This article was downloaded by: [Bibliothèques de l'Université de Montréal] On: 02 December 2014, At: 01:19 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Aquatic Animal Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uahh20
Screening Medicinal Plants for Use against Dactylogyrus intermedius (Monogenea) Infection in Goldfish a
b
b
b
b
c
Yang Hu , Jie Ji , Fei Ling , Yunhe Chen , Lin Lu , Qizhong Zhang & Gaoxue Wang a
ab
College of Science, Northwest A&F University, Yangling 712100, China
b
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China c
Institute of Hydrobiology, Jinan University, Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering Minister of Education, Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guangdong Higher Education Institutes, Guangzhou 510632, China Published online: 03 Jul 2014.
To cite this article: Yang Hu, Jie Ji, Fei Ling, Yunhe Chen, Lin Lu, Qizhong Zhang & Gaoxue Wang (2014) Screening Medicinal Plants for Use against Dactylogyrus intermedius (Monogenea) Infection in Goldfish, Journal of Aquatic Animal Health, 26:3, 127-136, DOI: 10.1080/08997659.2014.902872 To link to this article: http://dx.doi.org/10.1080/08997659.2014.902872
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 26:127–136, 2014 C American Fisheries Society 2014 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.902872
ARTICLE
Screening Medicinal Plants for Use against Dactylogyrus intermedius (Monogenea) Infection in Goldfish Yang Hu Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
College of Science, Northwest A&F University, Yangling 712100, China
Jie Ji, Fei Ling, Yunhe Chen, and Lin Lu College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Qizhong Zhang* Institute of Hydrobiology, Jinan University, Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering Minister of Education, Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guangdong Higher Education Institutes, Guangzhou 510632, China
Gaoxue Wang* College of Science, Northwest A&F University, Yangling 712100, China; and College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Abstract Methanol extracts of 24 traditional medicinal plants with potential anthelmintic activity against Dactylogyrus intermedius (Monogenea) in Goldfish Carassius auratus were investigated. Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum exhibited 100% activity and were selected for further evaluation by applying five solvents (petroleum ether, chloroform, ethyl acetate, methanol, and water) for the extraction of the samples, followed by an in vivo bioassay. Among the plants tested, water, methanol, and ethyl-acetate extracts of P. multiflorum showed the highest efficacies; EC50 values (median concentration that results in 50% of its maximal effect) were 1.9, 5.4, and 9.1 mg/L, respectively, and extracts showed 100% efficacy against Dactylogyrus intermedius at 100, 12.5, and 25 mg/L. This was followed by ethyl-acetate, chloroform, and methanol extracts of Dioscorea collettii, which demonstrated 100% efficacy at 80, 80, and 120 mg/L and had EC50 values of 19.7, 27.1, and 37.8 mg/L, respectively, after 48 h of exposure. Chloroform and ethyl-acetate extracts of C. medica, which exhibited 100% efficacy against Dactylogyrus intermedius at 100 and 125 mg/L, revealed similar activity and had EC50 values of 58.7 and 51.3 mg/L, respectively. The ethyl-acetate and methanol extracts of A. cantoniensis exhibited the lowest activity and had EC50 values of 279.4 and 64.3 mg/L. Acute toxicities of these active extracts were investigated on Goldfish for 48 h. The findings indicated that extracts of the four plants can be developed as a preferred natural antiparasitic for the control of D. intermedius.
According to the Food and Agriculture Organization of the United Nations (FAO 2008), global aquaculture has developed rapidly in recent years and grown at an annual rate of 6.4% from 2002 to 2006. In addition, aquaculture in developing countries will continuously grow in the next 10 years (Food Business Network 2008). China, the biggest developing country, is the
world’s largest producer of fish, which accounts for two-thirds of the global production in 2009 (FAO 2008), and thus plays a vital and decisive role in aquaculture. However, intensive fish production often results in increased incidences of diseases caused by viruses, bacteria, fungi, parasites, and other undiagnosed and emerging pathogens (Bondad-Reantaso et al. 2005).
*Corresponding authors: [email protected]; [email protected] Received June 15, 2013; accepted February 11, 2014
127
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
128
HU ET AL.
Dactylogyrus intermedius is an important monogenean parasite that invades the skin and gills of cyprinid fishes and thereby causes serious problems, such as gill inflammation, excessive mucous secretions, respiratory distress, and mixed infections with other parasites and secondary bacterial infections (Reed et al. 2009). Fish infected with D. intermedius can be treated effectively with a wide spectrum of parasiticides, such as formalin (Marshall 1999), trichlorfon (Goven and Amend 1982), a triazine derivative (Schmahl 1993), and the two most effective treatments in practice, praziquantel (Schmahl and Mehlhorn 1985) and mebendazole (Buchmann et al. 1993). However, these chemical parasiticides can present serious drawbacks through frequent use, including the threats of anthelmintic resistance, risk of residue, environmental contamination, and toxicity to the host (Goven et al. 1980; Klinger and Floyd 2002). Therefore, there is an urgent need for alternative therapies, including the use of natural products from medicinal plants, which are more environmentally acceptable and generally have lower toxicity than chemicals. Recently, medicinal plants and their extracts were tested for their ability to control D. intermedius, and different extracts from several medicinal plants had significant killing effects in vivo (Wang et al. 2009, 2010a, 2010b; Liu et al. 2010; Wu et al. 2011; Ji et al. 2012; Lu et al. 2012). In our research, we exploited the crude extracts of different medicinal plants extracted by five solvents (petroleum ether, chloroform, ethyl acetate, methanol, and water) for anthelmintic activity against D. intermedius in Goldfish Carassius auratus.
METHODS Parasite and host.—Goldfish weighing 5.8 ± 1.9 g (mean ± SD) and without any record of previous infestation with parasites were used throughout the study. All fish were collected from a Changxing fish farm, Xianyang, Shaanxi, China (34◦ 32 15 N, 108◦ 51 8 E). The fish were then acclimated in glass aquaria (200 fish per aquarium) containing 180 L groundwater at 25 ± 1◦ C (controlled by automatic aquarium heater) with aeration for 7 d and were fed once daily at 2% of their body weight with commercial pellet feed. One week later, all the fish were cohabitated with fish infected with D. intermedius, which were retained in our laboratory; the infection procedure was described in our previous study (Wang et al. 2008). Three weeks later, 10 fish were randomly sampled and killed by spinal severance, and the biopsies of eight gill filaments of each fish were used to determine the infestation level and intensity of adult D. intermedius under a light microscope (Olympus BX41, Tokyo, Japan) at 40 × magnification. Fish were chosen for the assays when the infection rate was 100% and the mean number of D. intermedius on gills was 40–50 parasites per fish. Preparation of plant materials.—The plant materials from each of the selected species (Table 1) were collected in September 2012 and the taxonomic identification was made by X. P. Song, Northwest A&F University, Shaanxi. The voucher spec-
imens have been deposited at the herbarium of the College of Life Science, Northwest A&F University. The plant materials were washed thoroughly with water and oven-dried at 45◦ C for 48 h, then they were crushed and reduced to a fine powder using a strainer (30–40 mesh) with an electrical disintegrator (model FW177, Tianjin Taisite Instrument Company). The powdered samples were freeze-dried at −45◦ C to ensure complete removal of water. Screening experiment.—A 50-g dry powder sample of each of 24 different plants was extracted three times with methanol (500 mL) for 48 h. To get more or less solidified crude extracts, the methanol filtrates were separately filtered and evaporated under reduced pressure in a vacuum rotary evaporator (model R-201, Shanghai Shenshen) until the solvents were completely evaporated. The resulting extracts of different plants were dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled water to obtain 0.6 g/mL (sample/solvent) of stock solutions, which were used for the preparations of the desired concentrations for the anthelmintic efficacy assay. An in vivo study was designed to determine the anthelmintic activity of extracts to D. intermedius according to the previous method of Ji et al. (2012). Five fish previously infected with the parasite were placed in plastic basins (N = 120) of 5 L capacity that were filled with 2 L of aerated groundwater. Extracts with designed concentration gradients of 100, 200, 300, 400, 500, and 600 mg/L were added to the basins. Negative control groups containing no plant extract were set up under the same conditions as used for the test groups. Another control containing the highest percentage of DMSO was included to assess the possible effects of DMSO on the parasites. After 48 h, the surviving fish in all of the treatment and control groups were killed by spinal severance and biopsied under a light microscope at 40 × magnification (Table 1). Experiment with selected anthelmintic plants.—Through previous experiments, material from four plant (Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum) that had 100% anthelmintic efficacy were selected from 24 different plants. Each plant material sample (50 g) was extracted with petroleum ether, chloroform, ethyl acetate, methanol, or water for 48 h for complete extraction, and the process was repeated three times. The ratio of sample to solvent was 1:10 (w/v). All the extracts were filtered, combined, and evaporated under reduced pressure in a vacuum rotary evaporator (model R-201, Shanghai Shenshen). The resulting extracts of different plants were dissolved in DMSO and diluted with distilled water to obtain 0.6 g/mL (sample/solvent) of stock solutions, which were used for the assay. Tests were conducted in each plastic basin (N = 100) (5 L capacity) that were filled with 2 L aerated groundwater. Each basin contained plant extract samples and five previously infected fish. Water pH ranged from 7 to 7.5, dissolved oxygen was between 6.2 and 7.8 g/mL (72–85% saturation), and the water temperature was constant at 24 ± 1◦ C. The negative control groups containing no plant extract were set up under the same
129
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
TABLE 1. Plants used in this study, plant part used, highest anthelmintic efficacy, concentration of the highest anthelmintic efficacy treatment, and concentration at which Goldfish died with Dactylogyrus intermedius infection. NA = not analyzed.
