ARTICLE IN PRESS

Experimental and Toxicologic Pathology 61 (2009) 123–132

www.elsevier.de/etp

The effects of royal jelly on liver damage induced by

paracetamol in mice$
Murat Kanbura, Gökhan Eraslana,, Latife Beyazb, Sibel Silicic, Bilal Cem Limana,
S- ule Altınordulua, Ayhan Ataseverb
a
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Erciyes, Kayseri, Turkey
b
Department of Pathology, Faculty of Veterinary Medicine, University of Erciyes, Kayseri, Turkey
c
Department of Animal Science, Vocational College of S. Çıkrıkçıoğlu, University of Erciyes, Kayseri, Turkey

Received 11 March 2008; accepted 18 June 2008

Abstract
The present study was undertaken to investigate the protective effect of royal jelly against paracetamol-induced liver
damage. The study was conducted in 90 female Swiss Albino mice, and six groups were established. While the first
group was maintained as control, Groups 2–6 were administered 200 mg/kg RJ for 1 day, 200 mg/kg RJ for 7 days,
400 mg/kg PAR for 1 day, 200 mg/kg RJ plus 400 mg/kg PAR for 1 day and 200 mg/kg RJ for 7 days and then second
400 mg/kg PAR on the 7th day, orally, respectively. It was shown that PAR significantly increased serum ALT, AST,
ALP, liver MDA levels and significantly decreased liver GSH-Px activity, when compared to the control group
(Group 1). On the other hand, meaningful changes were observed in the biochemical parameters of the group which
was administered long-term RJ (Group 6). The aforementioned parameters which were statistically significant were
determined to have drawn closer to values of the control group, and among these, the existing statistical differences for
MDA level and GSH-Px activity between the trial group (Group 6) and the control group disappeared (Group 1).
Compared to the pathological changes observed in the liver parenchyma, remark cords, sinusoids and hepatocytes in
the group which was administered paracetamol alone (Group 4), lesions were determined to be less severe particularly
in the group (Group 6) which received royal jelly for 7 days prior to paracetamol. In conclusion, the administration of
royal jelly as a hepatoprotective agent for 7 days against paracetamol-induced liver damage was determined to exhibit
marked protective effect on liver tissue.
r 2008 Elsevier GmbH. All rights reserved.

Keywords: Royal jelly; Liver damage; Paracetamol; Biochemical parameters; Oxidative stress; Mice

Abbreviations: ALT, alanine aminotransferase; AST, aspartate Introduction

aminotransferase; ALP, alkaline phosphatase; MDA, malondialdehyde;
GSH-Px, glutathione peroxidase; CAT, catalase; SOD, superoxide
dismutase; PAR, paracetamol; RJ, Royal Jelly.
$
This study was given as an oral presentation at the Second
National Veterinary Pharmacology and Toxicology Conference,
Samsun, Turkey.
Corresponding author. Fax: +90 352 3372740.
E-mail address: geraslan38@hotmail.com (G. Eraslan).
PAR is a drug of the para-aminophenol group, which is
considered quite safe at recommended doses, and is
commonly used in humans to relieve minor to moderate
pain, as well as to reduce fever (Savides and Oehme, 1983;
Meotti et al., 2006). When administered at normal doses,
PAR is metabolized extensively by conjugation with

0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2008.06.003
 ARTICLE IN PRESS
124 M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132

