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In vivo anthelmintic activity of ginger against gastrointestinal nematodes of sheep
Journal of Ethnopharmacology 106 (2006) 285–287
Ethnopharmacological communication
In vivo anthelmintic activity of ginger against gastrointestinal nematodes of sheep Zafar Iqbal a,∗ , Muhammad Lateef a , Muhammad Shoaib Akhtar b , Muhammad Nabeel Ghayur c , Anwarul Hassan Gilani c a Department of Veterinary Parasitology, University of Agriculture, Faisalabad 38040, Pakistan Department of Physiology and Pharmacology, University of Agriculture, Faisalabad 38040, Pakistan c Department of Biological and Biochemical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan b
Received 24 June 2005; received in revised form 16 November 2005; accepted 15 December 2005 Available online 26 January 2006
Abstract This paper describes the anthelmintic activity of Zingiber officinale Roscoe (family Zingiberaceae) rhizome, commonly known as ginger, to justify its traditional use in veterinary medicine. Crude powder (CP) and crude aqueous extract (CAE) of dried ginger (1–3 g/kg) were administered to sheep naturally infected with mixed species of gastrointestinal nematodes. Both CP and CAE exhibited a dose- and a time-dependent anthelmintic effect with respective maximum reduction of 25.6% and 66.6% in eggs per gram (EPG) of faeces on day 10 of post-treatment. Levamisole (7.5 mg/kg), a standard anthelmintic agent, exhibited 99.2% reduction in EPG. This study shows that ginger possesses in vivo anthelmintic activity in sheep thus justifying the age-old traditional use of this plant in helminth infestation. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Zingiber officinale; Ginger; Anthelmintic; Trichostrongylid nematodes; Sheep
1. Plant Dried rhizome of Zingiber officinale Roscoe (family Zingiberaceae), commonly known as ginger, were procured from a local market in Faisalabad, Pakistan and then identified and authenticated by a botanist using germplasm preserved in the herbarium, Department of Botany, University of Agriculture, Faisalabad, Pakistan. Crude powder (CP) and crude aqueous extract (CAE) of the dried ground rhizomes was prepared according to standard methods (Onyeyili et al., 2001) and tested for potential anthelmintic activity in sheep naturally infected with mixed infection of gastrointestinal nematodes. 2. Uses in traditional medicine Ginger has been used all over the world for since antiquity for relief from arthritis, rheumatism, sprains, muscular aches, pains, Abbreviations: CP, crude powder; CAE, crude aqueous extract; EPG, eggs per gram; PT, post-treatment; ECR, egg count percent reduction ∗ Corresponding author. Tel.: +92 41 9201106/9200161–70x3130. E-mail address: [email protected] (Z. Iqbal). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.12.031
sore throats, cramps, constipation, indigestion, vomiting, hypertension, dementia, fever, infectious diseases and helminthiasis (Nadkarni, 1976; Kapoor, 1990). Ginger has been widely studied for its pharmacological activities and has been reported to exhibit antiinflammatory, antipyretic, antimicrobial, hypoglycaemic, antimigraine, antischistosomal, antioxidant, hepatoprotective, diuretic, hypocholesterolaemic (Mascolo et al., 1989; Langner et al., 1998), hypotensive (Ghayur and Gilani, 2005a) and gastrointestinal prokinetic activities (Ghayur and Gilani, 2005b). 3. Previously isolated classes of constituents Phytochemical reports have shown that main constituents of ginger are the gingerols, shogaols, zingerone and paradol (for review see, Langner et al., 1998; Gilani and Ghayur, 2005). 6-Gingerol and 6-shogaol are the major gingerol and shogaol present in the rhizome (Connell and McLachlan, 1972). The main aroma-defining component is zingiberol while others as gingediol, monoacyldigalactosyl-glycerol, diarylheptanoids and phytosterols have also been identified (Varma et al., 1962). Even today, this plant still attracts extensive research both to elucidate
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Z. Iqbal et al. / Journal of Ethnopharmacology 106 (2006) 285–287