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
Species Polygonum multiflorum Thunb. Asparagus cochin-chinensis (Lour.) Merr. Sinapis alba L. Piper kadsura (Choisy) Ohwi. Aucklandia lappa Decne. Notopterygium incisum Ting ex H. T. Chang Lycium chinense Mill. Citrus medica L. Abrus cantoniensis Hance Perilla frutescens (L.) Britt. var. crispa (Thumb.) Hand.-Mand.-Mazz. Calvatia lilacina (Mont. et Berk.) Lloyd Typha angustifolia L. Buddleja officinalis Maxim. Platycladus orientalis (L.) Franco. Ilex pubescens Hook. et Arn. Citrus reticulata Blanco Gynostemma pentaphyllum (Thunb.) Makino Dioscorea collettii Hook. f. var. hypoglauca (Palibin) C. Pei and C. T. Ting Trichosanthes kirilowii Maxim., T. roswthornii Harms Dalbergia odorifera T. Chen Arnebia euchroma (Royle) Johnst. Aster tataricus L. F. Amomum villosum Lour. Morus alba L.
Best anthelmintic efficacy (%)
Concentration having the best anthelmintic efficacy (mg/L)
100 0
60 NA
125 500
30 0 0 0
500 NA NA NA
500 30 30 15
Root bark Fruit Herb Leaf
0 100 100 90
NA 250 200 350
400 300 250 400
Sporocarp
89
500
>1,000
Pollen Alabastrum Leaf
83 96 0
25 80 NA
30 125 125
Leaf and root Pericarp of immature fruit Rhizome
60 79
500 400
>1,000 500
70
450
500
100
45
Root
88
500
>1,000
Trunk and root Root
70 58
25 60
30 125
Root Fruit Twig
0 76 64
NA 230 225
>800 240 250
Plant part used for extract Stem Root tuber Seed Rattan Root Root
Rhizome
conditions as the test groups and included the DMSO control group. During the experiments, no food was offered to the fish. The death of each fish was recorded when opercular movement and tail beat stopped and the fish no longer responded to physical stimulus. To avoid deterioration of the water quality, the observed dead fish were removed from the water immediately. After 48 h, the surviving fish in all the treatments were killed by spinal severance and biopsied under a light microscope at 40 × magnification. Anthelmintic efficacy of each treatment and the negative control group was calculated according to the
Concentration at which Goldfish died (mg/L)
62.5
following formula: AE = (B − T )/B × 100, where AE is anthelmintic efficacy, B is the average number of surviving Dactylogyrus intermedius in the negative control, and T is the average number of surviving D. intermedius in the treatment groups.
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
130
HU ET AL.
FIGURE 1. Anthelmintic efficacy of different extracts of Abrus cantoniensis against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract; star indicates when fish mortality firstly occurred.
Acute toxicity test.—Aqueous static renewal 48-h bioassays were conducted to determine the acute toxicity of crude extracts to Goldfish. Healthy Goldfish were placed into several 5-L plastic basins (10 fish/basin) and crude extract concentrations for Goldfish- were added (a preliminary study had been performed to establish the concentrations [ranging from 0% to 100%] at which mortalities occurred). Control groups were set using the same test conditions but without extracts. Another control group containing the highest percentage of DMSO was also included. Mortality was observed in each aquarium every day. The fish were not fed during the exposure. This test was conducted two times. Statistical analysis.—The homogeneity of the replicates of the samples was checked by the Mann–Whitney U-test. Probit analysis was used for calculating the median lethal concentrations resulting in 50% and 90% mortality (LC50 and LC90) and the median effective concentrations resulting in 50% and 90% of its maximal effect (EC50 and EC90) and included the
95% CI with upper and lower confidence limits (Finney 1971). Since a chi-square test for goodness of fit was significant, a heterogeneity factor was used in the calculation of confidence limits. RESULTS The results of an in vivo study on the anthelmintic efficacies of selected plants against D. intermedius are shown in Table 1. Among the screened plants, A. cantoniensis, C. medica, D. collettii, and P. multiflorum had 100% anthelmintic efficacy at the concentrations of 200, 150, 45, and 60 mg/L. As a control the solvent DMSO showed no anthelmintic activity when used at the highest concentration. The anthelmintic efficacies of different extracts of A. cantoniensis, C. medica, D. collettii, and P. multiflorum are depicted in Figures 1–4, and the EC50 and EC90 values are shown in Table 2. The water extract of P. multiflorum was the most effective
FIGURE 2. Anthelmintic efficacy of different extracts of Citrus medica against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract; star indicates when fish mortality firstly occurred.
131
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
TABLE 2. Anthelmintic efficacy (EC50 and EC90 concentrations) of extracts from Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum against Dactylogyrus intermedius after 48 h of exposure. LCL and UCL are lower and upper 95% confidence limits.
Plants A. cantoniensis C. medica
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
D. collettii
P. multiflorum
Extraction solvent
EC50 (mg/L) (LCL–UCL)
EC90 (mg/L) (LCL–UCL)
χ2-value
P-value
Methanol Ethyl acetate Ethyl acetate Chloroform Methanol Ethyl acetate Chloroform Water Methanol Ethyl acetate
64.33 (57.23–71.23) 279.37 (264.03–294.14) 51.34 (26.13–62.96) 58.70 (55.89–61.44) 37.77 (7.36–74.07) 19.69 (18.10–21.29) 27.12 (15.81–33.84) 1.87 (12.47–7.70) 5.40 (4.70–6.10) 9.14 (6.12–11.64)
86.93 (78.96–99.93) 335.57 (317.04–367.93) 97.12 (84.21–128.60) 78.00 (74.14–83.22) 78.90 (54.60–273.52) 34.83 (32.23–38.23) 40.14 (33.45–55.17) 27.03 (19.76–48.67) 7.10 (6.35–8.73) 16.51 (13.40–26.57)
0.41 3.22 0.64 1.77 6.50 0.004 3.95 9.16 0 0.1
0.94 0.20 0.72 0.41 0.04 1.00 0.14 0.06 1.00 0.95
FIGURE 3. Anthelmintic efficacy of different extracts of Dioscorea collettii against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract star indicates when fish mortality firstly occurred.
FIGURE 4. Anthelmintic efficacy of different extracts of Polygonum multiflorum against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract; star indicates when fish mortality firstly occurred.
132
HU ET AL.
TABLE 3. Acute toxicity (LC50 and LC90 concentrations) of extracts from Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum to Goldfish during a 48-h exposure. LCL and UCL are lower and upper 95% confidence limits.
Plants A. cantoniensis C. medica D. collettii
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
P. multiflorum
Extraction solvent
LC50 (mg/L) (LCL–UCL)
LC90 (mg/L) (LCL–UCL)
χ2-value
P-value
Methanol Ethyl acetate Ethyl acetate Chloroform Methanol Ethyl acetate Chloroform Water Methanol Ethyl acetate
338.33 (308.58–361.83) 546.48 (474.11–593.59) 134.60 (103.37–152.50) 145.19 (62.74–194.06) 221.83 (192.97–243.57) 103.33 (82.79–111.96) 87.87 (83.64–92.21) 218.70 (1.35–273.99) 100.27 (39.84–171.66) 64.52 (32.00–87.01)
387.30 (363.40–455.15) 643.30 (595.60–816.77) 158.07 (144.84–323.13) 249.55 (199.18–443.33) 261.50 (240.66–334.07) 119.55 (111.14–161.50) 95.06 (91.08–106.82) 349.14 (290.51–709.80) 281.29 (198.81–740.15) 100.00 (80.16–196.04)
1.34 0.47 0 0.71 0.09 0.02 0.31 1.08 1.06 0
0.25 0.50 1.00 0.40 0.77 0.88 0.58 0.30 0.59 1.00
with EC50 and EC90 values of 1.9 and 27 mg/L, respectively. After a 48-h exposure, this extract exhibited 100% efficacy against D. intermedius at 100 mg/L. High anthelmintic activity against D. intermedius was also observed for the methanol and ethyl-acetate extracts with EC50 and EC90 values of 5.4 and 7.1 mg/L (methanol), and 9.1 and 16.5 mg/L (ethyl acetate), respectively. The chloroform and petroleum ether extracts, however, exhibited weak activity with the maximum anthelmintic efficacy of 81.8% and 77%, respectively, at 100 mg/L. For D. collettii, the ethyl-acetate extract was the most effective, and had EC50 and EC90 values of 19.7 and 34.8 mg/L, respectively, after 48 h of treatment. The extracts of methanol and chloroform exhibited 100% anthelmintic efficacy at 120 and 80 mg/L, and had EC50 and EC90 values of 37.8 and 78.9 mg/L (methanol), and 27.1 and 40.1 mg/L (chloroform), respectively. However, the extracts with water showed no anthelmintic activity at 120 mg/L when the fish began to die. Fish mortality occurred when the concentration reached 100 mg/L for petroleum-ether extract, which exhibited 4.9% anthelmintic efficacy. The methanol and ethyl-acetate extracts of A. cantoniensis displayed the optimal anthelmintic activity and showed 100% efficacy at the doses of 200 and 400 mg/L, respectively, while the C. medica extracts with ethyl acetate and chloroform demonstrated equivalent efficacy at 100 and 125 mg/L, respectively. The EC50 and EC90 values for A. cantoniensis were 64.3 and 86.9 mg/L for the methanol extract, and were 279.4 and 335.6 mg/L for ethyl-acetate extract. The EC50 and EC90 values for ethyl-acetate and chloroform extracts of C. medica were 51.3 and 97.1 mg/L (ethyl acetate) and 58.7 and 78 mg/L (chloroform), respectively. The highest activity was found in the remaining extracts of these two plants before fish began to die. Water, chloroform, and petroleum-ether extracts of A. cantoniensis showed 87.4, 77, and 20% activity at 600, 90, and 600 mg/L, respectively, and water, methanol, and petroleum-ether extracts of C. medica exhibited 13.6, 76.5, and 0% activity at 400, 300, and 300 mg/L, respectively.
