THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol.

15 (2), 2008
Page: 169-183
The Anti-Tumor Effect of Bee Honey in Ehrlich Ascite
Tumor Model of Mice is Coincided with Stimulation of
the Immune Cells
1
Attia, W.Y.,
2
Gabry, M.S.,
2
El-Shaikh, K.A.,
2
Othman, G.A
1
Zoology Department, Faculty of Science, Tanta University and
2
Zoology Department,
Faculty of Science, Helwan University, Egypt.
Honey is thought to exhibit a broad spectrum of therapeutic properties including antibacterial, antifungal,
cytostatic and anti-inflammatory activity and has been used for the treatment of gastric ulcers, burns, and for
storage of skin grafts. The present study investigated the antitumor effect of bee honey against Ehrlich
ascites tumor in mice and the possible mode of antitumor action. Peroral administration of mice with honey
(10, 100 or 1000 mg/ 100 g BW) every other day for 4 weeks before intraperitoneal inoculation with Ehrlich
ascites tumor (EAT, 1x10
6
cells) increased the number bone marrow cells as well as peritoneal macrophages,
but not peripheral blood leukocytes nor splenocytes. The phagocytic function of macrophages as well as the
T- and B-cell functions were also increased. Honey pre-treatment also recovered the total lipids, total
proteins, as well as liver and kidney enzyme activities in EAT-bearing mice. In vitro studies on EAT cells
demonstrated inhibitory effect of honey on tumor cell proliferation, viability % of tumor cells as well as the
size of solid tumor. The present results indicate that the preventive treatment with honey is considerably
effective against EAT in mice both in vivo and in vitro. The antitumor activity of honey may occur through the
activation of macrophages, T-cells and B-cells.
everal types of immunopotentiators
have been developed recently and are
being studied for possible use in the
treatment of patients suffering from malignant
diseases (Block & Mead, 2003; Sunila &
Kuttan, 2004). The increased interest in new
approaches to the immunotherapy of cancer,
and a considerable demand for therapeutic
agents which can modulate the several forms
of immunodeficiency have encouraged studies
on the immunomodulatory mechanism of
natural and synthetic substances (Mirandola et
al., 2002; Valadares et al., 2003).
Honey is one of the most complex
foodstuffs produced naturally, and certainly
the only sweetening agent that can be used by
humans without processing. Honey is
produced by honeybees from carbohydrate-
containing exudates produced by plants and
contains all of the trace minerals that are
essential to health (Terrab et al., 2004). A
large number of organic compounds have
been described as components of different
types of honeys (Perez et al., 2002). The main
components of honey volatiles belong, in
general, to three principal categories such as
terpenes, norisoprenoids, and benzene
derivatives (DArcy et al., 1997). Honey is
also characterized by containing different
flavonoids (Siess et al., 1996). Experimental
studies showed that flavonoids could inhibit
carcinogenesis in rodents (Suschetet et al.,
1990; Deschner et al., 1991). In addition,
many flavonoids are known to modify the
activities of drug metabolizing enzymes
(Smith & Yang, 1994).
To explore its immunomodulatory
properties, Duddukuri et al. (1997) reported
that intraperitoneal administration of honey in
low doses (100 l) suppressed the induction
of ovalbumin-specific murine humoral
antibody responses. Honey also suppressed
ovalbumin-specific immunoglobulin G (IgG)
subclasses and other classes of antibody
response (Duddukuri et al., 2001). T cell
proliferation induced by antigens was
significantly suppressed by various doses of
natural and commercial honeys (Duddukuri et
S
The anti-tumor effect of bee honey in ehrlich ascite tumor
is coincided with stimulation of the immune cells
170
al., 2002). On the other hand, Al-Waili & Haq
(2004) reported that oral honey (0.8 g/kg of
body weight/day) stimulated antibody
production during primary and secondary
immune responses against thymus-dependent
and thymus-independent antigens. Karmakar
et al. (2004) also found that honey
significantly increased the humoral immunity
in immunocompetent and immunodeficient
mice. They confirmed the use of honey as an
immunomodulator in experimental rodents. A
recent study by Tonks et al. (2007) showed
that a 5.8-kDa component, isolated from
manuka honey could stimulate cytokine
production from human monocytes.
Honey potentiated the antitumor activity of
chemotherapeutic drugs such as 5-fluorouracil
and cyclophosphamide (Wattenberg, 1986).
Studies by Gribel & Pashiniski (1990)
indicated that honey possesses moderate
antitumor and pronounced antimetastatic
effects in five different strains of rat and
mouse tumors. Rao et al. (1993) reported that
caffeic acid derivatives, present in honey,
inhibited azoxymethane-induced colonic
preneoplastic lesions, which are relevant to
colon carcinogenesis in male F344 rats.
Honey might also prevent tumor implantation
when applied locally (Hamzaoglu et al.,
2000). Honey and Nigella grains together
protected 100% against methylnitrosourea-
induced oxidative stress and carcinogenesis
(Mabrouk et al., 2002). Swellam et al. (2003)
reported that bee honey is an effective agent
for inhibiting the growth of both human and
murine bladder cancer cell lines in vitro.
Honey is also effective when administered
intralesionally or orally in the murine bladder
cancer cell line. Moreover, Orsolic & Basic
(2004) found a pronounced antimetastatic
effect by oral application of honey given
before tumor cell inoculation. However, given
after tumor cell inoculation, honey enhanced
lung metastases. These findings indicate that
honey activated immune system and may be
advantageous with respect to cancer and
metastasis prevention.
The main objective of the present study
was to investigate the prophylactic effect of
bee honey against Ehrlich ascites tumor in
mice and the possible mode of antitumor
action.
Materials and Methods
Animals
The experimental animals used in this study were
female Swiss albino mice (8-10 weeks old, weighing
about 20 g each). Mice were obtained from Helwan
Research Animal Center, Cairo, Egypt, and were
maintained in a quite room at 28
o
C. Mice received
laboratory chow and water ad libitum and were allowed
a period of 10 days, prior to the initiation of
experiments, to acclimatize to the laboratory
conditions.
Bee Honey
Bee honey (Research Institute of Plant Protection,
Cairo, Egypt) was dissolved in distilled water, and
doses of 10, 100 or 1000 mg/100 g body weight were
orally administered to mice (0.2 ml/mouse) every other
day for consecutive 4 weeks. Control mice were orally
administered with 0.2 ml of distilled water only.
Ehrlich Ascites Tumor (EAT) cells
A line of Ehrlich ascites tumor was supplied through
the courtesy of Dr. G. Klien, Amsterdam, Holland. The
tumor line was maintained in The Cancer Institute
(Cairo, Egypt) in female Swiss albino mice by weekly
intraperitoneal transplantation of viable 2x10
6

