Parasitol Res DOI 10.1007/s00436-015-4416-0
ORIGINAL PAPER
Anthelmintic activity of ginger, curcumin, and praziquentel against Raillietina cesticillus (in vitro and in vivo) Nasr M. El-Bahy & Eman K. A. Bazh
Received: 27 February 2015 / Accepted: 4 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract This work evaluates the anthelminitic activity of ginger and curcumin on the cestode Raillietina cesticillus. Live parasites were collected from intestine of naturally infected chickens in PBS 0.9 % and then incubated at 37 °C in media containing ginger extract at three different concentrations (125, 250, and 500 mg); every concentration was dissolved in 10 ml media. The curcumin extract was used at three different concentrations (250, 500, and 1000 mg); each was dissolved in 10 ml media. Praziquantel at a concentration of 600 mg was added to 10 ml media. A control one without extract was reported. Regression of worms increased gradually in all concentrations. At 500 mg ginger (50±0 %), worms were regressed at 48 h post-exposure (h.p.e.). Also (50±1.8 %), worms were regressed at 1000 mg curcumin at the same time. On the other hand, praziquantel showed the highest regression (65±2.3 %). The extract efficacy was exhibit as concentration-time-dependent mainly at higher concentrations used after 48 h. In vivo effects of ginger and curcumin were lower than those in vitro. Keywords Chicken . Cestode . Ginger . Curcumin . Praziquentel
Introduction Nowadays, several in vitro research studies have been done to find new compounds that treat cestode infections. The natural products were used prospecting their anti-parasitic potential. N. M. El-Bahy Department of Parasitology, Faculty of Veterinary Medicine, Sadat City University, Sadat City, Egypt E. K. A. Bazh (*) Department of Pathology and Parasitology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt e-mail: [email protected]
In Egypt, Sinai is rich in herbal plants which are used for many years ago to treat some diseases and parasites and also to induce immunity and body health (Bazh and El-Bahy 2013). Traditional medicine relies on using natural products for the treatment of both veterinary and human helminths (Hammond et al. 1997; Roy et al. 2012; Dhakal et al. 2015). The family Zingiberaceae contains three genera (Zingiber officinale, Curcuma longa L., and Elettaria cardamomum (Daoud 2008). Ginger (Z. officinale Roscoe, Zingiberacae) is a medicinal plant that has been widely used for infectious diseases and helminthiasis (Ali et al. 2008). Z. officinale displayed some degree of anti-schistosomal activity through reduction of the Schistosoma mansoni egg output (53.8 %), moderate reduction of 16.5 % in the worm burden compared with control-infected animals, and reduction of size of the liver granuloma (Al-Sharkawi et al. 2007). Ginger extract at concentration of 150 mg/kg has no demonstrable anti-schistosomal activity in infected mice, while in vitro studies showed a significant effect against adult S. mansoni at concentration of 200 mg lˉ1 (Sanderson et al. 2002). Curcumin (C. longa) is used as anti-parasitic for many skin affections (Gupta and Balasubrahmanyam 1998). For helminths, curcumin treatment modulates cellular and humeral immune responses of infected mice and leads to a significant reduction of parasite burden and liver pathology in acute murine S. mansoni (Allam 2009). Also, the efficacy of ginger and curcumin methanolic extract was estimated against Ascaridia galli by Bazh and ElBahy (2013). There was no previous literature studying the effect of ginger and curcumin against Raillietina cesticillus. So the aim of this work was to evaluate the efficacy of Z. officinale and C. longa to avoid the side effects of medicinal products.
