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PHYTOTHERAPY RESEARCH

Phytother. Res. 22, 15111519 (2008)


Published online 7 August 2008 in Wiley
MANUKA InterScience
HONEY ON EXPERIMENTAL BOWEL DISEASE 1511
(www.interscience.wiley.com) DOI: 10.1002/ptr.2523

Effect of Different Doses of Manuka Honey in


Experimentally Induced Inammatory Bowel
Disease in Rats

A. Prakash1, B. Medhi1*, P. K. Avti2, U. N. Saikia3, P. Pandhi1 and K. L. Khanduja2


1
Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India
2
Department of Biophysics, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India
3
Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India

To evaluate the effect of different doses of Manuka honey in experimentally induced inammatory bowel
disease in rats. Adult Wistar rats of either sex were used (n = 30). Colitis was induced by a single intracolonic
administration of TNBS dissolved in 35% ethanol. The rats (n = 30) were divided into ve groups (n = 6) and
were treated with vehicle (ethanol), TNBS, Manuka honey (5 g/kg, p.o.), Manuka honey (10 g/kg, p.o.) or
sulfasalazine (360 mg/kg, p.o.) body weight for 14 days. After completion of treatment, the animals were killed
and the following parameters were assessed: morphological score, histological score and different antioxidant
parameters.
Manuka honey at different doses provided protection against TNBS-induced colonic damage. There was
signicant protection with Manuka honey 5 g/kg as well as with 10 g/kg body weight compared with the control
(p < 0.001). All the treated groups showed reduced colonic inammation and all the biochemical parameters
were signicantly reduced compared with the control in the Manuka honey treated groups (p < 0.001). Manuka
honey at different doses restored lipid peroxidation as well as improved antioxidant parameters. Morphologi-
cal and histological scores were signicantly reduced in the low dose Manuka honey treated group (p < 0.001).
In the inammatory model of colitis, oral administration of Manuka honey 5 g/kg and Manuka honey 10 g/kg
body weight signicantly reduced the colonic inammation. The present study indicates that Manuka honey is
efcacious in the TNBS-induced rat colitis model, but these results require further conrmation in human
studies. Copyright 2008 John Wiley & Sons, Ltd.

Keywords: antioxidant defence system; Manuka honey; colitis; inammatory bowel disease.

bowel disease are well established. One of the mecha-


INTRODUCTION nisms is through cytokines that are secreted from
macrophages such as TNF, IL-1 and IL-8. TNF-
Inammatory bowel disease is an idiopathic, chronic stimulates and induces the production of other inam-
inammatory condition, which affects the gastro- matory mediators such as ROS, and it also activates
intestinal tract. The incidence of inammatory bowel oxidative stress-responsive genes which amplify and
disease in Western countries is 11 cases of ulcerative prolong inammation during IBD (Larrick and Wright,
colitis per 100 000 population and 7 cases per 100 000 1990). Growing evidence also demonstrates the signi-
population of Crohns disease, but currently the incid- cance of oxidative stress both in the clinical and experi-
ence of each are estimated to be about equal. It has mental studies of IBD. The increase of ROS and the
been suggested that intestinal damage in inamma- impairment of antioxidant defence mechanisms were
tory bowel disease (IBD) is related both to increased postulated to be causative factors in inammatory dis-
free radical production resulting from respiratory burst eases (Han and Meydani, 2000). Under physiological
of inltrating phagocytic cells and to impaired antioxi- conditions, a ne balance is maintained between the
dant defence (DOdorico et al., 2001). The protective oxidant and antioxidant systems but it is impaired
functions of phagocytic cells are mediated by the in pathological circumstances such as IBD. Because of
toxic inammatory mediators that are released. Inam- increased oxidative stress, the antioxidant system be-
matory mediators, including reactive oxygen species comes insufcient and the susceptibility of target mole-
(ROS) and cytokines, contribute to the inammatory cules to oxidative stress increases. Oxidant/antioxidant
cascade in modulating the immune system of IBD balance has been suggested to be an important factor
(Murata et al., 1995; Nieto et al., 2000; Wendland et al., for initiation and progression of cancer (Miranda
2001). Pathophysiological changes in inammatory et al., 2000). Excessive production of ROS in mucosal
cells induced by inammatory and immune responses
could directly or indirectly cause damage to intestinal
* Correspondence to: Dr Bikash Medhi, Associate Professor, Depart- epithelial cells, subsequently inuence the mucosal
ment of Pharmacology, Research Block B, Postgraduate Institute of
Medical Education and Research, Sector-12, Chandigarh-160012, India.
integrity or initiate an inammatory signaling cascade
E-mail: drbikashus@yahoo.com and lead to severe impairment in experimental colitis
Contract/grant sponsor: PGIMER, Chandigarh, India. (Oz et al., 2005; Kurutas et al., 2005).
Copyright 2008 John Wiley & Sons, Ltd. Received
Phytother. Res. 6 August(2008)
22, 15111519 2007
Revised 29DOI:
December 2007
10.1002/ptr
Copyright 2008 John Wiley & Sons, Ltd. Accepted 4 February 2008
1512 A. PRAKASH ET AL.

