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Experimental Parasitology ■■ (2015) ■■–■■

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Experimental Parasitology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

1 Full length article

2
3 Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia
4 mangostana extract in hamster opisthorchiasis
5
6 Ratchadawan Aukkanimart a,b,c,d, Thidarut Boonmars a,b,c,*, Pranee Sriraj a,b,c,d,
7 Jiraporn Songsri a,b,c, Porntip Laummaunwai a,b, Sakda Waraasawapati e,
8 Q1 Chantana Boonyarat f, Panaratana Ratanasuwan g, Sirintip Boonjaraspinyo h
a
9 Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
10 b Neglected, Zoonosis and Vector-Borne Disease Group, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
11 c
Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen 40002, Thailand
12 d
Rajamangala University of Technology Isan, Sakonnakhon Campus, Sakonnakhon 47160, Thailand
13 e Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

14 f Faculty of Pharmaceutical Science, Khon Kaen University, Khon Kaen 40002, Thailand

15 g Department of Anesthesiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

16 h
Department of Community Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
17

18 H I G H L I G H T S G R A P H I C A L A B S T R A C T
19
20
21 • We tested the anti-O. viverrini
22 activity of G. mangostana extracts.
23 • G. mangostana extracts interfered
24 the O. viverrini tegument and
25 reproductive organs.
26 • SEM shows sloughing and blebbing
27 of O. viverrini tegument after
28 treatment.
29 • G. mangostana extracts might
30 useful for anthelmintics
31 development in the future.

32

33 A R T I C L E I N F O A B S T R A C T
34
35
36 Article history: Administration of praziquantel for treatment of liver fluke infection may affect the host, with mild and
37
38 Received 28 July 2014 severe effects after treatment caused by host immune response. Therefore, we focused on the antioxi-
39
40 Received in revised form 30 January 2015 dant property, inflammatory and anthelmintic effects of the traditional folk medicine, G. mangostana pericarp
41 Accepted 18 March 2015
42 extract, in hamster opisthorchiasis. Syrian hamsters were divided into four groups: normal (control) (N);
43 Available online
44 administered G. mangostana alone (GM); infected with Opisthorchis viverrini alone (OV); and infected with
45
46 O. viverrini and administered G. mangostana extract for 1.5 months (OVGM). Hamster livers were collect-
47 Keywords:
48
49 Anthelmintic
ed 45 days after infection to determine histopathological changes, i.e. aggregation of inflammatory cells.
50
51 Antioxidant effect The morphology of adult O. viverrini (body size and sizes of reproductive organs) was analyzed, as well
52
53 Anti-inflammatory effect as worm burden, eggs per worm and eggs per gram of feces. Toxicity was tested by kidney function (blood
54
55 Garcinia mangostana urea nitrogen and creatinine); the results demonstrated that G. mangostana had no renal toxic effect. ABTS
56
57 Opisthorchis viverrini radical-scavenging assay indicated that the extract had antioxidant property. Reduction in aggregation
58
59 Hamster of inflammatory cells surrounding the hepatic bile duct, especially at the hilar region, was found in the
60 Opisthorchiasis

61
62 * Corresponding author. Fax: +66 43 202475.
63 E-mail address: bthida@kku.ac.th (T. Boonmars).

http://dx.doi.org/10.1016/j.exppara.2015.03.007
0014-4894/© 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Ratchadawan Aukkanimart, et al., Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthor-
chiasis, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.007
 ARTICLE IN PRESS
2 R. Aukkanimart et al./Experimental Parasitology ■■ (2015) ■■–■■

OVGM group. Worm burden was similar in both infected groups (treated or untreated with G. mangostana),
but the average size of adults in the OV group was larger than in the OVGM group; moreover, eggs per
worm and eggs per gram of feces were also comparatively higher. The present study suggests that
G. mangostana extract possesses anti-inflammatory and antioxidant properties and can interfere with
parasite development by affecting adult size and egg production. This may be useful for controlling the
spread of OV infection and other parasites in endemic areas.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction 2. Materials and methods

