Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus cantonensis

Acta Tropica 115 (2010) 69–76

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Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus cantonensis Rong-Jyh Lin a,b , Chung-Yi Chen c , Li-Yu Chung a , Chuan-Min Yen a,∗ a b c

Department of Parasitology and Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan School of Medical and Health Sciences, Fooyin University, Kaohsiung County 831, Taiwan

a r t i c l e

i n f o

Article history: Available online 4 January 2010 Keywords: Larvicidal activity Angiostrongylus cantonensis Ginger Anthelmintic activity DPPH and peroxyl radical

a b s t r a c t In this study, we investigated the anthelmintic activity of [6]-gingerol, [10]-shogaol, [10]-gingerol, [6]shogaol and hexahydrocurcumin, a constituent isolate from the roots of ginger (Zingiber officinale), for the parasite Angiostrongylus cantonensis. This study found that the above constituents killed A. cantonensis larvae or reduced their spontaneous movements in a time- and dose-dependent manner. The larvicidal effect or ability to halt spontaneous parasite movement of [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]shogaol and hexahydrocurcumin at various concentrations was reached from 24 to 72 h, respectively. Further investigation to determine minimal effective doses of [10]-gingerol and hexahydrocurcumin revealed [10]-gingerol to have a greater maximum larvicidal effect and loss of spontaneous movements than hexahydrocurcumin, mebendazole and albendazole. These constituents of ginger showed effects against DPPH and peroxyl radical under larvicidal effect. Together, these findings suggest that these constituents of ginger might be used as larvicidal agents against A. cantonensis. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Ginger (Zingiber officinale L., Zingiberaceae) is a widely used ingredient in various foods and beverages (Ali et al., 2008) and is used in traditional Chinese medicine. For centuries, ginger has been used in such traditional medicines as Kankyo (dried, steamed rhizomes), Kanshokyo (dried rhizomes), or Shokyo (fresh rhizomes) (Goto et al., 1990). These medicines are purported to be effective treatments for asthma, common cold disorders, nervous disease, stroke, toothache, gastrointestinal constipation, inflammation, migraine, oxidant stress, hypercholesterolaemia, helminthiasis and schistosomiasis (Langmead and Rampton, 2001; Sanderson et al., 2002; Sekiya et al., 2004; Iqbal et al., 2006; Ali et al., 2008; Chohan et al., 2008; Ghayur et al., 2008; Islam and Choi, 2008). Many pharmacological properties of ginger have been identified, including essential oil, zingiberol, zingiberone, zingiberene, and pungent components such as [6]-gingerol and [6]-shogaol (Goto et al., 1990; Shoji et al., 1982; Connell and Sutherland, 1969). Phytochemical reports have shown that the main constituents of ginger are zingerone, paradol and the gingerols and shogaols. The nonvolatile pungent ingredients from ginger include shogaol,

∗ Corresponding author. Tel.: +886 7 3121101x2169; fax: +886 7 3218309. E-mail addresses: [email protected] (R.-J. Lin), [email protected] (C.-Y. Chen), [email protected] (L.-Y. Chung), [email protected] (C.-M. Yen). 0001-706X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2009.12.007

zingerone and gingerol. These agents are known to have the ability to suppress the hyperproliferative, inflammatory, and transformative processes of carcinogenesis. The phenolic compounds derived from ginger (e.g. [6]-gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol) have many interesting physiological and pharmacological activities. Although it has been used for centuries, this plant still attracts extensive research attention. Angiostrongylus cantonensis, a nematode parasite, also known as the rat lungworm, dwells in the rat pulmonary artery where it develops to sexual maturity (Courdurier et al., 1968; Guilhon et al., 1973). The complex life cycle of the rat lungworm, A. cantonensis, including details of the migration of the third-stage larvae in rats, has been described (Alicata, 1965; Jindrak, 1968). After being ingested by the final host, the third-stage larvae penetrate the gastric and intestinal walls and are carried by the venules to the lungs and other tissues over the body. Then the larvae migrate to the brain and congregate in the spinal cord, cerebellum, medulla, and diencephalon, then where they gradually spread to the telencephalon and migrate into the subarachnoid space where they moult twice to become immature adults (fifth-stage larvae of A. cantonensis; AcL5). These L5 larvae of A. cantonensis have been strongly associated with the human A. cantonensis infections. However, the infection of A. cantonensis in human and other non-permissive hosts will cause eosinophilic meningitis or meningoencephalitis (Chye et al., 2004). Rats serve as the definitive host of the nematode. If an infection occurs in non-permissive hosts, including humans and

