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In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-

gingerol

Article  in  Asian Pacific Journal of Tropical Medicine · June 2016

DOI: 10.1016/j.apjtm.2016.06.013

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Asian Pacific Journal of Tropical Medicine 2016; ▪(▪): 1–8 1

56
H O S T E D BY
Contents lists available at ScienceDirect 57
58
Asian Pacific Journal of Tropical Medicine 59
60
journal homepage: http://ees.elsevier.com/apjtm 61
62
1 63
2 Original research http://dx.doi.org/10.1016/j.apjtm.2016.06.013 64
3 65
4 In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol 66
5 67
6 Q3 Manel Amri, Chafia Touil-Boukoffa* 68
7 University of Sciences and Technology Houari Boumediene, Faculty of Biological Science, Laboratory of Cellular and Molecular Biology, Team 69
8 ‘Cytokines and NO Synthases’, PB 32 El-Alia, Algiers 16111, Algeria 70
9 71
10 A R TI C L E I N F O ABSTRACT 72
11 73
12 Article history: Objective: To study in vitro anti-hydatic and immunomodulatory effects of ginger and 74
13 Received 15 May 2016 [6]-gingerol as an alternative therapy for cystic echinococcosis. 75
14 Received in revised form 16 Jun 2016 Methods: Effect of a commonly used herbal product and ginger (Zingiber officinale) 76
15 Accepted 21 Jun 2016 towards protoscoleces (PSC) and cyst wall in vitro was studied. The effect of [6]- 77
16 Available online xxx gingerol, and the pungent constituent of ginger, was also evaluated on PSC culture. 78
17 Furthermore, the activity of both extracts in association with interferon-gamma (IFN-g)
79
18 Keywords:
on PSC co-cultured with mononuclear cells of hydatic patients was evaluated. The nitric
80
19 oxide (NO) production was measured in each co-culture.
Ginger 81
20 Results: Ginger exhibited a concentration- and time-dependent cytotoxic effect against
[6]-Gingerol 82
21 PSC and cyst wall. Interestingly, ginger was more effective than the [6]-gingerol.
Echinococcosis
Moreover, additional parasitic effect between extracts and IFN-g are also observed in 83
22 Cytotoxic activity 84
23 co-cultures. Furthermore, both extracts attenuated the NO production elicited by this
Nitric oxide 85
infection or by the IFN-g.
24 86
Conclusions: Ginger has an important anti-hydatic effect in vitro. This effect is ampli-
25 87
fied in the presence of IFN-g. Moreover, this herbal product may protect against host's
26 88
cell death by reducing the high levels of NO. Ginger may act, at least, through the [6]-
27 gingerol. All our data suggest the promising use of ginger in the treatment of Echino- 89
28 coccus granulosus infection. 90
29 91
30 92
31 93
32 94
33 95
34 1. Introduction However, the protoscoleces dissemination can occur during
96
35 surgery leading to a high frequency of relapse. Chemotherapy
97
36 Cystic echinococcosis is a zoonotic disease in humans and with benzimidazole carbamate derivatives, amphotericin B and
98
37 sheep. This infection is caused by the larval stage of Echino- praziquantel is also used. This is the only option in many
99
38 coccus granulosus (E. granulosus). Man is being infected after inoperable cases like cyst in brain or multiple cysts and in the
100
39 ingestion of disseminated eggs in water, plants or on over the immune-depressed host. Nevertheless, these drugs are not effi-
101
40 definitive hosts (dogs). Each egg, containing an oncosphere, cient in some cases and leads to liver toxicity and other side
102
41 gives a unilocular cyst especially in the liver and lungs. Sur- effects [2]. All these data prompted us to find new drugs for
103
42 rounded by a thin wall: cyst wall (CW), the cyst is filled with therapy.
104
43 fluid containing brood capsules and protoscoleces (PSC). The natural products are attracting attention as new thera-
105
44 The development of hydatic cyst for a long period induces peutic drugs against a number of diseases. Among their effects
106
45 many complications like organ malfunction and can lead to we found antiparasitic and antioxidant activities [3]. One of
107
46 death [1]. The preferred therapy is cyst removal by surgery. commonly used herbal product in traditional medicine all over
108
47 the world is ginger (Zingiber officinale) [4,5]. There are few
109
48 studies about the direct effect of this rhizome or other natural
*Corresponding author: Chafia Touil-Boukoffa, University of Sciences and 110
49 products against the helminthes. Some of these studies are
Technology Houari Boumediene, Faculty of Biological Science, Laboratory of 111
50 Cellular and Molecular Biology, Team ‘Cytokines and NO Synthases’, PB 32 El-Alia, related to E. granulosus and Echinococcus multilocularis
112
51 Algiers 16111, Algeria. using ginseng derivates or other herbs or natural products [6].
113
Tel: +213 550819857
52 E-mail: touilboukoffa@yahoo.fr
However, as far as we know, there are no studies about the
114
53 Peer review under responsibility of Hainan Medical College. effect of ginger on E. granulosus metacestode in culture or
115
54 Foundation project: This work was supported by National Project in Health coculture with mononuclear cells (PBMC) of hydatic patients.
116
(PNR-Santé-2011-2014).
55 Q1
117
1995-7645/Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Amri M, Touil-Boukoffa C, In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol, Asian Pacific Journal of Tropical Medicine (2016), http://
dx.doi.org/10.1016/j.apjtm.2016.06.013
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2 Manel Amri, Chafia Touil-Boukoffa/Asian Pacific Journal of Tropical Medicine 2016; ▪(▪): 1–8

