Anthelmintic constituents from ginger (Zingiber officinale) against Hymenolepis nana

G Model

ARTICLE IN PRESS

ACTROP 3419 1–11

Acta Tropica xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Acta Tropica journal homepage: www.elsevier.com/locate/actatropica

Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana

1

2

3 4 5 6 7 8

Q1

Rong-Jyh Lin a , Chung-Yi Chen b , Chin-Mei Lu a,c,d,1 , Yi-Hsuan Ma a,d,1 , Li-Yu Chung a , Jiun-Jye Wang a , June-Der Lee a , Chuan-Min Yen a,d,∗ a

Department of Parasitology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan School of Medical and Health Sciences, Fooyin University, Kaohsiung 83102, Taiwan c Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan d Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan b

9

10 25

a r t i c l e

i n f o

a b s t r a c t

11 12 13 14 15 16

Article history: Received 25 February 2014 Received in revised form 14 June 2014 Accepted 15 July 2014 Available online xxx

17

24

Keywords: Cestocidal activity Hymenolepis nana Ginger Anthelmintic activity Oxygen radical absorbing capacity Cytokine

26

1. Introduction

18 19 20 21 22 23

27 28 29 30

This study investigated the anthelmintic activity of gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, hexahydrocurcumin, 3R,5S-[6]-gingerdiol and 3S,5S-[6]-gingerdiol, a constituent isolate from the roots of ginger, for the parasite Hymenolepis nana. The cestocidal activity or ability to halt spontaneous parasite movement (oscillation/peristalsis) in H. nana of above constituents was reached from 24 to 72 h in a time- and dosedependent manner, respectively. The [10]-shogaol and [10]-gingero1 have maximum lethal efficacy and loss of spontaneous movement than the others at 24–72 h. In addition, worms treated with 1 and 10 ␮M [10]-gingero1, more than 30% had spontaneous movement of oscillation at 72 h but [10]-shogaol at 72 h only about 15–20% of oscillation. This showing that [10]-gingero1 had less loss of spontaneous movement efficacy than [10]-shogaol. After exposure to 200 ␮M [10]-shogaol, 100% of H. nana had died at 12 h rather than died at 24 h for [10]-gingerol, showing that [10]-gingero1 had less lethal efficacy than [10]shogaol. In addition, these constituents of ginger showed effects against peroxyl radical under cestocidal activity. In order to evaluate the cestocidal activity and cytokine production caused by ginger’s extract R0 in the H. nana infected mice, we carried out in vivo examination about H. nana infected mice BALB/c mice were inoculated orally with 500 eggs. After post-inoculation, R0 (1 g/kg/day) was administered orally for 10 days. The R0 exhibited cestocidal activity in vivo of significantly reduced worms number and cytokines production by in vitro Con A-stimulated spleen cells showed that INF-␥ and IL-2 were significantly increases by R0. IL-4, IL-5, IL-6, IL-10 and IL-13 were significantly decreases and Murine KC and IL-12 were not significantly changes by R0. Together, these findings first suggest that these constituents of ginger might be used as cestocidal agents against H. nana. © 2014 Published by Elsevier B.V.

Widely used in various foods and drinks as a spice, ginger (Zingiber officinale L., Zingiberaceae) is also used in traditional Chinese medicine (Ali et al., 2008). Ginger has been used for centuries in traditional medicine such as Shokyo (fresh rhizomes), Kankyo

∗ Corresponding author at: Department of Parasitology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan. Q2 Tel.: +886 7 3121101x2169; fax: +886 7 3218309. E-mail addresses: [email protected] (R.-J. Lin), [email protected] (C.-Y. Chen), [email protected] (C.-M. Lu), [email protected] (Y.-H. Ma), [email protected] (L.-Y. Chung), [email protected] (J.-J. Wang), [email protected] (J.-D. Lee), [email protected] (C.-M. Yen). 1 These authors contributed equally to this work.

(dried, steamed rhizomes) or Kanshokyo (dried rhizomes) (Goto et al., 1990). Such medicinal treatments are considered effective for the common cold, asthma, nervous disease, stroke, gastrointestinal constipation, inflammation, oxidant stress, hypercholesterolaemia, schistosomiasis and helminthiasis (Ali et al., 2008; Chohan et al., 2008; Haniadka et al., 2013; Iqbal et al., 2006; Sanderson et al., 2002). Ginger has been identified as containing essential oil, zingiberol, zingiberone, zingiberene, and pungent components such as [6]-gingerol and [6]-shogaol, including pharmacological properties (Goto et al., 1990). According to several literature reviews (Ali et al., 2008), the main constituents of ginger are paradol, zingerone, gingerols and shogaols. Found in nonvolatile pungent ingredients from ginger, the above-mentioned shoagol, zingerone and gingerol can suppress the hyperproliferative, inflammatory and tumor growth. The

http://dx.doi.org/10.1016/j.actatropica.2014.07.009 0001-706X/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

G Model ACTROP 3419 1–11 2 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

phenolic compounds derived from ginger such as [6]-gingerol, [10]-shogaol, [10]-gingerol and [6]-shogaol have many common activities and therapeutic applications involving its physiological and pharmacological propriety. Despite its widespread use for many centuries, this plant still attracts considerable attention with respect to its parasitology, yet lesser interest in its parasiticidal activity. As the most common cause of all cestode infections, Hymenolepis nana is a globally widespread zoonosis disease. Although H. nana infections are typically asymptomatic, related heavy infections can cause headaches, weakness, anorexia, abdominal pain, and diarrhea (Sirivichayakul et al., 2000). As the only cestode capable of completing its cycle without an intermediate host, H. nana infection occurs in different stages eggs, onchosphere or an infected intermediate host such as beetles or fleas even without it. Additionally, eggs of H. nana are infected immediately when passed from the host stool and transported in contaminated food. Ingestion eggs hatch in the duodenum, releasing oncospheres (hexacanth larvae), which penetrate the mucosa and lie in the lymph channels of the villi then develops into a cysticercoid. After 5–6 days, cysticercoids emerge into the lumen of the small intestine, to which they attach before maturing. Upon rupture of the villus, the cysticercoids return to the intestinal lumen, evaginate their scoleces, attach to the intestinal mucosa and mature into adults that reside in the ileal portion of the small intestine. The eggs are then either passed in the stool when released from the proglottids through their genital atrium or disintegrate of proglottids in the small intestine. Alternatively, internal autoinfection occurs, in which the eggs release their hexacanth embryo, which penetrates the villus and continues the infective cycle without passing through the external environment (Andreassen et al., 2004; Ito, 1997). Previous studies have established that ginger can destroy Angiostrongylus cantonensis (Lin et al., 2010a), Dirofilaria immitis (Merawin et al., 2010), Anisakis simplex (Goto et al., 1990; Lin et al., 2010b), Schistosoma mansoni (Aly and Mantawy, 2013) and gastrointestinal nematodes (Iqbal et al., 2006). Whereas some components of ginger (e.g., [6]-gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin (HHC)) exert an anthelmintic effect against some parasitic species in vitro and in vivo, the mechanism underlying their cestocidal effects on H. nana is unclear. The underlying mechanisms of the activities of ginger components such as gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC, 3R, 5S-[6]-gingerdiol and 3S, 5S-[6]-gingerdiol must be thoroughly evaluated with respect to H. nana. T cells mediate many responses to immunity against parasitic infections. Recent studies have elucidated the role of the T helper (Th) subset and the cytokines that they release. Th cells can be classified based on cytokine production: Th1 cells release IL-2, IFN-␥ and lymphotoxin, and Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 and Murine KC. Although previous studies have demonstrated that type II immunity protects helminthes (Palmas et al., 1997; Wang and McKay, 2005), recent studies on mice indicate that Th1 (IFN-␥, IL-2) also play an important role in cestodes such as H. nana and H. diminuta, especially in some stages of infection (Asano et al., 1993). IFN-␥ is a more important cytokine in producing immunity to H. nana reinfection than IFN-␣ or IL-1␤ during H. nana egg infection in mice (Asano and Muramatsu, 1997). Cytokine production such as IFN-␥, IL-2, IL-3, IL-4 and IL-5 by in vitro Concanavalin A (Con A) stimulated mesenteric lymphnode cells (as measured daily after egg or cyst infection of mice with H. nana), indicating that cytokine production varies during parasite development and between different host strains such as BALB/c and C3H/He mice (Conchedda et al., 1997). Results of another study suggested that helper T cells, especially the Th1 subtype, are involved in protective immunity against H. nana. According to another study, Th1 and Th2 play important roles in H. nana infection (Ajami and Rafiei, 2007). Therefore, this