A number of drugs are available for the treatment positive control), Group IV (Manuka honey at a dose
of ulcerative colitis. Drugs such as 5-aminosalicylic acid of 5 g/kg b.wt) and Group V (Manuka honey at a dose
(5-ASA), sulfasulfapyridine (SASP) and glucocorticoids of 10 g/kg b.wt). The animals were given the different
could inhibit the inammatory mediators through dif- doses of honey for 2 weeks. The animals were allowed
ferent mechanisms which are engaged in the down- free access to water and a normal pellet diet. They
regulation of the immune and inammatory responses were housed in polypropylene cages bedded with steri-
of IBD, their adverse reactions during prolonged treat- lized rice husks in at 12 h light and dark cycle.
ment and the high relapse rate limit their use (Joshi
et al., 2005; Auphan et al., 1995). Induction of colitis. Male Wistar rats weighing 150
There are several herbal products including honey 250 g were used for the induction of colitis. Animals
that showed antioxidant properties in various disease received humane care and the experimental protocol
conditions (Bora et al., 2007). Additionally, honey of complied with the guidelines of our institution. Colitis
various origins was evaluated for its analgesic, anti- was induced according to a previously described method
pyretic and ulcerogenic properties alone and in com- (Morris et al., 1989). Animals were anaesthetized with
bination (Azim et al., 2007; Mythilypriya et al., 2007; ethyl ether after which a single intracolonic application
Lusby et al., 2006). The present study includes Manuka of 20 mg of TNBS dissolved in 0.25 mL of either 35%
honey which is obtained from the plant Leptospermum ethanol in saline (v/v) or saline, referred to as TNBS-
scopariu and used therapeutically for its antibacterial ethanol or TNBS-saline, respectively, into the descend-
properties. Based on earlier studies, it is suggested ing colon was given. The control rats received either
that Manuka honey has various biological activities and the same volume of 35% ethanol diluted with saline
might have different pharmacological effects including (v/v) or just saline. The animals were killed 14 days
wound healing, fungal infections, ophthalmology, dia- after TNBS-ethanol administration and the colonic
betes, gastrointestinal tract, skin ulcers and infections. mucosa was obtained for evaluation of damage micro-
Honey is a popular natural sweetener and a powerful scopically as well as the antioxidant status.
antioxidant used in the day to day life. Varieties of
enzymes are present in honey such as oxidase, invertase, Tissue preparation. The tissues were excised and perfused
amylase, catalase etc. So the present study was under- with ice-cold perfusion solution (0.15 M KCl, 2 mM
taken to evaluate the ameliorative effect of different EDTA, pH 7.4). The tissues were homogenized in Tris-
doses of Manuka honey in a TNBS-induced colitis HCl buffer (50 mM, pH 7.4), and the homogenates were
model of IBD and to analyse its effects on MPO, lipid centrifuged at 10 000 g at 4 C for 30 min to obtain
peroxidation, glutathione level and different anti- a post-mitochondrial supernatant (PMS). The PMS
oxidant enzymes including morphological and histolo- was used for the estimation of the antioxidant defence
gical parameters. system.

