Antiparasitic Effect of Cynatratoside-C from Cynanchum atratum

Jul 1, 2014 - Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Engineering Research Center of ...
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Antiparasitic Effect of Cynatratoside‑C from Cynanchum atratum against Ichthyophthirius multifiliis on Grass Carp Yao-Wu Fu,† Qi-Zhong Zhang,*,† De-Hai Xu,‡ Jing-Han Liang,† and Bin Wang† †

Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering Ministry of Education, Institute of Hydrobiology, Jinan University, Guangzhou 510632, People’s Republic of China ‡ Agricultural Research Service, Aquatic Animal Health Research Unit, United States Department of Agriculture, 990 Wire Road, Auburn, Alabama 36832-4352, United States ABSTRACT: Ichthyophthirius multifiliis (Ich), a fish ectoparasite, comprises an important challenge in the aquaculture industry. In this study, a steroidal glycoside, cynatratoside-C, isolated from Cynanchum atratum roots by bioassay-guided fractionation was used to treat I. multifiliis. The cynatratoside-C at 0.25 mg/L demonstrated a 100% mortality of I. multifiliis in vitro after 5 h exposure. The 5 h median effective concentration (EC50) of cynatratoside-C to nonencysted tomonts was 0.083 mg/L. In addition, cynatratoside-C at concentrations of 0.125 and 0.06 mg/L could completely terminate the reproduction of encysted tomonts. The cynatratoside-C at 2 mg/L could cure the infected grass carp within 48 h. The exact mechanism of cynatratoside-C for killing I. multifiliis is unknown, but it manifests itself microscopically through loss of membrane integrity of nonencysted tomonts or through releasing immature theronts from encysted tomonts. The immature theronts finally died before infecting fish. On the basis of these results, cynatratoside-C could be used as a natural anti-I. multifiliis agent. KEYWORDS: cynatratoside-C, Cynanchum atratum, antiparasitic activity, Ichthyophthirius multif iliis

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INTRODUCTION Ichthyophthirius multifiliis (Ich) is a ciliated parasite with a global distribution; it causes “white spot disease” of freshwater fish1 and results in a significant economic loss in the aquaculture industry.2 The life cycle of I. multifiliis consistes of an infective theront, a parasitic trophont, and a reproductive tomont. Theronts penetrate into the epidermis of a fish and become parasitic trophonts. The mature trophonts leave the host into the water and become nonencysted tomonts. The nonencysted tomonts adhere to substrates in water and transform into encysted tomont with a cyst wall. An encysted tomont divides into several hundreds to thousand tomites in its cyst, which differentiate into infective theronts. The theronts are then released from the cyst, infect fish in water, and start another life cycle.3,4 Parasiticides commonly used to control I. multifiliis have potential environmental and mammalian toxicities. Since malachite green has been banned for use in food fish due to its carcinogenic and teratogenic effects on humans, there are limited chemicals effective in treating I. multifiliis.5,6 Thus, there is an urgent need to screen new therapeutic agents to treat I. multifiliis with low toxicity and high efficacy. Recently, efficacy of plant extracts or their active ingredients to control I. multifiliis has been extensively studied.7−11 Compounds isolated and purified from plant extracts, such as pentagalloylglucose,12 cinnamaldehyde,13 sanguinarine,14 dihydrosanguinarine, and dihydrochelerythrine,15 demonstrated significant parasiticidal efficacy against I. multifiliis. The plant-derived compounds are secondary metabolites and can be easily biodegradable into nontoxic products, thus avoiding potential drug residues in fish farms.16 © 2014 American Chemical Society

The roots of Cynanchum atratum (Asclepiadaceae), commonly called “Bai Wei” in Chinese, have been used in traditional medicine for the treatment of hectic fevers, acute urinary infection, and abscesses.17,18 It has been demonstrated that this plant has cytotoxic, anti-inflammatory, and acetylcholinesterase-inhibitory activities.19−21 However, the anti-I. multifiliis activity of the C. atratum root has not been determined. This study isolated antiparasitic compounds from the C. atratum root with bioassay-guided fractionation and evaluated the antiparasitic effects of cynatratoside-C against I. multifiliis on grass carp.