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mice, the development of the parasites will terminate at the youngadult worm stage (fifth-stage larvae of A. cantonensis; AcL5) in the brain of the hosts leading to eosinophilic meningoencephalitis or eosinophilic meningitis (Hwang and Chen, 1988; Chye et al., 2004). Infection can be acquired by the consumption of fresh produce, such as lettuce, contaminated with these intermediate or transport hosts. Humans are infected by ingestion of freshwater and terrestrial snails and slugs, or transport hosts, such as freshwater prawns, frogs, fish, and planarians (Tsai et al., 2001a). The major intermediate hosts in Taiwan are the African giant snail (Achatina fulica) (Hwang and Chen, 1991; Neuhauss et al., 2007) and the golden apple snail (Ampullarium canaliculatus) (Yen et al., 1985). A. cantonensis is the major cause of human eosinophilic meningitis or meningoencephalitis in Taiwan, the Pacific Islands, Southeast Asia and Japan (Bisseru, 1971; Hwang and Chen, 1991). The use of anthelmintic agents and corticosteroids to treat A. cantonensis remains controversial and no anthelmintic drug alone has been proven to be effective (Tsai et al., 2001b). Ginger (Z. officinale) has been able to destroy Dirofilaria immitis (Datta and Sukul, 1987), Anisakis larvae (Goto et al., 1990), Schistosoma mansoni (Adewunmi et al., 1990; Sanderson et al., 2002), and gastrointestinal nematodes (Iqbal et al., 2006). Although some components of ginger such as [6]-gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin (HHC) have been demonstrated to have significant larvicidal activity against some parasitic species in vitro and in vivo, little is known about mechanism underlying their larvicidal effects on A. cantonensis. Free radical scavenging activities have been implicated in a number of pathological conditions such as atherosclerosis, inflammatory diseases, ischaemia–reperfusion, AIDS, cancer and aging (Al-Abrash et al., 2000; Favier, 2006). Some agents have been found to have antibacterial and antiprotozoae activity, but they have also been found to have free radical scavaging activity, though they were not sure to what extent this affected larvicidal activity (Lopes et al., 1998; Diallo et al., 2001; Tiew et al., 2003; Ahmad et al., 2005). Another study has suggested that free radical scavenging may reduce larvicidal activity by permitting larvae survival (Hierro et al., 2004). Therefore, it is still not clear how free radical scavaging may affect larvicidal activity of certain anthelmintic agents. In this study, we first identified which components of ginger root have antiparasitic activities on larvae of A. cantonensis in vitro, and then used DPPH scavenging assay and oxygen radical absorbance capacity (ORAC) assay to determine whether or not there was an association between the possible scavenger activity of these components and their anthelmintic activity against A. cantonensis L5 (AcL5). 2. Materials and methods 2.1. Plant material The roots of ginger (Z. officinale) were purchased from a local market of Kaohsiung in Taiwan in July 2006, which were identified by Dr. Yen-Ray Hsui of the Division of Silviculture, Taiwan Forestry Research Institute, Taipei, Taiwan. A voucher specimen (Hsui-Zo-1) was deposited at Fooyin University. 2.2. Extraction and isolation The roots (25.6 kg) of Z. officinale were chipped, air-dried, and extracted repeatedly with CHCl3 at room temperature. The combined CHCl3 extracts were evaporated and further separated into 20 fractions by column chromatography on silica gel with gradients of n-hexane/CHCl3 . Each fraction was rechromatographed over silica gel eluting with a gradient of CHCl3 /MeOH to obtain [10]-shogaol (210 mg, 0.0008%), [6]-shogaol (328 mg, 0.0013%), [10]-gingerol