cells/animal. Tumor cell suspensions were prepared in
balanced salt solution at pH 7.4 to a final concentration
of 5x10
6
viable cells/ml (Bincoletto et al., 2005). All
experimental animals were inoculated with EAT cells
intraperitoneally (i.p.) in a volume of 0.2 ml (1x10
6

cells) 24 hour after the last injection of honey. One
week later, all mice were sacrificed and lymphoid cells,
tumor cells as well as sera were obtained for
immunological, carcinogenic and biochemical analysis.
Determination of Lymphoid Cell Counts
Normal, EAT-bearing mice, and EAT-bearing mice
pretreated with honey were sacrificed, and thymuses
(Thy), spleens (Spl), peripheral lymph nodes (PLN)
and mesenteric lymph nodes (MLN) were excised,
cleaned and weighed. Single cell suspensions were
prepared in Hanks balanced salt solution (HBSS),
followed by filtering through a nylon sieve. Bone
marrow (BM) was obtained from femurs and tibiae,
THE EGYPTIAN JOURNAL OF IMMUNOLOGY 171
suspended in HBSS and filtered. Red blood cells from
all these tissues, in addition to peripheral blood (PBl)
were lysed by addition of Tris/NH
4
Cl buffer (0.017 M
Tris-hydroxymethyl aminomethane and 0.16 M
NH4Cl, pH 7.2). The respective cell suspensions were
washed three times, resuspended in HBSS, and cells
were counted with a haemocytometer and calculated
per gram of tissue.
Harvesting of Peritoneal Exudates Cells (PEC)
To obtain inflammatory peritoneal phagocytes, normal,
EAT-bearing mice and EAT-bearing mice pre-treated
with honey were i.p. injected with 2 ml of starch
suspension (1% starch in saline). Three days later, mice
were sacrificed and the peritoneal exudates cells (PEC)
were obtained by peritoneal lavage with 5 ml of HBSS.
Cells were washed and resuspended in HBSS. Total
and differential counts of PEC were determined using
haemocytometer, by the uptake of 1% W/V neutral red
in saline (Hudson & Hay, 1989).
Carbon Clearance Assay
The phagocytic activity of PEC was measured by using
Pelikan special biological ink (Pelikan-Werke,
Hannover, Germany) according to Salem et al. (1996).
The original suspension was diluted 1:1 with 0.9%
NaCl solution, and 0.2 ml of the diluted ink was i.p.
injected into normal, EAT-bearing mice and EAT-
bearing mice pre-treated with honey after stimulation
with 2 ml of starch suspension, which was i.p. injected
three days earlier. Carbon challenged animals were
sacrificed 15, 30, 45 and 60 minutes after carbon
injection. Five ml of 0.1% EDTA-saline solution was
i.p. injected, and the peritoneal lavage was collected
and centrifuged at 1200 r.p.m. for 5 min. The resultant
supernatant was decanted into another tube, and the
precipitated cells were resuspended in 1 ml of equal
volumes of gelatin (2% gelatin in saline) and ethanol
potassium saline (5% KOH in 70% ethanol), and
incubated overnight at 37
o
C. Optical densities of both
supernatant and digested cells were measured using a
spectrophotometer (Spectronic 20, Bausch and Lomb
Inc., Rochester, NY, USA).
E-rosette-Forming Cells (RFC) Assay
The procedure was performed as described by Hsu et
al. (1975). Seven days before they are sacrificed, the
mice received an i.p. injection of 1x10
8
sheep red blood
cells (SRBCs) in 0.2 ml saline. Spleens from normal,
EAT-bearing mice and EAT-bearing mice pre-treated
with honey were excised and cleaned. Single cell
suspensions were prepared, washed twice by
centrifugation at 1200 r.p.m. for 10 min and
resuspended in HBSS to a concentration of 2x10
6
. A
volume of 0.2 ml of spleen cell suspension was mixed
with an equal volume of 0.5% SRBCs in a glass tube
and incubated for 2-4 hours at 37
o
C. The tubes
containing mixture were gently shaken to resuspend the
cells in the pellet. The rosettes were counted in
