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
Inammatory bowel disease is an idiopathic, chronic
inammatory condition, which affects the gastro-
intestinal tract. The incidence of inammatory bowel
disease in Western countries is 11 cases of ulcerative
colitis per 100 000 population and 7 cases per 100 000
population of Crohns disease, but currently the incid-
ence of each are estimated to be about equal. It has
been suggested that intestinal damage in inamma-
tory bowel disease (IBD) is related both to increased
free radical production resulting from respiratory burst
of inltrating phagocytic cells and to impaired antioxi-
dant defence (DOdorico et al., 2001). The protective
functions of phagocytic cells are mediated by the
toxic inammatory mediators that are released. Inam-
matory mediators, including reactive oxygen species
(ROS) and cytokines, contribute to the inammatory
cascade in modulating the immune system of IBD
(Murata et al., 1995; Nieto et al., 2000; Wendland et al.,
2001). Pathophysiological changes in inammatory
* Correspondence to: Dr Bikash Medhi, Associate Professor, Depart-
ment of Pharmacology, Research Block B, Postgraduate Institute of
Medical Education and Research, Sector-12, Chandigarh-160012, India.
E-mail: drbikashus@yahoo.com
Contract/grant sponsor: PGIMER, Chandigarh, India.
Copyright 2008 John Wiley & Sons, Ltd.
Copyright 2008 John Wiley & Sons, Ltd.
nisms is through cytokines that are secreted from
macrophages such as TNF, IL-1 and IL-8. TNF-
stimulates and induces the production of other inam-
matory mediators such as ROS, and it also activates
oxidative stress-responsive genes which amplify and
prolong inammation during IBD (Larrick and Wright,
1990). Growing evidence also demonstrates the signi-
cance of oxidative stress both in the clinical and experi-
mental studies of IBD. The increase of ROS and the
impairment of antioxidant defence mechanisms were
postulated to be causative factors in inammatory dis-
eases (Han and Meydani, 2000). Under physiological
conditions, a ne balance is maintained between the
oxidant and antioxidant systems but it is impaired
in pathological circumstances such as IBD. Because of
increased oxidative stress, the antioxidant system be-
comes insufcient and the susceptibility of target mole-
cules to oxidative stress increases. Oxidant/antioxidant
balance has been suggested to be an important factor
for initiation and progression of cancer (Miranda
et al., 2000). Excessive production of ROS in mucosal
cells induced by inammatory and immune responses
could directly or indirectly cause damage to intestinal
epithelial cells, subsequently inuence the mucosal
integrity or initiate an inammatory signaling cascade
and lead to severe impairment in experimental colitis
(Oz et al., 2005; Kurutas et al., 2005).
Received
Phytother. Res. 6 August(2008)
22, 15111519 2007
Revised 29DOI:
December 2007
10.1002/ptr
Accepted 4 February 2008
1512 A. PRAKASH ET AL.
A number of drugs are available for the treatment
of ulcerative colitis. Drugs such as 5-aminosalicylic acid
(5-ASA), sulfasulfapyridine (SASP) and glucocorticoids
could inhibit the inammatory mediators through dif-
ferent mechanisms which are engaged in the down-
regulation of the immune and inammatory responses
of IBD, their adverse reactions during prolonged treat-
ment and the high relapse rate limit their use (Joshi
et al., 2005; Auphan et al., 1995).
There are several herbal products including honey
that showed antioxidant properties in various disease
conditions (Bora et al., 2007). Additionally, honey of
various origins was evaluated for its analgesic, anti-
pyretic and ulcerogenic properties alone and in com-
bination (Azim et al., 2007; Mythilypriya et al., 2007;
Lusby et al., 2006). The present study includes Manuka
honey which is obtained from the plant Leptospermum
scopariu and used therapeutically for its antibacterial
properties. Based on earlier studies, it is suggested
that Manuka honey has various biological activities and
might have different pharmacological effects including
wound healing, fungal infections, ophthalmology, dia-
betes, gastrointestinal tract, skin ulcers and infections.
Honey is a popular natural sweetener and a powerful
antioxidant used in the day to day life. Varieties of
enzymes are present in honey such as oxidase, invertase,
amylase, catalase etc. So the present study was under-
taken to evaluate the ameliorative effect of different
doses of Manuka honey in a TNBS-induced colitis
model of IBD and to analyse its effects on MPO, lipid
peroxidation, glutathione level and different anti-
oxidant enzymes including morphological and histolo-
gical parameters.
MATERIALS AND METHODS
Materials. Hexadecyl-trimethylammonium bromide buffer
(HTAB), O-dianisidine dihydrochloride, glutathione
reductase, nicotinamide adenine dinucleotide phosphate
reduced (NADPH) and thiobarbituric acid (TBA) were
purchased from Sigma Chemical (St Louis, MO, USA).