Opisthorchis viverrini infection is found throughout Southeast Asia,
especially Laos, Cambodia, Vietnam, and particularly Thailand.
Humans are infected by ingesting raw cyprinid fish that are con-
taminated with the metacercariae of O. viverrini. After infection, the
juveniles move to the biliary tract and can cause several hepatobiliary
diseases, including cholangitis, hepatitis, lithiasis and
cholangiocarcinoma (CCA). To eradicate this parasite would seem
to be a relatively simple matter, but eating raw cyprinid fish is part
of the culture among people living in this region, and the practice
is difficult to change. Therefore, O. viverrini infection remains a major
health problem in northeast Thailand, even though in recent years
there has been mass therapy for treatment of this parasite
(Vivatanasesth et al., 1982). The high prevalence of O. viverrini in-
fection in this region is correlated with its having the highest
incidence of CCA in the world (IARC, 2011). CCA can arise from a
combination of pathological causes: infection by the parasite itself;
host immune response to the parasite; immunopathology; and other
factors, such as consumption of improperly fermented food, and long-
term infection without treatment (Manwong et al., 2013). Several
previous studies have focused on the reduction of pathology from
liver fluke infection by administering anthelmintic drugs or certain
medicinal plants which may possibly prevent or retard CCA devel-
opment (Mahavorasirikul et al., 2010; Plengsuriyakarn et al., 2012).
Curcumin (Sriraj et al., 2009) and Thunbergia laurifolia (Wonkchalee
et al., 2012) were reported to have anti-inflammatory effects in cases
of animal opisthorchiasis and CCA.
To date, there have been no reports on the anthelmintic activ-
ity of medicinal plants against O. viverrini. However, many other
plants have been studied for anthelmintic activity: i.e. onions, garlic,
chives, coconut, birch bark, pineapple, banana, mangosteen, chicory,
date palm fruit, fig, pumpkin, and neem tree seeds against nema-
todes (Trichuris muris and Angiostrongylus cantonensis) (Klimpel et al.,
2011) and trematodes (Fasciola hepatica and Echinostoma caproni)
(Abdel-Ghaffar et al., 2011). Other studies have reported on the effects
of ginger (Zingiber officinale) on different stages of Schistosoma
mansoni (Aly and Mantawy, 2013; Mostafa et al., 2011), and the
effects of water extract of black nightshade (Solanum nigrum) leaves
on Schistosoma mansoni (Ahmed and Rifaat, 2005). Piper chaba fruits
demonstrated strong activity against schistosome cercariae
(Atjanasuppat et al., 2009), while Lysimachia ramosa Wall.
(Primulaceae) was found to have an effect on the helminth para-
sites Fasciolopsis buski, Ascaris suum and Raillietina echinobothrida
(Challam et al., 2010). G. mangostana pericarp, the hull of the man-
gosteen fruit, is a traditional folk medicine that has been shown to
be beneficial in the treatment of infected wounds (Pedraza-Chaverri
et al., 2008), has anti-colon cancer effects (Aisha et al., 2012), and
possesses anti-inflammatory (Chen et al., 2008), antimicrobial
(Iinuma et al., 1996) and cholesterol-lowering (Williams et al., 1995)
properties. Due to these previously demonstrated properties, and