its chemical and biological aspects. Jolad et al. (2004) reported 25 new gingerol derivatives and 7 gingerol thermal degradation products from fresh ginger extract with antiinflammatory activity while have again recently reported another 31 new gingerol derivatives from dry ginger (Jolad et al., 2005), indicating the vast amount of scope still alive in this age-old plant. 4. Materials and methods Sheep (natural host of the tested nematodes, Soulsby, 1982) of both sexes (female and male young stock ≤1 year), weighing 18–24 kg were used for in vivo trials conducted at Livestock Experiment Station, Rakh Kherewala (Punjab, Pakistan). Before the start of experiment, the animals were confirmed to be naturally infected with mixed species of gastrointestinal nematodes by qualitative and quantitative fecal examination using standard parasitological procedures (Soulsby, 1982). Identification of nematode eggs in the faeces was done using standard description of MAFF (1979). The selected animals were suffering from mixed gastrointestinal nematodes species including mainly: Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, Oesophagostomum columbianum, Strongyloides papillosus and Trichuris ovis. The sheep used for experiment were randomly divided into six groups of four animals each and assigned to different treatments. Group 1 served as negative control and received no treatment while Group 2 was a positive control which was given a single dose of levamisole HCl 7.5 mg/kg (ICI Pakistan Limited). Groups 3 and 4 were administered single oral doses of CP 1.0 and 3.0 g/kg, respectively while Groups 5 and 6 were given CAE 1.0 and 3.0 g/kg, respectively. The experimental doses were adjusted in the light of local ethnoveterinary practices (personal communication). Fecal samples of each group were collected in the morning, starting from day 0 pre-treatment and at days 5 and 10 post-treatment. These Fig. 1. Bar diagram showing the dose-dependent (1.0–3.0 g/kg) anthelmintic activity of Zingiber officinale crude powder (CP) in sheep naturally infected samples were then evaluated for the presence of worm eggs with mixed species of gastrointestinal nematodes at: (A) 5 days post-treatment by salt floatation technique (MAFF, 1979). The eggs were (PT) and (B) 10 days PT. Activity of CP is compared with that of positive control counted by the McMaster method (Soulsby, 1982). Egg count levamisole (7.5 mg/kg). Values shown are mean ± S.E.M., n = 4; * P < 0.01 and per cent reduction (ECR) was calculated using the following ** P < 0.001, vs. negative control. formula: pre-treatment egg count per gram − post-treatment egg count per gram ECR (%) = × 100 pre-treatment egg count per gram The data were statistically analyzed using SAS software (SAS, 1998). Results were expressed as mean ± standard error of mean (S.E.M., n = number of experiments). 5. Results and discussion The crude powder of ginger (1–3 g/kg) exhibited moderate anthelmintic activity in sheep (Fig. 1). The dose of 1.0 g/kg showed a maximum reduction of 16.3% in EPG on day 5 post-treatment while the next higher dose of 3.0 g/kg showed a time-dependent anthelmintic effect and significantly reduced the EPG to 16.6% (P < 0.01) and 25.6% (P < 0.001) for 5 days (Fig. 1A) and 10 days (Fig. 1B) post-treatment, respectively. No further increase in the anthelmintic activity of CP against
gastrointestinal nematodes of sheep was noted beyond 10 days post-treatment (data not shown). No evidence of toxicity of ginger was recorded on the experimental doses during or after the treatment. The alcoholic extract of ginger has been studied in human against a specific helminth infestation (Ascaris lumbricoides) and found active (Kalesaraj, 1975). However, it has not been widely studied for its anthelmintic activity particularly in dairy animals despite its use in the ethno-veterinary medicine as an anthelmintic (personal communication). Thus, this is the first study which has shown the activity of ginger against gastrointestinal nematodes in sheep. Likewise, the ginger crude aqueous extract was also tested in vivo for its anthelmintic activity in sheep. CAE exhibited a dosedependent (1.0–3.0 g/kg) anthelmintic activity (Fig. 2). The
Z. Iqbal et al. / Journal of Ethnopharmacology 106 (2006) 285–287
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of ginger is responsible for the anthelmintic activity seen in this study. The result show that ginger rhizome possesses significant anthelmintic activity thus justifying its traditional use by the sheep farmers as an anthelmintic agent in the ethno-veterinary system of Pakistan. Acknowledgement This research was funded by the University of Agriculture, Faisalabad (Pakistan) under Promotion of Research Scheme. References
Fig. 2. Bar diagram showing the dose-dependent (1.0–3.0 g/kg) anthelmintic activity of Zingiber officinale crude aqueous extract (CAE) in sheep naturally infected with mixed species of gastrointestinal nematodes at: (A) 5 days posttreatment (PT) and (B) 10 days PT. Activity of CAE is compared with that of positive control levamisole (7.5 mg/kg). Values shown are mean ± S.E.M., n = 4; * P < 0.05 and ** P < 0.001, vs. negative control.