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MATERIALS AND METHODS

General Methods. Nuclear magnetic resonance (NMR) experiments were performed on an Avance III 500 instrument (Bruker, Karlsruhe, Germany) operated at 500 MHz for 1H and at 125 MHz for 13 C, respectively. A sample was dissolved in d4-methanol. Electrospray ionization mass spectrometry (ESI−MS) was conducted on a 4000 QTRAP liquid chromatography−tandem mass spectrometry (LC− MS/MS) instrument (AB Sciex, Foster City, CA). High-resolution electrospray ionization mass spectrometry (HR-ESI−MS) was performed with a Triple TOF 5600+ mass spectrometer (AB Sciex). Column chromatography was performed using 200−300 mesh or 100−200 mesh silica gel (Qingdao Marine Chemical, Qingdao, China). Preparative high performance liquid chromatography (PHPLC) was conducted on an Agilent 1100 series instrument (Agilent, CA) equipped with a XB-C18 column (5 μm, 21.2 mm × 250 mm, Welch, MD). Another Agilent 1100 series instrument Received: Revised: Accepted: Published: 7183

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Figure 1. Bioassay-guided fractionation for identification of parasiticidal compound. *: Best antiparasitic activity from the same row. (Agilent) equipped with a TC-C18 column (5 μm, 4.6 mm × 250 mm, Agilent) was used to analyze the purity of active compound. The thin layer chromatography (TLC) plate was used to identify the obtained semblable fractions with similar TLC patterns for further mixing the similar fractions before bioassay by means of visualizing the plate with a UV lamp (λmax 254 and 365 nm) and anisaldehyde spray reagent (EtOH/anisaldehyde/sulfuricacid, 90:5:10, v/m/v). A Sartorius ME5 microelectronic balance (readability, 0.001 mg) (Sartorius, Niedersachsen, Germany) was used to weigh the fractions and active compound. An inverted microscope (Nikon, Tokyo, Japan) was used to observe and photograph the treated tomonts. Granule feed (Haid, Guangzhou, China) was used to feed grass carp daily to apparent satiation. Extraction and Isolation of Bioactive Compound. The roots of C. atratum (5 kg) were purchased from the Chinese medicinal market at Guangzhou, China, and kept in an oven at 55 °C until completely dried. The dried plant materials were then powdered by a pulverizer with 50 mesh strainer. The root powder was extracted with 95% ethanol (3 × 20 L) at 50 °C. After removal of ethanol in vacuo at 50 °C by a rotary vacuum evaporator, the extract (580 g) was suspended in water (1 L) and partitioned with petroleum ether (3 × 1 L), ethyl acetate (3 × 1 L), and butanol (3 × 1 L). A bioassay-guided fractionation was designed for the isolation and identification of compounds with anti-I. multifiliis effect as shown in Figure 1 and Table 1. Ethyl acetate fraction (100 g) was subjected to column chromatography and continuously eluted using a stepwise gradient of petroleum ether/ethyl acetate (10:0, 4:1, 1:1, and 0:10, v/ v) and methanol to get five fractions (A−E). The most active fraction B was further fractionated on a silica gel column and eluted with petroleum ether/ethyl acetate (4:1, 1:1, 1:4, and 0:10, v/v) to obtain fractions B1−B4. The active fraction B2, acquired from the elution of a

Table 1. Effects of Fractions against I. multifiliis Nonencysted Tomontsa antitomont efficacy (mg/L) fraction

100

50

25

0

petroleum ether fraction ethyl acetate fraction butyl alcohol fraction aqueous fraction fraction A fraction B fraction C fraction D fraction E fraction F fraction B1 fraction B2 fraction B3 fraction B4 fraction B2-1 fraction B2-2 fraction B2-3