study demonstrates the role of ginger against H. nana with respect to Th1 and Th2 subtypes immunity by determining the level of IFN␥, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and Murine KC cytokines in Con A stimulated spleen cells (lymphocytes) in vitro of H. nana infected BALB/c. Free radical scavenging activities have been implicated in some inflammatory diseases (Haniadka et al., 2013). Despite having antiprotozoae activity, some agents also have free radical scavenging activity (Lopes et al., 1998). However, free radical scavenging activity failed to reduce their larvicidal activity (Diallo et al., 2001). Other studies have suggested that free radical scavenging may reduce larvicidal activity by permitting larvae survival. Therefore, exactly how free radical scavenging affects the cestocidal activity of certain anthelminthic agents against H. nana still remains unclear. This study first identified which components of ginger root exhibit cestocidal activity on adult worms of H. nana in vitro. H. nana infection of mouse in vivo is then treated using ginger extract. Next, cestocidal activity and cytokine production are analyzed. Additionally, whether or not a correlation is found between the possible scavenger activity of these components and their cestocidal activity against H. nana is determined using oxygen radical absorbance capacity (ORAC) assay.

112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

2. Materials and methods

134

2.1. Plant material

135

The rhizomes of ginger used in this study were purchased from Chiayi in eastern Taiwan in April 2008, which were identified by Dr. Fu-Yuan Lu of the Department of Forestry and Natural Resources, College of Agriculture, National Chiayi University, Chiayi, Taiwan. A voucher specimen was deposited at the School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan. 2.2. Extraction and isolation of ginger The rhizomes of ginger were chipped and airdried, as well as extracted repeatedly with MeOH (40 L × 5) at room temperature. The combined MeOH extracts (R0, 631.9 g) were then evaporated and further separated into two fractions by column chromatography on silica gel (70–230 mesh) with gradients of CH2 Cl2 /MeOH. Part of fraction 1 (R1, 23.47 g) was subjected to silica gel chromatography by eluting with n-hexane-acetone (50:1), followed by enrichment with acetone to furnish three further fractions (11–1-3). Fraction 1-1 (6.25 g) was further purified on a silica gel column by using n-hexane/acetone mixtures to obtain [4]-shogaol (45 mg) and [6]-dehydrogingerdione (20 mg) (Fig. 1). Fraction 12 (10.25 g) was subjected to silica gel chromatography by eluting with n-hexane-acetone (40:1), followed by enrichment with acetone to furnish three further fractions (1-2-1–1-2-3). Fraction 1-2-1 (3.28 g) was further purified on a silica gel column by using n-hexane/acetone mixtures to obtain [6]-gingerol (50 mg) and [10]shogaol (50 mg) (Fig. 1). Fraction 1-2-2 (3.45 g) was further purified on a silica gel column by using n-hexane/acetone mixtures to obtain [6]-shogaol (30 mg) and [10]-gingerol (83 mg) (Fig. 1). Fraction 12-3 (3.27 g) was further purified on a silica gel column by using n-hexane/acetone mixtures to obtain gingerenone A (21.3 mg) (Fig. 1). Part of fraction 2 (R2, 12.35 g) was subjected to silica gel chromatography by eluting with n-hexane-acetone (20:1), followed by enrichment with acetone to furnish four further fractions (2-1–24). Fraction 2-1 (3.5 g) was subjected to silica gel chromatography by eluting with n-hexane-acetone (30:1), followed by enrichment with acetone to furnish three further fractions (2-1-1–2-1-3). Fraction 2-1-1 (1.13 g) was further purified on a silica gel column by

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

136 137 138 139 140 141

142

143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171

G Model ACTROP 3419 1–11

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

3

(Fig. 1). Fraction 2-3 (5.34 g) was further purified on a silica gel column by using n-hexane/acetone mixtures to obtain 3R, 5S-[6]gingerdiol (14 mg) and 3S,5S-[6]-gingerdiol (11 mg) (Fig. 1). Finally, these compounds were identified using spectroscopic analysis of data and compared with values in the literature (Shoji et al., 1982). 2.3. Experimental procedures of components of ginger Optical rotations were determined with a JASCO DIP-370 digital polarimeter. UV spectra were obtained in CH3 CN by using a JASCO V-530 spectrophotometer, and IR spectra were evaluated on a Hitachi 260-30 spectrophotometer. 1 H NMR (500 MHz, by using CDCl3 as solvent for determination), 13 C NNR (125 MHz), DEPT, HETCOR, COSY, NOESY, and HMBC spectra were obtained on a Varian (Unity Plus) NMR spectrometer. Low-resolution electrospray ionization-mass spectrometry (ESI-MS) spectra were obtained on an API 3000 (Applied Biosystems). Silica gel 60 (Merck, 70–230 mesh, 230–400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254 , 0.50 mm, 0.20 mm) were used for analytical thin layer chromatography (TLC), and precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.50 mm) were used for preparative TLC. Spots were detected by spraying the plates with 50% H2 SO4 and then heating them on a hot plate. 2.4. Drugs and chemicals Dulbecco’s Modified Eagle Medium (DMEM), RPMI-1640, fetal bovine serum (FBS), l-glutamine, penicillin G, streptomycin, amphotericin B and all other cell culture reagents were obtained from Gibco BRL Life Technologies (Grand Island, NY). Concanavalin A (Con A), Dimethyl sulfoxide (DMSO), Fluorescein, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 2,2¢-Azobis(2-amidinopropane) dihydrochloride (AAPH) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). All drugs and reagents were dissolved in sterilized distilled H2 O unless otherwise specified. Gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC and 3R,5S-[6]-gingerdiol and 3S,5S-[6]-gingerdiol were dissolved in DMSO at 1 M stock and diluted serially with sterilized distilled H2 O. Additionally, a vehicle containing 1% DMSO in sterilized distilled H2 O. R0 was dissolved in DMSO at 0.08 g/ml stock and serially diluted with sterilized distilled H2 O and a vehicle containing 1% DMSO in sterilized distilled H2 O. ELISA kits of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and INF-␥ were obtained from eBioscience (San Diego) and Murine KC from Perprote. 2.5. Ethics statement

Fig. 1. The major chemical constituents of ginger (Zingiber officinale).