Myeloperoxidase activity (MPO). Myeloperoxidase acti-


vity was measured according to the method of Krawiesz
MATERIALS AND METHODS et al. (1984). The tissue samples were homogenized in
50 mmol/L potassium phosphate buffer (pH 6.0). The
Materials. Hexadecyl-trimethylammonium bromide buffer homogenate was centrifuged at 10 000 g for 15 min at
(HTAB), O-dianisidine dihydrochloride, glutathione 4 C. The supernatant was discarded and the pellet was
reductase, nicotinamide adenine dinucleotide phosphate resuspended in hexadecyl-trimethylammonium bromide
reduced (NADPH) and thiobarbituric acid (TBA) were buffer (HTAB 0.5% w/v in 50 mmol/L potassium phos-
purchased from Sigma Chemical (St Louis, MO, USA). phate buffer, pH 6.0). The suspension was sonicated
Phosphate buffered saline (PBS), Tris-HCl buffer and on ice, and again centrifuged at 12 000 g for 15 min at
ethylenediamine tetrachloroacetic acid (EDTA) were 4 C. Supernatants were diluted in potassium phosphate
purchased from M/s HiMedia Chemicals (Mumbai, buffer (pH 6.0) containing 0.167 mg/mL of O-dianisidine
India). Reduced glutathione, hydroxylamine hydrochlo- dihydrochloride and 0.0005% of H2O2. Changes in the
ride, trichloroacetic acid, 5,5-dithiobis-(2-nitrobenzoic absorbance at 450 nm, every 10 s over 2 min, were re-
acid) (DTNB) and nitroblue tetrazolium (NBT) were corded with a spectrophotometer (Spectronix Genesys
procured from M/s Sisco Research Laboratories Pvt 2, Rochester, New York, USA). One unit of MPO
Ltd (Mumbai, India). The pellet diet duly approved by activity was dened as the quantity of MPO degrading
the Institutes Animal Ethics Committee was obtained 1 mol H2O2/min/mg protein at 25 C.
from M/s Ashirwad Industries (Punjab, India).
Lipid peroxidation. Lipid peroxidation was estimated
Animals and treatment. The experiments performed by the method of Ohkawa et al. (1978) in tissue homo-
on pathogen-free young male Wistar rats weighing 150 genates. Briey, the reaction mixture contained Tris-
250 g after the study were cleared by the Institutes HCl buffer (50 mM, pH 7.4), ter-butyl hydroperoxide
Animal Ethics Committee. The animals were obtained (BHP) (500 M in ethanol) and 1 mM FeSO4. After
from the Central Animal House of the Postgraduate incubating the samples at 37 C for 90 min, the reaction
Institute of Medical Education and Research, Chandi- was stopped by adding 0.2 mL of 8% sodium dodecyl
garh, India. The animals were divided into ve groups sulphate (SDS) followed by 1.5 mL of 20% acetic acid
containing six animals in each group. Group I (ethanol (pH 3.5). The amount of malondialdehyde (MDA)
as vehicle), Group II (TNBS 20 mg in 35% ethanol, formed during incubation was estimated by adding
applied through the rectum as a single intracolonic 1.5 mL of 0.8% TBA and further heating the mixture
application into descending colon through a rubber at 95 C for 45 min. After cooling, the samples were
catheter, Group III (sulfasalazine 360 mg/kg b.wt as a centrifuged, and the TBA reactive substances (TBARS)
Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
MANUKA HONEY ON EXPERIMENTAL BOWEL DISEASE 1513

were measured in supernatants at 532 nm. The levels tissue). The colon was dissected longitudinally into three
of lipid peroxidation are expressed in terms of nmol pieces for morphological analysis, histological analysis
TBARS/90 min/mg protein. and antioxidant assay.