because of the abundance of mangosteen hulls available in Thai-
land during the rainy season, the present study investigated the
antioxidant and anti-inflammatory properties and anthelmintic
effects of G. mangostana pericarp extract on hamster opisthorchia-
sis through observation of pathological changes, worm recovery and
parasite development.
2.1. Plant materials and preparation
Garcinia mangostana pericarp (mangosteen hulls) was collect-
ed from a local market in Khon Kaen, Thailand. The hulls were
washed, dried in a hot-air oven at 60 °C for 48 h, ground, and then
kept at 4 °C in a refrigerator until used. For in vivo testing, about
500 mg of G. mangostana powder was incubated with 1000 mL meth-
anol for 3 weeks. After filtering through 0.45 μm filter paper,
G. mangostana solution was evaporated on a hotplate to remove the
methanol. About 10 mg of G. mangostana extract was dissolved in
1 mL of DMSO, and distilled water was added to 100 mL. Fresh prepa-
rations were fed to hamsters for 2 weeks at a daily dose of 100 mg/
kg of body weight.
2.2. ABTS radical-scavenging activity assay
Free radical-scavenging activity of G. mangostana extract was de-
termined by 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS; Fluka, Steinheim, Germany), following the method de-
scribed by Chun et al. (2005) with slight modification. ABTS was
dissolved in distilled water to a 7 mM aqueous solution of ABTS
(30 mL) with 25 mM potassium persulfate (a blue/green chromo-
phore with characteristic absorption at 700 nm). The mixture was
kept in the dark at room temperature for 12 h before use. For the
study, the ABTS working solution was diluted with ethanol to give
an absorbance of (0.70 ± 0.02) at 700 nm and was equilibrated at
room temperature. The reaction mixtures, incubated in 96-well
plates, consisted of 50 μL of various concentrations of
G. mangostana extract and 100 μL of ABTS/ethanol working solu-
tion (Yang et al., 2011). The mixture was stirred and incubated for
10 min in the dark; then the absorbance was measured at 700 nm.
Trolox (25 μM) was used as a standard solution. The percentage in-
hibition of ABTS was calculated for each concentration relative to
a blank absorbance (ethanol). The dose–effect results were deter-
mined using 10 different concentrations; experiments were
performed 5 times. The percentage scavenging effect was calcu-
lated as:
% ABTS scavenging inhibition = ( A1 − A 2 ) ∗ 100 ( A1 − A 0 )
where A0 is the absorbance of the blank, A1 is the absorbance of ABTS
working solution as a positive control, and A2 is the absorbance of
the sample. The scavenging ability of the sample was expressed as
scavenging concentration (SC50), i.e. the effective concentration at
which 50% of ABTS radicals were scavenged. Data were analyzed
using CalcuSyn 2.1 software (Biosoft, Cambridge, UK).
2.3. Experimental animals
Hamsters were supplied by the Animal Care Unit, Faculty of Med-
icine, Khon Kaen University. Randomly selected male Syrian hamsters
7 weeks old were used. Hamsters were fed with a commercial rodent
diet (C.P. Foods, Bangkok, Thailand) ad libitum and kept in galva-
nized metal cages containing 8 hamsters/cage. The maintenance
and care of the experimental animals was in compliance with