172 173 174 175 176 177 178 179 180

using n-hexane/acetone mixtures to obtain [6]-gingerol (10 mg) and [10]-shogaol (13 mg) (Fig. 1). Fraction 2-1-2 (1.01 g) was further purified on a silica gel column by using n-hexane/acetone mixtures to obtain [6]-shogaol (5 mg) and [10]-gingerol (8 mg) (Fig. 1). Fraction 2-1-3 (0.78 g) was further purified on a silica gel column by using n-hexane/acetone mixtures to obtain hexahydrocurcumin (HHC, 30 mg) and [4]-gingerol (5 mg) (Fig. 1). Fraction 2-2 (4.7 g) was further purified on a silica gel column by using nhexane/acetone mixtures to obtain 5-hydroxy-[6]-gingerol (13 mg)

The handling of animals in this study was reviewed and approval by the Institutional Animal Care and Use Committee of Kaohsiung Medical University (IACUC Approval Number: 97119). The animals were handled according to the protocol statement (Approval Number: NSC 98-2320-B-037-014-MY3) from the National Science Council of the Republic of China, Taiwan, in compliance with Taiwanese laws (Animal Protection Act, Amended Date: 2011.06.29. and Enforcement Rules of Animal Protection, Announced Date: 2000.01.19.). This study received approval from the IACUC at Kaohsiung Medical University. BALB/c mice, weighing 25–30 g were provided by the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). The mice were raised in the Laboratory Animal Center with air conditioning (at 22 ± 1 ◦ C with a relative humidity of 50 ± 10%) and illumination control (with lights on between 7:30 and 19:30). Animals were allowed food and water ad libitum.

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

181 182 183 184 185

186

187 188 189 190 191 192 193 194 195 196 197 198 199 200 201

202

203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222

223

224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239

G Model ACTROP 3419 1–11

R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

4 240

241 242 243 244 245 246 247 248 249 250 251 252 253 254 255

256 257

ARTICLE IN PRESS

2.6. Preparation of adult worms of H. nana H. nana adult worms were obtained from each part of the intestines of BALB/c mice, as purchased from Lin’s farm in Fengshan, Kaohsiung, Taiwan. These parts of the intestine were the duodenum, jejunum, ileum, colon and rectum. The H. nana was an average 5–50 mm in length and were collected using a needle with a blunt tip, before placed in Petri dishes with 0.9% NaCl and gentamycin (10 mg/ml). The worms were then washed several times. The adult worms were individually observed under an inverted microscope, with subsequent discarding of those that exhibited internal or external damage. Next, the adult worms were identified based on their morphological features, divided into groups and placed in 24-well plates (which contained cultivated media RPMI-1640 plus 20% FBS, pH 7.4, in an atmosphere of 95% O2 /5% CO2 , 37 ◦ C). These in vitro culture conditions have been shown to maximize the development and survival of H. nana. 2.7. Assay of cestocidal efficacy and loss of spontaneous mobility with respect to oscillation and peristalsis activity of H. nana

phosphate buffer. The analyzer was programmed to record the fluorescence of FL every minute after adding AAPH. All fluorescent measurements are expressed relative to the initial reading (excitation at 495 nm and emission at 530 nm). Final ORAC values were calculated using the regression equation between Trolox concentration and the net AUC, and were expressed as micromole Trolox equivalents per liter. The area under curve (AUC) was calculated as f1 fi f49 AUC = 50 + + ··· + ··· + + 50 f0 f0 f0

 f 50 

(a)

f0

where f0 = initial fluorescence reading at 0 min and fi = fluorescence reading at time i. The data were analyzed using a Microsoft Excel macro program (Microsoft) using Eq. (a) to calculate AUC. The net AUC is obtained by subtracting AUC of the blank from that of a sample. The relative Trolox equivalent ORAC value is calculated as



Relative ORAC value =

(AUCsample − AUCblank )





Molarity of Trolox Molarity of test sample

(b)

282

283

2.8. Morphological examination

2.11. Cultivation of spleen cells for cytokine assay

288

Adult worms of H. nana were incubated with 100 ␮M [10]shogaol and [10]-gingerol for 24, 48 and 72 h, respectively. For each preparation, the worm was fixed with a 10% formalin solution for 24 h and mounted as thin paraffin sections transversally cut through several levels of worms.

289

2.9. Evaluation of oxygen radical absorbing capacity (ORAC)

BALB/c mice were sacrificed at 10 days after post-inoculation with H. nana. The spleen of each mouse was removed aseptically and scratched with the tips of forceps in DMEM. The cells suspensions were treated with 0.085 mg/ml ammonium chloride solution for haemolysis of contaminated erythrocytes and then washed with DMEM twice by centrifugation at 3000 rpm 10 min g at room temperature. Following this step, cell viability exceeded 95%, as determined by trypan blue exclusion. The spleen cells were then adjusted to a final concentration of 107 cells/ml with DMEM containing 10% FBS, 72 ␮g/ml gentamycin (Sigma) and 2 × 10−3 M l-glutamine. This cell suspension (1 ml) was dispensed into each well of a 24-well culture plate and, then, cultivated with or without 10 ␮g/ml 1 mg/L Con A at 37 ◦ C in an atmosphere containing 5% CO2 . Cell-free supernatants were harvested after 24, 48 and 72 h of incubation and stored at −20 ◦ C until use. Cell-free supernatants were assayed for IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, INF-␥ and Murine KC and determined according to manufacturer directions.

259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281

284 285 286 287

290 291 292 293 294 295 296 297 298

Automated ORAC assay was performed on a FLUOstar Galaxy plate reader (Roche Diagnostic System Inc., Branchburg, NJ), as described elsewhere (Gillespie et al., 2007). The experiment was undertaken at 37 ◦ C under a condition of pH 7.0 with a blank sample in parallel. Briefly, AAPH was used as a peroxyl generator, and 1 ␮M Trolox, a water-soluble analog of vitamin E, was used as a control standard. The final reaction mixture for each black microplate in a 96-well microplate assay contained 0.06 ␮M fluorescent (FL), 18.75 mM AAPH and appropriate 1 ␮M test substance in 75 mM

301 302 303 304 305 306

307

308 309 310 311 312 313



H. nana was obtained as described above. The above media were supplemented with l-glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 mg/ml) and amphotericin B (0.25 ␮g/ml), and tested concentrations of gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC, 3R,5S-[6]-gingerdiol and 3S,5S[6]-gingerdiol at 100 ␮M. Survival and mobility of the worms were evaluated at 2, 4, 6, 12, 24, 48 and 72 h by using a stereomicroscope. Next, the status of adult worms was scored as oscillation and peristalsis by two investigators in a blinded manner. Cestode activity was scored oscillation and peristalsis. Oscillation was scored of movement at scolex and neck for each second for 30 s and the highest score was 30. Peristalsis was recorded as the real contraction at the scolex and neck. All data were compared with the time before adding test compounds. Death and complete standstill of the worms, as determined by no oscillation and peristalsis changes for 30 min (defined as death), were identified. The mortality was recorded after determining that the worms neither moved when shaken vigorously nor when dipped in a warm medium. Besides evaluating the time course between dosage propriety of compound-induced loss of oscillation and peristalsis activities on H. nana, this study assessed [10]-shogaol and [10]-gingerol at 1, 10, 100 and 200 ␮M. Based on the above methods, survival and spontaneous mobility of the worms were assessed at 2, 4, 6, 12, 24, 48 and 72 h.