Phosphate buffered saline (PBS), Tris-HCl buffer and
ethylenediamine tetrachloroacetic acid (EDTA) were
purchased from M/s HiMedia Chemicals (Mumbai,
India). Reduced glutathione, hydroxylamine hydrochlo-
ride, trichloroacetic acid, 5,5-dithiobis-(2-nitrobenzoic
acid) (DTNB) and nitroblue tetrazolium (NBT) were
procured from M/s Sisco Research Laboratories Pvt
Ltd (Mumbai, India). The pellet diet duly approved by
the Institutes Animal Ethics Committee was obtained
from M/s Ashirwad Industries (Punjab, India).
Animals and treatment. The experiments performed
on pathogen-free young male Wistar rats weighing 150
250 g after the study were cleared by the Institutes
Animal Ethics Committee. The animals were obtained
from the Central Animal House of the Postgraduate
Institute of Medical Education and Research, Chandi-
garh, India. The animals were divided into ve groups
containing six animals in each group. Group I (ethanol
as vehicle), Group II (TNBS 20 mg in 35% ethanol,
applied through the rectum as a single intracolonic
application into descending colon through a rubber
catheter, Group III (sulfasalazine 360 mg/kg b.wt as a
Copyright 2008 John Wiley & Sons, Ltd.
positive control), Group IV (Manuka honey at a dose
of 5 g/kg b.wt) and Group V (Manuka honey at a dose
of 10 g/kg b.wt). The animals were given the different
doses of honey for 2 weeks. The animals were allowed
free access to water and a normal pellet diet. They
were housed in polypropylene cages bedded with steri-
lized rice husks in at 12 h light and dark cycle.
Induction of colitis. Male Wistar rats weighing 150
250 g were used for the induction of colitis. Animals
received humane care and the experimental protocol
complied with the guidelines of our institution. Colitis
was induced according to a previously described method
(Morris et al., 1989). Animals were anaesthetized with
ethyl ether after which a single intracolonic application
of 20 mg of TNBS dissolved in 0.25 mL of either 35%
ethanol in saline (v/v) or saline, referred to as TNBS-
ethanol or TNBS-saline, respectively, into the descend-
ing colon was given. The control rats received either
the same volume of 35% ethanol diluted with saline
(v/v) or just saline. The animals were killed 14 days
after TNBS-ethanol administration and the colonic
mucosa was obtained for evaluation of damage micro-
scopically as well as the antioxidant status.
Tissue preparation. The tissues were excised and perfused
with ice-cold perfusion solution (0.15 M KCl, 2 mM
EDTA, pH 7.4). The tissues were homogenized in Tris-
HCl buffer (50 mM, pH 7.4), and the homogenates were
centrifuged at 10 000 g at 4 C for 30 min to obtain
a post-mitochondrial supernatant (PMS). The PMS
was used for the estimation of the antioxidant defence
system.
Myeloperoxidase activity (MPO). Myeloperoxidase acti-
vity was measured according to the method of Krawiesz
et al. (1984). The tissue samples were homogenized in
50 mmol/L potassium phosphate buffer (pH 6.0). The
homogenate was centrifuged at 10 000 g for 15 min at
4 C. The supernatant was discarded and the pellet was
resuspended in hexadecyl-trimethylammonium bromide
buffer (HTAB 0.5% w/v in 50 mmol/L potassium phos-
phate buffer, pH 6.0). The suspension was sonicated
on ice, and again centrifuged at 12 000 g for 15 min at
4 C. Supernatants were diluted in potassium phosphate
buffer (pH 6.0) containing 0.167 mg/mL of O-dianisidine
dihydrochloride and 0.0005% of H2O2. Changes in the
absorbance at 450 nm, every 10 s over 2 min, were re-
corded with a spectrophotometer (Spectronix Genesys
2, Rochester, New York, USA). One unit of MPO
activity was dened as the quantity of MPO degrading
1 mol H2O2/min/mg protein at 25 C.
Lipid peroxidation. Lipid peroxidation was estimated
by the method of Ohkawa et al. (1978) in tissue homo-
genates. Briey, the reaction mixture contained Tris-
HCl buffer (50 mM, pH 7.4), ter-butyl hydroperoxide
(BHP) (500 M in ethanol) and 1 mM FeSO4. After
incubating the samples at 37 C for 90 min, the reaction
was stopped by adding 0.2 mL of 8% sodium dodecyl
sulphate (SDS) followed by 1.5 mL of 20% acetic acid
(pH 3.5). The amount of malondialdehyde (MDA)
formed during incubation was estimated by adding
1.5 mL of 0.8% TBA and further heating the mixture
at 95 C for 45 min. After cooling, the samples were
centrifuged, and the TBA reactive substances (TBARS)
Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
 MANUKA HONEY ON EXPERIMENTAL BOWEL DISEASE
were measured in supernatants at 532 nm. The levels
of lipid peroxidation are expressed in terms of nmol
TBARS/90 min/mg protein.