Please cite this article in press as: Ratchadawan Aukkanimart, et al., Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthor-
chiasis, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.007
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R. Aukkanimart et al./Experimental Parasitology ■■ (2015) ■■–■■ 3

metacercariae were administered to each hamster via gastric 51

intubation. 52
53
2.5. Pathological studies 54
55
A photograph of each hamster liver was taken for comparison 56
of the gross anatomy. The hepatobiliary system (liver, gallbladder 57
and extrahepatic bile duct) was then processed for histopathologi- 58
cal study. In brief, whole liver tissue was dissected and fixed 59
immediately in 10% buffered formalin for at least 24 h. Tissue samples 60
were embedded in paraffin; each paraffin block was then cut with 61
a microtome into 4 μm sections. Tissue sections were deparaffinized 62
with xylene, rehydrated through a graded series of ethyl alcohol, 63
and stained with Harris’s hematoxylin and eosin. Finally, the tissue 64
sections were dehydrated in a series of ethyl alcohol and mounted 65
1 Fig. 1. Scheme of the treatment to animal groups. GM: G. mangostana; OV: Opist- on slides with Permount™ mounting medium (Thermo Fisher Sci- 66
2 horchis viverrini. entific, Waltham, MA, USA). Each tissue slide was photographed 67
under a microscope (BX51; Olympus, Tokyo, Japan). The histopa- 68
3
thology was graded using the criteria shown in Table 1. 69
4 National Laboratory Animal Center guidelines for the humane care 70
5 and use of laboratory animals. All protocols were approved by the 2.6. Kidney function test 71
6 Animal Ethics Committee, Khon Kaen University (AEKKU 62/ 72
7 2556). Hamsters were divided into four groups: normal (control) To clarify the renal toxic effect of G. mangostana, serum from every 73
8 hamsters (N); administered G. mangostana 100 mg/kg/day (GM); in- hamster in each group was measured for blood urea nitrogen (BUN) 74
9 fected with O. viverrini alone (OV); and infected with O. viverrini and and creatinine using an assay kit (Thermo Fisher Scientific). BUN 75
10 administered G. mangostana 100 mg/kg/day (OVGM). Hamsters were was detected by measurement of catalyzed NAD (nicotinamide 76
11 sacrificed on day 45 after infection (Fig. 1). adenine dinucleotide) at 340/380 nm, which is proportional to the 77
12 amount of BUN present. Creatinine was detected from the forma- 78
13 2.4. Preparation of O. viverrini metacercariae and hamster infection tion of the quinoneimine dye product at 550 nm (530–570 nm), 79
14 which is directly proportional to the creatinine concentration in the 80
15 Metacercariae of O. viverrini were obtained from naturally in- sample. 81
16 fected cyprinoid fishes in Laos. The fishes were minced with an 82
17 electric blender in pepsin solution. The mixture was incubated at 2.7. Worm recovery and adult size 83
18 37 °C in a shaking water bath for 1 h and then filtered through a 84
19 set of four sieves with 1000, 300, 250 and 106 μm apertures, re- To directly determine O. viverrini reproductive organ develop- 85
20 spectively. The remainder was sedimented and washed several times ment, hamster livers were squeezed and minced into small pieces. 86
21 with saline solution until the supernatant was clear. The sedi- All worms collected were counted. Ten adult worms were 87
22 ment was examined for O. viverrini metacercariae, which appeared carmine stained and measured for parasite size and reproductive 88
23 as a double-walled cyst with an oval shape. The average size of en- organ size. 89
24 cysted metacercariae was 200 μm × 167 μm. Oral and ventral suckers 90
25 were clearly seen, with the oral sucker close to the posterior end 2.8. Scanning electron microscopy 91
26 of the worm. The excretory bladder appeared as an oval area con- 92
27 taining a dense mass of dark granules; brownish-yellow pigments To observe the surface ultrastructure of the adult flukes post- 93
28 were scattered throughout the body. Metacercariae were collect- treatment with G. mangostana extract, ten adult worms from each 94
29 ed and identified under a dissecting microscope. Fifty O. viverrini group were washed several times with 0.2 M cacodylate buffer (pH 95

30

31 Table 1
32 Histopathological grading for hepatic tissue inflammation by microscopic observation.

33 Histopathological features Score Month (s) post infection and groups of experiment

34 N (n = 3) % (n) GM (n = 3) % (n) OV (n = 3) % (n) OVGM (n = 3) % (n)

35 Periportal inflammation 0 100 (3) 100 (3)

36 Q5 1 66.67 (2)*
37 2 33.33 (1)
38 3 66.67 (2)
39 4 33.33 (1)
40 Focal inflammation 0 100 (3) 100 (3)
41 1 66.67 (2)*
42 2 33.33 (1)
43 3 66.67 (2)
44 4 33.33 (1)

45 Criteria for histological grading of hepatic inflammatory activity (modified from Boonjaraspinyo et al., 2011; Wonkchalee et al., 2012; Sriraj et al., 2013).
46 Periportal or periseptal interface hepatitis (piecemeal necrosis), 0: none, 1: mild (focal, few portal areas), 2: mild/moderate (focal, most portal areas), 3: moderate (contin-
47 uous around <50% of tracts or septa), 4: severe (continuous around >50% of tracts or septa).
48 Focal inflammation: 0 = none, 1 = one focus or less per × 10 objective, 2 = two to four foci per × 10 objective, 3 = four to ten foci per × 10 objective, 4 = more than ten foci
49 per × 10, N; normal control, GM; administered with Garcinia mangostana, OV; Opisthorchis viverrini, OVGM; O. viverrini administered with Garcinia mangostana.
50 The statistical analysis of the histopathological grading of OVGM group showed significantly decreased (P = 0.043) compared with OV group.

