Parasitol Res (2013) 112:2331–2340 DOI 10.1007/s00436-013-3397-0
ORIGINAL PAPER
Antiprotozoal screening of traditional medicinal plants: evaluation of crude extract of Psoralea corylifolia against Ichthyophthirius multifiliis in goldfish Fei Ling & Cheng Lu & Xiao Tu & Yanglei Yi & Aiguo Huang & Qizhong Zhang & Gaoxue Wang
Received: 9 December 2012 / Accepted: 12 March 2013 / Published online: 5 April 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Ichthyophthirius multifiliis (also called “ich”) is an external protozoan parasite that may infest almost all freshwater fish species and caused significant economic damage to the aquaculture industry. Since the use of malachite green was banned, there have been relatively few effective alternative strategies for controlling I. multifiliis infections. The present study was designed to screen potential antiparasitic medicinal plants based on our previous studies, and comprehensively evaluate in vitro and in vivo anti-ich activity of selected plant extracts. The screening results showed that the methanol extract of Psoralea corylifolia had the highest activity against I. multifiliis theronts. In vivo theront trials demonstrated that 1.25 mg/L or more concentrations of P. corylifolia methanol extract caused 100 % mortality during the 4-h exposure period, and the Electronic supplementary material The online version of this article (doi:10.1007/s00436-013-3397-0) contains supplementary material, which is available to authorized users. F. Ling : C. Lu : X. Tu : A. Huang : G. Wang (*) College of Animal Science and Technology, Northwest A&F University, Yangling 712100, People’s Republic of China e-mail:
[email protected] G. Wang e-mail:
[email protected] Y. Yi College of Science, Northwest A&F University, Yangling 712100, People’s Republic of China Q. Zhang 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, Institute of Hydrobiology, Jinan University, Guangzhou 510632, People’s Republic China
subsequent in vitro trials indicated that the minimum concentration of P. corylifolia methanol extract that prevented the initial infestation was 2.50 mg/L. Protomonts and encysted tomonts surviving trials suggested that encysted tomonts were less susceptible to P. corylifolia methanol extract than protomonts, and the methanol extract of P. corylifolia at a concentration of 5.00 mg/L could kill 100 % of protomonts and 88.89 % of encysted tomonts. It was also observed that after 12-h exposure of protomonts or encysted tomonts to 2.50 mg/L of P. corylifolia methanol extract, the theronts emerged from encysted tomonts led to more infection level than the ones in the other groups. The results suggested that whether the protomonts finish encystment is crucial to the survival, reproduction, and theronts infectivity. In addition, our results showed that long duration (24 h) and high concentration (5.00 mg/L) significantly reduced the survival and reproduction of I. multifiliis tomont exited from the fish after in-bath treatment, and it is indicated that P. corylifolia methanol extract had a potential detrimental effect on I. multifiliis trophont in situ.