Reduced glutathione (GSH). Reduced glutathione
estimation in the tissue homogenate was performed by
the method of Paglia and Valentine (1967). The re-
quired amount of tissue homogenate was mixed with
25% of trichloroacetic acid and centrifuged at 2000 g
for 15 min to settle the precipitated proteins. The
supernatant was aspirated and diluted to 1 mL with 0.2 M
sodium phosphate buffer (pH 8.0). Later, 2 mL of 0.6 mM
DTNB was added. After 10 min the optical density of
the yellow-coloured complex formed by the reaction
of GSH and DTNB (Ellmans reagent) was measured
at 405 nm. A standard curve was obtained with standard
reduced glutathione. The levels of GSH are expressed
as g GSH/mg protein.
Superoxide dismutase (SOD). Superoxide dismutase was
estimated according to the method of Kono (1978).
Briey, the reaction mixture containing solution A
(50 mM sodium carbonate, 0.1 mM EDTA, pH 10.0),
solution B (96 M nitroblue tetrazolium (NBT) in solu-
tion A) and solution C (0.6% Triton X-100 in solution
A) were incubated at 37 C for 10 min. The reaction was
initiated by adding 100 L solution D (20 mM hydroxyla-
mine hydrochloride, pH 6.0). The rate of NBT dye re-
duction by O2 anion generated due to photoactivation
of hydroxylamine hydrochloride was recorded at 560 nm
in the absence of PMS. Later, small aliquots of PMS
were added to the reaction mixture and 50% inhibition
in the rate of NBT reduction by SOD present in the
enzyme source was recorded. One unit of enzyme
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
PMS by the method of Luck (1963). The nal reaction
volume of 3 mL contained 0.05 M Tris-buffer, 5 mM EDTA
(pH 7.0) and 10 mM H2O2 (in 0.1 M potassium phosphate
buffer, pH 7.0). About 50 or 100 L aliquots of the
tissue PMS was added to the above mixture. The rate
of change of absorbance per min at 240 nm was re-
corded. The level of CAT is expressed in terms of mol
H2O2 consumed/min/mg protein.
Glutathione peroxidase (GPx). The GPx activity was
measured by the method of Paglia and Valentine (1967).
The reaction mixture contained 50 mM potassium phos-
phate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide,
0.2 mM NADPH, 1 U glutathione reductase and 1 mM
reduced glutathione. The sample, after its addition, was
allowed to equilibrate for 5 min at 25 C. The reaction
was initiated by adding 0.1 mL of 2.5 mM H2O2. The
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-
efcient of 6.2 103 M1 cm1 at 340 nm.
Assessment of inammation. The rats were killed
under ether anaesthesia by cervical dislocation 2 weeks
after the TNBS administration. The distal 10 cm of the
colon was quickly excised. Adherent adipose tissue was
made free and the colon incubated in the Tris-buffer
for 30 min at 37 C in a shaking water bath (1 mL/100 g
Copyright 2008 John Wiley & Sons, Ltd.
1513
tissue). The colon was dissected longitudinally into three
pieces for morphological analysis, histological analysis
and antioxidant assay.
Morphological analysis. The gross inammatory index
was assessed visually for inammation according to the
enteritis gross morphological score (Vogel and Goethe,
2002): 0, no sign of inammation in the whole 10 cm
length of intestine; 1, slight inammation, slight red-
ness, villi visible under 15-fold magnication; 2, in-
termediate inammations, discontinuous hyperaemia
intermediate redness of villi; 3, intensive inammation,
hyperaemia, intensive redness of villi.
Histological analysis. Microscopic examinations were
done by a qualied blinded pathologist using haemato-
xylin and eosin staining in a blinded fashion as described
by Levine et al. (2002), in the group treated with TNBS
in 35% ethanol and ethanol only treated group and
also in other groups after pharmacological intervention
using the following score: 0, normal; 1, mild mixed
inltrates in the lamina propria; 2, focal supercial
ulceration of the mucosa only, moderate cryptitis and
crypt abscess; 3, deep ulceration penetrating colonic
wall through mucosa till muscularis mucosa and severe
inammation; 4, necrosis through large bowel wall.