Please cite this article in press as: Ratchadawan Aukkanimart, et al., Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthor-
chiasis, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.007
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1 Q6 Fig. 2. ABTS radicals-scavenging effect of GM. Data are expressed as mean ±SD (n = 5).

3 7.2) and fixed with Karnovsky’s solution at 4 °C overnight. After 2.10. Eggs per worm of O. viverrini adult 31
4 washing for 10 min, three times, with the phosphate buffer, samples 32
5 were post-fixed with 1% OsO4 in 0.1 M phosphate buffer (pH 7.2) Five worms from each hamster group were minced and added 33
6 for 1 h, followed by washing again for 10 min, three times, in phos- to 5 mL of 70% alcohol. Then 50 μL of each solution was smeared 34
7 phate buffer. Samples were then dehydrated through a graded on a glass slide and the number of O. viverrini eggs per worm was 35
8 ethanol series (30%, 50%, 70%, 90%, 95% and 100% alcohol, two times), counted. The number of eggs per worm was calculated as follows: 36
9 dried with a critical point dryer, coated with gold using a JFC-
Number of O. viverrini eggs × 5000 37
10 1100E ion sputtering device (JEOL, Tokyo, Japan), and observed with Eggs per worm (EPW ) =
11 a JEOL JSM-7800F scanning electron microscope (SEM) at a 1.0 kV 0.05
38
12 accelerating voltage.
13 3. Results 39
14 2.9. Eggs per gram of feces 40
15 3.1. ABTS radical-scavenging activity assay 41
16 To indirectly determine the reproductive organ development of 42
17 O. viverrini, feces from each infected hamster were collected for de- Trolox, a water-soluble analog of vitamin E, is an antioxidant that 43
18 tection of eggs per gram of feces and for determining the relationship is used in biological or biochemical applications to reduce oxida- 44
19 to parasite size and reproductive development. Modified formalin tive stress or damage. The reactivity of the various antioxidants tested 45
20 technique was performed for the quantitative O. viverrini egg count. was compared to that of Trolox. G. mangostana demonstrated an- 46
21 Two pellets of feces from the rectum were weighed, fixed and mixed tioxidant activity, with an effective concentration (EC50) = 142.04 μg/ 47
22 thoroughly with 1000 μL of 10% formalin. Then 20 μL of the solu- mL, R = 0.9854 (Fig. 2). 48
23 tion was smeared with 1% iodine solution on a glass slide and the 49
24 number of O. viverrini eggs was counted. The eggs per gram were 3.2. Gross pathology of the hepatobiliary system 50
25 calculated as follows: 51
26 Based on gross appearance, livers of the group of hamsters ad- 52
Number of O. viverrini eggs × 1000
Eggs per gram (EPG) = ministered G. mangostana alone (GM; Fig. 3B) were similar in terms 53
Feces weight ( g ) × 20 of color, liver surface, hepatic bile duct and gallbladder to those of 54

27

28 Fig. 3. Representative gross appearances of liver, gall bladder and extrahepatic ducts in hamster; normal group (A; N), normal administered G. mangostana group (B; GM),
29 OV infected group (C; OV), OV administered G. mangostana group (D; OVGM). The thickened and dilated extrahepatic bile ducts were shown in OV and OVGM groups (arrow
30 indicates extrahepatic biliary tree).