sulphate and glucuronic acid. A small fraction of the drug Table 1. Free amino acid content of the royal jelly used in the
is subject to oxidation reactions catalyzed by cytochrome trial (mg/100 g)
P-450 enzymes in the liver, resulting in the generation of
Aspartic acid 17.33
hepatotoxic N-acetyl-p-benzo-quinoneimine. Exposure to
Serine 1.39
high doses of PAR results in increased levels of N-acetyl- Glycine 1.66
p-benzo-quinoneimine. Normally, toxic oxidation meta- Lysine 62.43
bolites generated in the liver are converted into non-toxic Cysteine 1.29
metabolites excreted in urine via conjugation with GSH, Glutamic acid 2.99
which contains sulphydryl groups. However, the intake of Threonine 1.15
high doses of PAR limits the capacity of GSH to detoxify Alanine 1.14
N-acetyl-p-benzo-quinoneimine, and results in the con- Proline 58.76
sumption of liver GSH stores (Mitchell et al., 1973; Valine 3.29
Savides and Oehme, 1983). Oxidative stress is reported to Methionine –
constitute a major mechanism in the pathogenesis of Tyrosine 1.29
Tryptophan –
PAR-induced liver damage (Ozdemirler et al., 1994;
Histidine –
Bessems and Vermeulen, 2001). Arginine –
RJ is a traditional product commonly used to Cystine 21.76
supplement the medical treatment of various diseases. Phenylalanine 1.49
RJ is secreted from the hypopharyngeal gland of young Hydroxyproline 1.61
worker (nurse) bees, to feed young larvae and the adult Leucine–isoleucine 1.51
queen bee. In addition to proteins, carbohydrates, fats, Glutamine 1.46
free amino acids, vitamins (biotin, folic acid, inositol,
niacin, pantothenic acid, pyridoxine, riboflavin, thiamine,
inary Medicine was sought, and the present study was
vitamin E) and minerals (copper, zinc, iron, calcium,
conducted in accordance with the Ethics Board Guide-
manganese, potassium, sodium), RJ also contains bioac-
line. Female Swiss albino mice, weighing from 35 to
tive substances including 10-hydroxy-2-decenoic acid,
40 g, were used. The mice were transferred to the
antibacterial proteins, reproductive system development
experimental environment 1 week prior to the initiation
stimulating factor (350-kDa protein) and monocyte
of the trial so as to ensure their environmental
stimulating factor (Lercker et al., 1981; Nagai and Inoue,
adaptation. The mice were housed under controlled
2004). RJ is ascertained to exhibit anti-inflammatory
heating and ventilation conditions, and 10 mice were
(Kohno et al., 2004), DNA-protective (Inoue et al.,
placed into each cage. Feed and water were provided ad
2003), and anti-tumor (Towsend et al., 1959) effects
libitum to the animals. Six groups were established in
in experimental animals. RJ also has antioxidant
the study as follows:
(El-Nekeety et al., 2007) and hepatoprotective effects
(Zimmermann, 2002; Uzbekova et al., 2006). RJ has been
determined to contain the 57-kDa glycoprotein which is  Group 1: This group comprised 10 mice, and served as
considered to stimulate hepatocyte development and liver the control group.
regeneration (Zimmermann, 2002).  Group 2: This group comprised 10 mice. These mice
The present study was undertaken to assess the effects were administered a single dose of 200 mg/kg RJ
of single or multiple-dose administration of RJ in mice (El-Nekeety et al., 2007) via gavage directly into the
with PAR-induced acute liver damage. stomach.
 Group 3: This group comprised 10 mice. These mice
were administered RJ at a dose of 200 mg/kg/day for
7 days via gavage directly into the stomach.
Materials and methods  Group 4: This group comprised 20 mice. These mice
were administered a single dose of 400 mg/kg PAR
Determination of the amino acid content of RJ (Douidar et al., 1985; Corcoran et al., 1985) via
gavage directly into the stomach.
The amino acid content of RJ was determined by
 Group 5: This group comprised 20 mice. These mice
LC–MS, as described by Ozcan and Senyuva (2006) and
were administered a single combined dose of
is given in Table 1.
400 mg/kg PAR and 200 mg/kg RJ via gavage directly
into the stomach.
Study design  Group 6: This group comprised 20 mice. These mice
were administered RJ at a dose of 200 mg/kg/day for
Prior to the initiation of the study, the approval of the 7 days and a single dose of 400 mg/kg PAR on the 7th
Ethics Board of Erciyes University, Faculty of Veter- day via gavage directly into the stomach.
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M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132
The mice included in the groups that were given
paracetamol and royal jelly (Groups 2–6) were weighed
daily, and accordingly the dose to be administered per