Statistical analysis. The statistical signicance of differ-
ences between the various groups was determined by
using one-way analysis of variance (ANOVA) followed
by the post-hoc test Bonferroni test, Dennett t (2 sided)
and Tukey test used for data analysis with respect
to the control. Values of p < 0.05 were considered
signicant.
RESULTS
The assessment was done by gross morphology and
microscopic histopathology studies together with vari-
ous biochemical parameters including myeloperoxidase,
lipid peroxidation, superoxide dismutase, catalase, glu-
tathione peroxidase activities and reduced glutathione.
During the study there was no mean weight loss/gain
and none of the rats were excluded from the study for
any reason. More than 50% of animals in the groups
had diarrhoea after the induction of inammation
in the colon. Although the rats continued to have
diarrhoea for 67 days, in the treatment group the diar-
rhoea were obsolete. After the rats were killed and the
colons opened, it was seen that some of the animals
had local peritonitis compatible with transmural necrosis
and inammatory masses in the region of the descend-
ing colon.
Morphological ndings
Fourteen days after TNBS treatment, the morphology
of the colon revealed inammatory change in the
mucosa. The gross inammatory index along the 10 cm
length of colon was assessed for inammation accord-
ing to the scoring system. The morphological score
in the control group animals was signicantly lower
(p < 0.001) compared with the TNBS groups (Fig. 1).
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-

Figure 2. Normal microphotograph showing lymphoid follicles
in the vessel of colon tissue (H&E, 280).
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
after 14 days of the single dose treatment of TNBS in rats.
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
Figure 5. Treatment of Manuka honey (5 g/kg/day) shows cystic
dilatation of the crypt with abscess in the colon of rats after
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-
pared with the control. Treatment with Manuka honey
(5 mg/kg) and 10 mg/kg signicantly reduced the TNBS-
induced myeloperoxidase activity 2 fold. Although there
were signicant decreases (p < 0.05) in TNBS-induced
MPO activity in all the treated groups, Manuka honey
Figure 6. Treatment with sulfasalazine showing leucostasis in
the vessel of the rat colon after 14 days of single dose treat-
ment with TNBS (H&E 280).
Copyright 2008 John Wiley & Sons, Ltd.
1515
Figure 7. Manuka honey (10 mg/kg) showing gastric metaplasia
at the base of crypts (H&E 280) of the rat colon after 14 days
of the single dose treatment of TNBS (H&E 280).
Figure 8. Effect of different doses of Manuka honey (MH) on
the myeloperoxidase activity after 14 days following single
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).
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
TNBS treatment signicantly (p < 0.001) decreased
the SOD activity by 2 fold compared with the con-
trol, whereas with either Manuka honey (5 mg/kg) or
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
lipid peroxidation after 14 days following single intracolonic
administration of TNBS-induced colitis in rats. Values are mean
SD, n = 6, * p < 0.05 considered significant with respect to
the control, # p < 0.05 considered with respect to TNBS group.
(LMH, low dose Manuka honey (5 g/kg, po); HMH, high dose
Manuka honey (10 g/kg, p.o.); SSZ, sulphasalazine; TNBS,
trinitrobenzene sulphonic acid).
Figure 10. Effect of different doses of Manuka honey (MH) on
the superoxide dismutase activity after 14 days following
single intracolonic administration of TNBS-induced colitis in
rats. Values are mean SD, n = 6, * p < 0.05 considered sig-
nificant with respect to the control, # p < 0.05 considered
with respect to TNBS group. (LMH, low dose Manuka honey
(5 g/kg, po); HMH, high dose Manuka honey (10 g/kg, p.o.); SSZ,
sulphasalazine; TNBS, trinitrobenzene sulphonic acid).
sulfasalazine alone the superoxide dismutase activity
reached the control values. On the other hand, Manuka
honey (10 mg/kg) signicantly increased (p < 0.05) the
TNBS-mediated decreased SOD activity (Fig. 10) by
2 fold compared with the control.
Catalase activity
In the Manuka honey (10 mg/kg) group, catalase activ-
ity was comparatively higher than the Manuka honey
5 mg/kg groups (p < 0.05) but signicantly lower than
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-
mediated decreased CAT activity.
Glutathione peroxidase activity
TNBS treatment signicantly decreased the GPx activ-
ity by 3 fold. Treatment with Manuka honey (5 mg/kg)
Copyright 2008 John Wiley & Sons, Ltd.
Figure 11. Effect of different doses of Manuka honey (MH) on
the Catalase activity after 14 days following single intracolonic
administration of TNBS-induced colitis in rats. Values are mean
SD, n = 7, * p < 0.05 considered significant with respect to the
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 12. Effect of different doses of Manuka honey (MH) on
the glutathione peroxidase activity after 14 following single
intracolonic administration of TNBS-induced colitis in rats.