258

300

314

(AUCTrolox − AUCblank ) ×

299

315 316

2.10. In vivo cestocidal activity in H. nana infected mouse The H. nana strain used in all experiments was maintained in our laboratory as Section 2.6. Adult worms were obtained from the small intestine, and eggs were released and collected by artificial dissection of gravid proglotids. Eggs were suspended in a saline solution of 0.85% and stored at 5 ◦ C. BALB/c mice, weighing 25–30 g, were inoculated orally with 500 eggs in 0.2 ml 0.9% NaCl via an oral stomach tube and maintained under standard environmental control. The mice were raised with commercial rodent diet food and water ad libitum in an air-conditioned laboratory animal center (22 ± 1 ◦ C and 50% ± 10% relative humidity). To confirm the infection, the presence of an egg was determined by fecal analysis after post-inoculation. The infected mice were distributed in groups of six animals in separate cages, and treatment with a test compound was initiated. R0 was administered by an oral stomach tube in a single dose of 1 g/kg/day for 10 days. Ten days after the final administration, mice were sacrificed and adult worms in the small intestine (including duodenum, jejunum, ileum, colon and rectum) were recovered and counted. The cestocidal activity was expressed as the recovery number of adult worms.

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

317

318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337

338

339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355

G Model ACTROP 3419 1–11

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

359

Cytokines were quantitated by reference to standard curves derived from known concentrations of recombinant interleukins, and indicated as the ration versus control group (H. nana infected group without R0, vehicle or Con A).

360

2.12. Statistical evaluation of data

356 357 358

367

The results were expressed as mean ± standard deviation (SD). Statistical differences were estimated by one-way analysis of variance (ANOVA), followed by Dunnett’s test or the Tukey–Kramer test. A p value of 0.05 was considered significant. Based on software (SigmaPlot Version 8.0 and SigmaStat Version 2.03, Chicago, IL), the data were analyzed and the figures were plotted.

368

3. Results

369

3.1. Cestocidal activity against H. nana

361 362 363 364 365 366

370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414

In the first series of experiments, the ability of the gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC, 3R,5S[6]-gingerdiol and 3S,5S-[6]-gingerdiol (Fig. 1) to alter the survival of adult worms for H. nana was studied using the cestocidal effects. According to Fig. 2A and B, the above ginger’s constitute reduced the survival of worms and spontaneous mobility of oscillation and peristalsis activities of H. nana in vitro. The above compounds decreased oscillation activity about 10–100%, respectively. The maximum lethal efficacy of [10]-gingerol and [10]-shogaol was approximately 100% (ranging from 90 to 100%) at 24 h. For lethal efficacy, loss oscillation percentage values of the compounds were in the following order: [10]-shogaol  [10]gingerol > [6]-gingerol > [6]-shogaol  [4]-shogaol > 3S,5S[6]-gingerdiol  5-hydroxy-[6]-gingerol > gingerenone A > 3R,5S-[6]-gingerdiol  [6]-dehydrogingerdione  HHC at 24 h. The above compounds decreased oscillation activity by about 10–100%, respectively. For 48 and 72 h, the [10]-shogaol and [10]-gingero1 have a maximum lethal efficacy and higher loss of oscillation activity than the others. In the oscillation activity assay, vehicle (0.1% DMSO) was decrease 35–40% after 72 h of cultivation (Fig. 2A). However, in peristalsis activity assay, vehicle was decrease 73–78% from 72 h cultivation (Fig. 2B). The mean peristalsis of H. nana was more sensitive to the vehicle than oscillation. In peristalsis activity assay, the percentage of peristalsis was more complex than oscillation. However, the loss of peristalsis increased with an increasing incubation time of the compound. In this study, peristalsis activity disappeared before oscillation activity was lost when H. nana died. In fact, H. nana has no peristalsis or oscillation effect when it dies. Our results further indicated that treatment with 100 ␮M gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC, 3R,5S-[6]-gingerdiol and 3S,5S-[6]-gingerdiol resulted in a more pronounced effect of peristalsis than during oscillation at 24, 48 and 72 h (Fig. 2B). Peristalsis activity first disappeared before oscillation activity was lost when H. nana died. Actually, H. nana was without both peristalsis and oscillation effects when dead. Loss peristalsis percentage values of the compounds were in the following order: [10]-shogaol  [10]gingerol > [6]-dehydrogingerdione  [4]-shogaol > [6]shogaol  [6]-gingerol  5-hydroxy-[6]-gingerol > gingerenone A  3S,5S-[6]-gingerdiol  3R,5S-[6]-gingerdiol > HHC at 24 h. The loss of oscillation and peristalsis activity (cestocidal effect) of these compounds was time-dependent (Figs. 2A and 2B).

5

Fig. 2. Time course of cestocidal activity with respect to loss of spontaneous movements of oscillation (A) and peristalsis (B) activities of gingerenone A, [6]dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, hexahydrocurcumin, 3R,5S-[6]-gingerdiol and 3S,5S[6]-gingerdiol on Hymenolepis nana. Effect of above constituents (100 ␮M) for 2, 4, 6, 12, 24, 48 and 72 h on H. nana, respectively. Vehicle is 0.1% DMSO solvent. Each value represents the mean ± SD of three individual experiments. Statistically significant, *p < 0.05 to vehicle group.

3.2. [10]-shogaol- and [10]-gingerol-induced cestocidal activity and loss of spontaneous mobility of oscillation and peristalsis activities The [10]-shogaol and [10]-gingero1 have maximum lethal efficacy and a higher loss of oscillation and peristalsis activity than the others at 24, 48 and 72 h. This study also compared the lethal efficacy and the loss of both oscillation and peristalsis activity induced by minimal effective dose of [10]-shogaol and [10]-gingero1 (i.e. 1–200 M and 1.0% DMSO as vehicle) for 72 h. In 100 and 200 ␮M [10]-shogaol, 100% of the worms were dead at 12 h, owing to the loss of both oscillation and peristalsis activity (Fig. 3A and B). The loss of peristalsis activity was occurred before the loss of oscillation activity in all concentrations. In 200 ␮M [10]-shogaol, despite the

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

415 416 417

418 419 420 421 422 423 424 425 426 427

G Model ACTROP 3419 1–11 6

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

Fig. 3. Effect of [10]-shogaol on H. nana. Treatment with various concentrations of [10]-shogaol (1, 10, 100 and 200 ␮M) with incubation times of 2, 4, 6, 12, 24, 48, and 72 h on H. nana, respectively. Time course of effect on oscillation (A) and peristalsis (B) of Hymenolepis nana of [10]-shogaol presented as percentages. Vehicle is 0.1% DMSO solvent. Each value is presented as mean ± SD of three individual experiments; *p < 0.05 indicates) a significant difference from the result for vehicletreated worms.