haemocytometer and calculated per million
mononuclear cells. The cells surrounded by three or
more SRBCs were counted as E-rosette-forming T-
cells.
Plaque-Forming Cells (PFC) Assay
The procedure was performed as described by
Brousseau et al. (1999). Primary humoral immune
responses against SRBCs were measured after one i.p.
injection of 1x10
8
SRBCs in 0.2 ml saline. Five days
later, normal, EAT-bearing mice and EAT-bearing
mice pre-treated with honey were sacrificed and spleen
were excised and cleaned. Single cell suspensions were
prepared, washed twice by centrifugation at 1200 r.p.m.
for 10 min and resuspended in HBSS to a concentration
of 2x10
6
/ml. The liquid assay mixture was prepared by
adding 50 l of 25% SRBCs and 50 l of guinea pig
complement to 100 l of spleen cell suspension. The
assay mixture was plated to a slide chamber and
incubated for 30-45 min at 37
o
C. The plaques were
scored microscopically and calculated per million
mononuclear cells.
Preparation of Serum
At the end of each experiment, blood was drawn from
the abdominal vein in centrifuge tubes and left to clot.
Thereafter, blood samples were centrifuged at 3000
r.p.m. for 30 minutes and then serum was separated and
kept at -20C until analysis.
Biochemical Analysis
Total lipids content was determined as described by
Zollner et al. (1966) using total lipids kit, LABKIT
Co., Spain. Total protein was determined as described
by Domas (1975) using protein kit, LABKIT Co.,
Spain. Aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) activities were determined as
described by Varliy, (1974) using AST and ALT kits,
LABKIT Co., Spain. Alkaline phosphatase (ALP) and
acid phosphatase (ACP) activities were determined as
described by Delfield & Goldberg (1971) using ALP
and ACP kits, LABKIT Co., Spain. Urea was
determined as described by Weatherbum (1987) using
urea kit, LABKIT Co., Spain. Creatinine was
determined as described by Kostir & Sonka (1952)
using creatinine kit, LABKIT Co., Spain.
Determination of EAT Cell Count
Control mice as well as mice pre-treated with honey
were i.p. inoculated with 1x10
6
EAT cells/mouse. One
week later, normal, EAT-bearing mice as well as EAT-
The anti-tumor effect of bee honey in ehrlich ascite tumor
is coincided with stimulation of the immune cells
172
bearing mice pre-treated with honey were sacrificed.
EAT cells were obtained by peritoneal lavage with 5 ml
of HBSS. Cells were washed three times by
centrifugation at 1200 r.p.m. for 10 min, resuspended in
HBSS, and counted with a haemocytometer.
Viability of EAT Cells
EAT cells (1x10
5
, 5x10
5
, 1x10
6
, 5x10
6
and 1x10
7

cells/well) were seeded into 96-well culture plates
(Falcon, Oxnard, CA) in RPMI-1640 medium (100
l/well). Honey (1, 10 and 100 mg/ml) was added to
each EAT cell concentration in a volume of 100 l/well
and incubated over night at 37
o
C. The respective cell
suspensions were washed three times, and resuspended
in RPMI-1640 medium. The viable cells were counted
with a haemocytometer using trypan blue dye. The
viability % was calculated according to the following
formula:
Viability % =
No. of viable cells
X 100
Total No. of cells
Measurement of Solid Tumor
EAT cells were suspended in normal saline and
adjusted to a concentration of 20x10
6
cells/ml. 0.2 ml
of the cell suspension (4x10
6
cells) was inoculated s.c.
in the right thigh of control mice and in mice pre-
treated with honey. Palpable tumors were measured
after one week using Vernier calipers (Tricle Brand,
Shanghai, China). Tumor volume was calculated so as
to monitor the response to treatment. According to
Papadopoulos et al. (1989), the formula used for this
calculation was:
Tumor volume (mm)=
4 (A/2)
2
(B/2)
3