body weight was calculated and administration by
gavage was considered to be more accurate and safety.
Collection and processing of samples
Twenty-four hours after the last administration of the
indicated substances, blood samples were collected by
cardiac puncture into dry tubes from 10 mice in each
group. The mice, from which blood samples were
collected, were euthanized by cervical dislocation, and
their liver tissue was excised. Blood samples were
centrifuged at 3000 rpm for 10 min for the separation
of sera. The serum samples obtained were transferred
into eppendorf tubes and were preserved in a deep
freezer at 80 1C. Part of the liver tissue was transferred
into 10% buffered formalin for histopathological
examination, and the remainder tissue was used for
the analyses of oxidative stress parameters. Liver tissue
samples were homogenized after being mixed with 1:9
phosphate buffer (pH 7.2), in an ice-containing medium.
The homogenates were centrifuged in a cooling cen-
trifuge with a temperature adjusted to +4 1C, at
20,000 rpm for 1 h. The supernatants obtained were
transferred into eppendorf tubes, and preserved at
80 1C in a deep freezer until used for analysis.
Biochemical analysis
Serum ALT, AST, ALP, bilirubin, total protein,
albumin, cholesterol and triglyceride levels were mea-
sured using Johnson & Johnson label kits and a Vitros
750 model autoanalyser. Protein levels in liver homo-
genates were measured as described by Lowry et al.
(1951). MDA analyses were performed in accordance
with the method described by Yoshioka et al. (1979).
GSH-Px activity was measured as described by Paglia
and Valentine (1967). CAT activity was determined in
accordance with the method described by Luck (1965).
SOD activity was determined as described by Sun et al.
(1988).
Preparation of tissues for histopathological
examination
Tissue samples were taken from the liver of the
necropsied animals and fixed in 10% formalin neutral
buffer solution. The trimmed tissues were first washed
with tap water followed by dehydration through a
graded alcohol series and then passed though xylol and
paraffin series before finally blocked in paraffin. The
paraffin blocks were cut into 5–6 mm sections using a
125
microtome stained using hematoxylin and eosin and
examined under a light microscope.
Statistical analysis
Data were presented as arithmetic mean7S.D. The
one-way analysis of variance was used for statistical
analyses, and the differences between the groups were
determined using the Tukey test.
Results
Clinical findings
No clinical disorder was observed in the mice included
in Groups 1–3. Eight of the 20 mice included in Group 4
were determined to die within 24 h. The mice which
survived the first 24 h displayed weakness, inactiveness
and unresponsiveness to external stimuli. Seven of the 20
mice in Group 5 were determined to die within 24 h. The
remainder mice were weak, inactive and unresponsive to
external stimuli. In Group 6, 2 of the 20 mice were
determined to die within 24 h. The survivor mice were
observed to be active and to respond to external stimuli.
Histopathological findings
 Group 1: No significant lesion was determined in the
liver cross-sections of the mice included in the control
group (Fig. 1A).
 Groups 2 and 3: Lymphoid cell infiltration in liver
parenchyma was observed in the groups which were
given RJ (Fig. 1B and C).
 Group 4: Blood vessels were determined to be severely
hyperaemic and large haemorrhagic areas were
present in the parenchyma (Fig. 1D). The Remark
cords were dissociated. Alterations ranging from
degenerative to necrotic were ascertained in hepato-
cytes, particularly in the periphery of Vv. centrales
(Fig. 2A). Pink-coloured roll-shaped erythrocyte
accumulations were present in the large haemorrha-
gic areas and some of the sinusoids (Fig. 2D). The
number of Kupffer cells had increased. The central
necrotic regions were more evident in some of the
cross-sections (Fig. 2B). Paranchymal and vacuolar
degeneration were marked in hepatocytes. Areas of
necrosis were marked particularly in the periacinar
and partially in the portal regions. Pink-coloured
homogeneous hyalinized areas were determined to be
present in the proximity of haemorrhagic areas.
Slight increase in fibrous tissue and limited lymphoid
cell infiltrations were observed in the portal region
(Fig. 2C).
 Group 5: Cross-sections pertaining to Group 5 were
similar to those of the group which was administered
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126 M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132