The results of the acute toxicity assay for methanol and ethylacetate extracts of A. cantoniensis, the ethyl-acetate and chloroform extracts of C. medica, the methanol, ethyl-acetate, and chloroform extracts of D. collettii, and the water, methanol, and ethyl-acetate extracts of P. multiflorum are summarized in Table 3. The results indicated thatthese extracts had low toxicity to Goldfish. The 48-h LC50 values of methanol extracts of A. cantoniensis, ethyl-acetate and methanol extracts of D. collettii, and the ethyl-acetate, methanol, and water extracts of P. multiflorum were approximately fivefold (A. cantoniensis), fiveand sixfold (D. collettii), and 7-, 20-, and 100-fold (P. multiflorum) higher than the corresponding EC50s, which had values of 338.3 (A. cantoniensis), 103.3 and 221.8 (D. collettii), and 64.5, 100.3, and 218.7 mg/L (P. multiflorum), respectively. Results of the acute toxicity tests are provided in the Appendix (Tables A.1–A.4). DISCUSSION Dactylogyrus, a genus of gill parasites that causes serious damage to fish, has become a common parasite in Chinese aquaculture (Ogawa 2002). Traditionally, a number of effective chemotherapeutic agents for the treatment of Dactylogyrus disease in aquaculture have been gradually prohibited because of their side effects, such as accumulation of drugs in tissues, development of drug resistance, and the potential deleterious effects on the environment and human consumers (e.g., formalin, malachite green). Therefore, it is logical to look for novel aquaculture medicines that are both environmentally friendly and highly efficient. Extracts from medicinal plants that could offer possible alternatives to control D. intermedius infection have been reported (Liu et al. 2010; Wu et al. 2011; Ji et al. 2012; Lu et al. 2012). In the present study, 24 medicinal plants were evaluated for the in vivo anthelmintic activity against D. intermedius (Monogenea) in Goldfish. Extracts from four plants—A. cantoniensis, C. medica, D. collettii, and P. multiflorum—resulted in 100% parasite elimination rate at low concentrations. As far
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
as we know, this is the first report on anthelmintic activity of A. cantoniensis, C. medica, D. collettii, and P. multiflorum. Among the four potent plants, extracts from P. multiflorum exhibited the strongest efficacy with the lowest EC50 and EC90 values. Dried stem of P. multiflorum, known as Ye Jiao Teng, is one of the most popular traditional medicinal herbs in China and is officially listed in the Chinese Pharmacopoeia (China Pharmacopoeia Committee 2004). We have previously reported that the root of P. multiflorum showed no anthelmintic activity against D. intermedius (Ji et al. 2012). Wong et al. (2006) found that the water extract from the stem of P. multiflorum showed a potent antioxidant activity and might exhibit a greater antioxidant-mediated, anti-aging effect than its extract from the root. This finding may explain the high anthelmintic activity from the extract of the stem and the lack of activity from the extract from the root of this plant. Stems of P. multiflorum also have a history of use in the treatment of traumatic arthritis, aching of the loins and knees, and traumatic injuries (Tu et al. 1992). Recently, Wong et al. (2006) and Li et al. (2007) found that P. multiflorum has the highest antioxidant capacity because of their rich phenolic content (including gallic acid, catechin, and stilbene glycosides). Additionally, it has been suggested that 2,3,5,4 -tetra-hydroxystilbene-2-O-β-D-glucoside, which is isolated from the aqueous extract of P. multiflorum, could be considered, among future therapeutic drugs, for the treatment of Alzheimer’s disease (Um et al. 2006; Zhang et al. 2006). Considering the major bioactive constituents of P. multiflorum, some of the substances mentioned above may contribute to the efficacy of this extract against D. intermedius independently or in combination. The rhizome of D. collettii is a traditional Chinese herb used for the treatment of cervical carcinoma, carcinoma of the urinary bladder, and renal tumor in China. He et al. (2006) reported that methyl protodioscin, a furostanol saponin, is a preclinical drug that has potent antiproliferative activities against most cell lines from leukemia and solid tumors. Kobayashi et al. (1996) showed that 14 steroidal saponins, which were isolated from the rhizome of D. collettii, performed antifungal activity inducing morphological deformation of mycelia and conidia of Pyricularia oryzae (a phytopathogenic fungus responsible for rice blast). Among them, 11 steroidal saponins showed cytotoxicity against the human acute myeloid leukemia K562 (AML) cell line in vitro (Hu et al. 1996, 1997). Meanwhile, according to our previous study, steroidal saponins from Paris polyphylla exhibited high anthelmintic activity (Wang et al. 2010a). Accordingly, anthelmintic activity of the methanol extract of D. collettii in this study might be related to the presence of steroidal saponins. Essien et al. (2008) reported that the essential oil of C. medica exhibited a wide spectrum of fungitoxicity (inhibiting 14 isolated fungus species) at the minimum inhibitory concentration of 500 ppm. Moreover, the n-hexane extract of C. medica has significant antioxidant activity, hypoglycaemic activity, and an anticholinesterase effect which may benefit the treatment of diabetes and Alzheimer’s disease (Conforti et al. 2007). Lota
133
et al. (1999) found that the oil of C. medica exhibited a high content of limonene (70.4%) and the proportions of geranial and neral were 14.4% and 7.6%, respectively. In our study, the chloroform extract of C. medica showed 100% activity at the concentration of 100 mg/L1 and had relatively low EC50 and EC90 values. Therefore, the main component limonene might be the anthelmintic compound in these plants. Although there have been no attempts to identify the anthelmintic compounds in this plant, some of the substances mentioned earlier are believed to contribute jointly or independently to its anthelmintic activity. Abrus cantoniensis, or Ji gu cao in Chinese, has long been used in southern China and southeastern Asia as an effective folk medicine for the treatment of infectious hepatitis (Kiangsu Hsin Yi Medical College 1977). According to Li et al. (2005), the ethanol extract from the aerial parts of A. cantoniensis exhibited the strongest growth inhibitions (minimum inhibitory concentration, ∼40 mg/mL) against six test strains of Helicobacter pylori among 30 Chinese herbal medicines. Meanwhile, the ethanol extracts of A. cantoniensis can have an obvious inhibitory effect on the hepatitis B surface antigen (HBsAg) and the hepatitis B e antigen (HBeAg) in serum and against Escherichia coli and Pseudomonas aeruginous (Cheng et al. 2006; Chen et al. 2009). Phytochemical results ascertained that A. cantoniensis contains triterpenic saponins, anthraquinones, alkaloids, and flavonoids (Xiao et al. 2002). The methanol extract that showed 100% anthelmintic activity at the concentration of 200 mgL in this study contains β-sitosterol, stigmasterol, tannins, anthraquinone glycosides, and saponin. Therefore, triterpenic saponins and alkaloids in the plant could be the anthelmintic substances since trierpenic saponins were the main compounds, and some of the alkaloids were found to be effective against D. intermedius in Goldfish (Wang et al. 2010c). The results of the acute toxicity assays for the extracts of A. cantoniensis, C. medica, D. collettii, and P. multiflorum indicated that these extracts had low toxicity to Goldfish. The 48-h LC50 values of these extracts were higher than the corresponding EC50 values. In summary, these four plants have the potential for the development of a novel therapy for the treatment against D. intermedius infection. However, more investigations, such as pharmacological evaluations before clinical trials, assessment of ecological risk posed by practical usage, and the detailed mechanisms of anthelmintic activity (i.e., against D. intermedius) must be performed. Further bioassay-guided isolation and purification of compounds responsible for the observed anthelmintic efficacy are in progress.
ACKNOWLEDGMENTS Authors Yang Hu and Jie Ji contributed equally to this work. This work was supported by the National High Technology Research and Development Program of China (863 Program; number 2011AA10A216), the National Natural Science Foundation of China (number 31072242), and the
134
HU ET AL.
National Training Programs of Innovation and Entrepreneurship for Undergraduates.