+ + − − − + + − − − + + − − − − +

− + − − − + − − − − − + − − − − +

− + − − − + − − − − − + − − − − +

− − − − − − − − − − − − − − − − −

−: The fraction at the listed concentration could not kill 100% nonencysted tomonts after 4 h exposure. +: The fraction at the listed concentration killed 100% of nonencysted tomonts by 4 h exposure. a

1:1 petroleum ether/ethyl acetate, was separated by PHPLC to collect fraction B2-1, fraction B2-2, and fraction B2-3. The mobile phase used 7184

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for PHPLC was methanol/H2O (80:20, v/v), applied with a flow rate of 10 mL/min, and ultraviolet detection was a 210 nm. The active fraction B2-3 was further purified by PHPLC under the same condition to yield an active compound (70 mg, tR = 23 min) at purity >98%. The active compound was recrystallized in methanol and identified by various spectroscopic analyses, including ESI−MS, HRESI−MS, and 1D NMR (1H,13C NMR, and DEPT), and characterized as cynatratoside-C. The other 256 mg of cynatratoside-C was purified from 15 kg of C. atratum roots by means of the same method mentioned above (showed in Figure 1) to meet the needs of trials, and stored at 4 °C in a refrigerator prior to use. The spectroscopic data of the active compound are as follows. ESI−MS (positive) m/z: 801.7 [M + Na]+. ESI−MS (negative) m/ z: 813.8 [M + Cl]− and 777.9 [M − H]−. HR-ESI−MS m/z: [M + Na]+ 801.4039. 1H NMR (500 MHz, MeOD) δ: 0.96 (3H, s, 19-CH3), 1.24 (3H, d, J = 6.3 Hz, 6′-CH3), 1.27 (3H, d, J = 6.2 Hz, 6″-CH3), 1.29 (3H, d, J = 6.2 Hz, 6‴-CH3), 1.49 (3H, s, 21-CH3), 3.44 (3H, s, 3′-O CH3), 3.45 (3H, s, 3‴-O CH3), 3.91 (1H, dd, J = 9.6, 6.2 Hz, 15CHβ), 4.13 (1H, dd, 3″-CH), 4.17 (1H, dd, J = 8.5, 7.0 Hz, 15-CHα), 4.67 (1H, dd, J = 9.7, 1.8 Hz, 1′-CH), 5.01 (1H, br d, J = 3.4 Hz, 1‴CH), 5.04 (1H, dd, J = 9.7, 1.9 Hz, 1″-CH), 5.32 (1H, ddd, J = 9.6, 7.5, 7.5 Hz, 16-CH), 5.45 (1H, d, J = 5.5 Hz, 6-CH), 6.33 (1H, br s, 18-CH). 13C NMR (125 MHz, MeOD) δ: 18.1 (19-C), 18.4 (6″-C), 18.5 (6‴-C), 18.7 (6′-C), 24.6 (11-C), 24.9 (21-C), 29.1 (12-C), 30.6 (2-C), 30.8 (7-C), 36 (2‴-C), 37.5 (1-C), 38.1 (2′-C), 39.6 (2″-C), 39.7 (10-C), 39.9 (4-C), 41.7 (9-C), 54.5 (8-C), 57.1 (17-C), 57.4 (3‴-OMe), 57.7 (3′-OMe), 68.5 (5″-C), 68.7 (15-C), 69.9 (5‴-C), 69.8 (3″-C), 72.4 (5′-C), 76.5 (16-C), 77.5 (4‴-C), 79 (3-C), 79.2 (3‴-C), 80.1 (3′-C), 82.6 (4″-C), 84.2 (4′-C), 99 (1′-C), 99.6 (1″-C), 101.1 (1‴-C), 115.6 (20-C), 119.4 (13-C), 121.4 (6-C), 141.6 (5C),144.5 (18-C), 177 (14-C). Fish and Parasite. Healthy grass carp (Ctenopharyngodon idellus) weighing 22.6 ± 3.5 g was obtained from a commercial fish farm at Huadu, Guangzhou City, Guangdong Province. I. multifiliis was isolated from goldfish obtained from the ornamental fish market of ̈ grass carp Guangzhou. The infected goldfish were kept with five naive in a 30 L opaque tank for 7 d to allow infection of the grass carp by I. multifiliis. The temperature of the water was controlled at 23.0 ± 0.3 °C, and the fish were fed daily with diets at 1% of fish weight. When grass carp were heavily infected with mature trophonts, the skin was gently scraped to dislodge the trophonts after the fish were anesthetized by 150 mg/L tricaine methanesulfonate (MS-222, Sigma). Mature trophonts were rinsed several times with dechlorinated water to wipe off fish mucus. Usage of grass carp was approved by the Animal Experimentation Ethics Committee of Jinan University. Bioassay of Fractions against I. multifiliis Nonencysted Tomonts. Each fraction weighing 5 mg was dissolved in 2.5 mL of dechlorinated freshwater containing 1% (v/v) dimethyl sulfoxide (DMSO) to make a 2000 mg/L stock solution. The stock solution was diluted with dechlorinated freshwater in a 24-well issue culture plate to concentrations of 200, 100, and 50 mg/L. The control (0 mg/L) was made following the method described by Fu et al. (2014)11 to avoid the influence of residual solvent in each fraction. Briefly, 50 mL of each solvent used in the different fraction was also concentrated under reduced pressure in a vacuum rotary evaporator until the solvent was invisible, and then 50 mL of dechlorinated freshwater containing 1% (v/v) DMSO was added to the evaporator to dissolve the residual solvent, and the solution was used as antitomont trial control. Nonencysted tomonts came from trophonts obtained above. The average number of nonencysted tomonts per 100 μL of water was determined with the assistance of a microscope (4×) according to Zhang et al. (2013).12 Approximately 80 nonencysted tomonts in 300 μL of dechlorinated freshwater were placed into each well of a 24-well tissue culture plate. Next, 300 μL of each fraction solution at different concentrations was added to each well in triplicate to make concentrations of 100, 50, 25, and 0 (control) mg/L, respectively. Survival or dead nonencysted tomonts were identified on the basis of movement (live) or no motion (paralysis or dead) of the parasite under a microscope at 4× magnification.