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448

loss of peristalsis activity at 2 h, the worms still had oscillation activity, the oscillation and peristalsis activity did not occur at 12, 24, 48 and 72 h, respectively. This finding suggests that worms stopped their spontaneous movement and died. As for loss of movement involving oscillation and peristalsis activity, [10]-shogaol induced a loss of spontaneous movement in H. nana in a time- and dosedependent manner (Figs. 3A and B). Additionally, for worms treated with 1 and 10 ␮M [10]gingero1, more than 30% had spontaneous movement of oscillation at 72 h (Fig. 4A). However, [10]-shogaol at 72 h had only around 15–20% of oscillation (Fig. 3A). This finding suggests that [10]gingero1 had a lower loss of spontaneous movement efficacy than that of [10]-shogaol. Figs. 2 and 3 reveal that, after exposure to 200 ␮M, all of H. nana had died at 12 h during treatment of [10]-shogaol. However, H. nana died at 24 h during treatment of [10]-gingerol, indicating that [10]-gingero1 had a lower lethal efficacy than that of [10]-shogaol. In 100 and 200 ␮M [10]-gingero1, all of the worms died at 24 h, owing to the loss of both oscillation and peristalsis activity (Fig. 4A and B). In 100 and 200 ␮M [10]-gingero1, although peristalsis activity was lost at 2 h, worms still had oscillation activity until

Fig. 4. Effect of [10]-gingerol on Hymenolepis nana. Treatment with various concentrations of [10]-gingerol (1, 10, 100 and 200 ␮M) with incubation times of 2, 4, 6, 12, 24, 48, and 72 h on H. nana, respectively. Time course of effect on oscillation (A) and peristalsis (B) of H. nana of [10]-gingerol presented as percentages. Vehicle is 0.1% DMSO solvent. Each value is presented as mean ± SD of three individual experiments; *p < 0.05 indicates) a significant difference from the result for vehicle-treated worms.

12 h. Moreover, [10]-gingero1 exhibited dose- and time-dependent losses of spontaneous movement of oscillation and peristalsis in H. nana. The vehicle control (1% DMSO) slightly affected the loss of oscillation and mild efficacy with respect to loss of peristalsis on H. nana. However in vehicle control, no worms died between 0 and 72 h of the oscillation and peristalsis experiments. 3.3. Determination of oxygen radical absorbing capacity (ORAC) This study also evaluated the antioxidant activity of gingerenone A, [6]-dehydrogingerdione, 5-hydroxy-[6]-gingerol, [4]-shogaol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC and 3R,5S-[6]-gingerdiol by performing ORAC assay. Fig. 5 shows the peroxyl radical absorbing capabilities of Trolox, ascorbic acid and above compounds, as determined by oxygen radical absorbance capacity (ORAC) fluorescein assay. Adding the test compounds decreased the relative fluorescence intensity. Fluorescein was exposed to excitation light at 495 nm without AAPH (no additive AAPH) over a 60-min period. Fluorescence intensity did not significantly change over 60 min, implying that 0.06 ␮M fluorescein is photostable under such conditions. Table 1 reveals that, at a concentration of 1.0 ␮M, the

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

449 450 451 452 453 454

455

456 457 458 459 460 461 462 463 464 465 466 467 468

G Model ACTROP 3419 1–11

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

7

Table 1 Relative ORAC (TE) values of compounds. Compound

ORAC (TE)

Trolox Ascorbic acid Gingerenone A [6]-Dehydrogingerdione [4]-Shogaol 5-Hydroxy-[6]-gingerol [6]-Shogaol [6]-Gingerol [10]-Shogaol [10]-Gingerol Hexahydrocurcumin 3R,5S-[6]-gingerdiol

1.00 0.51 ± 0.04 6.78 ± 0.34 2.83 ± 0.13 6.24 ± 0.47 0.85 ± 0.08 4.47 ± 0.72 5.42 ± 0.46 3.83 ± 0.88 4.18 ± 0.72 5.03 ± 0.40 5.48 ± 0.43

The ORAC (TE) value was calculated by dividing the area under the sample curve by the area under the Trolox curve, with both areas being corrected by subtracting the area under the blank curve. Trolox and ascorbic acid were used as a positive control. The results represent the mean ± SD for three independent experiments.

Fig. 5. Time course of changes in fluorescence decay curve of fluorescein in the presence of 1 ␮M Trolox, ascorbic acid, gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]gingerol, HHC and 3R,5S-[6]-gingerdiol, respectively. Details on the calculation of AUC can be found in Eq. (b) of Section 2.9.

476

average ORAC values of Trolox, ascorbic acid, gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC and 3R,5S[6]-gingerdiol, for relative Trolox equivalents (TE) ORAC were 1.00, 0.51 ± 0.04, 6.78 ± 0.34, 2.83 ± 0.13, 6.24 ± 0.47, 0.85 ± 0.08, 4.47 ± 0.72, 5.42 ± 0.46, 3.83 ± 0.38, 4.18 ± 0.72, 5.03 ± 0.40 and 5.48 ± 0.43, respectively. These values exceeded those found for ascorbic acid and Trolox (Table 1).

477

3.4. Morphological examination

469 470 471 472 473 474 475

478 479 480

Morphology examination was performed to observe the damage caused by the different ginger’s derivatives of 100 ␮M [10]-shogaol and [10]-gingerol in the adult worms of H. nana dead at the end of

24, 48 and 72 h in vitro assays. Next, 48 and 72 h treatment of [10]shogaol and [10]-gingerol revealed that scolox was destroyed and ulcerated compared with 24 h, respectively (Fig. 6). Additionally, microscopical examinations of H. nana adult worms destroyed by [10]-shogaol or [10]-gingerol indicated that the other proglottid segment, especially in the arc or triangle proglottid segment margin portion, was severely damaged and ulcerated as time increased (Fig. 7). 3.5. In vivo cestocidal activity in H. nana Correspondingly, the cestocidal activity caused by extract R0 of ginger in the H. nana infected mice was evaluated by performing an in vivo examination involving infected H. nana BALB/c mice were inoculated orally with 500 eggs. Following post-inoculation, R0 was administered orally in a single dose of 1 g/kg/day for 10 days. Ten days after the final administration, mice were sacrificed and adult worms were recovered according to the procedures of the above methods. The R0 exhibited cestocidal activity in mice (Fig. 8). The worms of R0 treatment revealed significantly reduced number of

Fig. 6. Morphology of scolex of Hymenolepis nana at various time intervals of drug treatment. H. nana was incubated with vehicle and 100 ␮M [10]-shogaol and [10]-gingerol for 24, 48 and 72 h, respectively. In 48 and 72 h treatment of [10]-shogaol and [10]-gingerol, scolox was destroyed and ulcerated compared with 24 h, respectively.