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
REFERENCES Bondad-Reantaso, M. G., R. P. Subasinghe, J. R. Arthur, K. Ogawa, S. Chinabut, R. Adlard, Z. L. Tan, and M. Shariff. 2005. Disease and health management in Asian aquaculture. Veterinary Parasitology 132:249–272. Buchmann, K., H. C. Slotved, and D. Dana. 1993. Epidemiology of gill parasite infections in Cyprinus carpio in Indonesia and possible control methods. Aquaculture 118:9–21. Chen, X, Y. Han, and P. Xu. 2009. The inhibitory effect of Abrus cantoniensis Hance on Hepatitis B virus in vitro. Herald of Medicine 28:418–420. Cheng, Y. K., Y. Chen, L. Wang, M. Li, L. L. Zhong, and L. R. Teng. 2006. Study on the antibacterial activities of the ethanol extracts of Abrus cantoniensis. Research and Practice on Chinese Medicines 2:39–42. China Pharmacopoeia Committee. 2004. Pharmacopoeia of the People’s Republic of China (the first division of 2005 edition). China Chemical Industry Press, Beijing. Conforti, F., G. A. Statti, R. Tundis, M. R. Loizzo, and F. Menichini. 2007. In vitro activities of Citrus medica L. cv. Diamante (Diamante citron) relevant to treatment of diabetes and Alzheimer’s disease. Phytotherapy Research 21:427–433. Essien, E. P., J. P. Essien, B. N. Ita, and G. A. Ebong. 2008. Physicochemical properties and fungitoxicity of the essential oil of Citrus medica L. against groundnut storage fungi. Turkish Journal of Botany 32:161–164. FAO (Food and Agriculture Organization of the United Nations). 2008. The state of world fisheries and aquaculture. FAO, Rome. Finney, D. J. 1971. Probit analysis, 3rd edition. Cambridge University Press, Cambridge, UK. Food Business Network. 2008. World aquaculture development prospects and international status of aquaculture for China. (In Chinese.) Available: http://www.21food.cn/html/news/12/260400.htm. (March 2011). Goven, B. A., and D. F. Amend. 1982. Mebendazole/trichlorfon combination: a new anthelmintic for removing monogenetic trematodes from fish. Journal of Fish Biology 20:373–378. Goven, B., J. Gilbert, and J. Gratzek. 1980. Apparent drug resistance to the organophosphate dimethyl (2, 2, 2-trichloro-1-hydroxyethyl) phosphonate by monogenetic trematodes. Journal of Wildlife Disease 16:343–346. He, X. J., A. M. Qiao, X. L. Wang, B. Liu, M. M. Jiang, L. N. Su, and X. S. Yao. 2006. Structural identification of methyl protodioscin metabolites in rats’ urine and their antiproliferative activities against human tumor cell lines. Steroids 71:828–833. Hu, K., A. J. Dong, X. S. Yao, H. Kobayashi, and S. Iwasaki. 1996. Antineoplastic agents. I. Three spirostanol glycosides from rhizomes of Dioscorea collettii var. hypoglauca, Planta Medica 62:573–575. Hu, K., A. J. Dong, X. S. Yao, H. Kobayashi, and S. Iwasaki 1997. A furostanol glycoside from rhizomes of Dioscorea collettii var. hypoglauca. Phytochemistry 44:161–165. Ji, J., C. Lu, Y. J. Kang, G. X. Wang, and P. Chen. 2012. Screening of 42 medicinal plants for in vivo anthelmintic activity against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus). Parasitology Research 111:97–104. Kiangsu Hsin Yi Medical College. 1977. Dictionary of Chinese drugs. Shanghai People’s Press, Shanghai. (In Chinese.) Klinger, R., and R. F. Floyd. 2002. Introduction to freshwater fish parasites. University of Florida, Institute of Food and Agricultural Science, Document CIR716, Gainesville. Kobayashi, H., M. Namikoshi, T. Yoshimoto, and T. Yokochi. 1996. A screening method for antimitotic and antifungal substances using conidia of Pyricularia oryzae, modification and application to tropical marine fungi. Journal of Antibiotics 49:873–879. Li, H. B., Y. Jiang, C. C. Wong, K. W. Cheng, and F. Chen. 2007. Evaluation of two methods for the extraction of antioxidants from medicinal plants. Analytical and Bioanalytical Chemistry 388:483–488.
Li, Y., C. Xu, Q. Zhang, J. Y. Liu, and R. X. Tan. 2005. In vitro anti-Helicobacter pylori action of 30 Chinese herbal medicines used to treat ulcer diseases. Journal of Ethnopharmacology 98:329–333. Liu, Y. T., F. Wang, G. X. Wang, J. Han, Y. Wang, and Y. H. Wang. 2010. In vivo anthelmintic activity of crude extracts of Radix angelicae pubescentis, Fructus bruceae, Caulis spatholobi, Semen aesculi, and Semen pharbitidis against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 106:1233–1239. Lota, M. L., D. De Rocca Serra, F. Tomi, J. M. Bessiere, and J. Casanova. 1999. Chemical composition of peel and leaf essential oils of Citrus medica L. and C. limonimedica Lush. Flavour and Fragrance Journal 14:161–166. Lu, C., H. Y. Zhang, J. Ji, and G. X. Wang. 2012. In vivo anthelmintic activity of Dryopteris crassirhizoma, Kochia scoparia, and Polygala tenuifolia against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 110:1085–1090. R Marshall, C. J. 1999. Use of Supaverm for the treatment of monogenean infestation in koi carp (Cyprinus carpio). Fish Veterinary Journal 4:33–37. Ogawa, K. 2002. Impacts of diclidophorid monogenean infections on fisheries in Japan. International Journal Parasitology 32:373–380. Reed, P., R. Francis-Floyd, R. Klinger, and D. Petty. 2009. Monogenean parasites of fish. University of Florida, Institute of Food and Agricultural Sciences Extension FA28, Gainesville. Available: http://edis.ifas.ufl.edu/FA033. (May 2009). Schmahl, G. 1993. Treatment of fish parasites 10. Effects of a new triazine derivative, HOE 092V, on Monogenea: a light and transmission electron microscopy study. Parasitology Research 79:559–566. Schmahl, G., and H. Mehlhorn. 1985. Treatment of fish parasites. 1. Praziquantel effective against Monogenea (Dactylogyrus vastator, Dactylogyrus extensus, Diplozoon paradoxum). Zeitschrift fur Parasitenkunde 71:727–737. Tu, G. S., Q. Z. Fang, and S. C. Yuan. 1992. Pharmacopoeia of the People’s Republic of China, English edition. Gaungdong Science and Technology Press, Guangzhou, China. Um, M. Y., W. H. Choi, J. Y. Aan, S. R. Kim, and T. Y. Ha. 2006. Protective effect of Polygonum multiflorum Thunb. onamyloid β-peptide 25–35 induced cognitive deficits in mice. Journal of Ethnopharmacology 104:144–148. Wang, G. X., J. Han, T. T. Feng, F. Y. Li, and B. Zhu. 2009. Bioassay-guided isolation and identification of active compounds from Fructus arctii against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 106:247–255. Wang, G. X., J. Han, L. W. Zhao, D. X. Jiang, Y. T. Liu, and X. L. Liu. 2010a. Anthelmintic activity of steroidal saponins from Paris polyphylla. Phytomedicine 17:1102–1105. Wang, G. X., D. X. Jiang, J. Li, J. Han, Y. T. Liu, and X. L. Liu. 2010b. Anthelmintic activity of steroidal saponins from Dioscorea zingiberensis C. H. Wright against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 107:1365–1371. Wang, G. X., Z. Zhou, C. Cheng, J. Y. Yao, and Z. W. Yang. 2008. Osthol and isopimpinellin from Fructus cnidii to control Dactylogyrus intermedius in Carassius auratus. Veterinary Parasitology 158:144–151. Wang, G. X., Z. Zhou, D. X. Jiang, J. Han, and J. F. Wang. 2010c. In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa (Maxim) Fedde against Dactylogyrus intermedius in Carassius auratus. Veterinary Parasitology 171:305–313. Wong, C. C., H. B. Li, K. W. Cheng, and F. Chen. 2006. A systematic survey of antioxidant activity of 30 Chinese medicinal plants using the ferric reducing antioxidant power assay. Food Chemistry 97:705–711. Wu, Z. F., B. Zhu, Y. Wang, C. Lu, and G. X. Wang. 2011. In vivo evaluation of anthelmintic potential of medicinal plant extracts against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 108:1557–1563. Xiao, P. G., D. P. Li, and S. L. Yang. 2002. Modern Chinese materia medica. Chemical Industry Press, Beijing. Zhang, L., Y. Xing, C. F. Ye, H. X. Ai, H. F. Wei, and L. Li. 2006. Learningmemory deficit with aging in APP transgenic mice of Alzheimer’s disease and intervention by using tetrahydroxystilbene glucoside. Behaviour Brain Research 173:246–254.
135
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
Appendix: Acute Toxicities of Plant Extracts to Goldfish
TABLE A.1.
Acute toxicity of different extracts of Abrus cantoniensis to Goldfish during 48 h exposure.
Number dead
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
Extraction solvent None None Ethyl acetate
Methanol
TABLE A.2.
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 500 600 700 300 360 400
10 10 10 10 10 10 10 10
0 0 1 3 1 0 0 7
0 0 2 0 5 0 4 3
0 0 0 4 4 2 2 0
100 100 70 30 0 80 40 0
Acute toxicity of different extracts of Citrus medica to Goldfish during 48 h exposure.
Number dead Extraction solvent None None Ethyl acetate
Chloroform
TABLE A.3.
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 125 150 250 100 200 300
10 10 10 10 10 10 10 10
0 0 0 0 0 0 2 6
0 0 0 2 9 3 5 3
0 0 3 6 1 0 0 1
100 100 70 20 0 70 30 0
Acute toxicity of different extracts of Dioscorea collettii to Goldfish during 48 h exposure.
Number dead Extraction solvent None None Ethyl acetate
Methanol
Chloroform
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 100 120 140 200 250 300 80 90 100
10 10 10 10 10 10 10 10 10 10 10
0 0 1 3 4 0 0 7 0 0 0
0 0 0 4 5 0 8 0 0 5 6
0 0 3 2 1 2 0 3 1 1 4
100 100 60 10 0 70 20 0 90 40 0
136 TABLE A.4.
HU ET AL. Acute toxicity of different extracts of Polygonum multiflorum to Goldfish during 48 h exposure.
Number dead Extraction solvent None None Ethyl acetate
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
Water
Methanol
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 50 100 200 200 300 400 50 100 200 400
10 10 10 10 10 10 10 10 10 10 10 10
0 0 3 5 3 0 0 0 0 0 0 4
0 0 0 4 3 0 1 9 2 4 3 3
0 0 0 0 4 5 6 1 1 2 4 3
100 100 70 10 0 50 30 0 70 40 30 0
Journal of Aquatic Animal Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uahh20
Screening Medicinal Plants for Use against Dactylogyrus intermedius (Monogenea) Infection in Goldfish a
b
b
b
b
c
Yang Hu , Jie Ji , Fei Ling , Yunhe Chen , Lin Lu , Qizhong Zhang & Gaoxue Wang a
ab
College of Science, Northwest A&F University, Yangling 712100, China
b
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China c
Institute of Hydrobiology, Jinan University, Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering Minister of Education, Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guangdong Higher Education Institutes, Guangzhou 510632, China Published online: 03 Jul 2014.