1 In this sense, we assessed here the effect of crude aqueous finally maintained in RPMI-1640 medium supplemented with 63
2 extract of dried ginger on E. granulosus CW and PSC in 5% of heat-inactivated FCS and antibiotics until culture. 64
3 culture. Furthermore, we evaluated the activity of this extract PSC were prepared as previously described [8,9]. Briefly, free 65
4 in association with IFN-g on PSC viability and nitric oxide PSC were obtained by centrifugation of hydatid fluid followed 66
5 (NO) production in coculture and culture performed with by washing in sterile PBS. The viability of PSC was determined 67
6 PBMC of hydatic patients and healthy donors. The effects of prior the cultures by microscopic examination using 0.1% eosin 68
7 ginger were also compared with those of [6]-gingerol, one of as a vital stain. While viable PSC still unstained, dead PSC 69
8 the active components of ginger. become stained. The appropriate percentage of viability was 70
9 95% or more. PSC were then maintained in RPMI-1640 me- 71
10 2. Materials and methods dium supplemented with 5% of heat-inactivated FCS until culture 72
11 and coculture. 73
12 2.1. Ginger and [6]-gingerol 74
13 2.5. CW and PSC culture and treatment 75
14 The dried rhizome of Zingiber officinale Roscoe (family 76
15 Zingiberaceae), was procured from a local market in Algiers, Two pieces of CW and 2 × 103 viable PSC were placed in 77
16 Algeria and then has been identified and authenticated by a each well of 24-well plastic trays. They were cultured in RPMI- 78
17 botanist. The bioactive compound [6]-gingerol was obtained 1640 medium supplemented with antibiotics and 10% heat- 79
18 from Calbiochem (San Diego, CA). inactivated FCS and treated with different concentrations of 80
19 ginger (100 mg/mL for CW and 1, 10 and 100 mg/mL for PSC). 81
20 2.2. Preparation of ginger crude aqueous extract and PSC cultures were also treated with [6]-gingerol (100 mg/mL). 82
21 [6]-gingerol The untreated cultures were used as a negative control. PSC 83
22 treated with 0.1% DMSO vehicle without [6]-gingerol were also 84
23 considered as a control. Cultures were then incubated at 37  C in 85
The crude aqueous extract was prepared by dissolving 1 mg
24 a humid atmosphere and 5% CO2 for 72 h for CW and 24 h and 86
of the dried extract of ginger in 1 ml of RPMI-1640 medium
25 48 h for PSC. 87
supplemented with 5% of heat-inactivated FCS (fetal calf serum,
26 Cultures were observed under microscope before and after 88
sigma) and antibiotics. Tubes were then mixed and centrifuged.
27 89
The aqueous extract was then filtered through 0.22 mm (Milli- incubation. Percentages of viable protoscoleces were determined
28 90
pore) and stored at −20  C until use. by microscopic examination using 0.1% eosin as a vital stain.
29 91
The [6]-gingerol was diluted from the stock solution (dis-
30 2.6. PBMC culture and coculture with PSC 92
solved in DMSO) with the culture medium (RPMI-1640) at the
31 93
maximum final concentration of DMSO at 0.1%.
32 PBMC were prepared from blood of hydatic patients and 94