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

481 482 483 484 485 486 487 488

489

490 491 492 493 494 495 496 497 498

G Model ACTROP 3419 1–11

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

8

Fig. 7. Morphology of immature sagment of Hymenolepis nana at various time intervals of drug treatment. H. nana was incubated with vehicle and 100 ␮M [10]-shogaol and [10]-gingerol for 24, 48 and 72 h, respectively. Notably, 48 and 72 h treatment of H. nana adult worms destroyed by [10]-shogaol or [10]-gingerol demonstrated that the other proglottid segment, especially in the arc or triangle proglottid segment margin portion, was severely damaged and ulcerated with increasing time.

499 500 501 502

503

504 505 506 507 508 509 510 511 512

worms blow 1 of male/female mice for 10 days of post-inoculation, respectively. In the vehicle treatment group, the number of recovered worms from male/female mice did not differ from that of the infective group, despite a slightly reduced number of worms (Fig. 8). 3.6. Cultivation of spleen cell for cytokine assay Correspondingly, in vivo mice were sacrificed at 10 days after post-inoculation with H. nana. The spleen of each mouse was treated to harvest spleen cells in order to determine the concentrations of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, INF-␥ and Murine KC after 24, 48 and 72 h of incubation. Fig. 9 illustrates the levels of lymphokine production elicited by in vitro Con A stimulation of spleen cells from BALB/c mice infected with 500 eggs per mouse. According Fig. 9A, IFN-␥ ration more significantly increased at 24, 48 and 72 h of incubation on R0 treatment than that of control + Con

Fig. 8. Recovery of Hymenolepis nana worm numbers after male/female BALB/c mice were inoculated orally with 500 eggs. Following post-inoculation, R0 was administered orally in a single dose of 1 g/kg/day for 10 days in vivo. Infected groups without any treatment. Vehicle and R0 were infected groups with treatment of vehicle or R0 for 10 days, respectively. Each value is presented as mean ± SD of eight individual experiments; *p < 0.05 indicates a significant difference from infective groups.

A. Otherwise, INF-␥ ration did not significantly increase at 24, 48 and 72 h of incubation for the solvent groups. Additionally, solvent slight elevated IFN-␥ ration at 24, 48 and 72 h of incubation yet did not appear to more significantly change than that of control + Con A. In the control, solvent and R0 groups, all INF-␥ rations significantly increased with Con A stimulation than no treatment. According Fig. 9B, although the pattern of increase in IL-2 ration resembled that of IFN-␥ ration (Fig. 9A), the percentage increase ration was low. Additionally, IL-2 ration more significantly increased at 24 and 48 h than that of control + Con A, respectively. Moreover, IL-2 ration of solvent at 24, 48 and 72 h of incubation did not appear to more significantly change than that of control + Con A. According Fig. 9C, IL-4 ration of R0 more significantly decreased at 24 and 48 h of incubation than that of control + Con A, respectively. Also, IL-4 ration of solvent at 24, 48 and 72 h of incubation did not appear to more significantly change than that of control + Con A. According Fig. 9D, IL-5 ration of R0 more significantly decreased at 48 h of incubation than that of control + Con A, respectively. Additionally, IL-5 ration of solvent at 48 h of incubation did not appear to more significantly change than that of control + Con A. According Fig. 9E, IL-6 ration of R0, more significantly decreased at 48 and 72 h of incubation than that of the control group, respectively. Moreover, IL-6 ration of solvent at 48 and 72 h incubation did not appear to more significantly change than that of control + Con A. IL-10 ration of R0 more significantly decreased at 48 and 72 h of incubation than that of the control + Con A, respectively (Fig. 9F). According Fig. 9G, IL-13 ration of R0 more significantly decreased at 48 and 72 h of incubation than that of control + Con A, respectively. Furthermore, IL-13 ration of solvent at 48 and 72 h of incubation did not appear to more significantly change than that of control + Con A (Fig. 9G). IL12 were slight elevated yet did not significantly differ between the control, solvent and R0, even with or without Con A at 24, 48 and 72 h (data not shown). Murine KC were not significantly elevated in the experiments, including the addition of Con A (data not shown). 4. Discussion This study attempted to more thoroughly elucidate the effects underlying cestocidal activity of ginger against H. nana by performing assays to observe the motility and loss of spontaneous

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546

547

548 549 550

G Model ACTROP 3419 1–11

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

9

Fig. 9. Cytokine production ration in spleen cells after post-inoculation with Hymenolepis nana (500 eggs). The spleen of each mouse was treated to harvest spleen cells in order to determine cytokine of INF-␥, IL-2, IL-4, IL-5, IL-6, IL-10 and IL-13 after 24, 48 and 72 h incubation. Ctrl was infected groups without any treatment in vivo. Solvent and R0 were infected groups with treatment of vehicle or R0 (1 g/kg/day) for 10 days in vivo, respectively. Con A mean spleen cells were cultured with Con A. INF-␥, IL-2, IL-4, IL-5, IL-6, IL-10 and IL-13 ration were showed at 24, 48 and 72 h of incubation at (A), (B), (C), (D), (E), (F) and (G). Each value is presented as mean ± SD of eight individual experiments; *p < 0.05 indicates a significant difference from ctrl + Con A.

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

G Model ACTROP 3419 1–11 10 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

movement of oscillation and peristalsis on adult worms of H. nana. According to our results, gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC, 3R,5S-[6]-gingerdiol and 3S,5S[6]-gingerdiol displayed a time-dependent cestocidal effect and capability to halt spontaneous parasite oscillation and peristalsis movement of H. nana. Our further studies demonstrated [10]shogaol and [10]-gingerol exhibited dose- and time-dependent cestocidal effect with respect to spontaneous parasite oscillation and peristalsis movement and have a greater maximal effect than others. [10]-shogaol had a higher lethal efficacy and loss of spontaneous movement efficacy than that of [10]-gingerol against H. nana. However, [10]-gingerol had a higher lethal efficacy and loss of spontaneous movement efficacy than that of [10]-shogaol against A. cantonensis and A. simplex. Anthelmintic activity differs from nematodes (A. cantonensis and A. simplex) and cestodes (H. nana), owing to the chemical structure manner and different worm species (Liang et al., 2013; Lin et al., 2010a,b). In A. cantonensis assay, the order of larvicidal activity and loss of spontaneous movement activity of the tested compounds for a 24 h treatment period were: [10]-gingerol > [10]-shogaol (Lin et al., 2010a). In another study in A. simplex, the order of larvicidal activity and loss of spontaneous movement activity of the tested compounds were: [10]-shogaol > [10]-gingerol for a 24 h treatment period (Lin et al., 2010b). In H. nana assay, for lethal efficacy, loss oscillation and peristalsis percentage values of the compounds were: [10]shogaol  [10]-gingerol. However, [10]-shogaol and [10]-gingerol had more benefit than the above ginger component of lethal and loss of spontaneous movement efficacy against H. nana. Additionally, in morphological examination, H. nana adult worms destroyed by [10]-shogaol or [10]-gingerol especially in scolox or arc or triangle proglottid segment as time increased (Fig. 7). These constituents of ginger were found in ORAC assays to free radical scavenging activity, which did not appear to adversely affect their cestocidal effects. In ORAC fluorescein assay, gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC, 3R,5S[6]-gingerdiol and 3S,5S-[6]-gingerdiol were evaluated as radical scavengers compared to ascorbic acid and Trolox. A previous study demonstrated that not only is scavenging activity not involved in larvicide activity for A. simplex, but also that free radicals could harm the A. simplex; in this case, the scavenging of these free radicals allows for larvae survival (Hierro et al., 2004; Lin et al., 2010b). However, in this study, gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC, 3R,5S[6]-gingerdiol and 3S,5S-[6]-gingerdiol not only have cestocidal activity for adult worms of H. nana, but also have inhibitory radical scavenging activity against peroxyl radical. This finding is resembled of A. cantonensis and A. simplex (Lin et al., 2010a,b). Previous studies have established that ginger represents anthelmintic activity against D. immitis, A. simplex, S. mansoni, and gastrointestinal nematodes (Datta and Sukul, 1987; Iqbal et al., 2006; Lin et al., 2010b; Merawin et al., 2010; Sanderson et al., 2002). According to the results of a previous study, [6]-shogaol, [6]-gingerol and ginger extracts have a significant nematocidal efficacy with respect to Ansakis in vitro. Extracts of the rhizomes of ginger have an anthelmintic activity against infective D. immitis in vivo and in vitro (Datta and Sukul, 1987; Merawin et al., 2010). Crude powder and aqueous extract of dried ginger were used in sheep naturally infected with mixed species of gastrointestinal nematodes, including Trichostrongylus colubriformis, Haemonchus contortus, Oesophagostomum columbianum, Trichostrongylus axei, Trichuris ovis and Strongyloides papillosus to investigate its anthelmintic activity (Iqbal et al., 2006). Gingerol