To cite this article: Yang Hu, Jie Ji, Fei Ling, Yunhe Chen, Lin Lu, Qizhong Zhang & Gaoxue Wang (2014) Screening Medicinal Plants for Use against Dactylogyrus intermedius (Monogenea) Infection in Goldfish, Journal of Aquatic Animal Health, 26:3, 127-136, DOI: 10.1080/08997659.2014.902872 To link to this article: http://dx.doi.org/10.1080/08997659.2014.902872
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 26:127–136, 2014 C American Fisheries Society 2014 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.902872
ARTICLE
Screening Medicinal Plants for Use against Dactylogyrus intermedius (Monogenea) Infection in Goldfish Yang Hu Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
College of Science, Northwest A&F University, Yangling 712100, China
Jie Ji, Fei Ling, Yunhe Chen, and Lin Lu College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Qizhong Zhang* Institute of Hydrobiology, Jinan University, Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering Minister of Education, Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guangdong Higher Education Institutes, Guangzhou 510632, China
Gaoxue Wang* College of Science, Northwest A&F University, Yangling 712100, China; and College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Abstract Methanol extracts of 24 traditional medicinal plants with potential anthelmintic activity against Dactylogyrus intermedius (Monogenea) in Goldfish Carassius auratus were investigated. Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum exhibited 100% activity and were selected for further evaluation by applying five solvents (petroleum ether, chloroform, ethyl acetate, methanol, and water) for the extraction of the samples, followed by an in vivo bioassay. Among the plants tested, water, methanol, and ethyl-acetate extracts of P. multiflorum showed the highest efficacies; EC50 values (median concentration that results in 50% of its maximal effect) were 1.9, 5.4, and 9.1 mg/L, respectively, and extracts showed 100% efficacy against Dactylogyrus intermedius at 100, 12.5, and 25 mg/L. This was followed by ethyl-acetate, chloroform, and methanol extracts of Dioscorea collettii, which demonstrated 100% efficacy at 80, 80, and 120 mg/L and had EC50 values of 19.7, 27.1, and 37.8 mg/L, respectively, after 48 h of exposure. Chloroform and ethyl-acetate extracts of C. medica, which exhibited 100% efficacy against Dactylogyrus intermedius at 100 and 125 mg/L, revealed similar activity and had EC50 values of 58.7 and 51.3 mg/L, respectively. The ethyl-acetate and methanol extracts of A. cantoniensis exhibited the lowest activity and had EC50 values of 279.4 and 64.3 mg/L. Acute toxicities of these active extracts were investigated on Goldfish for 48 h. The findings indicated that extracts of the four plants can be developed as a preferred natural antiparasitic for the control of D. intermedius.
According to the Food and Agriculture Organization of the United Nations (FAO 2008), global aquaculture has developed rapidly in recent years and grown at an annual rate of 6.4% from 2002 to 2006. In addition, aquaculture in developing countries will continuously grow in the next 10 years (Food Business Network 2008). China, the biggest developing country, is the
world’s largest producer of fish, which accounts for two-thirds of the global production in 2009 (FAO 2008), and thus plays a vital and decisive role in aquaculture. However, intensive fish production often results in increased incidences of diseases caused by viruses, bacteria, fungi, parasites, and other undiagnosed and emerging pathogens (Bondad-Reantaso et al. 2005).
*Corresponding authors: [email protected]; [email protected] Received June 15, 2013; accepted February 11, 2014
127
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
128
HU ET AL.
Dactylogyrus intermedius is an important monogenean parasite that invades the skin and gills of cyprinid fishes and thereby causes serious problems, such as gill inflammation, excessive mucous secretions, respiratory distress, and mixed infections with other parasites and secondary bacterial infections (Reed et al. 2009). Fish infected with D. intermedius can be treated effectively with a wide spectrum of parasiticides, such as formalin (Marshall 1999), trichlorfon (Goven and Amend 1982), a triazine derivative (Schmahl 1993), and the two most effective treatments in practice, praziquantel (Schmahl and Mehlhorn 1985) and mebendazole (Buchmann et al. 1993). However, these chemical parasiticides can present serious drawbacks through frequent use, including the threats of anthelmintic resistance, risk of residue, environmental contamination, and toxicity to the host (Goven et al. 1980; Klinger and Floyd 2002). Therefore, there is an urgent need for alternative therapies, including the use of natural products from medicinal plants, which are more environmentally acceptable and generally have lower toxicity than chemicals. Recently, medicinal plants and their extracts were tested for their ability to control D. intermedius, and different extracts from several medicinal plants had significant killing effects in vivo (Wang et al. 2009, 2010a, 2010b; Liu et al. 2010; Wu et al. 2011; Ji et al. 2012; Lu et al. 2012). In our research, we exploited the crude extracts of different medicinal plants extracted by five solvents (petroleum ether, chloroform, ethyl acetate, methanol, and water) for anthelmintic activity against D. intermedius in Goldfish Carassius auratus.
METHODS Parasite and host.—Goldfish weighing 5.8 ± 1.9 g (mean ± SD) and without any record of previous infestation with parasites were used throughout the study. All fish were collected from a Changxing fish farm, Xianyang, Shaanxi, China (34◦ 32 15 N, 108◦ 51 8 E). The fish were then acclimated in glass aquaria (200 fish per aquarium) containing 180 L groundwater at 25 ± 1◦ C (controlled by automatic aquarium heater) with aeration for 7 d and were fed once daily at 2% of their body weight with commercial pellet feed. One week later, all the fish were cohabitated with fish infected with D. intermedius, which were retained in our laboratory; the infection procedure was described in our previous study (Wang et al. 2008). Three weeks later, 10 fish were randomly sampled and killed by spinal severance, and the biopsies of eight gill filaments of each fish were used to determine the infestation level and intensity of adult D. intermedius under a light microscope (Olympus BX41, Tokyo, Japan) at 40 × magnification. Fish were chosen for the assays when the infection rate was 100% and the mean number of D. intermedius on gills was 40–50 parasites per fish. Preparation of plant materials.—The plant materials from each of the selected species (Table 1) were collected in September 2012 and the taxonomic identification was made by X. P. Song, Northwest A&F University, Shaanxi. The voucher spec-
imens have been deposited at the herbarium of the College of Life Science, Northwest A&F University. The plant materials were washed thoroughly with water and oven-dried at 45◦ C for 48 h, then they were crushed and reduced to a fine powder using a strainer (30–40 mesh) with an electrical disintegrator (model FW177, Tianjin Taisite Instrument Company). The powdered samples were freeze-dried at −45◦ C to ensure complete removal of water. Screening experiment.—A 50-g dry powder sample of each of 24 different plants was extracted three times with methanol (500 mL) for 48 h. To get more or less solidified crude extracts, the methanol filtrates were separately filtered and evaporated under reduced pressure in a vacuum rotary evaporator (model R-201, Shanghai Shenshen) until the solvents were completely evaporated. The resulting extracts of different plants were dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled water to obtain 0.6 g/mL (sample/solvent) of stock solutions, which were used for the preparations of the desired concentrations for the anthelmintic efficacy assay. An in vivo study was designed to determine the anthelmintic activity of extracts to D. intermedius according to the previous method of Ji et al. (2012). Five fish previously infected with the parasite were placed in plastic basins (N = 120) of 5 L capacity that were filled with 2 L of aerated groundwater. Extracts with designed concentration gradients of 100, 200, 300, 400, 500, and 600 mg/L were added to the basins. Negative control groups containing no plant extract were set up under the same conditions as used for the test groups. Another control containing the highest percentage of DMSO was included to assess the possible effects of DMSO on the parasites. After 48 h, the surviving fish in all of the treatment and control groups were killed by spinal severance and biopsied under a light microscope at 40 × magnification (Table 1). Experiment with selected anthelmintic plants.—Through previous experiments, material from four plant (Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum) that had 100% anthelmintic efficacy were selected from 24 different plants. Each plant material sample (50 g) was extracted with petroleum ether, chloroform, ethyl acetate, methanol, or water for 48 h for complete extraction, and the process was repeated three times. The ratio of sample to solvent was 1:10 (w/v). All the extracts were filtered, combined, and evaporated under reduced pressure in a vacuum rotary evaporator (model R-201, Shanghai Shenshen). The resulting extracts of different plants were dissolved in DMSO and diluted with distilled water to obtain 0.6 g/mL (sample/solvent) of stock solutions, which were used for the assay. Tests were conducted in each plastic basin (N = 100) (5 L capacity) that were filled with 2 L aerated groundwater. Each basin contained plant extract samples and five previously infected fish. Water pH ranged from 7 to 7.5, dissolved oxygen was between 6.2 and 7.8 g/mL (72–85% saturation), and the water temperature was constant at 24 ± 1◦ C. The negative control groups containing no plant extract were set up under the same
129
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
TABLE 1. Plants used in this study, plant part used, highest anthelmintic efficacy, concentration of the highest anthelmintic efficacy treatment, and concentration at which Goldfish died with Dactylogyrus intermedius infection. NA = not analyzed.