(197 mg, 0.0008%), [6]-gingerol (311 mg, 0.0012%) and hexahydrocurcumin (251 mg, 0.0010%), respectively. These compounds were identified using spectroscopic analysis of data and compared with the literature values (Shoji et al., 1982; Kikuzaki et al., 1991; Connell and Sutherland, 1969). 2.3. General experimental procedures Optical rotations were measured with a JASCO DIP-370 digital polarimeter. UV spectra were obtained in MeCN using a JASCO V530 spectrophotometer. The IR spectra were measured on a Hitachi 260-30 spectrophotometer. 1 H (400 MHz, using CDCl3 as solvent for measurement), 13 C (100 MHz), DEPT, HETCOR, COSY, NOESY, and HMBC NMR spectra were obtained on a Unity Plus Varian NMR spectrometer. LRFABMS and LREIMS were obtained with a JEOL JMS-SX/SX 102A mass spectrometer or a Quattro GC–MS spectrometer with a direct inlet system. Silica gel 60 (Merck, 230–400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.20 mm) were used for analytical 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), 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). Dimethyl sulfoxide (DMSO), Fluorescein, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 2,2 -Azobis(2-amidinopropane) dihydrochloride (AAPH), and 2,2-diphenyl-1-picrylhydrazyl (DPPH), mebendazole and albendazole were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO). All drugs and reagents were dissolved in distilled H2 O unless otherwise noted. [6]-Gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol, hexahydrocurcumin (HHC), mebendazole and albendazole were dissolved in DMSO at 1 M stock and serially diluted with distilled H2 O and vehicle (contains 1% DMSO in sterilized distilled H2 O). 2.5. Animal experiment This study was approved by the Animal Care and Use Committee at the Kaohsiung Medical University. Male Sprague–Dawley rats and male BALB/c mice, weighing 250–350 g and 25–40 g, respectively, were provided by the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). They were housed under constant temperature and controlled illumination (lights on between 7:30 and 19:30). Rats were allowed food and water ad libitum. 2.6. Angiostrongylus contonensis larval preparation The Taiwanese strain of A. contonensis used in this study was originally isolated from the naturally infected giant African snail (A. fulica) collected in Pingtung County in southern Taiwan. A. contonensis is maintained in our laboratory by cycling through the planorbid snail (Biomphalaria glabrata) and Sprague–Dawley rats. First-stage larvae of A. cantonensis were recovered from infected rat feces and fed to snails. Larvae within planorbid snail tissues were recovered using the method described by Chye et al. (2004) with slight modifications. Briefly, the snails shells were crushed and tissues were homogenized and digested with 0.6% pepsin–HCl solution (pH 2–3, 500 I.U. pepsin/g tissue), and incubated with agitation at 37 ◦ C in a water bath for 1 h. Host cellular debris was removed by centrifugation at 1400 × g for 10 min and larvae in the sediment

R.-J. Lin et al. / Acta Tropica 115 (2010) 69–76

were observed under a microscope. Morphological criteria for identification of the infective L3 (third-stage larvae) of A. cantonensis were a length of 422–525 mm and a width of 24–35 mm and the tail always terminating in a fine point (Ash, 1970; Shih et al., 2007). Hybrid mice were individually infected with 50 larvae (AcL3) via an oral stomach tube. The mice were raised in an air-conditioned laboratory animal center (22 ± 1 ◦ C and 50 ± 10% relative humidity). Three weeks after infestation, each mouse was sacrificed with an excess of ether, and fifth-stage larvae A. cantonensis (AcL5) were recovered from the rat brains.

rescent, 18.75 mM AAPH and appropriate test substance (1 ␮M) in 75 mM phosphate buffer. The analyzer was programmed to record the fluorescence of FL every minute after the addition of 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 are expressed as micromole Trolox equivalents per liter. The area under curve (AUC) was calculated as AUC = 50 +

2.7. Assay of lethal efficacy and loss of spontaneous mobility activity L5 viable larvae of A. cantonensis were obtained from the brain of mice as described above. The AcL5 worms were collected with the help of a needle with a blunt tip, placed in a Petri dish with 0.9% NaCl solution and washed several times. Most of the larvae were encysted, but those that were quickly became excysted in the washing NaCl solution, were observed individually under an inverted microscope and those which showed any kind of internal or external damage were discarded. The larvae were identified based on their morphological features, divided into groups, and placed in 24-well culture dishes (5 larvae each) containing DMEM plus 20% (v/v) heat-inactivated fetal bovine serum (FBS) at pH 7.4 in an atmosphere of 95% O2 /5% CO2 , at 37 ◦ C. These culture conditions have been shown to provide maximum development and survival of A. cantonensis. The 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 [6]-gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol, hexahydrocurcumin (HHC), mebendazole and albendazole (1, 10, 50, 100 and 200 ␮M). Survival and mobility of the larvae were assessed at 1, 4, 8, 12, 24, 48 and 72 h using a stereomicroscope. Larvae statuses were scored as death, poor mobility or normal mobility by two different investigators in a blinded fashion.