doses of 1.0 and 3.0 g/kg attenuated the nematode eggs/g of faeces by 12.1% and 24.7% (P < 0.05) on 5th day of post-treatment (Fig. 2A) while a further decrease in EPG was observed with 3.0 g/kg when it showed a maximum reduction of 66.6% on 10th day of post-treatment (Fig. 2B). No further decrease in EPG was noted by the 3.0 g/kg dose. Levamisol, a standard anthelmintic agent (Edwards and Breckenridge, 1988), also showed an inhibitory effect at the dose of 7.5 mg/kg against the nematodes (Figs. 1 and 2). Nematode muscles are known to contain excitatory neuromuscular junctions with acetylcholine as the neurotransmitter (Neal, 2002). Recently we have reported that ginger exhibits gastrointestinal prokinetic activity via activation of cholinergic receptors (Gilani and Ghayur, 2005) while levamisol is also known to activate these neuromuscular junctions causing a spastic paralysis that leads to the expelling of the worm (Neal, 2002). It could be that the cholinergic component
Connell, D.W., McLachlan, R., 1972. Natural pungent compounds: examination of gingerols, shogaols, paradols and related compounds by thin-layer and gas chromatography. Journal of Chromatography 67, 29–35. Edwards, G., Breckenridge, A.M., 1988. Clinical pharmacokinetics of anthelmintic drugs. Clinical Pharmacokinetics 15, 67–93. Ghayur, M.N., Gilani, A.H., 2005a. Ginger lowers blood pressure through blockade of voltage-dependent calcium channels. Journal of Cardiovascular Pharmacology 45, 74–80. Ghayur, M.N., Gilani, A.H., 2005b. Pharmacological basis for the medicinal use of ginger in gastrointestinal disorders. Digestive Diseases and Sciences 50, 1889–1897. Gilani, A.H., Ghayur, M.N., 2005. Ginger: from myths to reality. In: Gottschalk-Batschkus, C.E., Green, J.C. (Eds.), Ethnotherapies in the Cycle of Life, BOD – Books on Demand. Ethnomed Institut f¨ur Ethnomedizin e.V., Munchen, pp. 307–315. Jolad, S.D., Lantz, R.C., Solyom, A.M., Chen, G.J., Bates, R.B., Timmermann, B.N., 2004. Fresh organically grown ginger (Zingiber officinale): composition and effects on LPS-induced PGE2 production. Phytochemistry 65, 1937–1954. Jolad, S.D., Lantz, R.C., Chen, G.J., Bates, R.B., Timmermann, B.N., 2005. Commercially processed dry ginger (Zingiber officinale): composition and effects on LPS-stimulated PGE2 production. Phytochemistry 66, 1614–1635. Kalesaraj, R., 1975. Screening of some indigenous plants for anthelmintic action against human Ascaris lumbricoides. Part II. Indian Journal of Physiology and Pharmacology 19, 47–49. Kapoor, L.D., 1990. Handbook of Ayurvedic Medicinal Plants. CRC Press, Boca Raton, pp. 341–342. Langner, E., Greifenberg, S., Gruenwald, J., 1998. Ginger: history and use. Advances in Therapy 15, 25–44. MAFF, 1979. Parasitological Laboratory Techniques, Tech. Bull., No. 18. Ministry of Agriculture Fisheries and Food Manual of Veterinary. Her Majestey’s Stationary Office, London. Mascolo, N., Jain, R., Jain, S.C., Capasso, F., 1989. Ethnopharmacologic investigation of ginger (Zingiber officinale). Journal of Ethnopharmacology 27, 129–140. Nadkarni, K.M., 1976. Indian Materia Medica. Popular Prakashan, Bombay, pp. 1308–1315. Neal, M.J., 2002. Medical Pharmacology at a Glance. Blackwell Science, Oxford, pp. 88–89. Onyeyili, P.A., Nwosa, C.O., Amin, J.D., Jibike, J.I., 2001. Anthelmintic activity of crude aqueous extract of Nauclea latifolia stem bark against ovine nematodes. Fitoterapia 72, 12–21. SAS, 1998. Statistical Analysis System: User’s Guide. Statistical Institute, North Carolina. Soulsby, E.J.L., 1982. Helminths, Arthropods and Protozoa of Domesticated Animals. English Language Book Society, Baillere Tindall, London. Varma, K.R., Jain, T.C., Bhattacharyya, S.C., 1962. Structure and stereochemistry of zingiberol and juniper camphor. Tetrahedron 18, 979.