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
Species Polygonum multiflorum Thunb. Asparagus cochin-chinensis (Lour.) Merr. Sinapis alba L. Piper kadsura (Choisy) Ohwi. Aucklandia lappa Decne. Notopterygium incisum Ting ex H. T. Chang Lycium chinense Mill. Citrus medica L. Abrus cantoniensis Hance Perilla frutescens (L.) Britt. var. crispa (Thumb.) Hand.-Mand.-Mazz. Calvatia lilacina (Mont. et Berk.) Lloyd Typha angustifolia L. Buddleja officinalis Maxim. Platycladus orientalis (L.) Franco. Ilex pubescens Hook. et Arn. Citrus reticulata Blanco Gynostemma pentaphyllum (Thunb.) Makino Dioscorea collettii Hook. f. var. hypoglauca (Palibin) C. Pei and C. T. Ting Trichosanthes kirilowii Maxim., T. roswthornii Harms Dalbergia odorifera T. Chen Arnebia euchroma (Royle) Johnst. Aster tataricus L. F. Amomum villosum Lour. Morus alba L.
Best anthelmintic efficacy (%)
Concentration having the best anthelmintic efficacy (mg/L)
100 0
60 NA
125 500
30 0 0 0
500 NA NA NA
500 30 30 15
Root bark Fruit Herb Leaf
0 100 100 90
NA 250 200 350
400 300 250 400
Sporocarp
89
500
>1,000
Pollen Alabastrum Leaf
83 96 0
25 80 NA
30 125 125
Leaf and root Pericarp of immature fruit Rhizome
60 79
500 400
>1,000 500
70
450
500
100
45
Root
88
500
>1,000
Trunk and root Root
70 58
25 60
30 125
Root Fruit Twig
0 76 64
NA 230 225
>800 240 250
Plant part used for extract Stem Root tuber Seed Rattan Root Root
Rhizome
conditions as the test groups and included the DMSO control group. During the experiments, no food was offered to the fish. The death of each fish was recorded when opercular movement and tail beat stopped and the fish no longer responded to physical stimulus. To avoid deterioration of the water quality, the observed dead fish were removed from the water immediately. After 48 h, the surviving fish in all the treatments were killed by spinal severance and biopsied under a light microscope at 40 × magnification. Anthelmintic efficacy of each treatment and the negative control group was calculated according to the
Concentration at which Goldfish died (mg/L)
62.5
following formula: AE = (B − T )/B × 100, where AE is anthelmintic efficacy, B is the average number of surviving Dactylogyrus intermedius in the negative control, and T is the average number of surviving D. intermedius in the treatment groups.
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
130
HU ET AL.
FIGURE 1. Anthelmintic efficacy of different extracts of Abrus cantoniensis against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract; star indicates when fish mortality firstly occurred.
Acute toxicity test.—Aqueous static renewal 48-h bioassays were conducted to determine the acute toxicity of crude extracts to Goldfish. Healthy Goldfish were placed into several 5-L plastic basins (10 fish/basin) and crude extract concentrations for Goldfish- were added (a preliminary study had been performed to establish the concentrations [ranging from 0% to 100%] at which mortalities occurred). Control groups were set using the same test conditions but without extracts. Another control group containing the highest percentage of DMSO was also included. Mortality was observed in each aquarium every day. The fish were not fed during the exposure. This test was conducted two times. Statistical analysis.—The homogeneity of the replicates of the samples was checked by the Mann–Whitney U-test. Probit analysis was used for calculating the median lethal concentrations resulting in 50% and 90% mortality (LC50 and LC90) and the median effective concentrations resulting in 50% and 90% of its maximal effect (EC50 and EC90) and included the
95% CI with upper and lower confidence limits (Finney 1971). Since a chi-square test for goodness of fit was significant, a heterogeneity factor was used in the calculation of confidence limits. RESULTS The results of an in vivo study on the anthelmintic efficacies of selected plants against D. intermedius are shown in Table 1. Among the screened plants, A. cantoniensis, C. medica, D. collettii, and P. multiflorum had 100% anthelmintic efficacy at the concentrations of 200, 150, 45, and 60 mg/L. As a control the solvent DMSO showed no anthelmintic activity when used at the highest concentration. The anthelmintic efficacies of different extracts of A. cantoniensis, C. medica, D. collettii, and P. multiflorum are depicted in Figures 1–4, and the EC50 and EC90 values are shown in Table 2. The water extract of P. multiflorum was the most effective
FIGURE 2. Anthelmintic efficacy of different extracts of Citrus medica against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract; star indicates when fish mortality firstly occurred.
131
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
TABLE 2. Anthelmintic efficacy (EC50 and EC90 concentrations) of extracts from Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum against Dactylogyrus intermedius after 48 h of exposure. LCL and UCL are lower and upper 95% confidence limits.
Plants A. cantoniensis C. medica
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
D. collettii
P. multiflorum
Extraction solvent
EC50 (mg/L) (LCL–UCL)
EC90 (mg/L) (LCL–UCL)
χ2-value
P-value
Methanol Ethyl acetate Ethyl acetate Chloroform Methanol Ethyl acetate Chloroform Water Methanol Ethyl acetate
64.33 (57.23–71.23) 279.37 (264.03–294.14) 51.34 (26.13–62.96) 58.70 (55.89–61.44) 37.77 (7.36–74.07) 19.69 (18.10–21.29) 27.12 (15.81–33.84) 1.87 (12.47–7.70) 5.40 (4.70–6.10) 9.14 (6.12–11.64)
86.93 (78.96–99.93) 335.57 (317.04–367.93) 97.12 (84.21–128.60) 78.00 (74.14–83.22) 78.90 (54.60–273.52) 34.83 (32.23–38.23) 40.14 (33.45–55.17) 27.03 (19.76–48.67) 7.10 (6.35–8.73) 16.51 (13.40–26.57)
0.41 3.22 0.64 1.77 6.50 0.004 3.95 9.16 0 0.1
0.94 0.20 0.72 0.41 0.04 1.00 0.14 0.06 1.00 0.95
FIGURE 3. Anthelmintic efficacy of different extracts of Dioscorea collettii against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract star indicates when fish mortality firstly occurred.
FIGURE 4. Anthelmintic efficacy of different extracts of Polygonum multiflorum against Dactylogyrus intermedius after 48 h. PEE = petroleum ether extract, CLE = chloroform extract, EAE = ethyl-acetate extract, MEE = methanol extract, WAE = water extract; star indicates when fish mortality firstly occurred.
132
HU ET AL.
TABLE 3. Acute toxicity (LC50 and LC90 concentrations) of extracts from Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum to Goldfish during a 48-h exposure. LCL and UCL are lower and upper 95% confidence limits.
Plants A. cantoniensis C. medica D. collettii
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
P. multiflorum
Extraction solvent
LC50 (mg/L) (LCL–UCL)
LC90 (mg/L) (LCL–UCL)
χ2-value
P-value
Methanol Ethyl acetate Ethyl acetate Chloroform Methanol Ethyl acetate Chloroform Water Methanol Ethyl acetate
338.33 (308.58–361.83) 546.48 (474.11–593.59) 134.60 (103.37–152.50) 145.19 (62.74–194.06) 221.83 (192.97–243.57) 103.33 (82.79–111.96) 87.87 (83.64–92.21) 218.70 (1.35–273.99) 100.27 (39.84–171.66) 64.52 (32.00–87.01)
387.30 (363.40–455.15) 643.30 (595.60–816.77) 158.07 (144.84–323.13) 249.55 (199.18–443.33) 261.50 (240.66–334.07) 119.55 (111.14–161.50) 95.06 (91.08–106.82) 349.14 (290.51–709.80) 281.29 (198.81–740.15) 100.00 (80.16–196.04)
1.34 0.47 0 0.71 0.09 0.02 0.31 1.08 1.06 0
0.25 0.50 1.00 0.40 0.77 0.88 0.58 0.30 0.59 1.00
with EC50 and EC90 values of 1.9 and 27 mg/L, respectively. After a 48-h exposure, this extract exhibited 100% efficacy against D. intermedius at 100 mg/L. High anthelmintic activity against D. intermedius was also observed for the methanol and ethyl-acetate extracts with EC50 and EC90 values of 5.4 and 7.1 mg/L (methanol), and 9.1 and 16.5 mg/L (ethyl acetate), respectively. The chloroform and petroleum ether extracts, however, exhibited weak activity with the maximum anthelmintic efficacy of 81.8% and 77%, respectively, at 100 mg/L. For D. collettii, the ethyl-acetate extract was the most effective, and had EC50 and EC90 values of 19.7 and 34.8 mg/L, respectively, after 48 h of treatment. The extracts of methanol and chloroform exhibited 100% anthelmintic efficacy at 120 and 80 mg/L, and had EC50 and EC90 values of 37.8 and 78.9 mg/L (methanol), and 27.1 and 40.1 mg/L (chloroform), respectively. However, the extracts with water showed no anthelmintic activity at 120 mg/L when the fish began to die. Fish mortality occurred when the concentration reached 100 mg/L for petroleum-ether extract, which exhibited 4.9% anthelmintic efficacy. The methanol and ethyl-acetate extracts of A. cantoniensis displayed the optimal anthelmintic activity and showed 100% efficacy at the doses of 200 and 400 mg/L, respectively, while the C. medica extracts with ethyl acetate and chloroform demonstrated equivalent efficacy at 100 and 125 mg/L, respectively. The EC50 and EC90 values for A. cantoniensis were 64.3 and 86.9 mg/L for the methanol extract, and were 279.4 and 335.6 mg/L for ethyl-acetate extract. The EC50 and EC90 values for ethyl-acetate and chloroform extracts of C. medica were 51.3 and 97.1 mg/L (ethyl acetate) and 58.7 and 78 mg/L (chloroform), respectively. The highest activity was found in the remaining extracts of these two plants before fish began to die. Water, chloroform, and petroleum-ether extracts of A. cantoniensis showed 87.4, 77, and 20% activity at 600, 90, and 600 mg/L, respectively, and water, methanol, and petroleum-ether extracts of C. medica exhibited 13.6, 76.5, and 0% activity at 400, 300, and 300 mg/L, respectively.