Fig. 1. (A) The appearance of normal liver tissue (Group 1, H  E, 200  ); (B) foci of lymphoid cell infiltration in the liver
parenchyma (arrows) (Group 2, H  E, 200  ); (C) focus of lymphoid cell infiltration in the liver parenchyma (arrow) (Group 3,
H  E, 100  ); (D) degeneration and large haemorrhagic areas in the liver parenchyma (Group 4, H  E, 100  ).

Fig. 2. (A) Haemorrhagic area localised at the periphery of a vena centralis and parenchymal degeneration in the liver (Group 4,
H  E, 100  ); (B) centroacinar necrosis in the liver parenchyma (central necrosis) (arrows) (Group 4, H  E, 100  ); (C) lipid
degeneration in the liver parenchyma (arrows) (Group 4, H  E, 200  ); (D) erythrocyte accumulations in liver sinusoids (roll-
shaped) (arrows) (Group 4, H  E, 200  ).
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M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132 127

PAR. Large haemorrhagic areas (Fig. 3A) and
degenerative alterations (Fig. 3B) were observed in
the liver parenchyma.
 Group 6: When compared to the group which received
PAR, lesions were determined to be less severe. Blood
vessels were hyperaemic, and being less severe than
Group 4, erythrocyte accumulations were observed in
parts of the sinusoids. Compared to the group which
received paracetamol alone, haemorrhages were
observed in the form of foci. The vacuolar degenera-
tion in some of the hepatocytes was much less severe
than that in Group 4. Periacinarly and portally
localised areas of hepatocyte necrosis were less
diffuse than in the fourth group. Positive findings
pertaining to the sixth group which were not
observed in the fourth group included perivascularly
localised pink-coloured homogeneous hyalinized
areas. These areas contained focal lymphoid cell
infiltrations (Fig. 3C and D).
Compared to Group 1, serum ALT and AST levels
(Table 2) and liver MDA level (Table 4) were
determined to have significantly increased, and the liver
GSH-Px level (Table 4) was demonstrated to have
significantly decreased in Group 4 (po0.05). In com-
parison to Group 1, Groups 5 displayed statistically
significant increases in serum ALT and AST levels
(Table 2), and liver MDA level (Table 4), and
statistically significant decrease in serum triglyceride
level (Table 3). Compared to Group 4, the albumin level
(Table 3) was determined to have increased in Group 5
(po0.05). Compared to Group 1, statistically significant
increases were determined in serum ALT and AST
activities (Table 2) in Group 6. Furthermore, when
compared to Group 4, serum ALT and AST levels
(Table 2) and total protein, albumin levels (Table 3) and
liver MDA level (Table 4) were determined to have
decreased and liver GSH-Px enzyme activity (Table 4)
was demonstrated to have increased in Group 6
(po0.05).

Biochemical findings
No statistically significant difference was determined
in serum ALT, AST and ALP levels (Table 2); serum
bilirubin, total protein, albumin, cholesterol and trigly-
ceride levels (Table 3), liver MDA level and SOD, CAT,
GSH-Px activities (Table 4) in Groups 1–3 (p40.05).
Discussion
PAR is one of the commonest drugs used for the
treatment of minor to moderate pain in humans.
However, the intake of a single dose of 10 g or higher
often causes centrolobular liver necrosis (Melli and

Fig. 3. (A) Large haemorrhagic areas in the liver parenchyma (Group 5, H  E, 100  ); (B) large haemorrhagic areas and
degenerative alterations in the liver parenchyma (Group 5, H  E, 200  ); (C) perivascularly localised hyalinized structure in the
liver parenchyma (arrows) (Group 6, H  E, 200  ); (D) foci of centroacinar necrosis in the liver parenchyma (arrows) (Group 6,
H  E, 200  ).
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128 M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132

Table 2. Serum ALT, AST and ALP activities of the control and trial groups

Groupsa ALT (U/L) AST (U/L) ALP (U/L)

Group 1 76.00723.29 a 210.33748.90 a 90.83710.06 a

Group 2 67.83712.36 a 158.16717.29 a 109.16717.97 a
Group 3 62.66713.42 a 147.16722.46 a 79.83726.20 a
Group 4 405971101.65 c 5575.3371293.90 c 176.16766.76 b
Group 5 3892.507615.05 c 5416.3371291.68 c 129.83737.65 ab
Group 6 1702.337553.65 b 1470.837536.64 b 93.1679.78 a

Groups indicated with different letters (a, b, c) in the same column are statistically significant (po0.05).
a
Group 1, control; Group 2, single dose of 200 mg/kg RJ; Group 3, RJ at a dose of 200 mg/kg/day for 7 day; Group 4, single dose of 400 mg/kg
PAR; Group 5, single combined dose of 400 mg/kg PAR plus 200 mg/kg RJ; Group 6, RJ at a dose of 200 mg/kg/day for 7 days and a single dose of
400 mg/kg PAR on the 7th day.