Introduction The ciliate Ichthyophthirius multifiliis Fouquet, 1876, commonly called “ich,” is the main parasitic threat to freshwater teleosts occurring in both temperate and tropical regions throughout the world (Buchmann et al. 2001). The disease ichthyophthiriasis, caused by I. multifiliis, results in considerable economic damage to the aquaculture industry, including the freshwater ornamental fish trade, and epizootics in wild fish populations can cause mass kills (Matthews 2005). The life cycle of this parasite is direct and temperature dependent and consists of five key stages: a parasitic
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trophont (1) that resides within the epidermis of fish; a freeswimming protomont (2) which exits the fish and settles on the substrate to transform an encysted tomont (3) within which the parasite multiplies by binary fission; tomoties (4) which are released from the encysted tomont emerge into the water and subsequently differentiate into infective free-swimming theronts (5) (Matthews 2005; Shinn et al. 2012; Picón-Camacho et al. 2012). In the past years, malachite green has been used to treat ichthyophthiriasis successfully because of its high efficacy against both the free-living stage of the parasite and the feeding parasite stage within the epithelium of fish (Wahli et al. 1993; Tieman and Goodwin 2001; Buchmann et al. 2003; Picón-Camacho et al. 2012). However, the application of malachite green has been banned by some government agencies, e.g., the Food and Drug Administration of the USA and the European Union, due to its potential harmful impacts on human health and environment. The search for an alternative drug to control ichthyophthiriasis has been accelerated through the years. At present, some chemicals, such as copper sulfate (Ling et al. 1993; Schlenk et al. 1998), sodium chloride (Selosse and Rowland 1990), and potassium permanganate (Straus and Griffin 2002), which are aimed at interrupting the life cycle by killing the freeswimming stages of the parasite, play an important role in current strategies for controlling this disease in food fish, though these agents have limited efficacy in the trophont in situ (Matthews 2005). Accordingly, it is urgent to develop an effective and safe therapy for treating ichthyophthiriasis in fish since the use of malachite green was banned. Recently, a lot of research activities have been shown on the utilization of medicinal plants to control parasitic diseases in fish, and its demonstrable efficacy and low environmental hazard have aroused increasing concern. Raw extracts from garlic (Allium sativum) was used to prevent I. multifiliis infestation with a good efficacy because the extracts can kill freeliving theronts and tomonts (Buchmann et al. 2003). Ekanem et al. (2004) evaluated the effects of crude extracts from Mucuna pruriens and Carica papaya against I. multifiliis, and they observed that parasite-induced fish mortality was significantly reduced after treatment in baths of each plant extract. Ling et al. (2012) reported antiprotozoal activity of aqueous extract of Capsicum frutescens, which is readily available and affordable, against I. multifiliis under in vitro and in vivo conditions. However, the efficacy of these plant extracts may be limited unless they are applied at high dose. Applying these treatments at high dose is probably to cause harmful effects on the fish and their surroundings, or be prohibitively expensive. Therefore, it is necessary to obtain the possible alternatives that may both be sustainable and environmentally acceptable by screening and proper evaluation of the claimed medicinal plants (Eguale et al. 2007).
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Until now, our previous works have screened a lot of traditional medicinal plants for antiparasitic activity, and a part of them demonstrate strong anthelmintic efficacy against Dactylogyrus intermedius in goldfish (Wang et al. 2010; Liu et al. 2010; Wu et al. 2011; Lu et al. 2012; Ji et al. 2012); however, the main targets of the current work in our lab are to find some medicinal plants for multiply antiparasitic activity and to isolate active compounds using a bioactivity-guided fractionation from the plants. The purposes of this study were to: (1) screen 30 potential antiparasitic plants to get a plant extract with the highest antiich activity, (2) investigate the effect of the crude extract on the free-living stages of I. multifiliis, (3) assess in vivo efficacy of crude extract of selected plant to prevent an initial infestation of I. multifiliis theront, and (4) evaluate the efficacy of the extract on I. multifiliis theront infectivity and the trophonts in situ.
Materials and methods Fish Goldfish (Carassius auratus), weighting 3.27±0.78 g, were utilized throughout the study. All fish, referred as “naïve fish,” were kept in several 200-L aquariums equipped with outside aquarium filters and air stones (water temperature 20.0–22.0 °C, pH 7.0±0.3, dissolved oxygen 5.0–7.0 mg/L). They were fed once at 1 % body weight daily with commercial fish pellet feed. Parasite A local strain of I. multifiliis was isolated from Astronotus ocellatus, obtained from a pet shop (Xi’an, Shaanxi, China), and its passage was described as in Ling et al. (2009, 2010). The parasitized fish and healthy goldfish were held at 20.0– 22.0 °C in a static 200-L aquarium under the same conditions as described above. I. multifiliis protomonts were collected using a method described by Clayton and Price (1988) and Shinn et al. (2012). The heavily infected fish were placed into several beakers containing filtered aquarium water for 30 min (100 mL/fish). Mature parasites were quite freely dislodged from the host by body movements of the fish. These were either used immediately, or were incubated at 21.0–22.0 °C until they reached either the encysted tomont (i.e., after ∼6 h) or theront stage (i.e., after 20– 24 h). The theront concentration was determined by pipetting several microliter droplets of the theront suspension onto a glass slide and counting the organisms (×40 magnification); the mean count in ten droplets was extrapolated to determine the final concentration (Schlenk et al. 1998; Straus and Griffin 2001).