and shogaol exhibited potent molluscicidal activity on Biomphalaria glabrata (Adewunmi et al., 1990). Their experiments examined the major constituents of ginger responsible for its molluscicidal activity and the effect of the active component on different stages of S. mansoni. Whereas other studies on ginger components have demonstrated larvicidal activity against A. cantonensis, D. immitis, A. simplex, S. mansoni (Datta and Sukul, 1987; Lin et al., 2010a,b; Merawin et al., 2010), the effects underlying effects of cestocidal activity of ginger on H. nana are relatively unknown. However, this study demonstrated that ginger’s constituents (e.g., gingerenone A, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC and 3S,5S-[6]-gingerdiol) exhibited cestocidal activities and a loss of spontaneous movement effects against H. nana (Fig. 2). To our knowledge, this study determines for the first time the cestocidal activity of ginger on H. nana. According to our results, gingerenone A, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC and 3S,5S-[6]-gingerdiol not only have cestocidal activity for worms of H. nana, but also radical scavenging activity against peroxyl radical. Therefore, those compounds have radical scavenging activity, not reduced cestocidal activity against H. nana. Nevertheless, further investigations are necessary for the mode of ginger constituents’s actions and/or mechanisms for its cestocidal effects between free radical scavenging activity. Our results further demonstrated that gingerenone A, [4]-shogaol, 5-hydroxy[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, HHC and 3S,5S-[6]-gingerdiol have cestocidal activities against H. nana reduced spontaneous movement of oscillation and peristalsis and also have free radical scavenging activity that did not adversely affect their cestocidal activity. Above results might contribute to the search for more selective and efficient naturally cestocidal compounds. The feasibility of using ginger to treat parasite infection has received considerable renewed interest. Previously, ginger was found to inhibit arachidonic acid metabolism via the cyclooxygenase and lipooxygenase pathways (Grzanna et al., 2005; Srivastava and Mustafa, 1992). More recent studies have indicated that ginger inhibits the induction of genes encoding cytokines and chemokines that are synthesized and secreted at inflammation sites (Grzanna et al., 2004, 2005). Despite the extensive attention paid to the anti-inflammatory properties of ginger, exactly how this natural product affects the immune responses in vivo has seldom been evaluated. This study evaluated how ginger affects cytokine in vivo/in vitro in a mouse model of H. nana infection. Previously analysis of cytokine production (IL-2, IL-3, IL-4, IL-5 and INF-␥) by in vitro Con A-stimulated mesenteric lymph node cells measured daily after egg or cyst infection of mice with H. nana indicated that cytokine production varies during parasite development and between different host strains (BALB/c and C3H/He mice) (Conchedda et al., 1997). As is well known, T-helper (Th)-lymphocytes profoundly impact the regulation of immune and inflammatory reactions through the release of cytokines. The roles of the Thl and Th2 subsets and of the cytokines they release have been investigated in a number of parasitic helminth models (e.g., Nippostrongylus brasiliensis, Schistosoma japonicum and S. mansoni, Trichinella spiralis, Trichuris muris) (Conchedda et al., 1997). Only limited data are available regarding cytokine activities during H. nana infection. Previously Conchedda et al., demonstrated that Th1 polarized response is elicited during infection by the development of the post-oncospheral stages in the intestinal villi, and that this is probably involved in the resistance to reinfection with eggs that is observed in both rapid and slow mouse strains. In addition, a Th2-polarized response appears to be elicited in the lumen development stage in BALB/c mice (Conchedda et al., 1997). Above results shown that cytokine production varied during H. nana infection from stage to stage of development and between

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682

G Model ACTROP 3419 1–11

ARTICLE IN PRESS R.-J. Lin et al. / Acta Tropica xxx (2014) xxx–xxx

698

different strains of host. Furthermore, ginger can inhibit the synthesis of several pro-inflammatory cytokines by various cell types in vitro (Grzanna et al., 2005), as well as affect Th1-/Th2-derived responses in vivo (Ahui et al., 2008; Shen et al., 2005; Yao et al., 2007). Whether ginger can also affect Th1-/Th2-derived inflammatory responses in vitro has not been evaluated with respect to the ginger against H. nana infection. This work demonstrates, for the first time, that ginger can elevate Th1-mediate immune responses and suppress the Th2-mediated immune responses of H. nana infection. In summary, this study demonstrates for the first time that ginger possesses cestocidal activity against H. nana by reduced spontaneous movement of oscillation and peristalsis. Elevated Th1 cytokines and the ability to reduce Th2 cytokines were found in vitro as well. Ginger is thus a highly promising therapeutic approach for combating H. nana infection.