Where A is the tumor diameter in the minor axis and B
is the tumor diameter in the major axis.
Histological Examination of The Solid Tumor
Tumor-bearing mice as well as tumor-bearing mice
pre-treated with honey were sacrificed one week after
inoculation of EAT (4x10
6
EAT cells/mouse) in the
right thigh of each mouse. The solid tumors were
excised and fixed in 10 % neutral buffered formalin.
The specimens were then dehydrated in ascending
grades of ethyl alcohol, cleared in terpinol, washed in
benzene, embedded in paraffin wax, sectioned at 5 ,
and stained with haematoxylin and eosin (Delafield,
1984). The stained sections were then examined under
light microscopy for assessment of the tumor cell
growth using X 160 microscopic magnification.
Statistical Analysis
All in vivo results are expressed as the mean SD of
groups consisting of 6 mice. The in vitro data are also
expressed as the mean SD of groups consisting of
four wells. Each experiment was performed
independently at least three times. All data were
analysed for significance using Students t-test.
Results
Effect on lymphoid cell count
Table 1 shows that the number of PBl
leukocytes from EAT-bearing mice were
slightly increased compared to that of normal
mice, however, pre-treatment of EAT-bearing
mice with honey (10, 100 or 1000 mg/ 100 g
BW, every other day for 4 weeks) elicited a
pronounced decrease in the number of PBl
leukocytes compared to that of EAT-bearing
mice. This decrease was statistically
significant with the dose 100 mg (P<0.01).
The total number of cells from thymus
(P<0.01), PLN and MLN of EAT-bearing
mice was decreased, while the total number of
cells from Spl was slightly increased
compared to those of normal mice. Pre-
treatment of EAT-bearing mice with honey
caused a significant decrease in the number of
splenocytes with doses 10 and 100 mg
(P<0.05). BM cells from EAT-bearing mice
were significantly decreased when compared
with the corresponding value of normal mice.
Pre-treatment of EAT-bearing mice with
honey caused a statistically significant
increase in the total number of BM cells
(P<0.01).
THE EGYPTIAN JOURNAL OF IMMUNOLOGY 173
Table 1. Total cell count/ gram of tissue in honey treated mice.
Treatment
Total cell count/ g of tissue (Mean SD)
PBI x10
6
Thy x10
9
Spl x10
9
PLN x10
9
MLN x10
9
BM x10
6

Normal 6.17 1.45 2.05 0.31 2.22 0.29 1.23 0.43 1.04 0.34 2.04 0.34
Tumor-bearing
+ Vehicle
7.30 0.99 0.74 0.21## 2.68 0.64 1.19 0.32 0.96 0.25 0.96 0.21##
Tumor-bearing
+ Honey
(10 mg)
6.00 1.40 0.87 0.28 1.84 0.49* 1.12 0.34 0.98 0.35 2.06 0.46**
Tumor-bearing
+ Honey
(100 mg)
4.48 1.22** 0.92 0.30 1.78 0.50* 0.98 0.19 0.92 0.29 2.23 0.53**
Tumor-bearing
+ Honey
(1000 mg)
5.72 1.27 0.89 0.31 1.96 0.52 1.14 0.33 0.99 0.29 1.73 0.37**
Total number of leukocytes from peripheral blood (PBl), as well as cells from thymus (Thy), spleen (Spl), peripheral lymph nodes
(PLN), mesenteric lymph nodes (MLN) and bone marrow (BM) were assessed in normal and in tumor-bearing mice pre-treated
orally with vehicle (0.2 ml distilled water) or honey (10, 100 or 1000 mg/ 100 g BW) every other day for 4 weeks. (## at P < 0.01 in
comparison with the control group; * at P < 0.05 and ** at P < 0.01 in comparison with the tumor-bearing group).