33 healthy donors as previously described [8]. Briefly, PBMC isolated 95
2.3. Patients
34 by density gradient were washed three times with sterile PBS. Cell 96
35 viability was checked using 0.2% trypan blue dye. The appropriate 97
The peripheral blood samples were obtained from twenty
36 percent of viable cells was always >98%. Viable of 106 cells/mL 98
Algerian patients carrying hydatic cyst in lungs (n = 20) at
37 were then resuspended in RPMI-1640 medium supplemented with 99
Department of Surgery, Mustapha Bacha Hospital, Algiers,
38 antibiotics and 10% of FCS. 100
Algeria. The blood samples were obtained before (no more than
39 Cells were immediately cocultured with PSC and stimulated 101
1 week) and after (24–72) h surgery. All patients having other
40 with 100 mg/mL of ginger or [6]-gingerol alone or with IFN-g 102
infection or inflammatory chronic diseases were excluded from
41 (100 U/mL). The unstimulated cocultures were used as negative 103
this study. Blood samples from healthy donors (n = 10, from the
42 control. The cocultures treated with 0.1% DMSO vehicle 104
same region of Algeria) were included in this study as healthy
43 without [6]-gingerol were also considered as a control. Cells 105
controls. All samples were used immediately after collection for
44 were also stimulated with ginger extract (1, 10 and 100 mg/mL) 106
PBMC preparation and culture.
45 and [6]-gingerol (100 mg/mL). 107
All participants (patients and healthy donors) gave informed
46 The effects of ginger and [6]-gingerol on nitrites production 108
consent for this study, which was in accordance with the
47 was demonstrated using L-NMMA (0.5 mM), (Sigma) and hu- 109
guidelines of the ethic committee of the Algerian agency of
48 man recombinant IFN-g (100 U/mL) [8] (HuIFN-g is a gift from 110
research and development in health (ATRSS). All procedures
49 Dr. J. Wietzerbin, INSERM U365 – ‘Interférons et cytokines’ – 111
were performed in accordance with Helsinki declaration of
50 Institut Curie, INSERM U932 – ‘Immunité et cancer’ – Institut 112
2008.
51 Curie, Paris, France). 113
52 Cocultures and cultures were then incubated at 37  C in 114
53 2.4. Preparation of PSC and CW 115
humid atmosphere and 5% CO2. After 20 h, percentages of
54 viable protoscoleces were determined in coculture as described 116
55 The hydatid cysts (n = 5) were obtained after surgery in lungs 117
previously in Section 2.4. Moreover, nitrites production was
56 and livers at Mustapha Bacha Hospital, Algiers, Algeria. Cysts 118
were washed several times with sterile PBS, pH 7.4 supple- evaluated in coculture and culture supernatants.
57 119
58 mented with antibiotics. 120
CW was prepared as previously described [7]. Briefly, cyst 2.7. Nitrites measurement
59 121
60 wall, obtained after hydatid fluid removal, was washed several 122
times with sterile PBS. This membrane was then cut into Nitric oxide production was evaluated by measuring nitrites
61 123
small pieces (1 cm × 1 cm) and washed again. CW was concentration (NO2 − ) in each supernatant by Griess method as
62 124