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f f1 f49 + ··· + i + ··· + + 50 f0 f0 f0

f  50

(1)

f0

where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. The data were analyzed using a Microsoft Excel macro program (Microsoft) using this Eq. (1) to calculate the AUC. The net AUC is obtained by subtracting the AUC of the blank from that of a sample. The relative Trolox equivalent ORAC value is calculated as



relative ORAC value =

(AUCsample − AUCblank )



(AUCTrolox − AUCblank ) ×

 molarity of Trolox  molarity of sample

(2)

2.10. Statistical evaluation of data The results are expressed as mean ± SE. Statistical differences were estimated by one-way analysis of variance (ANOVA) followed by Dunnett’s test. A p value of 0.05 was considered significant. Analysis of the data and plotting of the figures were performed with the aid of software (SigmaPlot Version 8.0 and SigmaStat Version 2.03, Chicago, IL) run on an IBM-compatible computer. 3. Results

2.8. Evaluation of DPPH radical scavenging activity

3.1. Larvicidal activity against A. cantonensis

The antioxidant activities of compounds were assessed on the basis of the radical scavenging effect of the stable DPPH free radical as described previously (Hwang et al., 2001). 200 ␮L of freshly prepared DPPH (100 ␮M) in ethanol solution was added to 2 ␮L tested sample solutions (0.1–100 ␮M) in 96-well plate. After incubation at room temperature for 30 min, the absorbance values of each solution were measured at 517 nm using a spectrophotometer microplate reader (model: Dynex MRX II) and converted into the percentage antioxidant activity (AA) as follows: AA% = 1 − [(Abssample − Absblank )/Abscontrol ], where Absblank , Abscontrol and Abssample are the absorbance of the blank and control and sample, respectively. Ethanol (200 ␮L) plus test sample vehicle (1% DMSO) was used as a blank. DPPH solution plus ethanol was used as a negative control. The IC50 value is the concentration of test sample required to scavenge 50% DPPH free radicals, respectively. Ascorbic acid and Trolox were used as positive controls.

[6]-Gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin (HHC) (Fig. 1) were purified from roots of ginger (Z. officinale), and the identity of the isolated material was confirmed by spectroscopic methods according the Materials and methods (Section 2.2) described. In the first series of experiments, the larvicidal effects were used to study the ability of the above compounds to alter survival of fifth-stage larvae of A. cantonensis (AcL5). As shown in Table 1, all drugs reduced the survival of larvae of AcL5 in vitro. The lethal efficacy (larvicidal effect) of these compounds was observed to be dose- and time-dependent. In addition, the maximum lethal efficacy of [10]-gingerol, [6]-gingerol, HHC and mebendazole was approximately 100% (range, 95–100%). For lethal efficacy the −log LD50 (␮M) of [10]-shogaol, [6]-shogaol, [10]-gingerol, [6]-gingerol and HHC for 72 h were 3.180 ± 0.019, 4.782 ± 0.018, 4.429 ± 0.031, 4.877 ± 0.023, and 4.351 ± 0.039, respectively. LD50 (␮M) values were on average lower than those observed for 24 h and 48 h treatments. For 24 h, the LD50 (␮M) values of [10]-shogaol, [6]-shogaol, [10]-gingerol, [6]gingerol and HHC were −log LD50 = 3.057 ± 0.023, 3.620 ± 0.026, 4.609 ± 0.031, 3.557 ± 0.023, and 3.584 ± 0.016, respectively. For 48 h, they were −log LD50 = 3.180 ± 0.029, 4.095 ± 0.105, 4.413 ± 0.034, 4.105 ± 0.033, and 4.287 ± 0.022, respectively. However, the LD50 (␮M) values of mebendazole for 24 h were −log LD50 = 3.624 ± 0.075, and albendazole was found to have only slight larvicidal activity. The order of larvicidal activity of the tested compounds for a 24 h treatment period was: [10]-gingerol > [6]shogaol  mebendazole > HHC > [6]-gingerol > [10]-shogaol.