Parasitol Res
Material and methods Collection of tested cestodes From naturally infected chicken, the intestinal contents were expelled in 0.9 % normal saline in petri dish and examined under dissecting microscope. The adult cestodes (R. cesticillus) were obtained freshly from small intestine of examined chicken and immediately put in petri dish containing 0.9 % phosphatebuffered saline. Active worms (chosen with frequent body contractions observed) were maintained at room temperature and used within 1 h. Twenty live and active worms were used and grouped for each replicate of the screening assay (Atjanasuppat et al. 2009). Biological tests were performed in duplicate and were repeated three times. Plant extract and drugs Plant extracts and drugs used in the present study are as follows: – – – –
Ginger root extract® containing 5 % gingerols, 250-mg capsules, manufactured by Now, USA Standarized Tumeric Curcumin® containing 95 % curcuminoids, 500-mg capsules, manufactured by Earthstream Herbs, USA Praziquantel (Epiquantel®), 600-mg tablet, manufactured by EIPICO, Egypt RPMI 1640 medium (Sigma) supplemented with 200 μg/ml of streptomycin and 200 IU/ml of penicillin
In vitro determination of anthelmintic activity Worms were grouped and treated as follows: The first group was incubated with 125 mg of ginger dissolved in 10 ml RPMI 1640 medium. The second group was incubated with 250 mg of ginger dissolved in 10 ml RPMI 1640 medium. The third group was incubated with 500 mg of ginger dissolved in 10 ml RPMI 1640 medium. The fourth group was incubated with 250 mg of curcumin dissolved in 10 ml RPMI 1640 medium. The fifth group was incubated with 500 mg of curcumin dissolved in 10 ml RPMI 1640 medium. The sixth group was incubated with 1000 mg of curcumin dissolved in 10 ml RPMI 1640 medium. The seventh group was incubated with 600 mg praziquantel (reference drug) dissolved in 10 ml RPMI 1640 medium. The eighth group of worms was incubated with RPMI 1640 medium free and left as control one.
All groups were incubated at 37±1 °C and examined at 6, 12, 24, and 48 h. After incubation time, the changes were observed and recorded. The worms were categorized and counted as motile with good movement, and non-motile with regressed movement that means that the motility needs stimulation by vigorous shaking, needle, or light. And, dead worms without any response (movement) or signs of life and appeared completely relaxed. In vivo studies of ginger and curcumin Chickens used The in vivo study was done to evaluate the efficacy of the plant extracts as well as praziquantel against R. cesticillus. The total number of naturally infected chicks with R. cesticillus was 80. These chickens were selected from open yard rearing system. The confirmation of infection was done by dissection of five chicks from different yards of the infected flock, which were positive fecal examinations. Also, 20 parasite-free chicks were purchased (negative control group). Experimental design and anthelmintic assay The infected chickens were divided into four groups each containing 20 chicks. Another 20 chicks which were parasite-free were left as control non-infected group. Group 1 was treated with ginger extract at dose of 500 mg per bird. Group 2 was treated with curcumin extract at dose of 1000 mg per bird. Group 3 was treated with praziquantel at dose of 600 mg per bird. Group 4 was represented as control-infected non-treated group. Group 5 was represented as non-infected non-treated control group. Each group was housed in clean cages and was provided with ration and nipples. Worm collection and counting Lastly, worms were recovered from each group of the infected chickens. Four chickens were killed at 6, 12, 24, and 48 h after dosing them with the plant extract or praziquantel and dissected to obtain worms from their intestines under a stereomicroscope. The grossly recognized worms were collected and efficacy percentage of each relict was estimated (Tangpu et al. 2006). Anti-parasitic efficacy of each treatment was calculated using the following equation (Wang et al. 2009):
Parasitol Res Table 1
In vitro effects of ginger, curcumin, and praziquantel on adult Raillietina cesticillus
Material used
Concentration
The mean % of the worms±SEM 6 h.p.e.
Ginger
Curcumin
Praziquantel Control
125 mg 250 mg 500 mg 250 mg 500 mg 1000 mg 600 mg 0
12 h.p.e.
24 h.p.e.
48 h.p.e.