Values are mean SD, n = 7, * p < 0.05 considered significant
with respect to the 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, sulpha-
salazine; TNBS, trinitrobenzene sulphonic acid).
and Manuka honey (10 mg/kg) signicantly increased
(p < 0.05) the TNBS-mediated decreased GPx activity
reaching almost the control levels (Fig. 12).
Reduced glutathione
Compared with the control group TNBS treatment
signicantly decreased the GSH level (p < 0.001). After
14 days in all treated groups (LMH and HMH) there
was a signicantly (p < 0.05) increased glutathione
reductase level at the site (Fig. 13).
DISCUSSION
The trinitrobenzene sulphonic acid (TNBS) colitis model
in rats is a well established experimental model for IBD
in rats. Rectal application of TNBS which is dissolved
in 35% ethanol produces acute colonic inammation
followed by chronic inammation. TNBS may induce
colitis by different mechanisms. It binds covalently
Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
 MANUKA HONEY ON EXPERIMENTAL BOWEL DISEASE
Figure 13. Effect of different doses of Manuka honey (MH) on
the reduced glutathione levels after 14 days following single
intracolonic administration of TNBS-induced colitis in rats.
Values are mean SD, n = 6, * p < 0.05 considered significant
with respect to the control, # p < 0.05 considered with respect
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-
phasalazine; TNBS, trinitrobenzene sulphonic acid).
to the E-amino acid group of lysine and thus it can
covalently modify cell surface proteins. Pre-sensitized
T lymphocytes can lyse haptan modied autologous cells
quite efciently. Whereas T lymphocytes will lyse haptan
modied autologous cells only if the animal has been
pre-sensitized, macrophages will destroy TNBS modi-
ed autologous cells in the absence of pre-sensitization.
The initial local inammation followed by development
of chronic inammation is due to activation of the
immune system. The colitis produced by this experi-
mental method mimics the similar disease condition
evidenced in human inammatory bowel disease. This
standard experimental model is well established for
inammatory bowel disease (Nieto et al., 1998a, 1998b;
Kruidenier and Verspaget, 1998; Grisham et al., 1991).
This suggests that TNBS-induced colitis may partially
be mediated by cytotoxic reactive oxygen metabolites
(The et al., 1978; Kunin and Gallily, 1983). Other sources
of TNBS-induced ROS production by the inamed
colonic mucosa include the mitochondrial and micro-
somal transfer chains, xanthine oxidase, cyclooxygenase
and lipoxygenase enzymes (Karayalcin et al., 1990;
Schumbert et al., 1989; Sedor, 1986; Slater, 1984). ROS
can also be produced by the phagocytic cells through
the NADPH pathway and by the overproduction of
proinammatory mediators (i.e. cytokines and arachi-
donate metabolites) (Karayalcin et al., 1990). It has been
suggested that neutrophils are predominantly responsi-
ble for the production of excessive amounts of ROS
by the tissue in colonic inammation (Hermanowicz
et al., 1985; Wallace et al., 1998). MPO, a constitutive
component of neutrophil azurophilic granules, is a
good marker of inammation and tissue injury (Nieto
et al., 1998b). A six-fold increase in MPO activity was
observed after TNBS treatment. The decrease in MPO
activity after Manuka honey treatment with different
doses could be explained by a reduction in the number
of neutrophils within the mucosa, which would then
decrease the risk of hypochlorous acid damage as evident
even from the histological studies. The LM was equally
effective to the HMH dose. This leads to the overall
concept of attempting to restore normal homeostasis.
Moreover, it is evident from our study that the func-
tional status of antioxidant defence system in the
colonic epithelial cells of rats challenged with TNBS
Copyright 2008 John Wiley & Sons, Ltd.
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
imbalance between cellular pro-oxidant and antioxidant
levels results in oxidative stress that leads to tissue
damage. The antioxidant enzymes react directly with
reactive oxygen species (ROS) to yield non-radical
products. Superoxide dismutase, a mitochondrial as well
as cytosolic enzyme, dismutes O2 to H2O2, which is
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
been reported that superoxide radicals inhibit CAT
activity and that H2O2 suppresses SOD activity (Hassan
and Fridovich, 1978), which might explain the inhibi-
tion of these enzymes after TNBS administration. There-
fore, the natural balance between TNBS-induced ROS
production and free radical scavengers may be down-
regulated, leading to tissue injury (Nieto et al., 2000).