2.9. Evaluation of oxygen radical absorbing capacity (ORAC) The automated ORAC assay was carried out on a FLUOstar Galaxy plate reader (Roche Diagnostic System Inc., Branchburg, NJ) as described in a previous report by Gillespie et al. (2007). The experiment was conducted at 37 ◦ C under pH 7.0 condition with a blank sample in parallel. Briefly, AAPH was used as a peroxyl generator, and 1 ␮M Trolox, a water-soluble analogue 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 fluo-

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R.-J. Lin et al. / Acta Tropica 115 (2010) 69–76 Table 1 Larvicidal activity (lethal efficacy) of the major constituents of ginger on fifth-stage larvae of Angiostrongylus cantonensis (AcL5). Compound

Dose (␮M) 200

50

10

1

[10]-Shogaol

1D 2D 3D

100 65.0* 50.0* 50.0*

4.2 12.0* 12.0*

ND ND ND

0.0 10.0* 10.0*

0.0 ND ND

[6]-Shogaol

1D 2D 3D

8.3* 16.7* 50.0*

0.0 27.4* 47.6*

0.0 21.4* 41.7*

0.0 8.34* 41.7*

0.0 8.3* 31.0*

[10]-Gingerol

1D 2D 3D

95.0* 93.8* 93.8*

82.5* 70.0* 75.0*

14.0 ND ND

12.0* 12.0* 12.0*

10.0* ND ND

[6]-Gingerol

1D 2D 3D

50.0* 66.7* 100.0*

0.0 33.3* 41.7*

0.0 23.3* 40.0*

0.0 20.0* 36.7*

0.0 0.0 16.7*

HHC

1D 2D 3D

30.0* 80.0* 100.0*

25.0* 51.7* 60.0*

11.1* 22.2* 44.4*

8.3* 16.7* 41.7*

5.6 14.4* 24.0*

Mebendazole

1D 2D 3D

80.0* 90.0* 100*

73.4* 90.0* 90.0*

0.0 30.0* 30.0*

0.0 0.0 0.0

0.0 0.0 0.0

Albendazole

1D 2D 3D

0.0 10.0* 20.0*

0.0 0.0 10.0

ND ND ND

ND ND ND

0.0 0.0 0.0

Data represent the percentage of dead worms. HHC: hexahydrocurcumin. ND: not done. 1D, 2D and 3D mean 24, 48 and 72 h, respectively. Data represent the mean ± SE of 3 different experiments. *p < 0.05 compared with the vehicle, ANOVA followed by Dunnett’s test.

Table 2 In vitro loss of spontaneous movements effects of several compounds on fifth-stage larvae of Angiostrongylus cantonensis (AcL5). Fig. 1. The major chemical constituents of ginger (Zingiber officinale). Compound

