 989.50±6.1c 977.00±4.0cd 1060.00±5.8bc 876.00±3.5de 812.00±96.6e (20%) (29%) (32%) (26%) (39%) (42%) 1111.00±0.4b 952.00±1.2cd 895.50±2.6d 1024.00±2.1bc 842.30±1.3d 618.60±121.0e (23%) (32%) (38%) (29%) (41%) (56%) Musa paradisiaca L. 1250.00±14.4a 1237.50±80.0a 1212.50±4.3a 1237.50±62.5a 1250.00±14.4a 1225.00±14.4a (0%) (0%) (0%) (0%) (0%) (0%) 1223.50±2.0a 1225.00±59.5a 1154.00±2.3a 1175.00±52.0a 1187.50±65.7a 1147.50±1.4a (2%) (1%) (5%) (6%) (5%) (6%) 1210.50±5.5ab 1187.50±65.7abc 1096.00±2.3c 1137.50±62.5abc 1131.00±0.6bc 1092.50±4.3c (10%) (10%) (11%) (3%) (4%) (8%) b bc d bc c 1090.50±5.5 1037.50±65.7 684.50±3.2d 723.00±4.0 1025.00±47.9 988.00±1.2 (13%) (40%) (21%) (44%) (16%) (17%) 1050.50±5.5b 1000.00±70.7bc 676.00±2.3d 987.50±42.7bc 952.50±1.4c 640.00±5.8d (16%) (44%) (20%) (24%) (48%) (19%) b b c b b 997.50±1.4 975.15±75.0 629.20±0.5 937.50±42.7 928.70±0.8 595.30±2.7c (20%) (21%) (48%) (24%) (26%) (51%) Mangifera indica L. 1325.00±14.4a 1337.50±136.0a 1300.00±0.0a 1350.00±17.3a 1312.50±7.2a 1330.00±11.5a (0%) (0%) (0%) (0%) (0%) (0%) 1252.00±1.2ab 1262.50±143.0ab 1177.00±1.7b 1275.50±14.7ab 1239.00±9.0ab 1204.00±2.3ab (10%) (6%) (10%) (6%) (6%) (6%) 1200.00±5.8ab 1187.50±138.0ab 1095.00±118.0b 1222.50±4.3ab 1165.50±9.0ab 1092.00±4.6b (9%) (11%) (18%) (9%) (11%) (16%)

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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 a d b bc 0.00±0.0 1130.00±5.8 1075.00±120.0 944.50±93.7c 1312.50±7.2 9 (3%) (100%) (15%) (27%) (20%) 0.00±0.0d 1128.00±1.2b 1050.00±117.0b 889.50±77.5c 1300.00±14.4a 12 (3.7%) (100%) (15%) (32%) (22%) a d b b 0.00±0.0 1064.25±2.5 1012.60±107.0 875.80±65.8c 1296.00±2.3 15 (4%) (100%) (20%) (33%) (24%) Albizia lebbeck (L.) Benth. 1550.00±14.4ª 1575.00±14.4ª 1600.00±54.0a 1562.50±7.2ª 1550.00±73.6ª 0 (0%) (0%) (0%) (0%) (0%) 1525.00±52.0ab 1486.00±8.1ab 1462.50±42.7b 1540.00±5.8ª 0.00±0.0c 3 (100%) (5%) (1%) (5%) (6%) 1475.00±52.0ab 1460.50±6.1bc 1450.00±35.4bc 1525.00±14.4ª 0.00±0.0e 6 (100%) (7%) (8%) (7%) (2%) 1312.50±37.5b 1067.00±9.8d 1025.00±32.3d 1510.00±5.8ª 0.00±0.0f 9 (100%) (32%) (18%) (34%) (3%) 0.00±0.0f 1225.00±32.3b 1042.00±4.6d 1012.50±31.5d 1500.00±0.0a 12 (3%) (100%) (33%) (23%) (35%) g b d 1176.00±15.0 1004.70±2.7 945.50±2.6e 1488.00±6.9ª 0.00±0.0 15 (100%) (36%) (39%) (27%) (4%) Digera muricata L. 962.50±7.2ª 950.00±28.9ª 1000.00±40.8ª 975.00±14.4ª 987.50±42.7ª 0 (0%) (0%) (0%) (0%) (0%) 0.00±0.0c 964.00±3.5a 937.50±31.5ab 937.50±31.5ab 950.00±14.4ab 3 (1%) (100%) (4%) (4%) (5%) c ab 940.00±5.8ª 912.50±42.7 912.50±37.5ab 940.00±5.8ª 0.00±0.0 6 (100%) (6%) (6%) (8%) (2%) 866.00±29.3ab 812.50±55.4b 800.00±54.0b 935.00±2.9ª 0.00±0.0d 9 (100%) (17%) (19%) (3%) (13%) e b bc 842.00±4.6 787.50±47.3 775.00±47.9bc 930.00±5.8ª 0.00±0.0 12 (100%) (16%) (19%) (22%) (3%) 829.30±12.0b 762.50±55.4bc 737.50±55.4c 924.00±2.3ª 0.00±0.0e 15 (100%) (17%) (22%) (25%) (4%)