Table 3. Serum bilirubin, total protein, albumin, cholesterol and triglyceride levels of the control and trial groups

Groupsa Bilirubin (mg/dL) Total protein (mg/dL) Albumin (mg/dL) Cholesterol (mg/dL) Triglyceride (mg/dL)

Group 1 0.3670.12 6.7370.35 ab 2.8370.19 ab 119.33719.05 101.6678.93 b

Group 2 0.3170.04 6.7070.35 ab 2.9370.15 b 119.50710.69 94.16729.80 ab
Group 3 0.3870.17 6.5370.67 ab 2.6570.40 ab 129.50710.36 87.83714.52 ab
Group 4 0.4370.20 5.9170.39 a 2.4570.24 a 106.00713.25 78.83712.81 ab
Group 5 0.3870.07 6.5070.50 ab 3.0570.27 b 123.00722.24 71.50075.00 a
Group 6 0.5870.45 6.8570.50 b 3.1170.33 b 123.16717.66 82.16717.88 ab

Groups indicated with different letters (a, b) in the same column are statistically significant (po0.05).
a
Group 1, control; Group 2, single dose of 200 mg/kg RJ; Group 3, RJ at a dose of 200 mg/kg/day for 7 day; Group 4, single dose of 400 mg/kg
PAR; Group 5, single combined dose of 400 mg/kg PAR plus 200 mg/kg RJ; Group 6, RJ at a dose of 200 mg/kg/day for 7 days and a single dose of
400 mg/kg PAR on the 7th day.

Table 4. Liver MDA levels and SOD, CAT, GSH-Px activities of the control and trial groups

Groupsa MDA (nmol/mg-protein) SOD (U/mg-protein) CAT (U/mg-protein) GSH-Px (U/mg-protein)

Group 1 1.0670.11 a 2.0570.43 ab 0.2570.09 abc 3.0270.57 a

Group 2 1.1370.25 a 2.1270.63 ab 0.2870.08 ab 2.8770.52 a
Group 3 1.1970.18 a 2.2470.12 a 0.3170.15 a 2.9370.71 a
Group 4 2.0770.13 b 1.2270.18 b 0.1070.013 c 1.7470.34 b
Group 5 1.8170.24 b 1.5370.68 ab 0.1370.02 bc 2.1770.69 ab
Group 6 1.3470.24 a 1.9070.53 ab 0.2370.06 abc 2.9070.31 a

Groups indicated with different letters (a, b, c) in the same column are statistically significant (po0.05).
a
Group 1, control; Group 2, single dose of 200 mg/kg RJ; Group 3, RJ at a dose of 200 mg/kg/day for 7 day; Group 4, single dose of 400 mg/kg
PAR; Group 5, single combined dose of 400 mg/kg PAR plus 200 mg/kg RJ; Group 6, RJ at a dose of 200 mg/kg/day for 7 days and a single dose of
400 mg/kg PAR on the 7th day.