In Vitro Bioactivity of Cynatratoside-C on I. multifiliis Tomonts. The stock solution of cynatratoside-C (2000 mg/L) was prepared with the same method mentioned above. The stock solution was diluted with dechlorinated water in a 24-well plate to concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg/L. Next, 300 μL of the cynatratoside-C solution at different concentrations was added to 300 μL of water including about 75 nonencysted tomonts in each well of a 24-well plate in triplicate, and the final concentrations of cynatratoside-C were 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, and 0.015 mg/L, respectively. The negative control (0 mg/L) was prepared with the method described above, and malachite green at 0.125 and 0.06 mg/L was used as positive control. The duration for killing all nonencysted tomonts was monitored at different time points for 5 h. The numbers of encysted tomonts and released theronts were determined at 6 and 22 h post exposure to cynatratoside-C, respectively. Theronts number in each well were determined under a microscope (10×) according to Zhang et al. (2013).12 To evaluate the effect of cynatratoside-C on encysted tomonts, a 300 μL solution that contained about 60 nonencysted tomonts was distributed to each well of a 24-well plate. The nonencysted tomonts were incubated for 6 h, and then encysted tomonts were enumerated in every well. The encysted tomonts were subsequently exposed to cynatratoside-C at concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, and 0 (negative control) mg/L, and positive controls (malachite green at 0.125 and 0.06 mg/L), respectively, and then maintained at 23 ± 0.3 °C for 16 h. After 22 h exposure, theronts in each well were counted as described above. To monitor pathological changes in tomonts after exposure to cynatratoside-C, approximately 50 nonencysted tomonts with 200 μL of dechlorinated freshwater were placed in one well of a 24-well plate, and 200 μL of cynatratoside-C solution at 16 mg/L was added into the well to make a final concentration of 8 mg/L. Fifty nonencysted tomonts with 200 μL of water were put into another well and incubated for 18 h. Next, 200 μL of cynatratoside-C solution was added to the well to make a 0.25 mg/L final concentration. The treated nonencysted and encysted tomonts were observed and photographed under a Nikon ECLIPSE Ti−S inverted microscope at 10×, 20×, and 40× magnification, respectively. Treatment of Ichthyophthiriasis with Cynatratoside-C. One hundred and twenty grass carp naturally infected with I. multifiliis (382 ± 35 trophonts on body surface per fish) were transferred to 12 20-L tanks with 10-L static dechlorinated water at 23 ± 0.3 °C, and randomly divided into four treatments: 0 (control), 0.5, 1, and 2 mg/L cynatratoside-C in triplicate (10 fish/tank). During the trial, water was aerated with air stones, and fish were fed every day. The dead fish were removed twice per day, and mortality was recorded daily. At 48 h post cynatratoside-C treatment, all test fish were moved from each tank, and numbers of trophont on the whole body surface were counted after the fish was anesthetized with 150 mg/L MS-222. Infection intensity was determined and expressed as total numbers of trophont on the fish body surface per infected fish. Statistical Analysis. All experimental data were expressed as the mean ± SD (standard deviation). Statistical analysis was performed with Student-Newman-Keul’s test using a statistical analysis system software package (SPSS 16.0). A probit procedure was used to determine median effective concentration (EC50) with 95% confidence intervals (CI). p-values < 0.05 were considered of significant difference.