CAME 1 g kg-1 CAME 4 g kg-1 CAME 8 g kg-1 1137.50±12.5b 1055.00±2.9bc 938.00±6.9c (20%) (30%) (16%) 1095.00±2.9b 1030.50±11.3b 868.00±18.5c (19%) (35%) (22%) b bc 1063.13±3.1 969.15±11.1 854.15±2.4c (21%) (26%) (36%) 1550.00±20.4ª 1562.50±7.2ª 1587.50±7.2a (0%) (0%) (0%) 1506.50±3.8ab 1485.50±8.4ab 1471.00±16.7ab (3%) (5%) (7%) 1419.50±11.3c 1362.00±6.9d 1310.50±6.1d (13%) (17%) (8%) 1204.50±2.6c 1160.00±5.8c 936.00±12.1e (22%) (26%) (41%) 1144.50±25.7c 1104.50±2.6c 869.00±12.0e (29%) (45%) (26%) b c 1144.30±25.6 1101.50±0.9 842.15±7.9f (30%) (47%) (26%) 1000.00±40.8ª (0%) 954.00±2.3ab (5%) 922.00±16.2ab (8%) 821.00±16.7b (18%) 766.00±9.2c (23%) 734.40±9.0c (27%) 987.50±7.2ª (0%) 939.50±6.1ab (5%) 907.50±4.3ab (8%) 674.00±15.0c (32%) 658.50±4.9d (33%) 634.28±9.1d (36%) 962.50±7.2ª (0%) 908.00±4.6b (6%) 861.50±6.6b (11%) 636.50±7.8c (34%) 629.00±12.1d (35%) 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

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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 Trianthema portulacastrum Legnaria sisrarria Tribulus tresstris Musa paradisiaca Albezia lebbeck Syzygium cumini Bambusa arrundinacea Digra muricata Mangifera indica Ziziphus mauritiana FECR with CP @ 8 g gk-1 body weight 62 38 46 48 39 47 47 25 33 33 FECR with CAME @ 8 g gk-1 body weight 70 56 52 51 47 46 43 40 36 35

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1550 1350 Eggs per gram (EPG) of faeces 1150 950 51 66 70 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 4 19 24 Day 0 EPG Day 15 EPG 750 550

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Control

Crude Powder

Crude Aqueous Methanolic Extract

Doses and forms of Trianthema portulacastrum L. used

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)

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1550 1350 Eggs per gram (EPG) of faeces 1150 950 4 Day 0 EPG 28 37 27 36 Day 15 EPG

750 550 350 150 -50 Untreated Treated 100 1g 4g 8g

47

46

1g

4g

8g

Control

Crude Powder

Crude Aqueous Methanolic Extract

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

Fig. 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.

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1750 1550 1350 Eggs per gram (EPG) of faeces 1150 36 950 750 550 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 40 46 52

4 20 27 Day 0 EPG Day 15 EPG

Control

Crude Powder

Crude Aqueous Methanolic Extract

Doses and forms of Tribulus terrestris L. used

Fig. 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.