Kayaalp, 2006). When administered at doses of
300 mg/kg and higher, PAR leads to severe acute liver
necrosis in mice (Douidar et al., 1985; Corcoran et al.,
1985). In case of the intake of high doses, PAR is first
converted into non-reactive metabolites in the liver by
sulphation and glucuranidation reactions. However,
these metabolites are later converted into a reactive
metabolite known to be toxic for the liver, namely
N-acetyl-p-benzo-quinoneimine, by the liver cytochrom
P-450 enzyme system. The resulting metabolite cova-
lently binds to oxidized lipids and sulphydryl groups in
the liver tissue, and thereby leads to the severe damage
of cell membranes (Kupeli et al., 2006). The major
finding observed in PAR intoxication in humans and
animals is acute centrolobular necrosis (Macnaughton,
2003; Hewawasam et al., 2003). In the present study, the
haemorrhagic and necrotic findings observed in the
histopathological examination of the liver of mice which
were administered PAR demonstrate PAR-induced
damage in the liver (Figs. 1D and 2). Sharp increase in
the serum ALT level is considered to be a significant
indicator of PAR-induced acute liver damage (Black,
 ARTICLE IN PRESS
M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132
1980; Sener et al., 2005). ALT is an enzyme specific to
liver damage, and is used routinely for the determination
of liver damage (Turgut, 2000). In the present study,
when compared to the control group, the sharp increase
detected in the serum ALT levels of mice which were
given PAR (Table 2), demonstrates PAR-induced acute
liver damage to have developed. Serum AST and ALP
levels increase not only in liver damage but also in case
of the damage of various other tissues and organs. For
this reason, increased ALT levels are considered to be
specific to liver damage (Meyer and Harvey, 1994;
Hajimehdipoor et al., 2006). In experimental acute PAR
intoxications, in addition to ALT levels, serum AST and
ALP levels also increase (Sener et al., 2005; Kupeli et al.,
2006). Similarly, in this study, serum AST and ALP
levels were determined to have increased, when com-
pared to the control group (Table 2). Taking into
consideration that the liver is the primary target organ
in acute PAR intoxications, it can be concluded that
increased serum AST and ALP levels result from liver
damage. Due to the changes determined in the other
biochemical parameters evaluated (bilirubin, total pro-
tein, albumin, cholesterol, triglyceride) being insignif-
icant when compared to the control group, the possible
effects of PAR could not be evaluated based on the
indicated parameters.
N-Acetyl-p-benzo-quinoneimine, a reactive metabo-
lite generated as a result of the metabolism of PAR by
liver cytochrom P-450 enzymes, is known to be
responsible for liver damage in acute PAR intoxications
(Dahlin et al., 1984; Potter and Hinson, 1987). The
decrease of the oxidation of PAR and the stimulation of
GSH synthesis, have been determined to decrease the
toxicity of PAR in mice (Tee et al., 1987). Tirmenstein
and Nelson (1990) have reported GSH-Px and thiol-
transferase enzyme activities to decrease in case of PAR-
induced liver damage in animals. Mirochnitchenko et al.
(1999) have demonstrated decrease in liver GSH-Px
activity upon the administration of a high dose of PAR.
In the present study, compared to the controls, liver
GSH-Px enzyme activity was determined to have
decreased and MDA levels were ascertained to have
increased in mice which were administered PAR
(Table 4). These results suggest that PAR may exhibit
effects that induce lipid peroxidation in the liver.
GSH-Px is an enzyme which prevents the generation
of hydrogen peroxide and alkyl hydroperoxides in
association with GSH and GSH-reductase, as well as
the generation of more harmful metabolites such as the
hydroxyl radical (Therond et al., 2000; Parodi, 2007).
Liver GSH stores are reported to consume in PAR
intoxications (Boobis et al., 1989). The consumption of
GSH stores in the body may directly cause oxidative
stress by means of GSH, whereas decreased antioxidant
enzyme acitivity may indirectly lead to a similar effect
(Roder, 2001). In the present study, in comparison to
129
the control group (Group 1), the decrease observed in
liver GSH-Px enzyme activity in the group which
received PAR (Group 4) can be explained with the
consumption of the enzyme during the detoxification of
reactive oxygen metabolites generated due to PAR, as
well as the consumption of liver GSH stores. No
significant change being determined in the activity of
other antioxidant enzymes, namely SOD and CAT,
further supports GSH-Px to be primarily involved in the
cellular defense mechanism against PAR.
RJ is quite a safe product, and is reported not to cause
any adverse effect when administered to rats and mice at
a dose of 3 g/kg in the subacute period (Hashimoto
et al., 1977). In the present study, the administration of
RJ as a single dose or for a period of 7 days was
determined not to cause any statistically significant
change in liver oxidative stress or serum biochemical
parameters. However, in some cross-sections pertaining
to the groups which were given RJ, lymphoid cell
infiltrations were observed in the liver parenchyma
(Fig. 1B and C), and this situation can be considered as
the reaction of the liver to RJ as a substance foreign to
the body. This is not considered to be an adverse effect
on the liver. For, it is previously reported that the liver