699

Acknowledgments

683 684 685 686 687 688 689 690 691 692 693 694 695 696 697

Q3 700 701 702 703 704

705

706 707 708 709 710 711 712 713 714 715 716 717

Q4 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732

The authors would like to thank the National Science Council of the Republic of China, Taiwan for financially supporting this research under Contract No. NSC98-2320-B-037-014-MY3 (http://web1.nsc.gov.tw/). Ted Knoy is appreciated for his editorial assistance. References Adewunmi, C.O., Oguntimein, B.O., Furu, P., 1990. Molluscicidal and antischistosomal activities of Zingiber officinale. Planta Med. 56, 374–376. Ahui, M.L., Champy, P., Ramadan, A., Pham Van, L., Araujo, L., Brou Andre, K., Diem, S., Damotte, D., Kati-Coulibaly, S., Offoumou, M.A., Dy, M., Thieblemont, N., Herbelin, A., 2008. Ginger prevents Th2-mediated immune responses in a mouse model of airway inflammation. Int. Immunopharmacol. 8, 1626–1632. Ajami, A., Rafiei, A., 2007. Cytokine production in Hymenolepis nana infection. Iran. J. Immunol. 4, 236–240. Ali, B.H., Blunden, G., Tanira, M.O., Nemmar, A., 2008. Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber officinale Roscoe): a review of recent research. Food Chem. Toxicol. 46, 409–420. Aly, H.F., Mantawy, M.M., 2013. Efficiency of ginger (Zingiber officinale) against Schistosoma mansoni infection during host-parasite association. Parasitol. Int. Andreassen, J., Ito, A., Ito, M., Nakao, M., Nakaya, K., 2004. Hymenolepis microstoma: direct life cycle in immunodeficient mice. J. Helminthol. 78, 1–5. Asano, K., Muramatsu, K., 1997. Importance of interferon-gamma in protective immunity against Hymenolepis nana cysticercoids derived from challenge infection with eggs in BALB/c mice. Int. J. Parasitol. 27, 1437–1443. Asano, K., Muramatsu, K., Okamoto, K., 1993. Lymphokine production by mesenteric lymph node cells from BALB/C mice during Hymenolepis nana infection. Int. J. Parasitol. 23, 51–56. Chohan, M., Forster-Wilkins, G., Opara, E.I., 2008. Determination of the antioxidant capacity of culinary herbs subjected to various cooking and storage processes using the ABTS(*+) radical cation assay. Plant Foods Hum. Nutr. 63, 47–52. Conchedda, M., Bortoletti, G., Gabriele, F., Wakelin, D., Palmas, C., 1997. Immune response to the cestode Hymenolepis nana: cytokine production during infection with eggs or cysts. Int. J. Parasitol. 27, 321–327.

11

Datta, A., Sukul, N.C., 1987. Antifilarial effect of Zingiber officinale on Dirofilaria immitis. J. Helminthol. 61, 268–270. Diallo, D., Marston, A., Terreaux, C., Toure, Y., Paulsen, B.S., Hostettmann, K., 2001. Screening of Malian medicinal plants for antifungal, larvicidal, molluscicidal, antioxidant and radical scavenging activities. Phytother. Res. 15, 401–406. Gillespie, K.M., Chae, J.M., Ainsworth, E.A., 2007. Rapid measurement of total antioxidant capacity in plants. Nat. Protoc. 2, 867–870. Goto, C., Kasuya, S., Koga, K., Ohtomo, H., Kagei, N., 1990. Lethal efficacy of extract from Zingiber officinale (traditional Chinese medicine) or [6]-shogaol and [6]gingerol in Anisakis larvae in vitro. Parasitol. Res. 76, 653–656. Grzanna, R., Phan, P., Polotsky, A., Lindmark, L., Frondoza, C.G., 2004. Ginger extract inhibits beta-amyloid peptide-induced cytokine and chemokine expression in cultured THP-1 monocytes. J. Altern. Complement. Med. 10, 1009–1013. Grzanna, R., Lindmark, L., Frondoza, C.G., 2005. Ginger – an herbal medicinal product with broad anti-inflammatory actions. J. Med. Food 8, 125–132. Haniadka, R., Saldanha, E., Sunita, V., Palatty, P.L., Fayad, R., Baliga, M.S., 2013. A review of the gastroprotective effects of ginger (Zingiber officinale Roscoe). Food Funct. Hierro, I., Valero, A., Perez, P., Gonzalez, P., Cabo, M.M., Montilla, M.P., Navarro, M.C., 2004. Action of different monoterpenic compounds against Anisakis simplex s.l. L3 larvae. Phytomedicine 11, 77–82. Iqbal, Z., Lateef, M., Akhtar, M.S., Ghayur, M.N., Gilani, A.H., 2006. In vivo anthelmintic activity of ginger against gastrointestinal nematodes of sheep. J. Ethnopharmacol. 106, 285–287. Ito, A., 1997. Basic and applied immunology in cestode infections: from Hymenolepis to Taenia and Echinococcus. Int. J. Parasitol. 27, 1203–1211. Liang, C.H., Chan, L.P., Chou, T.H., Chiang, F.Y., Yen, C.M., Chen, P.J., Ding, H.Y., Lin, R.J., 2013. Brazilein from Caesalpinia sappan L. antioxidant inhibits adipocyte differentiation and induces apoptosis through caspase-3 activity and anthelmintic activities against Hymenolepis nana and Anisakis simplex. Evid. Based Complement. Alternat. Med. 2013, 864892. Lin, R.J., Chen, C.Y., Chung, L.Y., Yen, C.M., 2010a. Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus cantonensis. Acta Trop. 115, 69–76. Lin, R.J., Chen, C.Y., Lee, J.D., Lu, C.M., Chung, L.Y., Yen, C.M., 2010b. Larvicidal constituents of Zingiber officinale (ginger) against Anisakis simplex. Planta Med. 76, 1852–1858. Lopes, N.P., Chicaro, P., Kato, M.J., Albuquerque, S., Yoshida, M., 1998. Flavonoids and lignans from Virola surinamensis twigs and their in vitro activity against Trypanosoma cruzi. Planta Med. 64, 667–668. Merawin, L.T., Arifah, A.K., Sani, R.A., Somchit, M.N., Zuraini, A., Ganabadi, S., Zakaria, Z.A., 2010. Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis. Res. Vet. Sci. 88, 142–147. Palmas, C., Bortoletti, G., Gabriele, F., Wakelin, D., Conchedda, M., 1997. Cytokine production during infection with Hymenolepis diminuta in BALB/c mice. Int. J. Parasitol. 27, 855–859. Sanderson, L., Bartlett, A., Whitfield, P.J., 2002. In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production. J. Helminthol. 76, 241–247. Shen, C.L., Hong, K.J., Kim, S.W., 2005. Comparative effects of ginger root (Zingiber officinale Rosc.) on the production of inflammatory mediators in normal and osteoarthrotic sow chondrocytes. J. Med. Food. 8, 149–153. Shoji, N., Iwasa, A., Takemoto, T., Ishida, Y., Ohizumi, Y., 1982. Cardiotonic principles of ginger (Zingiber officinale Roscoe). J. Pharm. Sci. 71, 1174–1175. Sirivichayakul, C., Radomyos, P., Praevanit, R., Pojjaroen-Anant, C., Wisetsing, P., 2000. Hymenolepis nana infection in Thai children. J. Med. Assoc. Thai. 83, 1035–1038. Srivastava, K.C., Mustafa, T., 1992. Ginger (Zingiber officinale) in rheumatism and musculoskeletal disorders. Med. Hypotheses 39, 342–348. Wang, A., McKay, D.M., 2005. Immune modulation by a high molecular weight fraction from the rat tapeworm Hymenolepis diminuta. Parasitology 130, 575–585. Yao, H., Tong, J., Wang, Z., Zhang, Z.Q., Chen, J.X., 2007. Effect of acupoint application therapy on expression of IL-4 and IFN-gamma in lung tissue of asthma rats. Zhen Ci Yan Jiu 32, 174–178.

Please cite this article in press as: Lin, R.-J., et al., Cestocidal activities of ginger (Zingiber officinale) against Hymenolepis nana. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.07.009

733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795