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Collection of plant materials and preparation of crude extracts A method for collection of plant materials and preparation of crude extracts was used according to Liu et al. (2010) and Yi et al. (2012). Fresh plant materials from each of selected species (Table 1) were collected in 2011. They were identified by Prof. X.P. Song (Northwest A&F University, Shaanxi, China), and voucher references have been deposited in the College of Science, Northwest A&F University, China. The plant materials were washed thoroughly, air-dried under the sunlight for a week, and finally oven-dried at 45 °C for 48 h. The dried plant materials were crushed manually with a mortar and pestle and reduced to fine powder using commercial electrical stainless steel blender (30–40 mesh). The powdered
samples were freeze-dried at −54 °C to ensure complete removal of water. The dry powder (100 g) of each species was extracted with 1,000 mL methanol for 48 h, and the process was repeated three times. Each extract was subsequently filtered and concentrated under reduced pressure in a vacuum rotary evaporator until the solvents were complete evaporated to get some solidified crude extracts. Each crude methanol extract was dissolved in 0.02 % dimethyl sulfoxide (DMSO) to get 500 mg/L (sample/solvent) of stocking solution for the next antiprotozoal efficacy assay. In vitro screening of plant extracts for antiprotozoal activity An in vitro trial was designed to ascertain the antiprotozoal activity against theronts according to an immobilization
Table 1 The tested medicinal plants and antiprotozoal efficacy of the methanol extracts against I. multifiliis theronts Species
Family
Part of the plant used
Antiprotozoal efficacy
Acanthopanax gracilistylus W.W. Smith Acanthopanax senticosus (Rupr. et Maxim.) Harms Aconitum carmichaeli Debx. Acorus tatarinowii Schott Alpinia officinarum Hance Arctium lappa L. Artemisia anomala S. Moore Aucklandia lappa Decen.
Arallaceae Arallaceae Ranunculaceae Araceae Zingiberaceae Compositae Compositae Compositae
Root bark Rhizome Root Rhizome Rhizome Root The whole Root
+ − − − − − − −
Bupleurum chinense DC. Citrus medica L. Citrus reticulata Blanco Crataegus pinnatifida Bge. Curculigo orchioides Gaertn. Dysosma versipellis (Hance.) M. Cheng Houttuynia cordata Thumb. Isatis indigotica Fort. Lilium brownii F.E.Brown var. viridulum Baker Magnolia biondii Pamp. Momordica cochinchinensis (Lour.) Spreng. Morus alba L. Polygonatum kingianum Coll.et Hemsl. Polygonum cuspidatum Sieb. et Zucc. Psoralea corylifolia L. Phytolacca acinosa Roxb. Raphanus sativus L. Sabina vulgaris Ant.
Umbelliferae Rutaceae Rutaceae Rosaceae Amaryllidaceae Berberidaceae Saururaceae Cruciferae Liliaceae Magnoliaceae Cucurbitaceae Moraceae Liliaceae Polygonaceae Leguminosae Phytolaccaceae Cruciferae Cupressaceae
Root Fruit Seed Fruit Root and stem Root and stem Shoot Leaf Scale leaf Flower bud Fruit Twig Rhizome Root and stem Fruit Root Seed Branch and leaf
− − + − − − − − + + − − − − ++ − − +
Salvia miltiorrhiza Bunge. Sophora tonkinensis Gapnep. Trichosanthes kirilowii Maxim. Zingiber officinale Rosc.