Please cite this article in press as: Amri M, Touil-Boukoffa C, In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol, Asian Pacific Journal of Tropical Medicine (2016), http://
dx.doi.org/10.1016/j.apjtm.2016.06.013
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Manel Amri, Chafia Touil-Boukoffa/Asian Pacific Journal of Tropical Medicine 2016; ▪(▪): 1–8 3

1 previously described [10]. Briefly, 100 mL of supernatant ginger induced a strong morphological change with loss of the 63
2 were incubated for 5 min with 0.05% naphthyldiamine membrane integrity, the order of hooks, and the homogeneity of 64
3 dihydrochloride and 0.5% sulfanilamide prepared in 5% HCl. the calcareous corpuscles. Moreover, contractile movements of 65
4 The OD was measured by spectrophotometer at 543 nm. The membrane and suckers disappear gradually followed by com- 66
5 data were expressed as mM of nitrites per 106 cells. plete PSC degeneration and death. In contrast, no appreciable 67
6 change was detected in the PSC viability of untreated culture 68
7 2.8. Statistical analysis (Figure 3). 69
8 To confirm these observations, we also evaluated the PSC 70
9 The results obtained were expressed as mean ± SD. Data viability using 0.1% eosin as a vital stain (Figure 3). Figure 4A 71
10 analysis was performed using the Minitab 16. The comparison shows the percentage of viable PSC after ginger exposure for 72
11 between different groups was performed using Student's t-test. 24 h and 48 h of culture. We notice that scolicidal effect of 73
12 Differences were considered to be statistically significant at ginger is strongly related to concentration and exposure times. 74
13 P < 0.05. Interestingly, concentrations of 1 mg/mL and 100 mg/mL of 75
14 ginger kill respectively 51.80% and 89.72% (P < 0.000 1) of 76
15 parasite after 24 h of culture (Figure 4). Ginger exhibited also a 77
16 3. Results time-dependent protoscolocidal effect after 24 h and 48 h with 78
17 respective maximum reduction of 89.72% and 100% 79
18 3.1. Cytotoxic effect of ginger on cyst wall (P < 0.000 1) in PSC viability for 100 mg/mL (Figure 4A). 80
19 In the presence of 100 mg/mL [6]-gingerol, time-dependently 81
20 The effect of ginger on CW was examined in vitro. In this reduced cell viability was also observed (Figure 4B). Interest- 82
21 sense, morphological change in CW was evaluated by micro- ingly, [6]-gingerol was less effective than ginger (P < 0.000 1). 83
22 scopic examination in the absence and presence of crude For note, PSC treated with vehicle alone remained viable 84
23 aqueous extract of dried ginger (100 mg/mL) for 72 h. throughout the incubation period (24 and 48 h with respectively 85
24 Our results showed that ginger effect correlated with expo- P = 0.897 and P = 0.371). 86
25 sure times. In fact, between 12 and 24 h, the germinal layer (GL) 87
26 becomes darker and small areas of degeneration are observed 3.3. Scolicidal effect of ginger and [6]-gingerol in 88
27 after 36 h of exposure. After 48 h, GL loses completely its mononuclear cells-protoscoleces cocultures 89
28 integrity followed by the laminated layer (LL). CW loses its 90
29 integrity after 60–72 h (Figure 1).
In the second part of our study, we investigate the scolicidal 91
30 effect of ginger and [6]-gingerol on PSC cocultured with 92
31 3.2. Cytotoxic effect of ginger and [6]-gingerol on mononuclear cells (PBMC) in presence or absence of IFN-g. For 93
32 protoscoleces note, we have previously highlighted the role of this cytokine in 94
33 host defense and parasite killing [8]. 95
34 In the PSC culture, our results show that ginger exhibited a Our results show that 100 mg/mL of ginger and [6]-gingerol 96
35 dose-dependent cytotoxic effect on PSC (Figure 2). Remarkably, reduces protoscoleces viability in vitro from 61.94% ± 2.72% 97
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60 Figure 1. Progressive morphological alterations of CW after treatment with 100 mg/mL of ginger for 72 h showing by light microscopy. 122
A: CW culture at t = 0. B: CW culture after 72 h without ginger. C, D, E, F, G and H: CW culture with ginger (100 mg/mL) after 12 h, 24 h, 36 h, 48 h, 60 h
61 123
and 72 h, respectively. Discontinued arrow: Altered germinal layer (GL); Arrow: degeneration of GL. Arrowhead: loss of layers in the laminated layer
62 (LL) (Ob× 05). Q4 124

Please cite this article in press as: Amri M, Touil-Boukoffa C, In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol, Asian Pacific Journal of Tropical Medicine (2016), http://
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4 Manel Amri, Chafia Touil-Boukoffa/Asian Pacific Journal of Tropical Medicine 2016; ▪(▪): 1–8