Please cite this article in press as: Ratchadawan Aukkanimart, et al., Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthor-
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1 Fig. 4. Representative histopathological changes of the groups of uninfected normal control (N; A,B), administered with G. mangostana (GM; C,D), infected with O. viverrini
2 (OV; E–H), infected with O. viverrini and administered with G. mangostana (OVGM; I–L). Bd; bile duct, P; parasite and inflammatory cell infiltrates (indicated yellow star).
3 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

5 the control group (N; Fig. 3A). In the O. viverrini-infected group (OV), 3.5. Worm recovery, body size and sizes of internal organs (ovary, 46
6 the gallbladders were enlarged. The hepatic bile ducts were also uterus and testes) of adult worms 47
7 slightly enlarged and opaque, with thickening of the walls. 48
8 Livers appeared normal, with a red color and smooth surface (Fig. 3C). To determine the effect of G. mangostana extract on worm re- 49
9 Liver surfaces of hamsters infected with O. viverrini and adminis- covery, size and reproductive organ development of O. viverrini, adult 50
10 tered G. mangostana (OVGM group) appeared normal (Fig. 3D); worms collected from each group were counted and stained. Worms 51
11 the gallbladders were clear and yellow, but the hepatic bile were measured and represented in terms of width and length, whole 52
12 ducts were enlarged and opaque compared with the normal control body size, and sizes of the ovary, uterus and testes. Worm recov- 53
13 group. ery from Syrian hamsters treated with G. mangostana was lower than 54
14 from that of the untreated group, but there was no statistically sig- 55
nificant difference (Table 3). Overall body sizes of O. viverrini adults 56
15 3.3. Histopathological changes of the hepatobiliary system
from Syrian hamsters treated with G. mangostana were statistical- 57
16
ly significantly smaller compared with the untreated group 58
17 To evaluate the anti-inflammatory property, histopathological
(Fig. 5A–F). 59
18 changes with focus on the inflammatory cell in liver tissue were ob-
19 served. We could not observed the inflammatory cells in liver tissue 60
20 especially intrahepatic bile ducts of N (Fig. 4A, B) and GM group 3.6. Effect of G. mangostana extract on adult worms 61
21 (Fig. 4C, D) which correlated with the gross pathology results. In 62
22 the group of hamsters infected with O. viverrini (OV group), there Opisthorchis viverrini adults recovered from the hepatobiliary tract 63
23 were many inflammatory cells surrounding the hepatic bile duct of treated hamsters showed normal movements. Five O. viverrini 64
24 at 45 days post-infection (Fig. 4E–H). Statistical analysis showed a adults from each group were used for SEM analysis. All O. viverrini 65
25 significant decrease of hepatic tissue inflammation in the OVGM adults recovered from untreated hamsters had a normal appear- 66
26 group (P = 0.043) compared with the OV group (Table 1 and Fig. 4I–L). ance, and no damage of the ventral and oral suckers or the tegument 67
was visible (Fig. 6A–D). SEM analysis revealed evidence of disrup- 68
27
tion of the tegument. Three specimens showed eruption and 69
28 3.4. Toxicity of G. mangostana sloughing at the dorsal and ventral tegumental surface and the oral 70
29 and ventral suckers (Fig. 7A–D), and on two specimens blebbing was 71
30 Table 2 shows that G. mangostana had no renal toxicity, as in- observed at the dorsal and ventral tegumental surface and the oral 72
31 dicated by normal levels of blood urea nitrogen and creatinine in and ventral suckers (Fig. 8A–D). 73
32 the G. mangostana-treated groups compared with the untreated
74
33 groups.
3.7. Eggs per gram of feces and eggs per worm 75
34 76
Fig. 9 shows the effect of G. mangostana extract on parasite egg 77
35 Table 2
production, by eggs per gram of feces. The fecal egg counts from 78
36 Kidney function tests; blood urea nitrogen (BUN), creatinine (Cr) levels in the group hamsters infected with O. viverrini (4598 ± 596.4) and infected 79
37 of normal control (N), normal administered with Garcinia mangostana. (GM), O. viverrini
38 infection alone (OV), O. viverrini infection and administered with G. mangostana 80
39 (OVGM).
Table 3 81
40 Group BUN (mg/dL) Cr (g/dL)
Worm recovery in the group of O. viverrini infection alone (OV) or O. viverrini in- 82
41 mean ± SEM mean ± SEM
fection and administered with G. mangostana (OVGM). 83
42 N 22.88 ± 0.22 0.46 ± 0.02
Experimental group Worm burden mean ± SD 84
43 GM 20.23 ± 0.58 0.39 ± 0.03
44 OV 21.96 ± 2.20 0.42 ± 0.02 OVGM 31 ± 8.33 85
45 OVGM 19.35 ± 0.63 0.41 ± 0.03 OV 37.6 ± 6.42 86