Labiatae Leguminosae Cucurbitaceae Zingiberaceae
Root and Stem Root and rhizome Fruit Rhizome
− − + −
− the extract at a concentration of 10.00 mg/L cannot kill 100 % I. multifiliis theronts by 4 h; + the extract at a concentration of 10.00 mg/L can kill 100 % I. multifiliis theronts by 4 h, but cannot kill 100 % I. multifiliis theronts at a concentration of 5.00 mg/L; ++ the extract at a concentration of 5.00 mg/L can kill 100 % I. multifiliis theronts by 4 h
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method (Straus and Griffin 2001; Buchmann et al. 2003; Ling et al. 2010). The theronts were placed into 96-well microtiter plates at a final concentration of 100 theronts per well with 100 μL of solution and exposed to 5.00 and 10.00 mg methanol extract/L, respectively. A positive control well with 0.02 % DMSO but no methanol extracts and a negative control well without any methanol extracts and DMSO were set up under the same conditions as the test wells. Antiprotozoal activity was assessed directly by microscopic examination (×40 magnification) after 4-h exposure. The theronts with the absence of motility and integrity were regarded as dead. The trial was performed at 21.0–22.0 °C and replicated three times using separate populations of theronts for each treatment. The methanol extract at a concentration of 5.00 mg/L with 100 % mortality after 4-h exposure was considered to have the highest antiprotozoal activity and picked out for the further experiment. Prevention of an initial infestation of I. multifiliis According to the method described above, a trial was conducted to access a comprehensive antiprotozoal activity of selected methanol extract against I. multifiliis theronts. One hundred theronts were exposed to the methanol extract (0.16, 0.31, 0.62, 1.25, 2.50, and 5.00 mg/L), and microscopic examination (×40 magnification) was used to determine the antiprotozoal activity of each well at various intervals up to 4 h after exposure. The trial was repeated three times. An experiment was adapted from the method of Straus and Griffin (2001) to determinate minimum effective concentration of the methanol extract used to prevent an initial infestation with I. multifiliis. The treatment container were a 9-L opaque plastic tank containing 3 L of filtered aquarium water and six goldfish; after 24-h exposure to theronts, the water level was increased to 6 L and an air stone was used to maintain enough dissolved oxygen. In the first trial, six goldfish were exposed to 36,000 theronts (6,000 theronts/fish) followed by immediate treatment with 0.31, 0.62, 1.25, and 2.50 mg/L of the methanol extract. The negative controls were set up for each replication (N=3). The trial was terminated when each goldfish in control groups developed visible I. multifiliis trophonts, and infection level (no. trophont on the fins per infected fish) was recorded. In the second trial, six goldfish were exposed to 72,000 theronts (12,000 theronts/fish) and treated in the same way as mentioned above. The experiment (trials 1 and 2) was carried out at 20.0–22.0 °C. In vivo antiprotozoal activity against I. multifiliis protomonts and encysted tomonts For the protomont trial, 30 protomonts were placed into each well (N=18) of a 24-well tissue culture plate. After
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carefully discarding the water in each well with a pipette, 1 mL the methanol extract at a concentration of 0.31, 0.62, 1.25, 2.50, 5.00, and 10.00 mg/L was added to each well (three wells per each concentration), respectively. The solutions were replaced by filtered aquarium water with no methanol extract after 12-h exposure. The 24well plate with parasites was incubated at 21.0–22.0 °C throughout the trial. The positive controls (N=3) and the positive controls (N=3) were built under the same condition as the tests. The trial was allowed to stop until the parasites in the controls reached the theront stage. The number of dead parasites was recorded by microscope examination (×40 magnification) of each well at 3 h interval up to the theront stage after the solutions were changed. The parasites with the absence of internal cell motility or abnormal cell division (including the ones cannot produce the theronts) were considered dead. In addition, at the end of this trial, the