Dose (␮M) 50

10

1

[10]-Shogaol

1D 2D 3D

200 100.0* 100.0* 90.0*

39.2* 66.2* 83.7*

ND ND ND

40.0* 60.0* 70.0*

10.0* ND ND

[6]-Shogaol

1D 2D 3D

41.7* 83.4* 91.7*

20.8* 64.3* 76.8*

25.0* 54.7* 58.4*

14.3* 25.1* 56.0*

0.0 8.3* 31.0

[10]-Gingerol

1D 2D 3D

100.0* 100.0* 100.0*

87.5* 100.0* 95.0*

54.0* ND ND

72.0* 62.0* 100.0*

10.0* ND ND

[6]-Gingerol

1D 2D 3D

83.3* 83.4* 100.0*

45.8* 75.0* 85.0*

0.0 40.0* 81.7*

16.7* 28.3* 73.4*

0.0 16.7* 33.4*

HHC

1D 2D 3D

100.0* 100.0* 100.0*

75.0* 95.0* 95.0*

33.3* 66.6* 100.0*

70.5* 70.0* 100.0*

38.9* 47.7* 82.3*

Mebendazole

1D 2D 3D

80.0* 100.0* 100.0*

80.0* 90.0* 90.0*

20.0* 30.0* 30.0*

10.0* 10.0* 10.0*

0.0 0.0 0.0

Albendazole

1D 2D 3D

0.0 20.0* 20.0*

0.0 10.0* 10.0*

ND ND ND

ND ND ND

0.0 0.0 0.0

3.2. A. cantonensis loss of spontaneous mobility assay We further examined the time course of [6]-gingerol-, [10]gingerol-, [6]-shogaol-, [10]-shogaol-, HHC-, albendazole- and mebendazole-induced loss of mobility on A. cantonensis. As shown in Table 2, these compounds were observed to have doseand time-dependent effects. [10]-Shogaol, [6]-gingerol, [10]gingerol, [6]-shogaol, HHC, albendazole and mebendazole induced a maximum loss of spontaneous movement effects in various concentrations at 24 h, 48 h, and 72 h, respectively. However, both [10]-gingerol and HHC had induced greater immobility than the others, as maximum loss of spontaneous movement appeared in 10 ␮M at 72 h. The order of loss of spontaneous movement activity was for a 24 h treatment period was: HHC > [10]-gingerol > [10]shogaol > mebendazole > [6]-shogaol > [6]-gingerol > albendazole. However, albendazole was found to induce only a slight loss of spontaneous movement in AcL5 at 48 h to 72 h. 3.3. [10]-Gingerol- and HHC-induced larvicidal activity and loss of spontaneous mobility We compared AcL5 death and loss of spontaneous movement induced by minimal effective doses of [10]-gingerol, HHC and vehicle (DMSO) (i.e., 10–500 ␮M and 1.0% DMSO) over time. In 100 ␮M and 200 ␮M [10]-gingerol, a little more than 50% of the larvae were found to be dead at 4 h (Fig. 2A). Excluding 10 ␮M, [10]-gingerol

100

Data represent the percentage of loss spontaneous movement. HHC: hexahydrocurcumin. ND: not done. 1D, 2D and 3D mean 24, 48 and 72 h, respectively. Data represent the mean ± SE of 3 different experiments. *p < 0.05 compared with the vehicle, ANOVA followed by Dunnett’s test.

R.-J. Lin et al. / Acta Tropica 115 (2010) 69–76

Fig. 2. Time course of larvicidal activity (A) and loss of spontaneous movements (B) of [10]-gingerol on the L5 of Angiostrongylus cantonensis. Effect of [10]-gingerol (10, 100 and 200 ␮M) for 1, 4, 8, 12 and 24 h on fifth-stage larvae of Angiostrongylus cantonensis (AcL5), respectively. Each value represents the mean ± SE of three individual experiments. Statistically significant, *p < 0.05 to control (vehicle) group. ANOVA followed by Dunnett’s test.

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Fig. 3. Time course of larvicidal activity (A) and loss of spontaneous movements (B) of hexahydrocurcumin on the L5 of Angiostrongylus cantonensis. Effect of hexahydrocurcumin (10, 100, 200 and 500 ␮M) for 1, 4, 8, 12 and 24 h on fifth-stage larvae of Angiostrongylus cantonensis (AcL5), respectively. Each value represents the mean ± SE of three individual experiments. Statistically significant, *p < 0.05 to control (vehicle) group. ANOVA followed by Dunnett’s test.

3.4. Radical scavenging activity of constituents of ginger

(100–200 ␮M) treatment, up to 60% and 80% of larvae were dead at 4 h and 24 h, respectively. With regard to loss of movement, [10]gingerol induced loss of spontaneous movement in A. cantonensis in a time- and dose-dependent manner (Fig. 2B). Approximately more than 50% of the worms stopped moving at 8 h in all concentrations of [10]-gingerol treatment. By 24 h, up to 70% of larvae had ceased moving. As shown in Fig. 3A, after exposure to 500 ␮M HHC, 100% of A. cantonensis had died at 24 h, showing that HHC had less lethal efficacy than [10]-gingerol. In addition, in larvae treated with 10, 100 and 200 ␮M HHC, more than 70% had loss of spontaneous movement at 24 h (Fig. 3B). This shows that HHC had less loss of spontaneous movement efficacy than [10]-gingerol ([10]-gingerol at 4–24 h about range 95–100%) (Fig. 2B). In general, HHC exhibited a slightly greater dose- and time-dependent loss of spontaneous movement in the larvae. The vehicle control (1% DMSO) had no effect on loss of movement or lethal efficacy on A. cantonensis larvae. Next, none of the compounds, [10]-shogaol, [6]-gingerol, [10]gingerol, [6]-shogaol, HHC, albendazole or mebendazole, induced significant loss of spontaneous movement and death in either the male or female AcL5 at any test concentration for 24–72 h (data not shown).