Thus, the intestinal mucosa in IBD may be in a con-
stant state of oxidative stress, posing a serious threat
to intestinal tissue homeostasis. So increased oxidative
stress and impairment of antioxidant defences might
contribute to the pathogenesis of IBD. The present
study results with the TNBS-induced lipid peroxidation
are similar to a study conducted in the recent past
(Loquercio et al., 1996). This study showed the oxidative/
antioxidative status in the colon after TNBS adminis-
tration causes a signicant decrease in the antioxidant
enzyme activities and increased lipid peroxidation.
The effects of natural honey in IBD were studied
by the Bilsel et al. (2002) and Mahgoub et al. (2002)
acetic acid-induced and TNBS-induced IBD models.
The studies concluded that honey is effective in the
treatment of IBD. Honey given orally and per rectum
showed dose-dependently afforded protection against
acetic acid-induced colonic damage. There was almost
100% protection with the highest dose (5 g/kg) (Bilsel
et al., 2002). Our study also found similar results in
different parameters but the only difference is that
Manuka honey was used orally and increasing the doses
beyond 5 g/kg did not show more benets.
Treatment at different doses with Manuka honey
obtained from a plant from native New Zealand the
manuka tree, Leptospermum scoparium (Myrtaceae),
showed a marked improvement and both the doses
showed similar efcacy so there was no dose depend-
ent effect of Manuka honey in TNBS-induced IBD.
Recent reports suggest the presence of avonoids in
Manuka honey (Weston et al., 1999). Using the TNBS-
induced colitis model, a signicant enhancement in the
activities of antioxidants such as SOD, CAT, GPx and
reduced glutathione were observed after treatment with
Manuka honey. Of the non-enzymatic antioxidants,
reduced glutathione is the most abundant endogenous
thiol containing tripeptide present in millimolar con-
centrations in eukaryotic cells which has been observed
Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
1518 A. PRAKASH ET AL.
to be enhanced after Manuka honey treatment. It plays
a pivotal role in the maintenance of the balance of
cellular reduction and oxidation (redox) reactions and
acts as a radical scavenger due to redox-active sulphydryl
groups directly reacting with oxidants. In addition,
GSH is involved in protein and DNA synthesis, amino
acid transport, activation of metabolism, catalysis (as a
coenzyme) and detoxication of electrophiles either
through direct reaction with reactive intermediates
or via conjugation reactions catalysed by GST (Meister
and Anderson, 1983). Of these actions, protection against
oxidative damage caused by ROS is the most impor-
tant function of GSH. A lowered glutathione content
has generally been considered as an index of the in-
creased formation of ROS, and glutathione depletion
in mammalian cells causes cell damage by oxidative
stress (Jain et al., 1991; Martensson et al., 1991). It has
been reported that GSH levels are reduced in patients
with ROS related diseases (Djordjevic, 2004). A deple-
tion of GSH can lead to increased lipid peroxidation
with concomitant changes in membrane permeability
and cellular damage, but an increased GSH level en-
REFERENCES
Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. 1995.
Immunosuppression by glucocorticoids: inhibition of NF- B
activity through induction of IkB synthesis. Science 270:
286290.
Azim MK, Perveen H, Mesaik MA, Simjee SU. 2007. Anti-
nociceptive activity of natural honey in thermal-nociception
models in mice. Phytother Res 21: 194197.
Bilsel Y, Bugra D, Yamaner S, Bulut Y, Cevikbas U, Turkoglu U.
2002. Could honey have a place in colitis therapy? Effects
of honey, prednisolone, and disulfiram on inflammation,
nitric oxide, and free radical formation. Dig Surg 19(4): 306
311.
Bora U, Sahu A, Saikia AP, Ryakala VK, Goswami P. 2007.
Medicinal plants used by the people of Northeast India for
curing malaria. Phytother Res 21: 800804.
Djordjevic A, Spasic S, Jovanovic-Galovic A, Djordjevic R,
Grubor-Lajsic G. 2004. Oxidative stress in diabetic preg-
nancy: SOD, CAT and GSH-Px activity and lipid peroxidation
products. J Matern Fetal Neonatal Med 16(6): 367372.
DOdorico A, Bortolan S, Cardin R et al. 2001. Reduced plasma
antioxidant concentrations and increased oxidative DNA
damage in inflammatory bowel disease. Scand J Gastroenterol
136: 12891294.
Grant CM, Maclver FH, Dawes IW. 1996. Glutathione is an es-
sential metabolite required for resistance to oxidative stress
in the yeast saccharomyces cerevisiae. Curr Genet 29(6):
511515.
Grisham MB, Volkmer C, Tso P, Yamada T. 1991. Metabolism
of trinitrobenzenesulfonic acid by the rat colon produces
reactive oxygen species. Gastroenterology 101: 540547.