Reduced glutathione (GSH). Reduced glutathione Morphological analysis. The gross inammatory index
estimation in the tissue homogenate was performed by was assessed visually for inammation according to the
the method of Paglia and Valentine (1967). The re- enteritis gross morphological score (Vogel and Goethe,
quired amount of tissue homogenate was mixed with 2002): 0, no sign of inammation in the whole 10 cm
25% of trichloroacetic acid and centrifuged at 2000 g length of intestine; 1, slight inammation, slight red-
for 15 min to settle the precipitated proteins. The ness, villi visible under 15-fold magnication; 2, in-
supernatant was aspirated and diluted to 1 mL with 0.2 M termediate inammations, discontinuous hyperaemia
sodium phosphate buffer (pH 8.0). Later, 2 mL of 0.6 mM intermediate redness of villi; 3, intensive inammation,
DTNB was added. After 10 min the optical density of hyperaemia, intensive redness of villi.
the yellow-coloured complex formed by the reaction
of GSH and DTNB (Ellmans reagent) was measured Histological analysis. Microscopic examinations were
at 405 nm. A standard curve was obtained with standard done by a qualied blinded pathologist using haemato-
reduced glutathione. The levels of GSH are expressed xylin and eosin staining in a blinded fashion as described
as g GSH/mg protein. by Levine et al. (2002), in the group treated with TNBS
in 35% ethanol and ethanol only treated group and
Superoxide dismutase (SOD). Superoxide dismutase was also in other groups after pharmacological intervention
estimated according to the method of Kono (1978). using the following score: 0, normal; 1, mild mixed
Briey, the reaction mixture containing solution A inltrates in the lamina propria; 2, focal supercial
(50 mM sodium carbonate, 0.1 mM EDTA, pH 10.0), ulceration of the mucosa only, moderate cryptitis and
solution B (96 M nitroblue tetrazolium (NBT) in solu- crypt abscess; 3, deep ulceration penetrating colonic
tion A) and solution C (0.6% Triton X-100 in solution wall through mucosa till muscularis mucosa and severe
A) were incubated at 37 C for 10 min. The reaction was inammation; 4, necrosis through large bowel wall.
initiated by adding 100 L solution D (20 mM hydroxyla-
mine hydrochloride, pH 6.0). The rate of NBT dye re- Statistical analysis. The statistical signicance of differ-
duction by O2 anion generated due to photoactivation ences between the various groups was determined by
of hydroxylamine hydrochloride was recorded at 560 nm using one-way analysis of variance (ANOVA) followed
in the absence of PMS. Later, small aliquots of PMS by the post-hoc test Bonferroni test, Dennett t (2 sided)
were added to the reaction mixture and 50% inhibition and Tukey test used for data analysis with respect
in the rate of NBT reduction by SOD present in the to the control. Values of p < 0.05 were considered
enzyme source was recorded. One unit of enzyme signicant.
activity was dened by the 50% inhibition of NBT. The
levels of SOD are expressed in terms of IU/mg protein.

Catalase (CAT). Catalase activity was measured in the RESULTS


PMS by the method of Luck (1963). The nal reaction
volume of 3 mL contained 0.05 M Tris-buffer, 5 mM EDTA The assessment was done by gross morphology and
(pH 7.0) and 10 mM H2O2 (in 0.1 M potassium phosphate microscopic histopathology studies together with vari-
buffer, pH 7.0). About 50 or 100 L aliquots of the ous biochemical parameters including myeloperoxidase,
tissue PMS was added to the above mixture. The rate lipid peroxidation, superoxide dismutase, catalase, glu-
of change of absorbance per min at 240 nm was re- tathione peroxidase activities and reduced glutathione.
corded. The level of CAT is expressed in terms of mol During the study there was no mean weight loss/gain
H2O2 consumed/min/mg protein. and none of the rats were excluded from the study for
any reason. More than 50% of animals in the groups
Glutathione peroxidase (GPx). The GPx activity was had diarrhoea after the induction of inammation
measured by the method of Paglia and Valentine (1967). in the colon. Although the rats continued to have
The reaction mixture contained 50 mM potassium phos- diarrhoea for 67 days, in the treatment group the diar-
phate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide, rhoea were obsolete. After the rats were killed and the
0.2 mM NADPH, 1 U glutathione reductase and 1 mM colons opened, it was seen that some of the animals
reduced glutathione. The sample, after its addition, was had local peritonitis compatible with transmural necrosis
allowed to equilibrate for 5 min at 25 C. The reaction and inammatory masses in the region of the descend-
was initiated by adding 0.1 mL of 2.5 mM H2O2. The ing colon.
absorbance at 340 nm was recorded for 5 min. The
levels of GPx were expressed in terms of nmol NADPH
consumed/min/mg of protein using the extinction co- Morphological ndings
efcient of 6.2 103 M1 cm1 at 340 nm.
Fourteen days after TNBS treatment, the morphology
Assessment of inammation. The rats were killed of the colon revealed inammatory change in the
under ether anaesthesia by cervical dislocation 2 weeks mucosa. The gross inammatory index along the 10 cm
after the TNBS administration. The distal 10 cm of the length of colon was assessed for inammation accord-
colon was quickly excised. Adherent adipose tissue was ing to the scoring system. The morphological score
made free and the colon incubated in the Tris-buffer in the control group animals was signicantly lower
for 30 min at 37 C in a shaking water bath (1 mL/100 g (p < 0.001) compared with the TNBS groups (Fig. 1).
Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
1514 A. PRAKASH ET AL.