A DPPH assay and an oxygen radical absorbance capacity (ORAC) fluorescein assay was performed to evaluate of antioxidant activity of [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and HHC. As shown in Fig. 4, the effects of ascorbic acid, Trolox, [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and HHC on the scavenging activities of DPPH were compared to vehicle-treated controls. The above compounds were found to have significantly increased dose-dependent DPPH scavenging activity. In the DPPH assay system, the free radical scavenging activity of tested compounds was expressed as IC50 in Table 3. The order of antioxidant activity was: [10]-gingerol > [10]-shogaol  [6]-gingerol > [6]-shogaol > HHC. 3.5. Determination of oxygen radical absorbing capacity (ORAC) The peroxyl radical absorbing abilities of Trolox, ascorbic acid, [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and HHC as determined by oxygen radical absorbance capacity (ORAC) fluorescein assay are shown in Fig. 5. When the test compounds were added, the relative fluorescence intensity decreased. Fluorescein was exposed to excitation light at 495 nm in the absence of AAPH (no additive AAPH) over a 50-min period. There were no significant changes in fluorescence intensity over 50 min, suggesting that 0.06 ␮M fluorescein is photostable under such con-

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R.-J. Lin et al. / Acta Tropica 115 (2010) 69–76 Table 4 Relative ORAC (TE) values of compounds. Compound

ORAC (TE)

Trolox Ascorbic acid [10]-Shogaol [6]-Gingerol [10]-Gingerol [6]-Shogaol HHC

1.00 0.23 ± 0.05 2.65 ± 0.03 3.65 ± 0.05 2.38 ± 0.02 2.40 ± 0.04 3.51 ± 0.06

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. HHC: hexahydrocurcumin. The results represent the mean ± SE for three independent experiments. Fig. 4. DPPH scavenging activity of Trolox, ascorbic acid, [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin (HHC). Each value represents the mean ± SE of three individual experiments. Statistically significant, *p < 0.05 to vehicle group (control). ANOVA followed by Dunnett’s test.

Table 3 Free radical scavenging activity of several compounds, ascorbic acid, and Trolox in the DPPH assay. Compound [10]-Shogaol [6]-Shogaol [10]-Gingerol [6]-Gingerol HHC Ascorbic acid Trolox

Free radical scavenging activity IC50 (␮M) 37.21 75.03 23.04 41.75 94.45 9.68 8.35

± ± ± ± ± ± ±

1.15 3.36 2.46 4.24 5.54 0.12 0.15

The IC50 value is the concentration of test sample required to scavenge 50% DPPH free radicals, respectively. HHC: hexahydrocurcumin. Data represent the mean ± SE for three different experiments. Statistical analyses were performed using Student’s t-test.

ditions. As shown in Table 4, at a concentration of 1.0 ␮M, the average ORAC values of Trolox, ascorbic acid, [10]-shogaol, [6]gingerol, [10]-gingerol, [6]-shogaol and HHC for relative Trolox equivalents (TE) ORAC were determined to be 1.00, 0.23 ± 0.05, 2.65 ± 0.03, 3.65 ± 0.05, 2.38 ± 0.02, 2.40 ± 0.04 and 3.51 ± 0.06,

Fig. 5. Time course of changes in fluorescence decay curve of fluorescein in the presence of 1 ␮M Trolox, ascorbic acid, [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]shogaol and hexahydrocurcumin (HHC), respectively. For calculation of the AUC, please see Eq. (2) in Section 2.9.

respectively. These values were all higher than those found for ascorbic acid and Trolox (Table 4). The order of the ORAC values of the compounds was: [6]-gingerol > HHC > [10]-shogaol > [6]shogaol  [10]-gingerol > ascorbic acid. 4. Discussion To better understand effects underlying ginger’s larvicidal activity, we performed assays to observe motility and loss of spontaneous movement on AcL5. Finally, we found that [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin exhibited dose- and time-dependent larvicidal effect and ability to halt spontaneous parasite movement of AcL5. These constituents of ginger were found in DPPH and ORAC assays to free radical scavenging activity which did not appear to adversely affect their larvicidal effects. In the DPPH free radical scavenging test (Table 3) and an oxygen radical absorbance capacity (ORAC) fluorescein assay (Table 4), [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and HHC were evaluated as radical scavengers compared to ascorbic acid and Trolox. The previous evidence showed that not only that scavenging activity is not involved in larvicide activity for Anisakis simplex s.l., but also that free radicals could be harmful for A. simplex s.l. L3, in which case the scavenging of these free radicals would permit larvae survival (Hierro et al., 2004). However, in the present study, we found that [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]shogaol and HHC have not only larvicide activity for L5 larvae of A. cantonensis but also radical scavenging activity against DPPH and peroxyl radical, respectively. Analgesics, corticosteroids and careful removal of CSF at frequent intervals can relieve symptoms from increased intracranial pressure. Treatment of A. cantonensis is controversial and varies across endemic areas. No anthelmintic drug is proven to be effective and some patients have worsened with therapy. Albendazole and mebendazole, a benzimidazole derivative, has been used in human angiostrongyliasis. They kill a parasite by binding to parasite beta-tubulin, blocking worms polymerization and inhibiting glucose uptake. A combination of mebendazole and corticosteroid, however, appears to shorten the course of infection in eosinophilic meningitis caused by A. cantonensis (Tsai et al., 2001b). Albendazole has also relieved symptoms of angiostrongyliasis (Jitpimolmard et al., 2007). Previous studies have shown that ginger (Z. officinale) exhibits anthelmintic activity against D. immitis (Datta and Sukul, 1987), Anisakis larvae (Goto et al., 1990), S. mansoni (Adewunmi et al., 1990; Sanderson et al., 2002), and gastrointestinal nematodes (Iqbal et al., 2006). In a previous study, Goto et al. (1990) reported significant anti-nematodal efficacy of [6]-shogaol, [6]-gingerol, and Z. officinal’s extract in Ansakis larvae in vitro. Datta and Sukul