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RESULTS Isolation and Identification of the Anti-I. multifiliis Compound. The anti-I. multifiliis effects of different fractions are listed in Table 1. The active component (70 mg), a white amorphous powder, was isolated from fraction B2-3. The compound had a molecular weight of 778, which is consistent with a molecular formula of C41H62O14. In the 13C NMR and DEPT spectra, 41 carbons were displayed, including 10 methylene carbons and 5 quaternary carbons. After comparison 7185

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of its spectral data with the literature,22 the compound was identified as cynatratoside-C (glaucogenin C 3-O-α-D-oleandropyranosyl-(1 → 4)-β-D-digitoxopyranosyl-(1 → 4)-α-Doleandropyranoside) (Figure 2).

of 0.125 mg/L killed all nonencysted tomonts within 89 min and inhabited releasing infective theronts from all encysted tomonts. Pathological changes of nonencysted tomonts are shown in Figure 3. After exposure to cynatratoside-C at 8 mg/L for 23

Figure 2. Chemical structure of cynatratoside-C.

In Vitro Bioactivity of Cynatratoside-C on I. multifiliis Tomonts. Nonencysted tomonts showed a clear dose− response to cynatratoside-C (Table 2). Mean death duration for killing all nonencysted tomonts ranged from 14.7 min at concentration of 32 mg/L to 270.0 min at concentration of 0.25 mg/L. The mortality of nonencysted tomonts ranged from 63.2% at concentration of 0.125 mg/L to 0% at concentration of 0.015 mg/L or lower (negative control) 5-h post exposure to cynatratoside-C. The 5-h EC50 of cynatratoside-C to nonencysted tomonts was 0.083 mg/L with a 95% confidence interval 0.014−0.365. Moreover, the number of theronts released from encysted tomonts sharply decreased from 223 in the negative control to 0 at a concentration of 0.125 mg/L 22 h post exposures. Even though 0.125 mg/L cynatratoside-C killed 63.2% nonencysted tomont 5-h post treatment, the survival nonencysted tomonts did not produce infective theronts. In positive control, malachite green at a concentration

Figure 3. Morphology of nonencysted tomonts. Nonencysted tomont untreated with cynatratoside-C (a). The nonencysted tomont exposed to 8 mg/L cynatratoside-C for 23 min, showing a transparent bubble (black arrow) on plasma membrane (b). Cytoplasm spilled out of the nonencysted tomont from the bubble (c,d).