Effect on Peritoneal Exudate Cell (PEC)
Count
As shown in Table 2, the total number of PEC
as well as the absolute number and the
relative proportion of lymphocytes of EAT-
bearing mice were significantly increased
(P<0.01), while the absolute number and the
relative proportion of macrophages were
significantly decreased (P<0.01) when
compared to that of normal mice. Pre-
treatment of EAT-bearing mice with honey
recovered this effect and caused a significant
decrease in PEC count as well as the absolute
number and the relative proportion of
lymphocytes (P<0.01); and a significant
increase in the absolute number and the
relative proportion of macrophages (P<0.01)
with doses 100 and 1000 mg as compared
with the corresponding values of the EAT-
bearing mice.
Effect on the Phagocytic Function of
Peritoneal Exudate Cells (PECs)
Table 3 shows that carbon uptake by PECs of
EAT-bearing mice was significantly
decreased (P<0.01) when compared with that
of the normal mice. However, pre-treatment
of EAT-bearing mice with honey (10, 100 or
1000 mg/ 100 g BW, every other day for 4
weeks) caused a progressive increase in the
scavenger activity of PECs (P<0.05; P<0.01).
On the other hand, the carbon particles
remained in the peritoneal fluid of EAT-
bearing mice were significantly increased as
compared with those of normal mice
(P<0.01). Pre-treatment of EAT-bearing mice
with honey caused a gradual decrease carbon
contents as compared with those of the EAT-
bearing control mice (P<0.05; P<0.01).
The anti-tumor effect of bee honey in ehrlich ascite tumor
is coincided with stimulation of the immune cells
174
Table 2. Total and differential cell count of peritoneal exudates cells in honey treated mice.
Treatment
Total PEC count
(Mean SD x10
6
)
Macrophages Lymphocytes
Absolute No.
(Mean SD x10
6
)
%
Absolute No.
(Mean SD x10
6
)
%
Normal 6.41 1.03 4.32 0.49 67.4 2.09 0.54 32.6
Tumor-bearing +
Vehicle
27.89 1.49## 1.58 0.32## 5.7 26.31 1.40## 94.3
Tumor-bearing +
Honey (10 mg)
24.22 5.63 2.43 1.09 10.0 21.79 4.70
.
90.0
Tumor-bearing +
Honey (100 mg)
20.89 3.90** 2.52 0.46** 12.0 18.37
.
3.45** 88.0
Tumor-bearing +
Honey (1000 mg)
12.23 2.77** 3.14 0.59** 25.7 9.09 3.27** 74.3
Total peritoneal exudate cells (PEC) count, the absolute number and relative proportion (%) of both macrophages and lymphocytes
were assessed in normal and in tumor-bearing mice pre-treated orally with vehicle (0.2 ml distilled water) or honey (10, 100 or 1000
mg/ 100 g BW) every other day for 4 weeks. (## at P < 0.01 in comparison with the control group, and ** at P < 0.01 in comparison
with the tumor-bearing group).
Table 3. Phagocytic activity of peritoneal exudate cells in honey treated mice.
Treatment
Carbon uptake by PEC Carbon particles remained in fluid
15 min 30 min 45 min 60 min 15 min 30 min 45 min 60 min
Normal
0.85
0.08
1.05
0.07
1.11
0.25
1.51
0.14
1.00
0.18
0.81
0.04
0.47
0.08
0.43
0.15
Tumor-bearing +
Vehicle
0.32
0.06##
0.47
0.11##
0.51
0.12##
0.82
0.11##
1.77
0.15##
1.54
0.23##
1.45
0.20##
1.08
0.10##
Tumor-bearing +
Honey (10 mg)
0.45
0.13
0.56
0.10
0.78
0.07**
0.88
0.05
1.57
0.10*
1.27
0.06*
1.12
0.04**
0.85
0.14*
Tumor-bearing +
Honey (100 mg)
0.56
0.10**
0.75
0.08**
0.93
0.06**
0.98
0.23
1.34
0.14**
1.21
0.07*
0.97
0.07**
0.81
0.09**
Tumor-bearing +
Honey (1000 mg)
0.61
0.13**
0.68
0.11*
0.88
0.04**
0.95
0.06*
1.27
0.09**
1.06
0.10**
0.91
0.07**
0.59
0.07**
The phagocytic activity of peritoneal exudate cells (PEC), as determined by carbon uptake by PEC and carbon particles remained in
the peritoneal fluid was assessed in normal and in tumor-bearing mice pre-treated orally with vehicle (0.2 ml distilled water) or
honey (10, 100 or 1000 mg/ 100 g BW) every other day for 4 weeks. (## at P < 0.01 in comparison with the control group; * at P <
0.05 and ** at P < 0.01 in comparison with the tumor-bearing group).

Effect on Rosette-Forming Cells (RFCs)
Count
As shown in Table 4, the number of RFCs in
EAT-bearing mice was significantly
decreased when compared with that of the
normal mice (P<0.01). Pre-treatment of EAT-
bearing mice with honey (10, 100 or 1000 mg/
100 g BW, every other day for 4 weeks)
caused a statistically significant increase in
the number of RFCs as compared with that of
the corresponding EAT-bearing control mice
(P<0.01). The increment of this increase
reached about 0.56, 1.72 and 1.60 folds
respectively.
THE EGYPTIAN JOURNAL OF IMMUNOLOGY 175
Effect on Plaque-Forming Cells (PFCs)
Count
As shown in Table 4, the number of PFCs in
EAT-bearing mice was significantly
decreased when compared with that of the
normal mice (P<0.01). Pre-treatment of EAT-
bearing mice with honey (10, 100 or 1000 mg/
100 g BW, every other day for 4 weeks)
caused a progressive increase in the number
of PFCs as compared with that of the
corresponding EAT-bearing control mice.
However, this increase was statistically
significant with doses 100 and 1000 mg
(P<0.05), and reached about 0.40 and 0.35
folds respectively.
Table 4. T and B lymphocytes in spleen of honey treated mice.
Treatment
No. of RFCs/ million nucleated spleen
cells (Mean SD x10
3
)
No. of PFCs/ million nucleated spleen
cells (Mean SD x10
3
)
Normal 2.45 0.35 1.42 0.09
Tumor-bearing +
Vehicle
0.87 0.10## 1.10 0.13##
Tumor-bearing + Honey (10 mg) 1.36 0.09** 1.18 0.17
Tumor-bearing + Honey (100 mg) 2.37 0.24** 1.54 0.34*
Tumor-bearing + Honey (1000 mg) 2.26 0.27** 1.48 0.29*
Number of rosette-forming cells (RFCs) and plaque-forming cells (PFCs)/ 10
6
nucleated spleen cells were assessed in normal and
in tumor-bearing mice pre-treated orally with vehicle (0.2 ml distilled water) or honey (10, 100 or 1000 mg/ 100 g BW) every other
day for 4 weeks. All mice were immunized i.p. with 0.2 ml of SRBC 4 days before sacrifice. (## at P < 0.01 in comparison with the
control group; * at P < 0.05 and ** at P < 0.01 in comparison with the tumor-bearing group).