Han SN, Meydani SN. 2000. Antioxidants, cytokines, and influenza
infection in aged mice and elderly humans. J Infect Dis
182(suppl 1): S74S80.
Hassan HM, Fridovich I. 1978. Regulation and role of superoxide
dismutase. Biochem Soc Trans 6: 356361.
Hermanowicz A, Gibson PR, Jewell DP. 1985. The role of
phagocytes in inflammatory bowel disease. Clin Sci 69: 241
259.
Joshi R, Kumar S, Unnikrishnan M, Mukherjee T. 2005. Free radi-
cal scavenging reactions of sulfasalazine, 5-aminosalicylic
acid and sulfapyridine: mechanistic aspects and antioxidant
activity. Free Radic Res 39: 11631172.
Karayalcin SS, Sturbaum W, Wachsman JT, Cha JH, Powell DW.
1990. Hydrogen peroxide stimulates rat colonic prostaglandin
production and alters electrolyte transport. J Clin Invest 86:
6068.
Kono Y. 1978. Generation of superoxide radical during auto-
Copyright 2008 John Wiley & Sons, Ltd.
hances the antioxidant potential and cellular functions.
In the present study, the glutathione level increased
signicantly (p < 0.05) after Manuka honey treatment
suggesting its protective effect in the colon. The major
enzymatic antioxidants include SOD, CAT and GPx,
although present in small quantities in the colon com-
pared with the liver. It was found that the SOD and
GPx activities peaked and reached their control values
after treatment with the Manuka honey compared with
CAT. This indicates that these are primary enzymes
which play a major role in the detoxication of ROS.
Glutathione directly reacts with ROS, whereas GPx
catalyses the destruction of hydrogen peroxide and
hydroperoxide by utilizing GSH and NADPH (Meister
and Anderson, 1983; Grant and Dawes, 1996). It is likely
that the increased GPx activities due to Manuka honey
might provide a second line of cellular defence in the
colon against TNBS-induced oxidative stress.
In conclusion, Manuka honey is efcacious in TNBS-
induced IBD and there is no dose dependent effect of
Manuka honey, but these results require further con-
rmation in human studies.
oxidation of hydroxylamine and assay for SOD. Arch Biochem
Biophys 186: 189195.
Krawiesz JE, Sharan P, Stenson WF. 1984. Quantitative assay
to acute intestinal inflammation based on myeloperoxidase
activity: Assessment of inflammation in rat and hamster
model. Gastroenterology 87: 1344 1350.
Kruidenier L, Verspaget HW. 1998. Antioxidants and mucosa
protectives: realistic therapeutic options in inflammatory
bowel disease? Mediators Inflamm 7: 157162.
Kunin S, Gallily R. 1983. Recognition and lysis of altered
self-cells by macrophages, a modification of target cells
by 2,4,6-trinitrobenzenesulfonic acid. Immunology 48: 265
272.
Kurutas EB, Cetinkaya A, Bulbuloglu E, Kantarceken B. 2005.
Effects of antioxidant therapy on leukocyte myeloperoxidase
and Cu/Zn-superoxide dismutase and plasma malondialde-
hyde levels in experimental colitis. Mediators Inflamm 6:
390394.
Larrick JW, Wright SC. 1990. Cytotoxic mechanism of tumor
necrosis factor-. FASEB J 4: 32153223.
Levine A, Kenet G, Bruk R et al. 2002. Effect of heparin on
tissue binding activity of fibroblast growth factor and heparin
binding epidermal growth factor in experimental colitis in
rats. Pediatric Res 51: 635640.
Loquercio C, DArgenio G, Delle Cave M et al. 1996. Direct
evidence of oxidative damage in acute and chronic phases
of experimental colitis in rats. Dig Dis Sci 41: 1204 1211.
Luck H. 1963. Catalase. In Methods of Enzymatic Analysis,
Bergmeyer HW (ed.). Academic Press: New York, Section 3,
885894.
Lusby PE, Coombes AL, Wilkinson JM. 2006. A comparison of
wound healing following treatment with Lavandula allardii
honey or essential oil. Phytother Res 20: 755757.
Mahgoub AA, el-Medany Ah, Hagar HH, Sabah DM. 2002. Pro-
tective effect of natural honey against acetic acid-induced
colitis in rats. Trop Gastroenterol 23(2): 8287.
Mrtensson J, Jain A, Stole E, Frayer W, Auld PA, Meister A.
1991. Inhibition of glutathione synthesis in the newborn
rat: a model for endogenously produced oxidative stress.
Proc Natl Acad Sci USA 88(20): 93609364.