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15 Figure 2. Protoscolicidal effect of ginger showing by light microscopy.
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16 PSC were cultured and treated with ginger for 24 h, at 37  C, in 5% CO2. A: PSC culture at t = 0 (97% of viability). B: PSC culture after 24 h. C, D and E: 78
17 PSC culture after 24 h with ginger at 1, 10 and 100 mg/mL, respectively. cc: calcareous corpuscles; deg: degeneration; ev: evaginated PSC; H: hooks; inv: 79
18 invaginated PSC; R: rostellum; res: PSC residues; su: suckers; v: viable PSC. Arrow: Altered tegument; Arrowhead: loss of hooks. Black discontinued 80
19 arrow: evagination and invagination of the head; White discontinued arrow: tegument shift (×108). 81
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Figure 3. Morphological alterations of protoscoleces after treatment with
46 108
ginger showing by light microscopy.
47 A and B: viable PSC, C and D: dead PSC. A and C: invaginated PSC. B 109
48 and D: evaginated PSC. bo: body; cc: calcareous corpuscles; H: hooks; R: 110
49 rostellum; sc: scolex; su: suckers. Teg: tegument. Arrow: Altered tegument. 111
50 Arrowhead: loss of hooks (×269). 112
51 113
Figure 4. Killing of E. granulosus protoscoleces after exposure to ginger
52 (A) and [6]-gingerol (B) for 24 h and 48 h of culture. 114
53 to respectively 39.82% ± 0.57% (P < 0.000 1) and Each histogram represents the mean percentage of viable protoscoleces 115
54 46.63% ± 1.67% (P < 0.000 1) (for PBMC from patients before from five different experiments. Differences between different groups are 116
▵▵ ▵▵▵ ▵▵▵▵
55 surgery). Interestingly, synergistic scolicidal effects between indicated ( P < 0.01, P < 0.001, P < 0.000 1; NS: no signif- 117
56 these two extracts and IFN-g are also observed in PSC-PBMC icance). Triangles (▵) and NS above single bars indicate differences with 118
57 co-cultures. In fact, incubation with extracts and IFN-g the negative control (untreated cocultures) for 24 h and 48 h. Asterisks 119
above horizontal brackets indicate difference between 24 h and 48 h. Q2
58 induced the most damage to the protoscoleces as indicated by 120
59 viability loss in comparison with co-cultures treated with IFN-g 121
60 or extracts alone (Figure 5A). donors. However, incubation of PBMC from healthy donors 122
61 The same results are reported for co-cultures performed with with ginger or [6]-gingerol induced less damage as manifested 123
62 PBMC from patients before and after surgery and healthy by the high levels of PSC viability. 124

Please cite this article in press as: Amri M, Touil-Boukoffa C, In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol, Asian Pacific Journal of Tropical Medicine (2016), http://
dx.doi.org/10.1016/j.apjtm.2016.06.013
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Manel Amri, Chafia Touil-Boukoffa/Asian Pacific Journal of Tropical Medicine 2016; ▪(▪): 1–8 5

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44 Figure 5. Killing of E. granulosus protoscoleces. 106
45 (A) In coculture with mononuclear cells (PBMC) treated with ginger or [6]-gingerol (100 mg/mL) alone or with human recombinant IFN-g (100 U/mL) for 107
46 20 h. Correlation with nitrites production (B). Each histogram represents the mean percentage of viable protoscoleces from different experiments (n = 20 and 108
▵▵ ▵▵▵ ▵▵▵▵
47 n = 10 for patients and controls respectively). Differences between different groups are indicated ( P < 0.01, P < 0.001, P < 0.000 1; NS: no 109
48 significance). Triangles (▵) and NS above single bars indicate differences with the negative control (untreated cocultures). Asterisks above horizontal 110
brackets indicate difference between IFN-g alone or with ginger or [6]-gingerol. Gin: ginger, veh: vehicle, 6-ging: [6]-gingerol.
49 111
50 112
51 3.4. Inhibitory effect of ginger and [6]-gingerol on NO attenuate the increase in NO production elicited by IFN-g 113
52 production by mononuclear cells (Figure 5B). 114
53 The same results are also observed for culture with a dose- 115
54 In our previous study, we have reported that the scolicidal dependent inhibitory effect of ginger on nitrites production. 116
55 effect of IFN-g is associated with an increase in NO produc- However, the inhibition observed using 1 mg/mL of ginger is 117
56 tion [8]. On the basis of these results, we investigated here if statistically not significant for all cultures (Figure 6). 118
57 ginger and [6]-gingerol act by the same mechanism. During To confirm the effects of ginger on NO production, L-NMMA 119
58 our experiment, loss of viability in the presence of ginger and (NOSynthase inhibitor) and IFN-g are used in PBMC culture. 120
59 [6]-gingerol is associated with a decrease in nitrites levels. While L-NMMA decreases nitrites production, IFN-g has an 121
60 This effect contrasts with the increase observed in the presence opposite effect. Remarkably, ginger and [6]-gingerol decrease the 122
61 of IFN-g [8]. Interestingly, ginger and [6]-gingerol also levels on nitrites when used alone or with IFN-g (Figure 6). 123
62 124