The results of the acute toxicity assay for methanol and ethylacetate extracts of A. cantoniensis, the ethyl-acetate and chloroform extracts of C. medica, the methanol, ethyl-acetate, and chloroform extracts of D. collettii, and the water, methanol, and ethyl-acetate extracts of P. multiflorum are summarized in Table 3. The results indicated thatthese extracts had low toxicity to Goldfish. The 48-h LC50 values of methanol extracts of A. cantoniensis, ethyl-acetate and methanol extracts of D. collettii, and the ethyl-acetate, methanol, and water extracts of P. multiflorum were approximately fivefold (A. cantoniensis), fiveand sixfold (D. collettii), and 7-, 20-, and 100-fold (P. multiflorum) higher than the corresponding EC50s, which had values of 338.3 (A. cantoniensis), 103.3 and 221.8 (D. collettii), and 64.5, 100.3, and 218.7 mg/L (P. multiflorum), respectively. Results of the acute toxicity tests are provided in the Appendix (Tables A.1–A.4). DISCUSSION Dactylogyrus, a genus of gill parasites that causes serious damage to fish, has become a common parasite in Chinese aquaculture (Ogawa 2002). Traditionally, a number of effective chemotherapeutic agents for the treatment of Dactylogyrus disease in aquaculture have been gradually prohibited because of their side effects, such as accumulation of drugs in tissues, development of drug resistance, and the potential deleterious effects on the environment and human consumers (e.g., formalin, malachite green). Therefore, it is logical to look for novel aquaculture medicines that are both environmentally friendly and highly efficient. Extracts from medicinal plants that could offer possible alternatives to control D. intermedius infection have been reported (Liu et al. 2010; Wu et al. 2011; Ji et al. 2012; Lu et al. 2012). In the present study, 24 medicinal plants were evaluated for the in vivo anthelmintic activity against D. intermedius (Monogenea) in Goldfish. Extracts from four plants—A. cantoniensis, C. medica, D. collettii, and P. multiflorum—resulted in 100% parasite elimination rate at low concentrations. As far
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
as we know, this is the first report on anthelmintic activity of A. cantoniensis, C. medica, D. collettii, and P. multiflorum. Among the four potent plants, extracts from P. multiflorum exhibited the strongest efficacy with the lowest EC50 and EC90 values. Dried stem of P. multiflorum, known as Ye Jiao Teng, is one of the most popular traditional medicinal herbs in China and is officially listed in the Chinese Pharmacopoeia (China Pharmacopoeia Committee 2004). We have previously reported that the root of P. multiflorum showed no anthelmintic activity against D. intermedius (Ji et al. 2012). Wong et al. (2006) found that the water extract from the stem of P. multiflorum showed a potent antioxidant activity and might exhibit a greater antioxidant-mediated, anti-aging effect than its extract from the root. This finding may explain the high anthelmintic activity from the extract of the stem and the lack of activity from the extract from the root of this plant. Stems of P. multiflorum also have a history of use in the treatment of traumatic arthritis, aching of the loins and knees, and traumatic injuries (Tu et al. 1992). Recently, Wong et al. (2006) and Li et al. (2007) found that P. multiflorum has the highest antioxidant capacity because of their rich phenolic content (including gallic acid, catechin, and stilbene glycosides). Additionally, it has been suggested that 2,3,5,4 -tetra-hydroxystilbene-2-O-β-D-glucoside, which is isolated from the aqueous extract of P. multiflorum, could be considered, among future therapeutic drugs, for the treatment of Alzheimer’s disease (Um et al. 2006; Zhang et al. 2006). Considering the major bioactive constituents of P. multiflorum, some of the substances mentioned above may contribute to the efficacy of this extract against D. intermedius independently or in combination. The rhizome of D. collettii is a traditional Chinese herb used for the treatment of cervical carcinoma, carcinoma of the urinary bladder, and renal tumor in China. He et al. (2006) reported that methyl protodioscin, a furostanol saponin, is a preclinical drug that has potent antiproliferative activities against most cell lines from leukemia and solid tumors. Kobayashi et al. (1996) showed that 14 steroidal saponins, which were isolated from the rhizome of D. collettii, performed antifungal activity inducing morphological deformation of mycelia and conidia of Pyricularia oryzae (a phytopathogenic fungus responsible for rice blast). Among them, 11 steroidal saponins showed cytotoxicity against the human acute myeloid leukemia K562 (AML) cell line in vitro (Hu et al. 1996, 1997). Meanwhile, according to our previous study, steroidal saponins from Paris polyphylla exhibited high anthelmintic activity (Wang et al. 2010a). Accordingly, anthelmintic activity of the methanol extract of D. collettii in this study might be related to the presence of steroidal saponins. Essien et al. (2008) reported that the essential oil of C. medica exhibited a wide spectrum of fungitoxicity (inhibiting 14 isolated fungus species) at the minimum inhibitory concentration of 500 ppm. Moreover, the n-hexane extract of C. medica has significant antioxidant activity, hypoglycaemic activity, and an anticholinesterase effect which may benefit the treatment of diabetes and Alzheimer’s disease (Conforti et al. 2007). Lota
133
et al. (1999) found that the oil of C. medica exhibited a high content of limonene (70.4%) and the proportions of geranial and neral were 14.4% and 7.6%, respectively. In our study, the chloroform extract of C. medica showed 100% activity at the concentration of 100 mg/L1 and had relatively low EC50 and EC90 values. Therefore, the main component limonene might be the anthelmintic compound in these plants. Although there have been no attempts to identify the anthelmintic compounds in this plant, some of the substances mentioned earlier are believed to contribute jointly or independently to its anthelmintic activity. Abrus cantoniensis, or Ji gu cao in Chinese, has long been used in southern China and southeastern Asia as an effective folk medicine for the treatment of infectious hepatitis (Kiangsu Hsin Yi Medical College 1977). According to Li et al. (2005), the ethanol extract from the aerial parts of A. cantoniensis exhibited the strongest growth inhibitions (minimum inhibitory concentration, ∼40 mg/mL) against six test strains of Helicobacter pylori among 30 Chinese herbal medicines. Meanwhile, the ethanol extracts of A. cantoniensis can have an obvious inhibitory effect on the hepatitis B surface antigen (HBsAg) and the hepatitis B e antigen (HBeAg) in serum and against Escherichia coli and Pseudomonas aeruginous (Cheng et al. 2006; Chen et al. 2009). Phytochemical results ascertained that A. cantoniensis contains triterpenic saponins, anthraquinones, alkaloids, and flavonoids (Xiao et al. 2002). The methanol extract that showed 100% anthelmintic activity at the concentration of 200 mgL in this study contains β-sitosterol, stigmasterol, tannins, anthraquinone glycosides, and saponin. Therefore, triterpenic saponins and alkaloids in the plant could be the anthelmintic substances since trierpenic saponins were the main compounds, and some of the alkaloids were found to be effective against D. intermedius in Goldfish (Wang et al. 2010c). The results of the acute toxicity assays for the extracts of A. cantoniensis, C. medica, D. collettii, and P. multiflorum indicated that these extracts had low toxicity to Goldfish. The 48-h LC50 values of these extracts were higher than the corresponding EC50 values. In summary, these four plants have the potential for the development of a novel therapy for the treatment against D. intermedius infection. However, more investigations, such as pharmacological evaluations before clinical trials, assessment of ecological risk posed by practical usage, and the detailed mechanisms of anthelmintic activity (i.e., against D. intermedius) must be performed. Further bioassay-guided isolation and purification of compounds responsible for the observed anthelmintic efficacy are in progress.
ACKNOWLEDGMENTS Authors Yang Hu and Jie Ji contributed equally to this work. This work was supported by the National High Technology Research and Development Program of China (863 Program; number 2011AA10A216), the National Natural Science Foundation of China (number 31072242), and the
134
HU ET AL.
National Training Programs of Innovation and Entrepreneurship for Undergraduates.