Meister A, Anderson ME. 1983. Glutathione. Annu Rev Biochem
52: 711760.
Miranda A, Janssen L, Bosman CB et al. 2000. Superoxide
dismutases in gastric and esophageal cancer and the prog-
nostic impact in gastric cancer. Clin Cancer Res 6: 3183
3192.
Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR,
Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr
 MANUKA HONEY ON EXPERIMENTAL BOWEL DISEASE
Wallace JL. 1989. Heptan induced model of chronic inflam-
mation and ulceration in rat colon. Gastroenterology 96:
795.
Mulder TP, Verspaget HW, Janssens AR, de Bruin PA, Pena AS,
Lamers CB. 1991. Decrease in two intestinal copper/zinc
containing proteins with antioxidant function in inflamma-
tory bowel disease. Gut 32: 11461150.
Murata Y, Ishiguro Y, Itoh J, Munakata A, Yoshida Y. 1995. The
role of proinflammatory and immunoregulatory cytokines
in the pathogenesis of ulcerative colitis. J Gastroenterol
30(suppl 8): 5660.
Mythilypriya R, Shanthi P, Sachdanandam P. 2007. Analgesic,
antipyretic and Ulcerogenic properties of an indigenous
formulation Kalpaamruthaa. Phytother Res 21: 574 578.
Nieto N, Fernandez MI, Torres MI, Rios A, Suarez MD, Gil A.
1998a. Dietary n-3 and n-6 long-chain polyunsaturated fatty
acids affect cellular antioxidant defense system in rats with
experimental ulcerative colitis induced by trinitrobenzene-
sulfonic acid. Dig Dis Sci 43: 26762687.
Nieto N, Giron MD, Suarez MD, Gil A. 1998b. Changes in plasma
and colonic mucosa fatty acid profiles in rats with ulcera-
tive colitis induced by trinitrobenzenesulfonic acid. Dig Dis
Sci 43: 26882695.
Nieto N, Torres MI, Fernandez MI et al. 2000. Experimental
ulcerative colitis impairs antioxidant defense system in rat
intestine. Dig Dis Sci 45: 18201827.
Ohkawa H, Ohishi N, Yoge K. 1978. Assay of lipid peroxides in
animal tissue by thiobarbituric acid reaction. Anal Biol Chem
95: 351358.
Oz HS, Chen TS, McClain CJ, de Villiers WJ. 2005. Antioxidants as
novel therapy in a murine model of colitis. J Nutr Biochem
16: 297304.
Copyright 2008 John Wiley & Sons, Ltd.
1519
Paglia DE, Valentine WN. 1967. Studies on the quantitative and
qualitative characterization of erythrocyte glutathione per-
oxidase. J Lab Clin Med 701: 158169.
Schumbert R, Towner J, Zipser RD. 1989. Role of eicosanoids in
human and experimental colitis. Dig Dis Sci 33: 58S64S.
Sedor JR. 1986. Free radicals and prostanoid synthesis (edito-
rial). Lab Clin Med 108: 521522.
Slater TF. 1984. Free-radical mechanisms in tissue injury.
Biochem J 222: 14.
The HS, Phillips RA, Miller RG. 1978. Quantitative studies on
the precursors of cytotoxic lymphocytes. The cellular basis
of the cross reactivity of TNP-specific clones. J Immunol
121: 17111718.
Verspaget HW, Mulder TP, Van der Sluys Veer A, Pena A,
Lamers CB. 1991. Reactive oxygen metabolites and colitis:
a disturbed balance between damage and protection. A
selective review. Scand J Gastroenterol Suppl 188: 44 51.
Vogel HG, Goethe JW. 2002. Experimental colitis. In Drug
Discovery and Evaluation Pharmacological Assay, 2nd edn.
Springer: Berlin, 896899.
Wallace JL, McKnight W, Asfaha S, Lio DY. 1998. Reduction
of acute and reactivated colitis in rats by an inhibitor of
neutrophil activation. Am J Physiol 274: G802G808.
Wendland BE, Aghdassi E, Tam C et al. 2001. Lipid peroxidation
and plasma antioxidant micronutrients in Crohn disease.
Am J Clin Nutr 74: 259264.
Weston RJ, Mitchell KR, Allen KL. 1999. Antibacterial phenolic
components of Manuka Honey. Food Chem 64: 295301.
Zea-Iriarte WL, Makiyama K, Goto S et al. 1996. Impairment
of antioxidants in colonic epithelial cells isolated from
trinitrobenzene sulphonic acid-induced colitis rats. Scand J
Gastroenterol 31: 985992.
Phytother. Res. 22, 15111519 (2008)
DOI: 10.1002/ptr