Ethnopharmacological communication
In vivo anthelmintic activity of ginger against gastrointestinal nematodes of sheep Zafar Iqbal a,∗ , Muhammad Lateef a , Muhammad Shoaib Akhtar b , Muhammad Nabeel Ghayur c , Anwarul Hassan Gilani c a Department of Veterinary Parasitology, University of Agriculture, Faisalabad 38040, Pakistan Department of Physiology and Pharmacology, University of Agriculture, Faisalabad 38040, Pakistan c Department of Biological and Biochemical Sciences, The Aga Khan University Medical College, Karachi 74800, Pakistan b
Received 24 June 2005; received in revised form 16 November 2005; accepted 15 December 2005 Available online 26 January 2006
Abstract This paper describes the anthelmintic activity of Zingiber officinale Roscoe (family Zingiberaceae) rhizome, commonly known as ginger, to justify its traditional use in veterinary medicine. Crude powder (CP) and crude aqueous extract (CAE) of dried ginger (1–3 g/kg) were administered to sheep naturally infected with mixed species of gastrointestinal nematodes. Both CP and CAE exhibited a dose- and a time-dependent anthelmintic effect with respective maximum reduction of 25.6% and 66.6% in eggs per gram (EPG) of faeces on day 10 of post-treatment. Levamisole (7.5 mg/kg), a standard anthelmintic agent, exhibited 99.2% reduction in EPG. This study shows that ginger possesses in vivo anthelmintic activity in sheep thus justifying the age-old traditional use of this plant in helminth infestation. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Zingiber officinale; Ginger; Anthelmintic; Trichostrongylid nematodes; Sheep
1. Plant Dried rhizome of Zingiber officinale Roscoe (family Zingiberaceae), commonly known as ginger, were procured from a local market in Faisalabad, Pakistan and then identified and authenticated by a botanist using germplasm preserved in the herbarium, Department of Botany, University of Agriculture, Faisalabad, Pakistan. Crude powder (CP) and crude aqueous extract (CAE) of the dried ground rhizomes was prepared according to standard methods (Onyeyili et al., 2001) and tested for potential anthelmintic activity in sheep naturally infected with mixed infection of gastrointestinal nematodes. 2. Uses in traditional medicine Ginger has been used all over the world for since antiquity for relief from arthritis, rheumatism, sprains, muscular aches, pains, Abbreviations: CP, crude powder; CAE, crude aqueous extract; EPG, eggs per gram; PT, post-treatment; ECR, egg count percent reduction ∗ Corresponding author. Tel.: +92 41 9201106/9200161–70x3130. E-mail address: [email protected] (Z. Iqbal). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.12.031
sore throats, cramps, constipation, indigestion, vomiting, hypertension, dementia, fever, infectious diseases and helminthiasis (Nadkarni, 1976; Kapoor, 1990). Ginger has been widely studied for its pharmacological activities and has been reported to exhibit antiinflammatory, antipyretic, antimicrobial, hypoglycaemic, antimigraine, antischistosomal, antioxidant, hepatoprotective, diuretic, hypocholesterolaemic (Mascolo et al., 1989; Langner et al., 1998), hypotensive (Ghayur and Gilani, 2005a) and gastrointestinal prokinetic activities (Ghayur and Gilani, 2005b). 3. Previously isolated classes of constituents Phytochemical reports have shown that main constituents of ginger are the gingerols, shogaols, zingerone and paradol (for review see, Langner et al., 1998; Gilani and Ghayur, 2005). 6-Gingerol and 6-shogaol are the major gingerol and shogaol present in the rhizome (Connell and McLachlan, 1972). The main aroma-defining component is zingiberol while others as gingediol, monoacyldigalactosyl-glycerol, diarylheptanoids and phytosterols have also been identified (Varma et al., 1962). Even today, this plant still attracts extensive research both to elucidate
286
Z. Iqbal et al. / Journal of Ethnopharmacology 106 (2006) 285–287