min, nonencysted tomonts changed their shape, formed transparent bubbles on plasma membrane, and cytoplasm spilled out from the tomonts (Figure 3b−d). Reproduction of encysted tomonts was significantly (p < 0.05) reduced after exposure to cynatratoside-C at concentration of 0.03 mg/L or higher as compared to that in negative control after 16 h treatment (Table 3). When encysted tomonts after incubation for 18 h were treated with 0.25 mg/L cynatratoside-C for about 60 min, the tomites (immature theronts) were released from

Table 2. Effect of Cynatratoside-C on Survival and Reproduction of I. multifiliis Nonencysted Tomonta concentration (mg/L) 0 (control) 0.015 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 32 0.125 (malachite green)f 0.06 (malachite green)f

MDDNTb (min) − − − − − 270.0 95.3 56.7 41.7 29.0 20.3 17 14.7 89.0 −

± ± ± ± ± ± ± ± ±

MNTc (%) 0.0 0.0 11.9 28.0 63.2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 58.4

20.0 a 6.1 b 3.5 c 1.5 d 2.0 de 1.5 ef 1.0 ef 0.6 f 8.5 b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 a 0.0 a 40.3 a 12.7 b 8.7 c 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 0.0 d 5.0 c

MNETd 72 77 63 32 15 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 29

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.0 3.5 11.0 7.5 3.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5

MNTLETe 223.4 309.0 105.2 33.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 21.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

25.5 a 103.1 a 42.7 b 2.4 bc 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 4.7 bc

a Values are expressed as mean ± standard deviation of three replicates. Values with different letters in the same column are significant differences (p < 0.05). −: Cynatratoside-C at the listed concentrations could not kill 100% nonencysted tomonts after 5 h exposure. bMDDNT: mean death duration for killing all nonencysted tomonts within 5 h exposure. cMNT: mortality of I. multifiliis nonencysted tomonts at 5 h. dMNET: mean number of encysted tomonts transformed from nonencysted tomonts at 6 h. eMNTLET: mean number of theronts released from each live encysted tomont at 22 h exposure. fPositive control.

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Table 3. Reproduction of I. multifiliis Encysted Tomonts Post Cynatratoside-C Treatmenta number of encysted tomonts

concentration (mg/L) 0 (control) 0.015 0.03 0.06 0.125 0.25 0.125 (malachite green)b 0.06 (malachite green)b

52 47 63 49 54 62 48 47

± ± ± ± ± ± ± ±

5.3 6.1 9.0 7.8 8.2 13.7 7.6 12.7

of grass carp treated with 1 mg/L cynatratoside-C was 184 trophonts/fish, significantly (p < 0.05) lower than that of the fish treated with 0.5 mg/L (406 trophonts/fish) and 0 mg/L (357 trophonts/fish).

number of theronts released from each live encysted tomont 280.1 310.9 131.7 0.0 0.0 0.0 0.0 17.6