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
REFERENCES Bondad-Reantaso, M. G., R. P. Subasinghe, J. R. Arthur, K. Ogawa, S. Chinabut, R. Adlard, Z. L. Tan, and M. Shariff. 2005. Disease and health management in Asian aquaculture. Veterinary Parasitology 132:249–272. Buchmann, K., H. C. Slotved, and D. Dana. 1993. Epidemiology of gill parasite infections in Cyprinus carpio in Indonesia and possible control methods. Aquaculture 118:9–21. Chen, X, Y. Han, and P. Xu. 2009. The inhibitory effect of Abrus cantoniensis Hance on Hepatitis B virus in vitro. Herald of Medicine 28:418–420. Cheng, Y. K., Y. Chen, L. Wang, M. Li, L. L. Zhong, and L. R. Teng. 2006. Study on the antibacterial activities of the ethanol extracts of Abrus cantoniensis. Research and Practice on Chinese Medicines 2:39–42. China Pharmacopoeia Committee. 2004. Pharmacopoeia of the People’s Republic of China (the first division of 2005 edition). China Chemical Industry Press, Beijing. Conforti, F., G. A. Statti, R. Tundis, M. R. Loizzo, and F. Menichini. 2007. In vitro activities of Citrus medica L. cv. Diamante (Diamante citron) relevant to treatment of diabetes and Alzheimer’s disease. Phytotherapy Research 21:427–433. Essien, E. P., J. P. Essien, B. N. Ita, and G. A. Ebong. 2008. Physicochemical properties and fungitoxicity of the essential oil of Citrus medica L. against groundnut storage fungi. Turkish Journal of Botany 32:161–164. FAO (Food and Agriculture Organization of the United Nations). 2008. The state of world fisheries and aquaculture. FAO, Rome. Finney, D. J. 1971. Probit analysis, 3rd edition. Cambridge University Press, Cambridge, UK. Food Business Network. 2008. World aquaculture development prospects and international status of aquaculture for China. (In Chinese.) Available: http://www.21food.cn/html/news/12/260400.htm. (March 2011). Goven, B. A., and D. F. Amend. 1982. Mebendazole/trichlorfon combination: a new anthelmintic for removing monogenetic trematodes from fish. Journal of Fish Biology 20:373–378. Goven, B., J. Gilbert, and J. Gratzek. 1980. Apparent drug resistance to the organophosphate dimethyl (2, 2, 2-trichloro-1-hydroxyethyl) phosphonate by monogenetic trematodes. Journal of Wildlife Disease 16:343–346. He, X. J., A. M. Qiao, X. L. Wang, B. Liu, M. M. Jiang, L. N. Su, and X. S. Yao. 2006. Structural identification of methyl protodioscin metabolites in rats’ urine and their antiproliferative activities against human tumor cell lines. Steroids 71:828–833. Hu, K., A. J. Dong, X. S. Yao, H. Kobayashi, and S. Iwasaki. 1996. Antineoplastic agents. I. Three spirostanol glycosides from rhizomes of Dioscorea collettii var. hypoglauca, Planta Medica 62:573–575. Hu, K., A. J. Dong, X. S. Yao, H. Kobayashi, and S. Iwasaki 1997. A furostanol glycoside from rhizomes of Dioscorea collettii var. hypoglauca. Phytochemistry 44:161–165. Ji, J., C. Lu, Y. J. Kang, G. X. Wang, and P. Chen. 2012. Screening of 42 medicinal plants for in vivo anthelmintic activity against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus). Parasitology Research 111:97–104. Kiangsu Hsin Yi Medical College. 1977. Dictionary of Chinese drugs. Shanghai People’s Press, Shanghai. (In Chinese.) Klinger, R., and R. F. Floyd. 2002. Introduction to freshwater fish parasites. University of Florida, Institute of Food and Agricultural Science, Document CIR716, Gainesville. Kobayashi, H., M. Namikoshi, T. Yoshimoto, and T. Yokochi. 1996. A screening method for antimitotic and antifungal substances using conidia of Pyricularia oryzae, modification and application to tropical marine fungi. Journal of Antibiotics 49:873–879. Li, H. B., Y. Jiang, C. C. Wong, K. W. Cheng, and F. Chen. 2007. Evaluation of two methods for the extraction of antioxidants from medicinal plants. Analytical and Bioanalytical Chemistry 388:483–488.
Li, Y., C. Xu, Q. Zhang, J. Y. Liu, and R. X. Tan. 2005. In vitro anti-Helicobacter pylori action of 30 Chinese herbal medicines used to treat ulcer diseases. Journal of Ethnopharmacology 98:329–333. Liu, Y. T., F. Wang, G. X. Wang, J. Han, Y. Wang, and Y. H. Wang. 2010. In vivo anthelmintic activity of crude extracts of Radix angelicae pubescentis, Fructus bruceae, Caulis spatholobi, Semen aesculi, and Semen pharbitidis against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 106:1233–1239. Lota, M. L., D. De Rocca Serra, F. Tomi, J. M. Bessiere, and J. Casanova. 1999. Chemical composition of peel and leaf essential oils of Citrus medica L. and C. limonimedica Lush. Flavour and Fragrance Journal 14:161–166. Lu, C., H. Y. Zhang, J. Ji, and G. X. Wang. 2012. In vivo anthelmintic activity of Dryopteris crassirhizoma, Kochia scoparia, and Polygala tenuifolia against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 110:1085–1090. R Marshall, C. J. 1999. Use of Supaverm for the treatment of monogenean infestation in koi carp (Cyprinus carpio). Fish Veterinary Journal 4:33–37. Ogawa, K. 2002. Impacts of diclidophorid monogenean infections on fisheries in Japan. International Journal Parasitology 32:373–380. Reed, P., R. Francis-Floyd, R. Klinger, and D. Petty. 2009. Monogenean parasites of fish. University of Florida, Institute of Food and Agricultural Sciences Extension FA28, Gainesville. Available: http://edis.ifas.ufl.edu/FA033. (May 2009). Schmahl, G. 1993. Treatment of fish parasites 10. Effects of a new triazine derivative, HOE 092V, on Monogenea: a light and transmission electron microscopy study. Parasitology Research 79:559–566. Schmahl, G., and H. Mehlhorn. 1985. Treatment of fish parasites. 1. Praziquantel effective against Monogenea (Dactylogyrus vastator, Dactylogyrus extensus, Diplozoon paradoxum). Zeitschrift fur Parasitenkunde 71:727–737. Tu, G. S., Q. Z. Fang, and S. C. Yuan. 1992. Pharmacopoeia of the People’s Republic of China, English edition. Gaungdong Science and Technology Press, Guangzhou, China. Um, M. Y., W. H. Choi, J. Y. Aan, S. R. Kim, and T. Y. Ha. 2006. Protective effect of Polygonum multiflorum Thunb. onamyloid β-peptide 25–35 induced cognitive deficits in mice. Journal of Ethnopharmacology 104:144–148. Wang, G. X., J. Han, T. T. Feng, F. Y. Li, and B. Zhu. 2009. Bioassay-guided isolation and identification of active compounds from Fructus arctii against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 106:247–255. Wang, G. X., J. Han, L. W. Zhao, D. X. Jiang, Y. T. Liu, and X. L. Liu. 2010a. Anthelmintic activity of steroidal saponins from Paris polyphylla. Phytomedicine 17:1102–1105. Wang, G. X., D. X. Jiang, J. Li, J. Han, Y. T. Liu, and X. L. Liu. 2010b. Anthelmintic activity of steroidal saponins from Dioscorea zingiberensis C. H. Wright against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 107:1365–1371. Wang, G. X., Z. Zhou, C. Cheng, J. Y. Yao, and Z. W. Yang. 2008. Osthol and isopimpinellin from Fructus cnidii to control Dactylogyrus intermedius in Carassius auratus. Veterinary Parasitology 158:144–151. Wang, G. X., Z. Zhou, D. X. Jiang, J. Han, and J. F. Wang. 2010c. In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa (Maxim) Fedde against Dactylogyrus intermedius in Carassius auratus. Veterinary Parasitology 171:305–313. Wong, C. C., H. B. Li, K. W. Cheng, and F. Chen. 2006. A systematic survey of antioxidant activity of 30 Chinese medicinal plants using the ferric reducing antioxidant power assay. Food Chemistry 97:705–711. Wu, Z. F., B. Zhu, Y. Wang, C. Lu, and G. X. Wang. 2011. In vivo evaluation of anthelmintic potential of medicinal plant extracts against Dactylogyrus intermedius (Monogenea) in Goldfish (Carassius auratus). Parasitology Research 108:1557–1563. Xiao, P. G., D. P. Li, and S. L. Yang. 2002. Modern Chinese materia medica. Chemical Industry Press, Beijing. Zhang, L., Y. Xing, C. F. Ye, H. X. Ai, H. F. Wei, and L. Li. 2006. Learningmemory deficit with aging in APP transgenic mice of Alzheimer’s disease and intervention by using tetrahydroxystilbene glucoside. Behaviour Brain Research 173:246–254.
135
ANTHELMINTIC ACTIVITY OF MEDICINAL PLANTS
Appendix: Acute Toxicities of Plant Extracts to Goldfish
TABLE A.1.
Acute toxicity of different extracts of Abrus cantoniensis to Goldfish during 48 h exposure.
Number dead
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
Extraction solvent None None Ethyl acetate
Methanol
TABLE A.2.
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 500 600 700 300 360 400
10 10 10 10 10 10 10 10
0 0 1 3 1 0 0 7
0 0 2 0 5 0 4 3
0 0 0 4 4 2 2 0
100 100 70 30 0 80 40 0
Acute toxicity of different extracts of Citrus medica to Goldfish during 48 h exposure.
Number dead Extraction solvent None None Ethyl acetate
Chloroform
TABLE A.3.
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 125 150 250 100 200 300
10 10 10 10 10 10 10 10
0 0 0 0 0 0 2 6
0 0 0 2 9 3 5 3
0 0 3 6 1 0 0 1
100 100 70 20 0 70 30 0
Acute toxicity of different extracts of Dioscorea collettii to Goldfish during 48 h exposure.
Number dead Extraction solvent None None Ethyl acetate
Methanol
Chloroform
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 100 120 140 200 250 300 80 90 100
10 10 10 10 10 10 10 10 10 10 10
0 0 1 3 4 0 0 7 0 0 0
0 0 0 4 5 0 8 0 0 5 6
0 0 3 2 1 2 0 3 1 1 4
100 100 60 10 0 70 20 0 90 40 0
136 TABLE A.4.
HU ET AL. Acute toxicity of different extracts of Polygonum multiflorum to Goldfish during 48 h exposure.
Number dead Extraction solvent None None Ethyl acetate
Downloaded by [Bibliothèques de l'Université de Montréal] at 01:19 02 December 2014
Water
Methanol
Concentration (mg/L)
Total number tested
12 h
24 h
48 h
Survival (%)
Control DMSO (0.3%) 50 100 200 200 300 400 50 100 200 400
10 10 10 10 10 10 10 10 10 10 10 10
0 0 3 5 3 0 0 0 0 0 0 4
0 0 0 4 3 0 1 9 2 4 3 3
0 0 0 0 4 5 6 1 1 2 4 3
100 100 70 10 0 50 30 0 70 40 30 0