R
D
R
D
R
D
R
D
10±0 15±1.3* 20±1.3* 5±0 10±0 15±0 30±1.3* 0
20±0 25±1.3* 35±1.3* 15±1.3* 20±1.3* 30±1.8* 50±0 0
20±0 25±0 25±1.3* 5±0 10±0 15±0 40±1.8* 0
30±1.8* 35±1.3* 40±1.8* 20±0 30±0 40±2.6* 70±0 0
30±0 35±1.3* 35±0 20±2.3* 25±0 30±0 50±0 0
50±1.8* 60±1.8* 70±2.6* 40±0 50±1.8* 60±0 80±1.8* 0
35±0 40±0 50±0 30±1.8* 35±0 50±1.8* 65±2.3* 0
70±1.8* 80±2.6* 85±2.3* 65±2.3* 70±0 80±0 95±1.8* 0
Every concentration used was dissolved in 10 ml of RPMI 1640 media. Data represent the percentage of dead and regressed worms (relative to 20 worms) as the mean ± SEM of the different trials. ANOVA followed by Dunnett’s test with significant differences at *p
ORIGINAL PAPER
Anthelmintic activity of ginger, curcumin, and praziquentel against Raillietina cesticillus (in vitro and in vivo) Nasr M. El-Bahy & Eman K. A. Bazh
Received: 27 February 2015 / Accepted: 4 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract This work evaluates the anthelminitic activity of ginger and curcumin on the cestode Raillietina cesticillus. Live parasites were collected from intestine of naturally infected chickens in PBS 0.9 % and then incubated at 37 °C in media containing ginger extract at three different concentrations (125, 250, and 500 mg); every concentration was dissolved in 10 ml media. The curcumin extract was used at three different concentrations (250, 500, and 1000 mg); each was dissolved in 10 ml media. Praziquantel at a concentration of 600 mg was added to 10 ml media. A control one without extract was reported. Regression of worms increased gradually in all concentrations. At 500 mg ginger (50±0 %), worms were regressed at 48 h post-exposure (h.p.e.). Also (50±1.8 %), worms were regressed at 1000 mg curcumin at the same time. On the other hand, praziquantel showed the highest regression (65±2.3 %). The extract efficacy was exhibit as concentration-time-dependent mainly at higher concentrations used after 48 h. In vivo effects of ginger and curcumin were lower than those in vitro. Keywords Chicken . Cestode . Ginger . Curcumin . Praziquentel
Introduction Nowadays, several in vitro research studies have been done to find new compounds that treat cestode infections. The natural products were used prospecting their anti-parasitic potential. N. M. El-Bahy Department of Parasitology, Faculty of Veterinary Medicine, Sadat City University, Sadat City, Egypt E. K. A. Bazh (*) Department of Pathology and Parasitology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt e-mail: [email protected]
In Egypt, Sinai is rich in herbal plants which are used for many years ago to treat some diseases and parasites and also to induce immunity and body health (Bazh and El-Bahy 2013). Traditional medicine relies on using natural products for the treatment of both veterinary and human helminths (Hammond et al. 1997; Roy et al. 2012; Dhakal et al. 2015). The family Zingiberaceae contains three genera (Zingiber officinale, Curcuma longa L., and Elettaria cardamomum (Daoud 2008). Ginger (Z. officinale Roscoe, Zingiberacae) is a medicinal plant that has been widely used for infectious diseases and helminthiasis (Ali et al. 2008). Z. officinale displayed some degree of anti-schistosomal activity through reduction of the Schistosoma mansoni egg output (53.8 %), moderate reduction of 16.5 % in the worm burden compared with control-infected animals, and reduction of size of the liver granuloma (Al-Sharkawi et al. 2007). Ginger extract at concentration of 150 mg/kg has no demonstrable anti-schistosomal activity in infected mice, while in vitro studies showed a significant effect against adult S. mansoni at concentration of 200 mg lˉ1 (Sanderson et al. 2002). Curcumin (C. longa) is used as anti-parasitic for many skin affections (Gupta and Balasubrahmanyam 1998). For helminths, curcumin treatment modulates cellular and humeral immune responses of infected mice and leads to a significant reduction of parasite burden and liver pathology in acute murine S. mansoni (Allam 2009). Also, the efficacy of ginger and curcumin methanolic extract was estimated against Ascaridia galli by Bazh and ElBahy (2013). There was no previous literature studying the effect of ginger and curcumin against Raillietina cesticillus. So the aim of this work was to evaluate the efficacy of Z. officinale and C. longa to avoid the side effects of medicinal products.