theronts in each well were enumerated as described above. The mortality and reproduction of tomonts were determined for each well according to Xu et al. (2008). The reproduction was represented as number of theronts released by each live tomont, calculated by total theronts/live tomont. For the encysted tomont trial, protomonts were collected and distributed as above. Until the parasites had produced a cyst coat, the water in each well was removed carefully by a pipette, and 1 mL solution with the methanol extract (0.31, 0.62, 1.25, 2.50, 5.00, and 10.00 mg/L) was added to each well. After 12-h exposure, the solutions were changed by filtered aquarium water with no methanol extract. The next process was the same as the protomont trial. Effect on infectivity of I. multifiliis theronts The experiment consisted of two trials. Trial 1 was conducted to determine infectivity of I. multifiliis theronts after 12-h exposure of protomonts to the methanol extract. Thousands of protomonts were placed into several Petri dishes and exposed to the methanol extract at a concentration of 0.63, 1.25, and 2.50 mg/L for 12 h, respectively. The solutions in the Petri dishes were discarded, and filtered aquarium water was added to the Petri dishes. Until the parasites reached the theront stage, the theronts were collected and used for further subsequent infection. The infection protocol was referred to by Straus and Griffin (2001) as described previously. In each treatment, six goldfish and 24,000 theronts (4,000 theronts/fish) were transferred into a 9-L opaque plastic tank containing 3 L of filtered aquarium water; after 24-h exposure to theronts, 6 L filtered aquarium water was added to the tank. The negative control was built for each replication (N=3). When each fish in controls developed visible I. multifiliis trophonts, the trial
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was terminated and infection level was recorded. This trial was carried out at 20.0–22.0 °C. Trial 2 was designed to determine infectivity of I. multifiliis theronts after 12h exposure of encysted tomonts to the methanol extract. The theront collection and infection protocol were referred to the details described above. Effect on the trophont in situ An in vivo trial was designed to evaluate the effect of the methanol extract on the trophont in situ according to Ling et al (2011). Forty-five heavily infected fish were divided into nine groups (five fish per group) and exposed to the solutions containing different concentrations of the methanol extract [0 (control), 2.50, and 5.00 mg/L, three groups for each concentration] for 1, 12, and 24 h in opaque breakers, respectively. The fish were placed into nine opaque breakers containing filtered aquarium water without the methanol extract, and the parasites were collected as described above. For each treatment, 30 trophonts were distributed to each well (N=3) of 24-well tissue culture plate and allowed to attach. The water in each well was removed carefully, and 1 mL of filtered aquarium water was added to each well. Until the parasites in the control groups reached the theront stage, the mortality and reproduction of tomonts in all wells were determined and calculated as mentioned above. Statistic analysis All data in this study were performed using the software Predictive Analytics Software Statistic v. 18.0. Tomont survival, tomont reproduction, and infection level were compared with Student–Newman–Keuls test procedure for multiple comparisons (α=0.05).
Results In vitro screening of plant extracts for antiprotozoal activity Table 1 shows that the methanol extract of Psoralea corylifolia had the highest activity against I. multifiliis theronts, and the extract at a concentration of 5.00 mg/L resulted in 100 % mortality after 4-h exposure. It also demonstrates that methanol extracts of Acanthopanax gracilistylus, Citrus reticulata, Lilium brownii, Magnolia biondii, Sabina vulgaris, and Trichosanthes kirilowii at a concentration of 10.00 mg/L killed 100 % of theronts within 4 h, but the extracts at a concentration of 5.00 mg/L did not cause 100 % mortality. Based on the results above, the methanol extract of P. corylifolia was selected for the next study. In addition, no mortality was observed in the positive controls and in the negative controls.