its chemical and biological aspects. Jolad et al. (2004) reported 25 new gingerol derivatives and 7 gingerol thermal degradation products from fresh ginger extract with antiinflammatory activity while have again recently reported another 31 new gingerol derivatives from dry ginger (Jolad et al., 2005), indicating the vast amount of scope still alive in this age-old plant. 4. Materials and methods Sheep (natural host of the tested nematodes, Soulsby, 1982) of both sexes (female and male young stock ≤1 year), weighing 18–24 kg were used for in vivo trials conducted at Livestock Experiment Station, Rakh Kherewala (Punjab, Pakistan). Before the start of experiment, the animals were confirmed to be naturally infected with mixed species of gastrointestinal nematodes by qualitative and quantitative fecal examination using standard parasitological procedures (Soulsby, 1982). Identification of nematode eggs in the faeces was done using standard description of MAFF (1979). The selected animals were suffering from mixed gastrointestinal nematodes species including mainly: Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, Oesophagostomum columbianum, Strongyloides papillosus and Trichuris ovis. The sheep used for experiment were randomly divided into six groups of four animals each and assigned to different treatments. Group 1 served as negative control and received no treatment while Group 2 was a positive control which was given a single dose of levamisole HCl 7.5 mg/kg (ICI Pakistan Limited). Groups 3 and 4 were administered single oral doses of CP 1.0 and 3.0 g/kg, respectively while Groups 5 and 6 were given CAE 1.0 and 3.0 g/kg, respectively. The experimental doses were adjusted in the light of local ethnoveterinary practices (personal communication). Fecal samples of each group were collected in the morning, starting from day 0 pre-treatment and at days 5 and 10 post-treatment. These Fig. 1. Bar diagram showing the dose-dependent (1.0–3.0 g/kg) anthelmintic activity of Zingiber officinale crude powder (CP) in sheep naturally infected samples were then evaluated for the presence of worm eggs with mixed species of gastrointestinal nematodes at: (A) 5 days post-treatment by salt floatation technique (MAFF, 1979). The eggs were (PT) and (B) 10 days PT. Activity of CP is compared with that of positive control counted by the McMaster method (Soulsby, 1982). Egg count levamisole (7.5 mg/kg). Values shown are mean ± S.E.M., n = 4; * P < 0.01 and per cent reduction (ECR) was calculated using the following ** P < 0.001, vs. negative control. formula: pre-treatment egg count per gram − post-treatment egg count per gram ECR (%) = × 100 pre-treatment egg count per gram The data were statistically analyzed using SAS software (SAS, 1998). Results were expressed as mean ± standard error of mean (S.E.M., n = number of experiments). 5. Results and discussion The crude powder of ginger (1–3 g/kg) exhibited moderate anthelmintic activity in sheep (Fig. 1). The dose of 1.0 g/kg showed a maximum reduction of 16.3% in EPG on day 5 post-treatment while the next higher dose of 3.0 g/kg showed a time-dependent anthelmintic effect and significantly reduced the EPG to 16.6% (P < 0.01) and 25.6% (P < 0.001) for 5 days (Fig. 1A) and 10 days (Fig. 1B) post-treatment, respectively. No further increase in the anthelmintic activity of CP against
gastrointestinal nematodes of sheep was noted beyond 10 days post-treatment (data not shown). No evidence of toxicity of ginger was recorded on the experimental doses during or after the treatment. The alcoholic extract of ginger has been studied in human against a specific helminth infestation (Ascaris lumbricoides) and found active (Kalesaraj, 1975). However, it has not been widely studied for its anthelmintic activity particularly in dairy animals despite its use in the ethno-veterinary medicine as an anthelmintic (personal communication). Thus, this is the first study which has shown the activity of ginger against gastrointestinal nematodes in sheep. Likewise, the ginger crude aqueous extract was also tested in vivo for its anthelmintic activity in sheep. CAE exhibited a dosedependent (1.0–3.0 g/kg) anthelmintic activity (Fig. 2). The
Z. Iqbal et al. / Journal of Ethnopharmacology 106 (2006) 285–287
287
of ginger is responsible for the anthelmintic activity seen in this study. The result show that ginger rhizome possesses significant anthelmintic activity thus justifying its traditional use by the sheep farmers as an anthelmintic agent in the ethno-veterinary system of Pakistan. Acknowledgement This research was funded by the University of Agriculture, Faisalabad (Pakistan) under Promotion of Research Scheme. References
Fig. 2. Bar diagram showing the dose-dependent (1.0–3.0 g/kg) anthelmintic activity of Zingiber officinale crude aqueous extract (CAE) in sheep naturally infected with mixed species of gastrointestinal nematodes at: (A) 5 days posttreatment (PT) and (B) 10 days PT. Activity of CAE is compared with that of positive control levamisole (7.5 mg/kg). Values shown are mean ± S.E.M., n = 4; * P < 0.05 and ** P < 0.001, vs. negative control.