Parasitol Res
Material and methods Collection of tested cestodes From naturally infected chicken, the intestinal contents were expelled in 0.9 % normal saline in petri dish and examined under dissecting microscope. The adult cestodes (R. cesticillus) were obtained freshly from small intestine of examined chicken and immediately put in petri dish containing 0.9 % phosphatebuffered saline. Active worms (chosen with frequent body contractions observed) were maintained at room temperature and used within 1 h. Twenty live and active worms were used and grouped for each replicate of the screening assay (Atjanasuppat et al. 2009). Biological tests were performed in duplicate and were repeated three times. Plant extract and drugs Plant extracts and drugs used in the present study are as follows: – – – –
Ginger root extract® containing 5 % gingerols, 250-mg capsules, manufactured by Now, USA Standarized Tumeric Curcumin® containing 95 % curcuminoids, 500-mg capsules, manufactured by Earthstream Herbs, USA Praziquantel (Epiquantel®), 600-mg tablet, manufactured by EIPICO, Egypt RPMI 1640 medium (Sigma) supplemented with 200 μg/ml of streptomycin and 200 IU/ml of penicillin
In vitro determination of anthelmintic activity Worms were grouped and treated as follows: The first group was incubated with 125 mg of ginger dissolved in 10 ml RPMI 1640 medium. The second group was incubated with 250 mg of ginger dissolved in 10 ml RPMI 1640 medium. The third group was incubated with 500 mg of ginger dissolved in 10 ml RPMI 1640 medium. The fourth group was incubated with 250 mg of curcumin dissolved in 10 ml RPMI 1640 medium. The fifth group was incubated with 500 mg of curcumin dissolved in 10 ml RPMI 1640 medium. The sixth group was incubated with 1000 mg of curcumin dissolved in 10 ml RPMI 1640 medium. The seventh group was incubated with 600 mg praziquantel (reference drug) dissolved in 10 ml RPMI 1640 medium. The eighth group of worms was incubated with RPMI 1640 medium free and left as control one.
All groups were incubated at 37±1 °C and examined at 6, 12, 24, and 48 h. After incubation time, the changes were observed and recorded. The worms were categorized and counted as motile with good movement, and non-motile with regressed movement that means that the motility needs stimulation by vigorous shaking, needle, or light. And, dead worms without any response (movement) or signs of life and appeared completely relaxed. In vivo studies of ginger and curcumin Chickens used The in vivo study was done to evaluate the efficacy of the plant extracts as well as praziquantel against R. cesticillus. The total number of naturally infected chicks with R. cesticillus was 80. These chickens were selected from open yard rearing system. The confirmation of infection was done by dissection of five chicks from different yards of the infected flock, which were positive fecal examinations. Also, 20 parasite-free chicks were purchased (negative control group). Experimental design and anthelmintic assay The infected chickens were divided into four groups each containing 20 chicks. Another 20 chicks which were parasite-free were left as control non-infected group. Group 1 was treated with ginger extract at dose of 500 mg per bird. Group 2 was treated with curcumin extract at dose of 1000 mg per bird. Group 3 was treated with praziquantel at dose of 600 mg per bird. Group 4 was represented as control-infected non-treated group. Group 5 was represented as non-infected non-treated control group. Each group was housed in clean cages and was provided with ration and nipples. Worm collection and counting Lastly, worms were recovered from each group of the infected chickens. Four chickens were killed at 6, 12, 24, and 48 h after dosing them with the plant extract or praziquantel and dissected to obtain worms from their intestines under a stereomicroscope. The grossly recognized worms were collected and efficacy percentage of each relict was estimated (Tangpu et al. 2006). Anti-parasitic efficacy of each treatment was calculated using the following equation (Wang et al. 2009):
Parasitol Res Table 1
In vitro effects of ginger, curcumin, and praziquantel on adult Raillietina cesticillus
Material used
Concentration
The mean % of the worms±SEM 6 h.p.e.
Ginger
Curcumin
Praziquantel Control
125 mg 250 mg 500 mg 250 mg 500 mg 1000 mg 600 mg 0
12 h.p.e.
24 h.p.e.
48 h.p.e.
R
D
R
D
R
D
R
D
10±0 15±1.3* 20±1.3* 5±0 10±0 15±0 30±1.3* 0
20±0 25±1.3* 35±1.3* 15±1.3* 20±1.3* 30±1.8* 50±0 0
20±0 25±0 25±1.3* 5±0 10±0 15±0 40±1.8* 0
30±1.8* 35±1.3* 40±1.8* 20±0 30±0 40±2.6* 70±0 0
30±0 35±1.3* 35±0 20±2.3* 25±0 30±0 50±0 0
50±1.8* 60±1.8* 70±2.6* 40±0 50±1.8* 60±0 80±1.8* 0
35±0 40±0 50±0 30±1.8* 35±0 50±1.8* 65±2.3* 0
70±1.8* 80±2.6* 85±2.3* 65±2.3* 70±0 80±0 95±1.8* 0
Every concentration used was dissolved in 10 ml of RPMI 1640 media. Data represent the percentage of dead and regressed worms (relative to 20 worms) as the mean ± SEM of the different trials. ANOVA followed by Dunnett’s test with significant differences at *p