± ± ± ± ± ± ± ±

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DISCUSSION Medical plants have provided an abundant source of natural products of new parasiticides to control I. multifiliis.8,11,12 Since the crude extracts of Allium sativum,23 Mucuna pruriens, and Carica papaya7 were reported to be used to treat I. multifiliis, more studies have been conducted to discover new compounds from medical plants.8−12,24 C. atratum, a perennial herb native to East Asia, showed significant parasiticidal effect to I. multifiliis in this study. An ethanol extract of C. atratum was partitioned with petroleum ether, ethyl acetate, butanol, and water. A bioassay-guided fractionation, a proven method for the discovery of active principles from natural products,25−27 was used to isolate an anti-I. multifiliis compound from C. atratum. Finally, the anti-I. multifiliis compound was identified as cynatratoside-C by NMR, ESI−MS, and HR-ESI−MS. This is the first report on parasiticidal property of cynatratoside-C. Cynatratoside-C, one of the C-21 steroidal glycosides in C. atratum, has a 13,14:14,15-diseco-pregnane-type skeleton. Even though other structurally related analogues, such as cynatratoside-A, cynatratoside-B, cynatratoside-D, and cynatratoside-E, have the same skeleton of 13,14:14,15-diseco-pregnane-type,22 only cynatratoside-C was isolated by bioassay-guided fractionation. This fact suggested that cynatratoside-C was the main ingredient and the principal compound in C. atratum responsible for killing I. multifiliis. Glycosylation plays a key role in the activity of some natural compounds.28,29 For example, rebeccamycin’s inhibition of the topoisomerase I activity by the rebeccamycin analogues is more active than that of the corresponding aglycones lacking the sugar moiety.28 Compound properties are affected if the glycosyl is changed.30,31 For example, if the antitumor compound (25S)-ruscogenin 1-O-[O-β-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside] from Brodiaea californica has a D-xylose added at the C-3 position of its inner glucose, the antitumor activity is significantly reduced.30 Hence, we expect that the type of carbohydrate chain at C-3 position in the 13,14:14,15-disecopregnane-type skeleton of cynatratoside-C will influence the anti-I. multifiliis effect. Once a tomont of I. multifiliis settles on a suitable substrate, it reproduces hundreds to thousands of infective theronts.2,32 Once theronts penetrate into fish skin and gills, the control of the parasite becomes difficult.33 Therefore, killing the parasite at the reproductive stage is an important way to prevent releasing of infective theronts and protect fish from infection.12 On the basis of this consideration, trials were conducted to evaluate the parasiticidal effects of cynatratoside-C on nonencysted and encysted tomonts. The results indicated that cynatratoside-C at 0.125 mg/L markedly reduced nonencysted tomonts survival and at 0.06 mg/L completely inhibited encysted tomonts reproduction. Malachite green was used as a positive control, but its concentration of 0.06 mg/L could not completely stop theronts released from the encysted tomonts; the mean number of theronts released from each live tomont was 17. This suggested that cynatratoside-C was more toxic to encysted tomonts than malachite green. Previous studies demonstrated that pentagalloylglucose from Galla chinensis at a concentration of 40 mg/L was effective against nonencysted

17.0 a 12.2 b 16.9 c 0.0 d 0.0 d 0.0 d 0.0 d 2.1 d

a

Nonencysted tomonts were allowed to attach for 6 h, expose to cynatratoside-C for 16 h, and theronts were counted in every well. Values are expressed as mean ± SD of three replicated wells. Values with different letters in the same column are significant differences (p < 0.05). bPositive control.

the cyst and subsequently died (Figure 4a−d). The same phenomenon was also observed at concentrations of 0.06 and 0.125 mg/L after 22 h exposure.

Figure 4. Morphology of encysted tomonts. A normal encysted tomont (a). Encysted tomont exposed to 0.25 mg/L cynatratoside-C for 60 min, showing the first tomite (black arrow) released from the cyst (b). More tomites were released from an encysted tomont (c). Subsequently, a tomite was dead and its cytoplasm (black arrow) was spilled out (d).

Treatment of Ichthyophthiriasis with CynatratosideC. Cynatratoside-C at concentration of 2 mg/L completely eliminated all trophonts and cured the infected grass carp within 48 h (100% survival) (Table 4). The infective intensity Table 4. Treatment of Grass Carp Infected by I. multifiliis with Cynatratoside-C for 48 ha concentration (mg/L) 0 (control) 0.5 1 2

infective intensityb 357 406 184 0

± ± ± ±

126 a 48 b 88 c 0d

survival rate (%) 96.7 100 100 100

± ± ± ±

5.8 a 0a 0a 0a

Values are expressed as mean ± SD of three replicates. In the same column, values with different letters are significant differences (p < 0.05). bInfective intensity = number of trophonts on grass carp/ infected fish number. a