has been occurred to react to foreign substances which
are not synthesized in the body (Percy and Barthold,
2001; Salmi et al., 1998).
Due to the consumption of GSH stores, PAR
intoxications are treated with GSH precursors such as
n-acetyl cysteine (Boobis et al., 1989). Bee products are
reported to have beneficial effects in PAR intoxications:
Juzwiak (1993) has determined pollen extracts to
increase the survival rate and to improve both histo-
pathological lesions in the liver and liver enzymes in
mice with PAR-induced acute intoxication. Seo et al.
(2003) have demonstrated both the mortality rate and
severity of hepatic necrosis to decrease in mice which
were administered 400 mg/kg PAR following the admin-
istration of propolis at doses of 10 and 25 mg/kg for
7 days, and hence have indicated propolis to may have
hepatoprotective effects. Research has been conducted
on the hepatoprotective effects of RJ. Uzbekova et al.
(2006), following the administration of thyroxine
which is known to cause liver damage, and additionally
10 mg/kg RJ for 10 days, have demonstrated RJ to
exhibit protective effect against thyroxine-induced liver
damage. Following the administration of 200 mg/kg bw
fumonisin and 100 or 150 mg/kg bw RJ to rats for a
period of 3 weeks, El-Nekeety et al. (2007) have reported
RJ to improve fumonisin-induced histopathological
alterations in the liver, and hence have indicated RJ to
may have hepatoprotective effects against fumonisin
intoxication. Merino et al. (1996) have clarified that
Cuban red propolis exctract exhibits hepatoprotective
effects on carbon tetrachloride-induced liver damage in
rats, probably due to antioxidant effects. No previous
 ARTICLE IN PRESS
130 M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132
study exists on the effects of RJ on PAR-induced
liver damage. In the present study, particularly in the
group which was given 200 mg/kg bw RJ for 7 days
and a single dose of 400 mg/kg bw PAR on the 7th day
of the trial (Group 6), when compared to the group
which received a single dose of PAR (Group 4), the
low level of mortality determined to occur within
24 h, decrease in serum ALT, AST, ALP levels
(Table 2) and liver MDA level (Table 4), the increase
in serum total protein and albumin levels (Table 3) and
liver GSH-Px activity (Table 4), and the alteration of
haemorrhagic and necrotic histopathological findings of
the liver from diffuse to focal form (Fig. 3C and D)
demonstrate RJ to have hepatoprotective and antiox-
idant effects.
As can be seen in Table 1, the RJ used in the present
study contained biologically active amino acids includ-
ing aspartic acid, cystine, cysteine, tyrosine, glycine,
lysine, leucine, valine and isoleucine. De Toranzo et al.
(1983) have reported aspartic acid, cystine, methionine
and tyrosine to exhibit protective effect against carbon
tetrachloride-induced acute liver damage. Tanaka et al.
(1991) have determined cysteine to exhibit hepatopro-
tective effect against PAR-induced liver damage when
administered at a dose of 300 mg/kg bw to mice, and to
decrease the mortality rate in mice. Fodor et al. (1976)
have reported aspartic acid to provide protection
against D-galactosamine-induced liver damage in rats.
Glycine has also been indicated to may have protective
and curative effects on alcoholic hepatitis and alcohol-
induced hepatocellular carcinoma (Yamashina et al.,
2005). Jamall et al. (1979) have reported L-lysine to may
have protective effect against acute ethanol toxicity in
mice. Pisarenko (1996) has demonstrated increased
intracellular leucine, valine and isoleucine concentra-
tions in myocardial cells to stimulate the synthesis of
acetyl coenzyme A and succinyl coenzyme A, and to
deteriorate the oxidative metabolism, and methionine
and cysteine to may reduce myocardial damage caused
by oxygen radicals. Glycine has been proven to be an
antioxidant amino acid which protects cells against
various toxic substances, primarily metals (Paniagua-
Castro et al., 2006). Lysine, which is a nucleophilic
amino acid, is reported to facilitate the exchange of
electrophiles derived from lipid peroxidation (Jamall
et al., 1979). Cystine and cysteine take place in the
synthesis of GSH, an effective cellular antioxidant. GSH
breaks down reactive oxygen species and detoxifies
carcinogens, both directly and by antioxidant enzymes
with which it reacts (Parodi, 2007). In this context, GSH
precursors such as acetyl cysteine are used for the
treatment of PAR intoxications (Boobis et al., 1989).
The indicated researches show the hepatoprotective and
antioxidant effects of RJ in PAR intoxications to may
be related to the biological effects of its free amino acid
content, including glycine, aspartic acid, tyrosine, lysine,
leucine, valine and isoleucine, and mainly cystine and
cysteine which are GSH precursors.
In comparison to the administration of PAR alone,
the potential for biological effects of PAR, known to
have hepatotoxic effect, was determined to be reduced
when given to mice following the administration of
royal jelly at the indicated dose and particularly for a
period of 7 days. This can be explained by the values of
blood biochemical parameters and liver tissue oxidative
stress markers differing from those pertaining to the
group which received paracetamol alone and having
drawn closer to values of the control group. The
decrease in the severity of histopathological findings
further supports this conclusion.
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