Effect on Serum Total Lipids and Total
Proteins
As shown in Table 5, serum levels of total
lipids were decreased, while serum levels of
total proteins were increased in EAT-bearing
mice as compared with those of normal mice.
Pre-treatment of EAT-bearing mice with
honey (10, 100 or 1000 mg/ 100 g BW, every
other day for 4 weeks) caused an increase in
the serum levels of total lipids and a decrease
in the serum levels of total proteins when
compared with those of EAT-bearing control
mice.
Effect on Serum Enzyme Activities of the
Liver
As shown in Table 5, serum enzyme levels of
ALT, AST, ALP and ACP of EAT-bearing
mice were markedly increased as compared
with those of normal mice. Pre-treatment of
EAT-bearing mice with honey (10, 100 or
1000 mg/ 100 g BW, every other day for 4
weeks) ameliorated this effect and caused a
progressive decrease in serum enzyme levels
of the liver as compared with those of the
EAT-bearing control mice.

The anti-tumor effect of bee honey in ehrlich ascite tumor
is coincided with stimulation of the immune cells
176
Table 5. Serum chemistry profile in honey treated mice.
Treatment
Total lipids
Ref. Range:
0.4-1.0 g/dl
Total protein
Ref. Range:
6.5-8.3 g/dl
ALT
Ref Range:
up to 32 U/L
AST
Ref. Range:
up to 31 U/L
ALP
Ref. Range:
98-279 U/L
ACP
Ref. Range:
up to 11 U/L
Normal 0.8 6.2 10 63 237 3.7
Tumor-bearing + Vehicle 0.38 8.5 52 230 292 23
Tumor-bearing + Honey
(10 mg)
0.42 6.5 15 220 134 18
Tumor-bearing + Honey
(100 mg)
0.56 7.5 10 115 192 11
Tumor-bearing + Honey
(1000 mg)
0.40 7.6 10 134 296 21
Serum levels of total lipids, total proteins, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase
(ALP), and acid phosphatase (ACP) were assessed in normal and in tumor-bearing mice pre-treated orally with vehicle (0.2 ml
distilled water) or honey (10, 100 or 1000 mg/ 100 g BW) every other day for 4 weeks.

Effect on Serum Enzyme Activities of the
Kidney
As shown in Table 6, serum urea and
creatinine levels of EAT-bearing mice were
increased as compared with those of normal
mice. However, pre-treatment of EAT-bearing
mice with honey (10, 100 or 1000 mg/ 100 g
BW, every other day for 4 weeks) ameliorated
this effect and caused a progressive decrease
in serum urea and creatinine levels as
compared with those of the EAT-bearing
control mice.
Table 6. Serum urea and creatinine in honey treated mice.
Treatment
Urea
(Ref. range: 15-45 mg/dl)
Creatinine
(Ref. range: 0.5-1.4 mg/ dl)
Normal 33 0.9
Tumor-bearing + Vehicle 55 1.1
Tumor-bearing + Honey (10 mg) 48 1.2
Tumor-bearing + Honey (100 mg) 40 0.8
Tumor-bearing + Honey (1000 mg) 18 0.6
Normal and tumor-bearing mice were pre-treated orally with vehicle (0.2 ml distilled water) or honey (10, 100 or 1000 mg/ 100 g
BW) every other day for 4 weeks.

THE EGYPTIAN JOURNAL OF IMMUNOLOGY 177
Effect on EAT Cell Count
As shown in Table 7, the number of EAT
cells one week after incubation in the
abdominal cavity of mice pre-treated with
honey (10, 100 or 1000 mg/ 100 g BW, every
other day for 4 weeks) was progressively
decreased as compared with that of the
corresponding vehicle-treated control mice.
However, this decrease was statistically
significant with doses 100 and 1000 mg
(P<0.01).
Table 7. Number of tumor cells in honey treated mice.
Treatment Number of tumor cells (Mean SD x10
6
)
Vehicle 24.12 2.92
Honey (10 mg) 19.84 3.99
Honey (100 mg) 16.22 3.93##
Honey (1000 mg) 9.77 2.98##
Number of tumor cells was assessed one week after incubation of 1x10
6
tumor cells in the abdominal cavity of mice pre-treated
orally with vehicle (0.2 ml distilled water) or honey (10, 100 or 1000 mg/100 g BW) every other day for 4 weeks. (## at P < 0.01 in
comparison with the control group).