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1750 1550 1350 Eggs per gram (EPG) of faeces 1150 950 20 750 550 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 21 48 24 26 4

Day 0 EPG Day 15 EPG

51

Control

Crude Powder

Crude Aqueous Methanolic Extract

Doses and forms of Musa paradisiaca L. used

Fig. 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.

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1750 1550 4 1350 Eggs per gram (EPG) of faeces Day 0 EPG 1150 950 750 550 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 27 36 39 47 26 30 Day 15 EPG

Control

Crude Powder

Crude Aqueous Methanolic Extract

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

Fig. 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.

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1750 1550 1350 Eggs per gram (EPG) of faeces 1150 Day 0 EPG 950 750 550 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 23 29 32 38 41 56 Day 15 EPG 4

Control

Crude Powder

Crude Aqueous Methanolic Extract

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

Fig. 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.

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1750 1550 1350 Eggs per gram (EPG) of faeces 20 1150 950 750 550 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 26 37 47 Day 0 EPG Day 15 EPG

4

32

43

Control

Crude Powder

Crude Aqueous Methanolic Extract

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

Fig. 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.

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1150

950 4 Eggs per gram (EPG) of faeces Day 0 EPG 17 22 550 36 49 25 27 Day 15 EPG

750

350

150

100 -50 Untreated Treated 1g 4g 8g 1g 4g 8g

Control

Crude Powder

Crude Aqueous Methanolic Extract

Doses and forms of Digera muricata L. used

Fig. 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.

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1750 1550 1350 Eggs per gram (EPG) of faeces 1150 950 750 550 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 21 24 26 33 36

4 Day 0 EPG Day 15 EPG 20

Control

Crude Powder

Crude Aqueous Methanolic Extract

Doses and forms of Mangifera indica L. used

Fig. 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.

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1750 1550 1350 Eggs per gram (EPG) of faeces 4 1150 950 750 550 350 150 -50 Untreated Treated 100 1g 4g 8g 1g 4g 8g 16 23 28 32 28 35 Day 0 EPG Day 15 EPG

Control

Crude Powder

Crude Aqueous Methanolic Extract

Doses and forms of Ziziphus mauritiana Lam. used

Fig. 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.

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Chapter # 5 DISCUSSION

The discussion has been arranged in the following order: Subject 5.1. Survey 5.2. Tests used for evaluation of anthelmintic activity 5.2.1. Egg Hatch Test (EHT) 5.2.2. Adult Motility Assay (AMA) 5.2.3. Fecal Egg Count Reduction Test (FECRT) 5.3. In vitro and in vivo anthelmintic activity 5.3.1. Plants demonstrated anthelmintic activity in EHT, AMA and FECRT 5.3.1.1. Albizia lebbeck (L.) Benth. 5.3.1.2. Bambusa arundinacea (Retz.) Willd. 5.3.1.3. Digera muricata L. 5.3.1.4. Lagenaria siceraria (Molina) Standl. 5.3.1.5. Mangifera indica L. 5.3.1.6. Musa paradisiaca L. 5.3.1.7. Syzygium cumini (L.) Skeels 5.3.1.8. Trianthema portulacastrum L. 5.3.1.9. Tribulus terrestris L. 5.3.1.10. Ziziphus mauritiana Lam. 5.4. Phytoanthelmintics Page # 75 79 80 81 83 84 85 85 86 87 87 88 89 90 91 92 93 94

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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

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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).

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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),

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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 Cucumis melo L., Coriandrum sativum L.,

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

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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 nonplant 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

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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).

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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

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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

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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

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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 transcuticular 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

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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 and dose-dependent in vitro anthelmintic activity as well as in vivo anthelmintic activity with 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

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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 ([email protected], 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

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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 and dose-dependent in vitro anthelmintic activity as well as in vivo anthelmintic activity with 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

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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 -sitosterol-Dglucoside. 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.,

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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

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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.

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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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