Please cite this article in press as: Amri M, Touil-Boukoffa C, In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol, Asian Pacific Journal of Tropical Medicine (2016), http://
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6 Manel Amri, Chafia Touil-Boukoffa/Asian Pacific Journal of Tropical Medicine 2016; ▪(▪): 1–8

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23 Figure 6. Nitric oxide production in PBMC cultures treated with ginger (1, 10 and 100 mg/mL) for 20 h. 85
24 Human recombinant IFN-g (100 U/mL) and L-NMMA (0.5 mM) were also used to confirm the effect of ginger on NO production. Each histogram represents 86
25 the mean nitrite levels from different experiments (n = 20 and n = 10 for patients and controls respectively). Differences between different groups are 87
▵▵ ▵▵▵ ▵▵▵▵
indicated ( P < 0.01, P < 0.001, P < 0.000 1; NS: no significance). Triangles (▵) and NS above single bars indicate differences with the
26 88
negative control (untreated cultures). Asterisks above horizontal brackets indicate difference between IFN-g alone or with ginger. Gin 1, 10 and 100
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30 4. Discussion Our data show that in vitro treatment with ginger extract 92
31 induces significant alterations in cyst wall and PSC leading to 93
32 Nowadays, several studies have been done to find new nat- parasite death. In vivo, such damage to the cyst wall destroys the 94
33 ural compounds that treat parasitic diseases. However, hydati- defense system of the parasite. Thus, the PSC could easily be 95
34 dosis is among the most neglected parasitic infections, and attacked by the protective host immune responses. We have 96
35 development of new therapy and drugs received very little found that ginger induces a loss of spontaneous movement of 97
36 attention. In this study, the role of ginger (Zingiber officinale) PSC and damage to the hooks in a time- and dose-dependent 98
37 against E. granulosus larvae has been investigated. Interestingly, manner. These effects cause loss of PSC ability to adhere to 99
38 we found that ginger exhibited dose- and time-dependent lar- the host tissues. Moreover, the observed tegumental alterations 100
39 vicidal effect against E. granulosus metacestode and could be a committed mechanism by ginger to destroy the 101
40 protoscoleces. parasite. Our observations are more or less similar to the alter- 102
41 Our results are in the same line with previous studies showing ations observed after treatment with genistein as observed by 103
42 that ginger has an anthelmintic activity against some parasitic Naguleswaran et al. [6]. The alterations in the surface 104
43 species both in vitro and in vivo. In their report, Iqbal et al. reported architecture of E. granulosus metacestode were used by 105
44 that methanol extracts of ginger killed all the Haemonchus con- several investigators as a deleterious indicator for the 106
45 tortus worms within two hours post exposure [11]. These findings evaluation of anti-hydatic activities of different drugs. More- 107
46 agree with other works revealing that ginger extract affects the over, the reduction observed in PSC viability was considered by 108
47 viability of Schistosoma mansoni (S. mansoni) adult pairs [12] several authors as a strong evidence of the efficiency of anti- 109
48 and Ascaridia galli [13], both in vitro and in vivo in mice. hydatic drugs [6,18,19]. 110
49 Our present results are also in harmony with previous in vivo Interestingly, ginger acts synergistically with IFN-g to kill 111
50 studies conducted by Datta and Sukul showing a significant PSC. However, the immunomodulatory effect of ginger on host 112
51 anthelmintic efficacy of ginger alcoholic extract in dogs, naturally immune response has not been evaluated during E. granulosus 113
52 infected with Dirofilaria immitis [14]. Later, Iqbal et al. demonstrated infection. Based on our previous work showing a significant 114
53 that crude powder and crude aqueous extract of dried ginger exhibit induction of nitric oxide (NO) production after in vitro treatment 115
54 anthelmintic activity in sheep naturally infected with different with IFN-g [8], we wished to determine the effect of ginger on 116
55 species of gastrointestinal nematodes including: Haemonchus NO production. This present work demonstrates, for the first 117
56 contortus, Trichostrongylus colubriformis, Trichostrongylus axei, time, that ginger reduces NO production by host cells in 118
57 Oesophagostomum columbianum, Strongyloides papillosus and response to E. granulosus infection in vitro. 119
58 Trichuris ovis [15]. More recently, Mostafa et al. and Aly and It is known that pro-inflammatory cytokines (likes IFN-g and 120
59 Mantawy reported that the crude aqueous extract of ginger TNF-a) simulate iNOS expression and activity in macrophages 121
60 exhibited antischistosomal activity in mice as showed by the [20,21]. Thus, when the production of these cytokines is inhibited, 122
61 reduction in worm and egg burden in the hepatic tissue and faeces the generation of NO is likely to be reduced. Several studies 123
62 of infected mice [16,17]. have shown that ginger inhibited the expression of genes 124