Effect on the Viability of EAT Cells in vitro
As shown in Table 8, in vitro incubation of
honey (1, 10 or 100 mg/ ml) with serial
concentrations of EAT cells (1x10
5
, 5x10
5
,
1x10
6
, 5x10
6
and 1x10
7
) for 24 hours elicited
a progressive decrease in the % of viable
tumor cells as compared with those of vehicle
control.
Table 8. Tumor cell viability in honey treated mice.
Treatment
% of viable tumor cells
1 x10
5
5 x10
5
1 x10
6
5 x10
6
1 x10
7

Vehicle 92 81 76 86 90
Honey (1 mg/ml) 78 62 61 82 83
Honey (10 mg/ml) 67 53 40 55 72
Honey (100 mg/ml) 72 66 57 72 78
Viability % of tumor cells was assessed in vitro. Serial concentrations of tumor cells (1x10
5
-1x10
7
cells) were exposed to vehicle
(100 l RPMI medium) or honey (1, 10 or 100 mg/ ml) and incubated for 24 hours.

Effect on the Volume of Solid Tumor
As shown in Table 9, the volume of solid
Ehrlich tumor of mice pre-treated with honey
(10, 100 or 1000 mg/ 100 g BW, every other
day for 4 weeks) was markedly decreased
when compared with that of vehicle-treated
control group. The percentage of decrease
reached about 19.5, 66.2 and 84.9 %
respectively.
Histological Architecture of the Solid
Tumor Mass
Administration of EAT cells within the thigh
muscles of mice resulted in proliferation and
growth of the tumor cells that form tumor
masses infiltrating the muscle fibers (Fig. 1 a).
A gradual reduction in the tumor cell masses
was noticed when mice were pre-treated with
honey with doses 10, 100 and 1000 mg/100g
BW (Fig. 1 b-d) respectively.
The anti-tumor effect of bee honey in ehrlich ascite tumor
is coincided with stimulation of the immune cells
178
Table 9. Size of tumor in honey treated mice.
Treatment
Measurement of tumor
(A) Minor axis (mm) (B) Major axis (mm) Volume (mm
3
) %
Tumor-bearing + Vehicle 2.0 2.5 5.23
Tumor-bearing + Honey (10 mg) 2.0 2.0 4.21 19.5
Tumor-bearing + Honey (100 mg) 1.5 1.5 1.77 66.2
Tumor-bearing + Honey (1000 mg) 1.0 1.5 0.79 84.9
Percentage of solid Ehrlich carcinoma growth was assessed in mice pre-treated orally with honey (10, 100 or 1000 mg/ 100 g BW)
every other day for 4 weeks, compared to the tumor-bearing control group. Tumor volume (mm
3
) = [4 (A/2)
2
x (B/2)] /3


Figure 1. (a) Longitudinal section of the thigh muscles of EAT-bearing mice showing the proliferation and
growth of the tumor cells. (b-d) Longitudinal sections of the thigh muscles of tumor-bearing mice pre-treated with
honey (10, 100 and 1000 mg/100g BW) showing the marked reduction of the tumor cell masses. (H&E, X 160).
THE EGYPTIAN JOURNAL OF IMMUNOLOGY 179
Discussion
The present study showed that honey
pretreatment (10, 100 or 1000 mg/ 100 g BW)
every other day for 4 weeks to EAT-bearing
mice significantly increased the number of
BM lymphocytes as well as peritoneal
macrophages, but not peripheral blood
lymphocytes nor splenocytes. The phagocytic
function of macrophages as well as T cell and
B cell activities were significantly increased,
indicating that the preventive treatment with
honey could activate the immune system.
These results are consistent with the results by
Karmakar et al. (2004) who found that honey
significantly increased peritoneal
macrophages but significantly decreased
splenic lymphocyte count. However, Orsolic
& Basic (2004) have observed an increase in
the number of peripheral blood leukocytes
and increased activity of peritoneal
macrophages.
It is well known that macrophages are the
major factor of host defense. Macrophages
from mammary tumor-bearing mice have
impaired cytotoxic activity against syngeneic
tumor target (Sotomayor et al., 1995). Tumor-
associated macrophages were shown to
possess tumor growth-promoting abilities that
stimulate tumor growth and, in addition, these
macrophages suppress many T cell and NK
cell antitumor responses (Kono et al., 1996).
The present results are in relatively good
agreement with the findings described above
and suggest that the preventive treatment with
honey could reduce tumor growth by
activating macrophage function.
The present study showed a gradual
increase in PFC response in honey pre-treated
EAT-bearing mice, immunized with 1x10
8