doses of 1.0 and 3.0 g/kg attenuated the nematode eggs/g of faeces by 12.1% and 24.7% (P < 0.05) on 5th day of post-treatment (Fig. 2A) while a further decrease in EPG was observed with 3.0 g/kg when it showed a maximum reduction of 66.6% on 10th day of post-treatment (Fig. 2B). No further decrease in EPG was noted by the 3.0 g/kg dose. Levamisol, a standard anthelmintic agent (Edwards and Breckenridge, 1988), also showed an inhibitory effect at the dose of 7.5 mg/kg against the nematodes (Figs. 1 and 2). Nematode muscles are known to contain excitatory neuromuscular junctions with acetylcholine as the neurotransmitter (Neal, 2002). Recently we have reported that ginger exhibits gastrointestinal prokinetic activity via activation of cholinergic receptors (Gilani and Ghayur, 2005) while levamisol is also known to activate these neuromuscular junctions causing a spastic paralysis that leads to the expelling of the worm (Neal, 2002). It could be that the cholinergic component
Connell, D.W., McLachlan, R., 1972. Natural pungent compounds: examination of gingerols, shogaols, paradols and related compounds by thin-layer and gas chromatography. Journal of Chromatography 67, 29–35. Edwards, G., Breckenridge, A.M., 1988. Clinical pharmacokinetics of anthelmintic drugs. Clinical Pharmacokinetics 15, 67–93. Ghayur, M.N., Gilani, A.H., 2005a. Ginger lowers blood pressure through blockade of voltage-dependent calcium channels. Journal of Cardiovascular Pharmacology 45, 74–80. Ghayur, M.N., Gilani, A.H., 2005b. Pharmacological basis for the medicinal use of ginger in gastrointestinal disorders. Digestive Diseases and Sciences 50, 1889–1897. Gilani, A.H., Ghayur, M.N., 2005. Ginger: from myths to reality. In: Gottschalk-Batschkus, C.E., Green, J.C. (Eds.), Ethnotherapies in the Cycle of Life, BOD – Books on Demand. Ethnomed Institut f¨ur Ethnomedizin e.V., Munchen, pp. 307–315. Jolad, S.D., Lantz, R.C., Solyom, A.M., Chen, G.J., Bates, R.B., Timmermann, B.N., 2004. Fresh organically grown ginger (Zingiber officinale): composition and effects on LPS-induced PGE2 production. Phytochemistry 65, 1937–1954. Jolad, S.D., Lantz, R.C., Chen, G.J., Bates, R.B., Timmermann, B.N., 2005. Commercially processed dry ginger (Zingiber officinale): composition and effects on LPS-stimulated PGE2 production. Phytochemistry 66, 1614–1635. Kalesaraj, R., 1975. Screening of some indigenous plants for anthelmintic action against human Ascaris lumbricoides. Part II. Indian Journal of Physiology and Pharmacology 19, 47–49. Kapoor, L.D., 1990. Handbook of Ayurvedic Medicinal Plants. CRC Press, Boca Raton, pp. 341–342. Langner, E., Greifenberg, S., Gruenwald, J., 1998. Ginger: history and use. Advances in Therapy 15, 25–44. MAFF, 1979. Parasitological Laboratory Techniques, Tech. Bull., No. 18. Ministry of Agriculture Fisheries and Food Manual of Veterinary. Her Majestey’s Stationary Office, London. Mascolo, N., Jain, R., Jain, S.C., Capasso, F., 1989. Ethnopharmacologic investigation of ginger (Zingiber officinale). Journal of Ethnopharmacology 27, 129–140. Nadkarni, K.M., 1976. Indian Materia Medica. Popular Prakashan, Bombay, pp. 1308–1315. Neal, M.J., 2002. Medical Pharmacology at a Glance. Blackwell Science, Oxford, pp. 88–89. Onyeyili, P.A., Nwosa, C.O., Amin, J.D., Jibike, J.I., 2001. Anthelmintic activity of crude aqueous extract of Nauclea latifolia stem bark against ovine nematodes. Fitoterapia 72, 12–21. SAS, 1998. Statistical Analysis System: User’s Guide. Statistical Institute, North Carolina. Soulsby, E.J.L., 1982. Helminths, Arthropods and Protozoa of Domesticated Animals. English Language Book Society, Baillere Tindall, London. Varma, K.R., Jain, T.C., Bhattacharyya, S.C., 1962. Structure and stereochemistry of zingiberol and juniper camphor. Tetrahedron 18, 979.