Please cite this article in press as: Ratchadawan Aukkanimart, et al., Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthor-
chiasis, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.007
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1 Fig. 5. Representative adult size in OV (left) and OVGM (right) in length ( A) and width (B), morphology through area of whole parasite (C), area of ovary (D), area of whole
2 testes (E) and area of uterus (F).

3
4 hamsters that were administered G. mangostana (1536 ± 302.3) diarrhea, infected wounds, suppuration, leukorrhea, chronic ulcer, 16
5 showed a statistically significant decrease (P = 0.0004). Moreover, and gonorrhea (Gutierrez-Orozco and Failla, 2013; Kaomongkolgit 17
6 we analyzed the effect of G. mangostana extract on parasite egg pro- et al., 2009; Suksamram et al., 2006). In this study, G. mangostana 18
7 duction, by eggs per worm. Fig. 10 shows that the egg counts from pericarp methanolic extract displayed antioxidant and anti- 19
8 adult worms from hamsters infected with O. viverrini (1630 ± 174.4) inflammatory properties. Moreover, this study is the first report to 20
9 and infected hamsters that were administered G. mangostana show that G. mangostana has an inhibitory effect on body size and 21
10 (1060 ± 164.5) showed a statistically significant decrease (P = 0.04). reproductive organ development of O. viverrini. 22
11 ABTS radical-scavenging assay results showed that G. mangostana 23
12 4. Discussion pericarp methanolic extract has high antioxidant activity, which is sup- 24
13 ported by several previous reports (Chomnawang et al., 2007; 25
14 Garcinia mangostana, or mangosteen, has long been used as a tra- Ngawhirunpat et al., 2010; Pothitirat et al., 2010). Based on its anti- 26
15 ditional medicine for the treatment of abdominal pain, dysentery, oxidant and anti-inflammatory properties, G. mangostana pericarp 27

Please cite this article in press as: Ratchadawan Aukkanimart, et al., Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthor-
chiasis, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.007
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1 Fig. 6. Representative of SEM of Opisthorchis viverrini adult from untreated hamster (A) Oral sucker, Os (B); ventral sucker, Vs (C ) and tegumental surface (D).

3 methanolic extract could be effective in the treatment of hepatobiliary mangostana/acetone extract in a mouse model of inflammation (Chen 13
4 disease caused by O. viverrini. et al., 2008). In addition, another study found that α-mangostin from 14
5 Examination of pathological changes in hamster livers revealed anti- G. mangostana extract demonstrated anti-inflammatory property 15
6 inflammatory activity of G. mangostana against inflammatory cells (Pothitirat et al., 2010); this was in agreement with our results using 16
7 induced by O. viverrini infection. This finding is supported by a previ- thin-layer chromatography, which showed high α-mangostin levels in 17
8 ous report which showed the anti-inflammatory effect of Garcinia G. mangostana extract (Supplementary data). 18

10 Fig. 7. Representative of SEM of Opisthorchis viverrini adult from treated hamster (A) Oral sucker, Os (B); ventral sucker, Vs (C ), tegumental surface (D). Sloughing (s), arrow.