Figure 1. Effect of different doses of Manuka honey (MH) on


the morphological observations after 14 days of single dose
intracolonic administration of TNBS-induced colitis in rats.
Values are mean SD, n = 6, * p < 0.05 considered significant
with respect to control, # p < 0.05 considered with respect to
TNBS group. (LMH, low dose Manuka honey (5 g/kg, p.o.); HMH,
high dose Manuka honey (10 g/kg, p.o.); SSZ, sulphasalazine;
TNBS, trinitrobenzene sulphonic acid).
Figure 3. Microphotograph showing moderate inflammation in
the lamina propia of colon in the vehicle (ethanol) treated group.
Manuka honey (5 mg/kg, LMH) treatment signicantly L, lamina propria, the arrows indicate the infiltration of
eosinophils, neutrophils (H&E, 280).
reduced (p < 0.05) the morphological inammatory
changes compared with the TNBS group animals
(Fig. 1). Also, Manuka honey (10 mg/kg) (HMH) was
effective in treating the morphological changes induced
by TNBS (Fig. 1).

Histological ndings

The microscopic examination of the colon tissue showed


the presence of inammation in the mucosa which was
scored as described previously by Levine et al. (2002).
In the control group, there was no signicant change
(p < 0.001) at the cellular level, however, reactive
lymphoid follicles were present (Fig. 2). The scanner
view of the tissue shows intact mucosa inamed lamina
propria and widened submucosa. There was signicant
change in the vehicle treated group and the examina-
tion of tissue showed mixed inammation rich in
neutrophils and eosinophils in the lamina propria (Fig. 3).
There was also presence of lymphocytes inltration into
the crypt with features of cryptitis (Fig. 4a, b). These
ndings indicated severe inammation of the colon
and the histological score was very high compared with

Figure 4. (a) Microphotograph showing mild chronic inflam-


mation in lamina propria and focal cryptitis of the colon after
14 days of the single dose treatment of TNBS in rats. Arrows
indicate the infiltration of inflammatory cells (H&E 280). (b)
High power microphotograph showing cryptitis of the colon
Figure 2. Normal microphotograph showing lymphoid follicles after 14 days of the single dose treatment of TNBS in rats.
in the vessel of colon tissue (H&E, 280). Arrows indicate the infiltration of inflammatory cells (H&E, 550).

Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
MANUKA HONEY ON EXPERIMENTAL BOWEL DISEASE 1515

Figure 7. Manuka honey (10 mg/kg) showing gastric metaplasia


Figure 5. Treatment of Manuka honey (5 g/kg/day) shows cystic at the base of crypts (H&E 280) of the rat colon after 14 days
dilatation of the crypt with abscess in the colon of rats after of the single dose treatment of TNBS (H&E 280).
14 days of the single dose treatment of TNBS (H&E 550).

the vehicle treated group. In Manuka honey group


the tissue showed mild inammation of lamina propia
by lymphocytes and scattered eosinophils. There was
no evidence of cryptitis and no crypt abscess was
seen compared with the control (Figs 5, 6, 7). There
was mild inammation and lamina propria with occa-
sional neutrophils. No cryptitis was found in this group.
The histological score was equal for both doses of
Manuka honey.

Myeloperoxidase activity (MPO)

There was a 6-fold increase (p < 0.001) in the myeloper-


oxidase activity after 14 days of TNBS treatment com- Figure 8. Effect of different doses of Manuka honey (MH) on
pared with the control. Treatment with Manuka honey the myeloperoxidase activity after 14 days following single
(5 mg/kg) and 10 mg/kg signicantly reduced the TNBS- intracolonic administration of TNBS-induced colitis in rats.
induced myeloperoxidase activity 2 fold. Although there Values are mean SD, n = 6, * p < 0.05 considered significant
with respect to control, # p < 0.05 considered with respect to
were signicant decreases (p < 0.05) in TNBS-induced TNBS group. (LMH, low dose Manuka honey (5 g/kg, p.o.); HMH,
MPO activity in all the treated groups, Manuka honey high dose Manuka honey (10 g/kg, p.o.); SSZ, sulphasalazine;
TNBS, trinitrobenzene sulphonic acid).

at a dose of 10 mg/kg, p.o. signicantly decreased (p < 0.001)


the MPO activity reaching the control levels (Fig. 8).