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1 encoding pro-inflammatory cytokines by various cells [22,23]. fibrosis that affect tissue architecture and impair organ function 63
2 These findings are consistent with in vivo study showing that [35,36].Thus, we can conclude here, that ginger extract and [6]- 64
3 ginger extract inhibit the production of NO, TNF-a and IL-12 gingerol could minimize the deleterious effects of this parasite 65
4 in mice infected with S. mansoni [17]. on the vital functions of infected organs [1] through its 66
5 Although, our previous studies have demonstrated that IFN-g immunomodulatory effect on the iNOS pathway. This active 67
6 and iNOS pathways are involved in protective immunity against component of ginger may have a direct effect on the vitality 68
7 E. granulosus [8,9,24]. Our present study indicates that IFN-g and viability of PSC and NO production. However, ginger had 69
8 kills the parasite even although NO production was reduced a higher efficacy than that of [6]-gingerol. Another component 70
9 by ginger. Thus, our data suggest the commitment of another of ginger such as the [6]-shogaol may act in synergy with [6]- 71
10 mechanism by which IFN-g kills the protoscoleces in vitro. gingerol [17,25]. Further studies to explore the effect of these 72
11 In summary, our study indicates for the first time that ginger components on E. granulosus metacestode appear to be useful. 73
12 possesses cestocidal activity against E. granulosus. However, This study is the first to determine the larvicidal activity of 74
13 further studies are required to clarify the mechanisms involved ginger and [6]-gingerol on E. granulosus metacestode and pro- 75
14 in this effect. Sanderson et al. and Iqbal et al. reported that toscoleces. Interestingly, we found that ginger and its main 76
15 ginger kills S. mansoni and gastrointestinal nematode in vivo component have also an immunomodulatory effect that did not 77
16 through its binding to parasite beta-tubulin and inhibition of adversely affect their larvicidal activity. Further investigations 78
17 glucose uptake [12,15]. Another study attributed the significant for the mechanisms underlying ginger effects are necessary. 79
18 reduction in tissue egg after ginger treatment to the Above results might contribute to the use of ginger as new 80
19 disturbance in worm fecundity [25]. Nevertheless, the alternative therapy. However, in vivo studies on an animal model 81
20 bioactivity of ginger may be due to its constituents. In fact, should be done before using ginger or ginger derivatives for 82
21 phytochemical reports show that ginger contains over 400 treatment of human hydatidosis. 83
22 different compounds. The major constituents are zingerone, 84
23 paradol, gingerols and shogaols. According to several Conflict of interest statement 85
24 literature reviews, [6]-gingerol is the most abundant active 86
25 constituent of whole ginger. It has various pharmacological and We declare that we have no conflict of interest. 87
26 microbiological proprieties, including anti-parasitic, anti- 88
27 tumorigenic, anti-inflammatory and anti-oxidant activities. Acknowledgments 89
28 Interestingly, in many study, researchers used the [6]-gingerol as 90
29 a marker substance of ginger [4,26,27]. This work was supported by National Project in Health 91
30 These data prompted us to investigate the effect of [6]- (PNR-Santé-2011-2014). 92
31 gingerol on E. granulosus PSC and NO production. In this 93
32 study, we have found that [6]-gingerol significantly kills PSC References 94
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8 Manel Amri, Chafia Touil-Boukoffa/Asian Pacific Journal of Tropical Medicine 2016; ▪(▪): 1–8

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