11

12 Fig. 8. Representative of SEM of Opisthorchis viverrini adult from treated hamster (A) ventral sucker, Vs (B,C), tegumental surface (D). Blebbing, B.

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a reduction in the number, body size, and size of reproductive organs 37

(testes, ovaries and vitellaria) of the parasites. Moreover, SEM results 38
confirmed the effect of G. mangostana on O. viverrini, as evidenced 39
by the presentation of blebbing and sloughing at the tegument, and 40
oral and ventral suckers (Figs. 7 and 8). G. mangostana may act against 41
O. viverrini adults via induction of Ca2+ influx, which causes depo- 42
lymerization of the microtrabecular network; this leads to the 43
vacuolization, swelling, blebbing, disruption and detachment of the 44
tegument, similar to the actions of praziquantel and artemisinin 45
(Apinhasmit and Sobhon, 1996; Golenser et al., 2006). In trema- 46
todes, the tegument plays a key role in osmoregulation, protection, 47
secretion and synthesis (Keiser and Morson, 2008b; Keiser et al., 48
2008a). Thus, the parasite may die, or else survive but not grow well, 49
as represented by the small size and defective reproductive organs 50
of adult worms (Fig. 5). 51
1 Fig. 9. The effect of G. mangostana extracts on parasitic egg production evidenced The present study demonstrates the antioxidant and anti- 52
2 by egg per gram of feces on day 45 in the group of infected with O. viverrini (OV) inflammatory properties of G. mangostana extract, as well as its ability 53
3 and O. viverrini infected and administered with G. mangostana (OVGM). to limit parasite growth by inducing cell blebbing and disruption 54
4 and detachment of the tegument, which interferes with O. viverrini 55
reproductive organ development, resulting in lower egg produc- 56
5 The histopathology of Syrian hamsters infected with O. viverrini tion and a reduction in liver pathology. 57
6 was similar to a previous report (Boonmars et al., 2009). At 45 days
58
7 post-infection, aggregations of inflammatory cells were observed
8 surrounding the hepatic bile duct; there was also evidence of ep- Acknowledgements 59
9 ithelial hyperplasia, goblet cell metaplasia, and thickened periductal 60
10 fibrosis (Fig. 4E, H). A slight decrease in inflammatory cells was ob- This research was supported by: Research Affairs, Faculty of Med- Q2 61
11 served in the case of O. viverrini infection and G. mangostana icine, Khon Kaen University (Gold research) and the National Q3 62
12 administration (Fig. 4I, L). These results were consistent with a pre- Research University Initiative and Research Promotion in Higher Ed- 63
13 vious report showing that fingerroot reduced the number of ucation Project, Office of the Higher Education Commission, Thailand, Q4 64
14 inflammatory cells in hepatic tissue in hamsters subjected to through the Health Cluster (SHeP-GMS), Khon Kaen University. 65
15 O. viverrini infection and N-nitrosodimethylamine (NDMA) admin- 66
16 istration (Boonjaraspinyo et al., 2011). Similarly, a recent study Conflict of interest 67
17 (Wonkchalee et al., 2012) reported that T. laurifolia administered at 68
18 100 mg/kg/day demonstrated anti-inflammatory activity in ham- None. 69
19 sters with opisthorchiasis. 70
20 In hamsters in the OVGM group (O. viverrini infection and
Appendix: Supplementary material 71
21 G. mangostana administration), G. mangostana extract was able to
72
22 inhibit O. viverrini reproductive organ development by affecting fat
Supplementary data to this article can be found online at 73
23 metabolism and elimination via ATP-binding cassette (ABC) trans-
doi:10.1016/j.exppara.2015.03.007. 74
24 porters of the parasites (Lespine, 2013). The decreased size of
25 O. viverrini reproductive organs in turn leads to a decrease in the 75
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chiasis, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.007
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Please cite this article in press as: Ratchadawan Aukkanimart, et al., Anthelmintic, anti-inflammatory and antioxidant effects of Garcinia mangostana extract in hamster opisthor-
chiasis, Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.007