Lipid peroxidation

TNBS treatment signicantly increased the lipid per-


oxidation levels by 2 fold compared with the control.
Treatment with Manuka honey (5 mg/kg) signicantly
decreased the LPx levels (p < 0.05) to their control
values, whereas Manuka honey (10 mg/kg) showed less
effectiveness compared with the low dose treatment in
decreasing the lipid peroxidation levels (Fig. 9).

Superoxide dismutase activity

Figure 6. Treatment with sulfasalazine showing leucostasis in


TNBS treatment signicantly (p < 0.001) decreased
the vessel of the rat colon after 14 days of single dose treat- the SOD activity by 2 fold compared with the con-
ment with TNBS (H&E 280). trol, whereas with either Manuka honey (5 mg/kg) or
Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
1516 A. PRAKASH ET AL.

Figure 9. Effect of different doses of Manuka honey (MH) on the Figure 11. Effect of different doses of Manuka honey (MH) on
lipid peroxidation after 14 days following single intracolonic the Catalase activity after 14 days following single intracolonic
administration of TNBS-induced colitis in rats. Values are mean administration of TNBS-induced colitis in rats. Values are mean
SD, n = 6, * p < 0.05 considered significant with respect to SD, n = 7, * p < 0.05 considered significant with respect to the
the control, # p < 0.05 considered with respect to TNBS group. control, # p < 0.05 considered with respect to TNBS group.
(LMH, low dose Manuka honey (5 g/kg, po); HMH, high dose (LMH, low dose Manuka honey (5 g/kg, p.o.); HMH, high dose
Manuka honey (10 g/kg, p.o.); SSZ, sulphasalazine; TNBS, Manuka honey (10 g/kg, p.o.); SSZ, sulphasalazine; TNBS,
trinitrobenzene sulphonic acid). trinitrobenzene sulphonic acid).

Figure 10. Effect of different doses of Manuka honey (MH) on Figure 12. Effect of different doses of Manuka honey (MH) on
the superoxide dismutase activity after 14 days following the glutathione peroxidase activity after 14 following single
single intracolonic administration of TNBS-induced colitis in intracolonic administration of TNBS-induced colitis in rats.
rats. Values are mean SD, n = 6, * p < 0.05 considered sig- Values are mean SD, n = 7, * p < 0.05 considered significant
nificant with respect to the control, # p < 0.05 considered with respect to the control, # p < 0.05 considered with respect
with respect to TNBS group. (LMH, low dose Manuka honey to TNBS group. (LMH, low dose Manuka honey (5 g/kg, p.o.);
(5 g/kg, po); HMH, high dose Manuka honey (10 g/kg, p.o.); SSZ, HMH, high dose Manuka honey (10 g/kg, p.o.); SSZ, sulpha-
sulphasalazine; TNBS, trinitrobenzene sulphonic acid). salazine; TNBS, trinitrobenzene sulphonic acid).

sulfasalazine alone the superoxide dismutase activity and Manuka honey (10 mg/kg) signicantly increased
reached the control values. On the other hand, Manuka (p < 0.05) the TNBS-mediated decreased GPx activity
honey (10 mg/kg) signicantly increased (p < 0.05) the reaching almost the control levels (Fig. 12).
TNBS-mediated decreased SOD activity (Fig. 10) by
2 fold compared with the control.
Reduced glutathione

Catalase activity Compared with the control group TNBS treatment


signicantly decreased the GSH level (p < 0.001). After
In the Manuka honey (10 mg/kg) group, catalase activ- 14 days in all treated groups (LMH and HMH) there
ity was comparatively higher than the Manuka honey was a signicantly (p < 0.05) increased glutathione
5 mg/kg groups (p < 0.05) but signicantly lower than reductase level at the site (Fig. 13).
the control group (Fig. 11). Catalase activity in the TNBS
group was signicantly decreased (p < 0.001) by 1.5
fold compared with the control. Treatment with Manuka
honey (5 mg/kg) signicantly increased the TNBS- DISCUSSION
mediated decreased CAT activity.
The trinitrobenzene sulphonic acid (TNBS) colitis model
in rats is a well established experimental model for IBD
Glutathione peroxidase activity in rats. Rectal application of TNBS which is dissolved
in 35% ethanol produces acute colonic inammation
TNBS treatment signicantly decreased the GPx activ- followed by chronic inammation. TNBS may induce
ity by 3 fold. Treatment with Manuka honey (5 mg/kg) colitis by different mechanisms. It binds covalently
Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
MANUKA HONEY ON EXPERIMENTAL BOWEL DISEASE 1517