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Prevention of an initial infestation of I. multifiliis The results of an in vitro trial on the effect of the methanol extract of P. corylifolia against I. multifiliis theronts were listed in Table 2. P. corylifolia methanol extract at a concentration of 0.16 mg/L did not kill I. multifiliis theronts after 4-h exposure, and 1.25 mg/L or more concentrations of P. corylifolia methanol extract caused 100 % mortality during the 4-h exposure period. Some theronts exposed to 1.25 mg P. corylifolia methanol extract/L assumed a spherical shape by 1 h, though they were able to move, and during the next observation period, the swollen theronts were dead. No abnormal morphology and loss of motility were observed in the wells with 0.16 mg/L of P. corylifolia extract and control wells. Figure 1 illustrates that the efficacy of the methanol extract of P. corylifolia in preventing I. multifiliis theronts initial infestation in goldfish. The minimum concentration of P. corylifolia methanol extract that prevented the initial infestation was 2.50 mg/L. In additional, 0.63 and 1.25 mg/L of P. corylifolia methanol extract reduced significantly infection level of the fish infected with different concentrations of theronts (6,000 and 12,000 theronts/fish, respectively) than the controls, but infection level of the fish exposed to high concentration theronts was increased 3.7 times and 4.7 times over those to low concentration theronts, respectively. In vitro antiprotozoal activity against I. multifiliis protomonts and encysted tomonts The effects of P. corylifolia methanol extract on I. multifiliis protomonts and encysted tomonts are shown in Tables 3 and 4, respectively. The results of the two trials indicated that encysted tomonts were less susceptible to P. corylifolia methanol extract than protomonts. Exposure of I. multifiliis protomonts to 5.00 mg P. corylifolia methanol extract/L caused in 100 % mortality while the methanol extract at the same concentration killed barely 91.11 % of encysted tomonts until all surviving parasites in the controls reached the theront stage. Additionally, the reproduction of tomonts was remarkably reduced after the protomonts or tomonts exposed to 1.25 mg or more P. corylifolia methanol extract/L for 12 h. It was also noted that there was no significant difference in the survival and reproduction of protomonts or encysted tomonts between the positive controls and negative controls, and hence, the data from negative controls were listed in Tables 3 and 4. Effect on infectivity of I. multifiliis theronts Figure 2 shows that after exposure of protomonts or encysted tomonts to the highest concentration of P. corylifolia methanol
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Table 2 Effect of methanol extract of P. corylifolia on mortality of I. multifiliis theronts
Effect on the trophont in situ
Concentration Percent mortality (mg methanol extract/L) 30 min 1h
2h
4h
0 0.16 0.31 0.63 1.25 2.50 5.00
0 0 13.33±1.53 41.33±2.52 87.67±3.79 100 100
0 0 26.67±2.08 64.67±4.73 100 100 100
The results of this trial indicated that long duration (24 h) and high concentration (5.00 mg/L) significantly reduced I. multifiliis tomont survival and reproduction (Table 5). For the same concentration, the time of bath treatment against tomont survival or reproduction demonstrated a distinct time–response relationship. P. corylifolia methanol extract at a concentration of 2.50 mg/L significantly decreased I. multifiliis tomont survival and reproduction when the tomonts were collected from infested fish bathed for 24 h, whereas there was no significant difference in tomont survival and reproduction between the fish in the control groups and the fish bathed for 1 or 12 h. In addition, the results also demonstrated that for the same time of bath treatment, high concentration resulted in more distinct toxic effect on tomonts, whereas short duration (1 and 12 h) had no notable effect on tomont reproduction.
0 0 0 11.33±3.51 22.00±3.00 100 100
0 0 0 31.33±3.51 44.33±2.08 100 100
Number of dead theronts was expressed as mean (± SD) of three replicates
extract for 12 h, the theronts released from both of them had the capacity to infect the fish. It also illustrates that the theronts emerged from encysted tomonts led to more infection level than the ones in the other groups exposed to the same concentration of P. corylifolia methanol extract. The minimum concentration (1.25 mg/L) had no significant influence on infection level after 12-h exposure of protomonts or encysted tomonts.
Fig. 1 Efficacy of methanol extract of P. corylifolia in preventing the initial infestation of I. multifiliis in goldfish when exposed to 6,000 and 12,000 theronts/fish. Infection level and number of infected fish were represented as number of trophont on the fins per infected fish and total number of infected fish with three replicates. Bars were expressed as mean infection level (± SD) of three replicates, and statistically significant differences were indicated by the different lowercase letters (P