SRBCs 7 days before sacrifice, as compared
with that of the EAT-bearing control mice.
These results agree with the results by Al-
Waili & Haq (2004) who first reported that
honey increased antibody production during
primary and secondary immune responses
against thymus-dependent antigen (SRBCs).
The present results are also consistent with the
results by Karmakar et al. (2004) who
reported that honey significantly increased
anti-SRBC antibody titers in
immunocompetent mice.
The actual mechanism to stimulate
antibody production was not identified.
However, Al-Waili & Boni (2003) have found
that honey increased salivary nitric oxide in
humans. Also, honey given to sheep,
increased plasma and urinary nitric oxide
levels (Al-Waili, 2003a). Nitric oxide is a
very important mediator of immune responses
(Zeidek & Masek, 1998). It inhibits tumor
growth and metastasis (Zeidek & Masek,
1998). A single dose of L-arginine, a known
precursor of nitric oxide, caused a significant
increase in humoral immune response (Sunita
et al., 2000). Therefore, honey might increase
humoral immunity by means of enhancing
nitric oxide production.
The present data showed that honey
pretreatment to EAT-bearing mice recovered
the total lipids, total protein, ALT, AST, ALP,
ACP as well as urea and creatinine levels as
compared to those of tumor-bearing control
mice. These results confirm the results of Al-
Waili et al (2003b, c) who reported that slow
i.v. infusion or rapid i.v. injection of honey in
different concentrations was safe and could
improve renal and hepatic functions as well as
lipid profile. Honey decreased AST by 22 %,
ALT by 18 %, and creatinine kinase by 33 %.
The present results also confirm the results of
Al-Waili et al. (2006) who reported a
significant reduction in AST, ALT and
alkaline phosphatase and, and a significant
elevation of serum albumin with honey
feeding.
Several cellular components such as
polyamines and polyamine synthetic enzyme
activities including ornithine decarboxylase
are present at high levels in proliferating
The anti-tumor effect of bee honey in ehrlich ascite tumor
is coincided with stimulation of the immune cells
180
neoplastic cells (Heby, 1981; Spector &
Moore, 1988). In addition, many kinases, such
as tyrosine protein kinase, mediate
proliferative as well as metabolic signals in
the cells. Eicosanoids, the metabolites of
arachidonic acid through the lipo-oxygenase
and cyclo-oxygenase pathways, exerts a
variety of biological activities (Wattenberg et
al., 1980; Honn et al., 1989). The mechanism
of antitumor effect shown in the present study
may be related to the inhibitory effect of
honey on tyrosine protein kinase, lipo-
oxygenase and cyclo-oxygenase pathways
metabolites.
The present results clearly demonstrated
the inhibitory effect of honey on EAT cell
proliferation in the abdominal cavity of mice,
on the viability % of tumor cells as well as on
the size of solid tumor. These results are
consistent with the results by Swellam et al.
(2003) who found that is an effective agent
for inhibiting the growth of T24, RT4, 253J
and MBT-2 bladder cancer cell lines in vitro.
It is also effective in the MBT-2 bladder
cancer implantation model. The present
results also agree with the results by Orsolic
& Basic (2004) who demonstrated the
inhibitory effect of honey on metastasis
formation of a mammary carcinoma and
fibrosarcoma in CBA mice as well as on
adenocarcinoma of Y59 rats.
Honey contains a variety of compounds
including caffeic acid, benzoic acid and esters,
substituted phenolic acid and esters,
flavonoids glycones, and bee wax (Heby,
1981; Greenaway et al., 1987). Some of the
observed biological activities of honey may
be traced to its chemical constituents (Spector
& Moore, 1981; Honn et al., 1989). It is likely
that, polyphenolic components present in
honey stimulate host antitumor defense
(Orsolic & Basic, 2003). Wattenberg et al.
(1980) demonstrated that dietary
administration of hydroxycinnamates
(constituents of honey) significantly inhibited
benzopyrene-induced neoplasia of the
forestomach in mice. Caffeic acid ester
derivatives are thought to exhibit a broad
spectrum of activities that possibly include
tumor inhibition (Chinthalapally et al., 1993).
Apoptosis of tumor cells suggests one of
the ways by which honey induces cellular
death. Marked cellular and nuclear changes
suggestive of apoptosis were observed in
honey-treated cancer cell lines (Swellam et
al., 2003). By measuring the DNA content of
cancer cell lines, both ploidy and S-phase
fractions have a prognostic significance in
bladder cancer. Treatment of bladder cancer
cell lines with honey resulted in a significant
reduction in the S-phase fraction, and the
accumulation of large cell population behind
the G1 peak might be an indicator for
apoptosis (Swellam et al., 2003).
In conclusion, the present results indicate
that honey treatment to mice is considerably
effective against EAT model in vivo and in
vitro. Its antitumor activity may occur through
the activation of macrophages, T cells and B-
cells. The property of honey to stimulate both
humeral and cell-mediated immunity will
expand its clinical application and explain
some of its biological activities, particularly in
host defense against tumors.
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