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(1987) investigated the anthelmintic activity of alcoholic extracts of the rhizomes of ginger (Z. officinale) in dogs, naturally infected with D. immitis. By subcutaneous injections of the ginger extract reduced microfilarial concentration in blood by a maximum of 98% and fifty-five days after the last injection these was an 83% reduction in microfilarial concentration suggesting the partial destruction of adult worms. Recently, Iqbal et al. (2006) investigated the anthelmintic activity of crude powder and crude aqueous extract of dried ginger in sheep naturally infected with mixed species of gastrointestinal nematodes including: Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, Oesophagostomum columbianum, Strongyloides papillosus and Trichuris ovis. The authors of that study concluded that ginger exhibits anthelmintic activity in sheep in vivo, thus explaining the age-old traditional use of ginger in helminth infestation. Adewunmi et al. (1990) had reported that gingerol and shogaol exhibited potent molluscicidal activity on B. glabrata. Their experiments were conducted to study the major constituents of Z. officinale responsible for its molluscicidal activity and the effect of the active component on different stages of S. mansoni. Gingerol completely abolished the infectivity of S. mansoni miracidia and cercariae in B. glabrata and mice, respectively, indicating that the molluscicide is capable of interrupting schistosome transmission at a concentration lower than its molluscicidal concentrations. Whereas previous investigations on ginger components have shown larvicidal activity against D. immitis (Datta and Sukul, 1987), Anisakis larvae (Goto et al., 1990), S. mansoni (Adewunmi et al., 1990; Sanderson et al., 2002), and gastrointestinal nematodes (Iqbal et al., 2006), but little is known about the effects underlying ginger’s larvicidal effects on A. cantonensis, but in the present experiment, we found that ginger’s constituents (e.g. [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin) exhibited larvicidal and loss of spontaneous movement effects against AcL5 (Tables 1 and 2). This study is the first to determine the larvicidal activity of ginger (Z. officinale) on A. cantonensis. Thus we found that [10]shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and HHC have not only larvicide activity for L5 larvae of A. cantonensis but also radical scavenging activity against DPPH and peroxyl radical, respectively. So those compounds have radical scavenging activity not reduced their larvicide activity against AcL5 but further investigations for the mode of ginger constituents’s actions or/and mechanisms for its larvicidal effects between free radical scavenging activity are necessary. We found [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and HHC to have larvicidal against A. cantonensis reduced spontaneous movement of these parasites and to have free radical scavenging activity that did not adversely affect their larvicidal activity. These results might be useful in the search of more selective and efficient naturally larvicidal compounds. Acknowledgments This study was supported by grants from the National Science Council, Taiwan (NSC-95-2320-B-037-052-MY2) and by a grant from the Kaohsiung Medical University Research Foundation (Q096008 and Q097029) to R.-J.L. We would like to thank Miss Lu Chin-Mei and Miss Tzu Han-Fan for their assistance in experiments and recovery of worms from the brains of infected mice. The use of animals in this study was reviewed and permitted by the Committee of the Care and Use of Laboratory Animals, Kaohsiung Medical University, according to Taiwanese laws. References Adewunmi, C.O., Oguntimein, B.O., Furu, P., 1990. Molluscicidal and antischistosomal activities of Zingiber officinale. Planta Med. 56, 374–376.

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