is impaired (Zea-Iriarte et al., 1996). The efcacy of


the antioxidant defence system may be impaired
during inammation. It has also been described earlier
that the mammalian colon has only small amounts of
antioxidant enzymes such as CAT, SOD and GPX
(Verspaget et al., 1991; Mulder et al., 1991). Decreased
activities of antioxidant enzymes such as SOD and CAT
might be due to the overwhelming effects of free radicals,
as evidenced by the elevated levels of lipid peroxidation.
Cellular antioxidant enzymes such as SOD, CAT and
GPx and free radical scavengers such as GSH protect
cells and tissues against noxious free radicals. An
Figure 13. Effect of different doses of Manuka honey (MH) on
imbalance between cellular pro-oxidant and antioxidant
the reduced glutathione levels after 14 days following single levels results in oxidative stress that leads to tissue
intracolonic administration of TNBS-induced colitis in rats. damage. The antioxidant enzymes react directly with
Values are mean SD, n = 6, * p < 0.05 considered significant reactive oxygen species (ROS) to yield non-radical
with respect to the control, # p < 0.05 considered with respect products. Superoxide dismutase, a mitochondrial as well
to the TNBS group. (LMH, low dose Manuka honey (5 g/kg,
p.o.); HMH, high dose Manuka honey (10 g/kg, p.o.); SSZ, sul- as cytosolic enzyme, dismutes O2 to H2O2, which is
phasalazine; TNBS, trinitrobenzene sulphonic acid). decomposed by CAT to H2O. Overproduction of these
radicals has an inhibitory effect on the enzymes re-
sponsible for the removal of ROS such as CAT. It has
to the E-amino acid group of lysine and thus it can been reported that superoxide radicals inhibit CAT
covalently modify cell surface proteins. Pre-sensitized activity and that H2O2 suppresses SOD activity (Hassan
T lymphocytes can lyse haptan modied autologous cells and Fridovich, 1978), which might explain the inhibi-
quite efciently. Whereas T lymphocytes will lyse haptan tion of these enzymes after TNBS administration. There-
modied autologous cells only if the animal has been fore, the natural balance between TNBS-induced ROS
pre-sensitized, macrophages will destroy TNBS modi- production and free radical scavengers may be down-
ed autologous cells in the absence of pre-sensitization. regulated, leading to tissue injury (Nieto et al., 2000).
The initial local inammation followed by development Thus, the intestinal mucosa in IBD may be in a con-
of chronic inammation is due to activation of the stant state of oxidative stress, posing a serious threat
immune system. The colitis produced by this experi- to intestinal tissue homeostasis. So increased oxidative
mental method mimics the similar disease condition stress and impairment of antioxidant defences might
evidenced in human inammatory bowel disease. This contribute to the pathogenesis of IBD. The present
standard experimental model is well established for study results with the TNBS-induced lipid peroxidation
inammatory bowel disease (Nieto et al., 1998a, 1998b; are similar to a study conducted in the recent past
Kruidenier and Verspaget, 1998; Grisham et al., 1991). (Loquercio et al., 1996). This study showed the oxidative/
This suggests that TNBS-induced colitis may partially antioxidative status in the colon after TNBS adminis-
be mediated by cytotoxic reactive oxygen metabolites tration causes a signicant decrease in the antioxidant
(The et al., 1978; Kunin and Gallily, 1983). Other sources enzyme activities and increased lipid peroxidation.
of TNBS-induced ROS production by the inamed The effects of natural honey in IBD were studied
colonic mucosa include the mitochondrial and micro- by the Bilsel et al. (2002) and Mahgoub et al. (2002)
somal transfer chains, xanthine oxidase, cyclooxygenase acetic acid-induced and TNBS-induced IBD models.
and lipoxygenase enzymes (Karayalcin et al., 1990; The studies concluded that honey is effective in the
Schumbert et al., 1989; Sedor, 1986; Slater, 1984). ROS treatment of IBD. Honey given orally and per rectum
can also be produced by the phagocytic cells through showed dose-dependently afforded protection against
the NADPH pathway and by the overproduction of acetic acid-induced colonic damage. There was almost
proinammatory mediators (i.e. cytokines and arachi- 100% protection with the highest dose (5 g/kg) (Bilsel
donate metabolites) (Karayalcin et al., 1990). It has been et al., 2002). Our study also found similar results in
suggested that neutrophils are predominantly responsi- different parameters but the only difference is that
ble for the production of excessive amounts of ROS Manuka honey was used orally and increasing the doses
by the tissue in colonic inammation (Hermanowicz beyond 5 g/kg did not show more benets.
et al., 1985; Wallace et al., 1998). MPO, a constitutive Treatment at different doses with Manuka honey
component of neutrophil azurophilic granules, is a obtained from a plant from native New Zealand the
good marker of inammation and tissue injury (Nieto manuka tree, Leptospermum scoparium (Myrtaceae),
et al., 1998b). A six-fold increase in MPO activity was showed a marked improvement and both the doses
observed after TNBS treatment. The decrease in MPO showed similar efcacy so there was no dose depend-
activity after Manuka honey treatment with different ent effect of Manuka honey in TNBS-induced IBD.
doses could be explained by a reduction in the number Recent reports suggest the presence of avonoids in
of neutrophils within the mucosa, which would then Manuka honey (Weston et al., 1999). Using the TNBS-
decrease the risk of hypochlorous acid damage as evident induced colitis model, a signicant enhancement in the
even from the histological studies. The LM was equally activities of antioxidants such as SOD, CAT, GPx and
effective to the HMH dose. This leads to the overall reduced glutathione were observed after treatment with
concept of attempting to restore normal homeostasis. Manuka honey. Of the non-enzymatic antioxidants,
Moreover, it is evident from our study that the func- reduced glutathione is the most abundant endogenous
tional status of antioxidant defence system in the thiol containing tripeptide present in millimolar con-
colonic epithelial cells of rats challenged with TNBS centrations in eukaryotic cells which has been observed
Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
1518 A. PRAKASH ET AL.