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tomonts,12 and chelerythrine and chloroxylonine from Toddalia asiatica could be 100% effective against nonencysted tomonts at concentrations of 1.2 and 3.5 mg/L, respectively.34 As for other chemicals used to treat parasites, potassium ferrate(VI) at a concentration of 19.2 mg/L demonstrated 100% mortality on both nonencysted and encysted tomonts,35 and a mixture of hydrogen peroxide, peracetic, acetic, and peroctanoic acid at a concentration of 8 mg/L eliminates tomonts. 36 Thus, cynatratoside-C appears to be more effective against I. multifiliis than the reported parasiticides. Published reports demonstrated that nonencysted tomonts were more sensitive to drugs than encysted tomonts, because an encysted tomont secreted a thick cyst wall that prevents drugs from permeating into encysted tomont.10,35 The methanol extract from Psoralea corylifolia at a concentration of 5.00 mg/L kills 100% nonencysted tomonts, but killed only 88.89% encysted tomonts at the same concentration.10 The acetone extract from Morus alba root bark at a dose of 25 and 50 mg/L causes 100% mortality of nonencysted tomonts and encysted tomonts, respectively.11 However, the encysted tomonts were more susceptible to cynatratoside-C than nonencysted tomonts in this study. The reason for this phenomenon is unclear and needs further study. The trophonts are more difficult to be killed than the parasite at the free-living stage,12 because the trophonts are covered by a layer of host epidermis and a thick layer of mucus protects them from drugs in water.32 In this study, cynatratoside-C at 2 mg/L completely kills all trophonts and cures the infected grass carp within 48 h. The infective intensity of grass carp exposed to 1 mg/L cynatratoside-C was significantly lower than those at 0.5 and 0 mg/L. Other studies on parasiticides, nitazoxanide at 1.5, 2, or 3 mg/L, showed reduction of approximately 98% in the number of trophonts on infected silver catfish (Rhamdia quelen) at 96 h exposure.37 Bronopol utilized once a day for 27 days at concentrations of 2 and 5 mg/L significantly reduced the number of trophonts on juvenile rainbow trout (Oncorhynchus mykiss) by 90−98%.38 The plant-active drugs dihydrosanguinarine and dihydrochelerythrine from Macleaya microcarpa were effective against I. multifiliis in barbel chub (Squaliobarbus curriculus) with median effective concentration (EC50) values of 5.18 and 9.43 mg/L, respectively. 15 Pentagalloylglucose at 20 mg/L was effective for treating I. multifiliis infected catfish.12 These data indicate that concentrations of the therapeutic agents against trophonts are much higher than that of cynatratoside-C. Therefore, cynatratoside-C could be a promising alternative parasiticide against I. multifiliis. Pathological changes occurred after exposure to cynatratoside-C and the plasma membrane of nonencysted tomont was destroyed. This phenomenon was similar to the membrane of nonencysted tomont damaged by pentagalloylglucose.12 When an encysted tomont was treated with cynatratoside-C, the tomont released abnormal tomites from the cyst. These abnormal tomites were immature theronts, could not infect fish, and finally died. Therefore, we postulate that cyantratoside-C permeates into an encysted tomont across the cyst wall and triggers pathological changes of tomites. Even though the exact mechanism of cynatratoside-C for killing nonencysted tomonts is unknown, it manifests itself microscopically through loss of membrane integrity. In conclusion, cynatratoside-C isolated from C. atratum showed anti-I. multifiliis effects in vitro and in vivo. The compound at a very low concentration significantly decreased the survival of nonencysted tomonts and reproduction of

encysted tomonts. In addition, the compound can cure the ichthyophthiriasis under laboratory conditions. Therefore, cynatratoside-C is a potential alternative parasiticide to control I. multifiliis, and deserves further studies to elucidate the anti-I. multifiliis mechanism and evaluate its effectiveness under fish farming conditions.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86+20+85225808. E-mail: [email protected]. Funding

This work was supported by the National High Technology Research and Development Program of China (863 Program) (no. 2011AA10A216), Guangzhou Science and Technology Project (no. 2013J4100047), the Marine and Fishery Special Project of Science and Technology in Guangdong Province (A201301B05), and the Fundamental Research Funds for the Central Universities (21612111, 21613105). Notes

The authors declare no competing financial interest.

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REFERENCES

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