to be enhanced after Manuka honey treatment. It plays hances the antioxidant potential and cellular functions.
a pivotal role in the maintenance of the balance of In the present study, the glutathione level increased
cellular reduction and oxidation (redox) reactions and signicantly (p < 0.05) after Manuka honey treatment
acts as a radical scavenger due to redox-active sulphydryl suggesting its protective effect in the colon. The major
groups directly reacting with oxidants. In addition, enzymatic antioxidants include SOD, CAT and GPx,
GSH is involved in protein and DNA synthesis, amino although present in small quantities in the colon com-
acid transport, activation of metabolism, catalysis (as a pared with the liver. It was found that the SOD and
coenzyme) and detoxication of electrophiles either GPx activities peaked and reached their control values
through direct reaction with reactive intermediates after treatment with the Manuka honey compared with
or via conjugation reactions catalysed by GST (Meister CAT. This indicates that these are primary enzymes
and Anderson, 1983). Of these actions, protection against which play a major role in the detoxication of ROS.
oxidative damage caused by ROS is the most impor- Glutathione directly reacts with ROS, whereas GPx
tant function of GSH. A lowered glutathione content catalyses the destruction of hydrogen peroxide and
has generally been considered as an index of the in- hydroperoxide by utilizing GSH and NADPH (Meister
creased formation of ROS, and glutathione depletion and Anderson, 1983; Grant and Dawes, 1996). It is likely
in mammalian cells causes cell damage by oxidative that the increased GPx activities due to Manuka honey
stress (Jain et al., 1991; Martensson et al., 1991). It has might provide a second line of cellular defence in the
been reported that GSH levels are reduced in patients colon against TNBS-induced oxidative stress.
with ROS related diseases (Djordjevic, 2004). A deple- In conclusion, Manuka honey is efcacious in TNBS-
tion of GSH can lead to increased lipid peroxidation induced IBD and there is no dose dependent effect of
with concomitant changes in membrane permeability Manuka honey, but these results require further con-
and cellular damage, but an increased GSH level en- rmation in human studies.

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Copyright 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr