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Experimental and Toxicologic Pathology 61 (2009) 123–132


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
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Erciyes, Kayseri, Turkey
Department of Pathology, Faculty of Veterinary Medicine, University of Erciyes, Kayseri, Turkey
Department of Animal Science, Vocational College of S. Çıkrıkçıoğlu, University of Erciyes, Kayseri, Turkey

Received 11 March 2008; accepted 18 June 2008

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

0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
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.
M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132 125

The mice included in the groups that were given microtome stained using hematoxylin and eosin and
paracetamol and royal jelly (Groups 2–6) were weighed examined under a light microscope.
daily, and accordingly the dose to be administered per
body weight was calculated and administration by Statistical analysis
gavage was considered to be more accurate and safety.
Data were presented as arithmetic mean7S.D. The
one-way analysis of variance was used for statistical
Collection and processing of samples
analyses, and the differences between the groups were
determined using the Tukey test.
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 Results
collected, were euthanized by cervical dislocation, and
their liver tissue was excised. Blood samples were Clinical findings
centrifuged at 3000 rpm for 10 min for the separation
of sera. The serum samples obtained were transferred No clinical disorder was observed in the mice included
into eppendorf tubes and were preserved in a deep in Groups 1–3. Eight of the 20 mice included in Group 4
freezer at 80 1C. Part of the liver tissue was transferred were determined to die within 24 h. The mice which
into 10% buffered formalin for histopathological survived the first 24 h displayed weakness, inactiveness
examination, and the remainder tissue was used for and unresponsiveness to external stimuli. Seven of the 20
the analyses of oxidative stress parameters. Liver tissue mice in Group 5 were determined to die within 24 h. The
samples were homogenized after being mixed with 1:9 remainder mice were weak, inactive and unresponsive to
phosphate buffer (pH 7.2), in an ice-containing medium. external stimuli. In Group 6, 2 of the 20 mice were
The homogenates were centrifuged in a cooling cen- determined to die within 24 h. The survivor mice were
trifuge with a temperature adjusted to +4 1C, at observed to be active and to respond to external stimuli.
20,000 rpm for 1 h. The supernatants obtained were
transferred into eppendorf tubes, and preserved at Histopathological findings
80 1C in a deep freezer until used for analysis.  Group 1: No significant lesion was determined in the
liver cross-sections of the mice included in the control
Biochemical analysis group (Fig. 1A).
 Groups 2 and 3: Lymphoid cell infiltration in liver
Serum ALT, AST, ALP, bilirubin, total protein, parenchyma was observed in the groups which were
albumin, cholesterol and triglyceride levels were mea- given RJ (Fig. 1B and C).
sured using Johnson & Johnson label kits and a Vitros  Group 4: Blood vessels were determined to be severely
750 model autoanalyser. Protein levels in liver homo- hyperaemic and large haemorrhagic areas were
genates were measured as described by Lowry et al. present in the parenchyma (Fig. 1D). The Remark
(1951). MDA analyses were performed in accordance cords were dissociated. Alterations ranging from
with the method described by Yoshioka et al. (1979). degenerative to necrotic were ascertained in hepato-
GSH-Px activity was measured as described by Paglia cytes, particularly in the periphery of Vv. centrales
and Valentine (1967). CAT activity was determined in (Fig. 2A). Pink-coloured roll-shaped erythrocyte
accordance with the method described by Luck (1965). accumulations were present in the large haemorrha-
SOD activity was determined as described by Sun et al. gic areas and some of the sinusoids (Fig. 2D). The
(1988). number of Kupffer cells had increased. The central
necrotic regions were more evident in some of the
cross-sections (Fig. 2B). Paranchymal and vacuolar
Preparation of tissues for histopathological degeneration were marked in hepatocytes. Areas of
examination necrosis were marked particularly in the periacinar
and partially in the portal regions. Pink-coloured
Tissue samples were taken from the liver of the homogeneous hyalinized areas were determined to be
necropsied animals and fixed in 10% formalin neutral present in the proximity of haemorrhagic areas.
buffer solution. The trimmed tissues were first washed Slight increase in fibrous tissue and limited lymphoid
with tap water followed by dehydration through a cell infiltrations were observed in the portal region
graded alcohol series and then passed though xylol and (Fig. 2C).
paraffin series before finally blocked in paraffin. The  Group 5: Cross-sections pertaining to Group 5 were
paraffin blocks were cut into 5–6 mm sections using a similar to those of the group which was administered
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  ).
M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132 127

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

Biochemical findings
No statistically significant difference was determined
in serum ALT, AST and ALP levels (Table 2); serum PAR is one of the commonest drugs used for the
bilirubin, total protein, albumin, cholesterol and trigly- treatment of minor to moderate pain in humans.
ceride levels (Table 3), liver MDA level and SOD, CAT, However, the intake of a single dose of 10 g or higher
GSH-Px activities (Table 4) in Groups 1–3 (p40.05). 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  ).
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).
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).
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).
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 the liver tissue, and thereby leads to the severe damage
300 mg/kg and higher, PAR leads to severe acute liver of cell membranes (Kupeli et al., 2006). The major
necrosis in mice (Douidar et al., 1985; Corcoran et al., finding observed in PAR intoxication in humans and
1985). In case of the intake of high doses, PAR is first animals is acute centrolobular necrosis (Macnaughton,
converted into non-reactive metabolites in the liver by 2003; Hewawasam et al., 2003). In the present study, the
sulphation and glucuranidation reactions. However, haemorrhagic and necrotic findings observed in the
these metabolites are later converted into a reactive histopathological examination of the liver of mice which
metabolite known to be toxic for the liver, namely were administered PAR demonstrate PAR-induced
N-acetyl-p-benzo-quinoneimine, by the liver cytochrom damage in the liver (Figs. 1D and 2). Sharp increase in
P-450 enzyme system. The resulting metabolite cova- the serum ALT level is considered to be a significant
lently binds to oxidized lipids and sulphydryl groups in indicator of PAR-induced acute liver damage (Black,
M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132 129

1980; Sener et al., 2005). ALT is an enzyme specific to the control group (Group 1), the decrease observed in
liver damage, and is used routinely for the determination liver GSH-Px enzyme activity in the group which
of liver damage (Turgut, 2000). In the present study, received PAR (Group 4) can be explained with the
when compared to the control group, the sharp increase consumption of the enzyme during the detoxification of
detected in the serum ALT levels of mice which were reactive oxygen metabolites generated due to PAR, as
given PAR (Table 2), demonstrates PAR-induced acute well as the consumption of liver GSH stores. No
liver damage to have developed. Serum AST and ALP significant change being determined in the activity of
levels increase not only in liver damage but also in case other antioxidant enzymes, namely SOD and CAT,
of the damage of various other tissues and organs. For further supports GSH-Px to be primarily involved in the
this reason, increased ALT levels are considered to be cellular defense mechanism against PAR.
specific to liver damage (Meyer and Harvey, 1994; RJ is quite a safe product, and is reported not to cause
Hajimehdipoor et al., 2006). In experimental acute PAR any adverse effect when administered to rats and mice at
intoxications, in addition to ALT levels, serum AST and a dose of 3 g/kg in the subacute period (Hashimoto
ALP levels also increase (Sener et al., 2005; Kupeli et al., et al., 1977). In the present study, the administration of
2006). Similarly, in this study, serum AST and ALP RJ as a single dose or for a period of 7 days was
levels were determined to have increased, when com- determined not to cause any statistically significant
pared to the control group (Table 2). Taking into change in liver oxidative stress or serum biochemical
consideration that the liver is the primary target organ parameters. However, in some cross-sections pertaining
in acute PAR intoxications, it can be concluded that to the groups which were given RJ, lymphoid cell
increased serum AST and ALP levels result from liver infiltrations were observed in the liver parenchyma
damage. Due to the changes determined in the other (Fig. 1B and C), and this situation can be considered as
biochemical parameters evaluated (bilirubin, total pro- the reaction of the liver to RJ as a substance foreign to
tein, albumin, cholesterol, triglyceride) being insignif- the body. This is not considered to be an adverse effect
icant when compared to the control group, the possible on the liver. For, it is previously reported that the liver
effects of PAR could not be evaluated based on the has been occurred to react to foreign substances which
indicated parameters. are not synthesized in the body (Percy and Barthold,
N-Acetyl-p-benzo-quinoneimine, a reactive metabo- 2001; Salmi et al., 1998).
lite generated as a result of the metabolism of PAR by Due to the consumption of GSH stores, PAR
liver cytochrom P-450 enzymes, is known to be intoxications are treated with GSH precursors such as
responsible for liver damage in acute PAR intoxications n-acetyl cysteine (Boobis et al., 1989). Bee products are
(Dahlin et al., 1984; Potter and Hinson, 1987). The reported to have beneficial effects in PAR intoxications:
decrease of the oxidation of PAR and the stimulation of Juzwiak (1993) has determined pollen extracts to
GSH synthesis, have been determined to decrease the increase the survival rate and to improve both histo-
toxicity of PAR in mice (Tee et al., 1987). Tirmenstein pathological lesions in the liver and liver enzymes in
and Nelson (1990) have reported GSH-Px and thiol- mice with PAR-induced acute intoxication. Seo et al.
transferase enzyme activities to decrease in case of PAR- (2003) have demonstrated both the mortality rate and
induced liver damage in animals. Mirochnitchenko et al. severity of hepatic necrosis to decrease in mice which
(1999) have demonstrated decrease in liver GSH-Px were administered 400 mg/kg PAR following the admin-
activity upon the administration of a high dose of PAR. istration of propolis at doses of 10 and 25 mg/kg for
In the present study, compared to the controls, liver 7 days, and hence have indicated propolis to may have
GSH-Px enzyme activity was determined to have hepatoprotective effects. Research has been conducted
decreased and MDA levels were ascertained to have on the hepatoprotective effects of RJ. Uzbekova et al.
increased in mice which were administered PAR (2006), following the administration of thyroxine
(Table 4). These results suggest that PAR may exhibit which is known to cause liver damage, and additionally
effects that induce lipid peroxidation in the liver. 10 mg/kg RJ for 10 days, have demonstrated RJ to
GSH-Px is an enzyme which prevents the generation exhibit protective effect against thyroxine-induced liver
of hydrogen peroxide and alkyl hydroperoxides in damage. Following the administration of 200 mg/kg bw
association with GSH and GSH-reductase, as well as fumonisin and 100 or 150 mg/kg bw RJ to rats for a
the generation of more harmful metabolites such as the period of 3 weeks, El-Nekeety et al. (2007) have reported
hydroxyl radical (Therond et al., 2000; Parodi, 2007). RJ to improve fumonisin-induced histopathological
Liver GSH stores are reported to consume in PAR alterations in the liver, and hence have indicated RJ to
intoxications (Boobis et al., 1989). The consumption of may have hepatoprotective effects against fumonisin
GSH stores in the body may directly cause oxidative intoxication. Merino et al. (1996) have clarified that
stress by means of GSH, whereas decreased antioxidant Cuban red propolis exctract exhibits hepatoprotective
enzyme acitivity may indirectly lead to a similar effect effects on carbon tetrachloride-induced liver damage in
(Roder, 2001). In the present study, in comparison to rats, probably due to antioxidant effects. No previous
130 M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132

study exists on the effects of RJ on PAR-induced leucine, valine and isoleucine, and mainly cystine and
liver damage. In the present study, particularly in the cysteine which are GSH precursors.
group which was given 200 mg/kg bw RJ for 7 days In comparison to the administration of PAR alone,
and a single dose of 400 mg/kg bw PAR on the 7th day the potential for biological effects of PAR, known to
of the trial (Group 6), when compared to the group have hepatotoxic effect, was determined to be reduced
which received a single dose of PAR (Group 4), the when given to mice following the administration of
low level of mortality determined to occur within royal jelly at the indicated dose and particularly for a
24 h, decrease in serum ALT, AST, ALP levels period of 7 days. This can be explained by the values of
(Table 2) and liver MDA level (Table 4), the increase blood biochemical parameters and liver tissue oxidative
in serum total protein and albumin levels (Table 3) and stress markers differing from those pertaining to the
liver GSH-Px activity (Table 4), and the alteration of group which received paracetamol alone and having
haemorrhagic and necrotic histopathological findings of drawn closer to values of the control group. The
the liver from diffuse to focal form (Fig. 3C and D) decrease in the severity of histopathological findings
demonstrate RJ to have hepatoprotective and antiox- further supports this conclusion.
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. Bessems JG, Vermeulen NP. Paracetamol (acetaminophen)-
(1983) have reported aspartic acid, cystine, methionine induced toxicity: molecular and biochemical mechanisms,
and tyrosine to exhibit protective effect against carbon analogues and protective approaches. Crit Rev Toxicol
tetrachloride-induced acute liver damage. Tanaka et al. 2001;31:55–138.
(1991) have determined cysteine to exhibit hepatopro- Black W. Acetaminophen hepatotoxicity. Gastroenterology
tective effect against PAR-induced liver damage when 1980;78:382–92.
administered at a dose of 300 mg/kg bw to mice, and to Boobis AR, Fawthrop DJ, Davis DS. Mechanisms of cell
decrease the mortality rate in mice. Fodor et al. (1976) deaths. Trends Pharmacol Sci 1989;10:275–80.
have reported aspartic acid to provide protection Corcoran GB, Racz WJ, Smith CV, Michell JR. Effects of
N-acetylcysteine on acetaminophen covalent binding and
against D-galactosamine-induced liver damage in rats.
hepatic necrosis in mice. J Pharmacol Exp Ther 1985;232:
Glycine has also been indicated to may have protective
and curative effects on alcoholic hepatitis and alcohol- Dahlin DC, Miwa GT, Lu AYH, Nelson SD. N-acetyl-
induced hepatocellular carcinoma (Yamashina et al., p-benzoquinoneimine: a cytochrome P-450 mediated oxida-
2005). Jamall et al. (1979) have reported L-lysine to may tion product of acetaminophen. Proc Natl Acad Sci 1984;
have protective effect against acute ethanol toxicity in 81:1327–31.
mice. Pisarenko (1996) has demonstrated increased De Toranzo EG, De Ferreyra EC, De Fenos OM, Castro JA.
intracellular leucine, valine and isoleucine concentra- Prevention of carbon tetrachloride-induced liver necrosis
tions in myocardial cells to stimulate the synthesis of by several amino acids. Brit J Exp Pathol 1983;64:166–71.
acetyl coenzyme A and succinyl coenzyme A, and to Douidar SM, Boor PJ, Ahmed AE. Potentiation of the
deteriorate the oxidative metabolism, and methionine hepatotoxic effect of acetaminophen by prior administra-
tion of salicylate. J Pharmacol Exp Ther 1985;233:242–8.
and cysteine to may reduce myocardial damage caused
El-Nekeety AA, El-Kholy W, Abbas NF, Ebaid A, Amra HA,
by oxygen radicals. Glycine has been proven to be an
Abdel-Wahhab MA. Efficacy of royal jelly against the
antioxidant amino acid which protects cells against oxidative stress of fumonisin in rats. Toxicon 2007;50:
various toxic substances, primarily metals (Paniagua- 256–69.
Castro et al., 2006). Lysine, which is a nucleophilic Fodor O, Schvartz M, Suciu A, Barbarino F, Duca S, Lezeu
amino acid, is reported to facilitate the exchange of R. Protecting action of aspartate on the hepatic changes
electrophiles derived from lipid peroxidation (Jamall induced by D-galactosamine. Arzneimittelforschung 1976;
et al., 1979). Cystine and cysteine take place in the 26:855–8.
synthesis of GSH, an effective cellular antioxidant. GSH Hajimehdipoor H, Sadeghi Z, Elmi S, Elmi A, Ghazi-Khansari
breaks down reactive oxygen species and detoxifies M, Amanzadeh Y, et al. Protective effects of Swertia
carcinogens, both directly and by antioxidant enzymes longifolia Boiss. and its active compound, swerchirin, on
paracetamol-induced hepatotoxicity in mice. J Pharm
with which it reacts (Parodi, 2007). In this context, GSH
Pharmacol 2006;58:277–80.
precursors such as acetyl cysteine are used for the
Hashimoto T, Takeuchi K, Hara M, Akatsuka K. Pharmaco-
treatment of PAR intoxications (Boobis et al., 1989). logical study on royal jelly (RJ). 1. Acute and subacute
The indicated researches show the hepatoprotective and toxicity tests on RJ in mice and rats. Bull Meiji Pharm
antioxidant effects of RJ in PAR intoxications to may 1977;7:1–13.
be related to the biological effects of its free amino acid Hewawasam RP, Jayatilaka KA, Pathirana C, Mudduwa LK.
content, including glycine, aspartic acid, tyrosine, lysine, Protective effect of Asteracantha longifolia extract in mouse
M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132 131

liver injury induced by carbon tetrachloride and paraceta- free amino acids in various foods. J Chromatogr A 2006;
mol. J Pharm Pharmacol 2003;55:1413–8. 1135:179–85.
Inoue S, Koya-Miyata S, Ushio S, Iwaki K, Ikeda M, Ozdemirler G, Aykac G, Uysal M, Oz H. Liver lipid
Kurimoto M. Royal jelly prolongs the life span C3H/HeJ peroxidation and glutathione-related defence enzyme sys-
mice: correlation with reduced DNA damage. Exp Ger- tems in mice treated with paracetamol. J Appl Toxicol
ontol 2003;38:965–9. 1994;14:297–9.
Jamall IS, Mignano JE, Lynch VD, Bidanset JH, Lau-Cam C, Paglia PE, Valentine WN. Studies on the quantitative and
Greening M. Protective effects of zinc sulfate and L-lysine qualitative characterization of erythrocyte glutathione
on acute ethanol toxicity in mice. Environ Res 1979;191: peroxidase. J Lab Clin Med 1967;70:158–69.
112–20. Paniagua-Castro N, Escalona-Cardoso G, Chamorro-Cevallos
Juzwiak S. Experimental evaluation of the effect of pollen G. Glycine reduces cadmium-induced teratogenic damage
extract on the course of paracetamol poisoning. Ann Acad in mice. Reprod Toxicol 2006;23:92–7.
Med Stetin 1993;39:57–69. Parodi PW. A role for milk proteins and their peptides in
Kohno K, Okamoto I, Sano O, Arai N, Iwaki K, Ikeda M, cancer prevention. Curr Pharm Des 2007;13:813–28.
et al. Royal jelly inhibits the production of proinflamma- Percy DH, Barthold SW. Pathology of laboratory rodents/
tory cytokines by activated macrophages. Biosci Biotechnol rabbit, 2nd ed. Iowa State Press; 2001. p. 90.
Biochem 2004;68:138–45. Pisarenko OI. Mechanisms of myocardial protection by amino
Kupeli E, Orhan DD, Yesilada E. Effect of Cistus laurifolius acids: facts and hypotheses. Clin Exp Pharmacol Physiol
L. leaf extracts and flavonoids on acetaminophen-induced 1996;23:627–33.
hepatotoxicity in mice. J Ethnopharmacol 2006;103:455–60. Potter DW, Hinson JA. Mechanisms of acetaminophen
Lercker GP, Capella L, Conte S, Ruini F, Giordani G. oxidation to N-acetyl p-benzoquinoneimine by horseradish
Components of royal jelly. I. Identification of the organic peroxidase and cytochrome P-450. J Biol Chem 1987;262:
acids. Lipids 1981;16:912–9. 966–73.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein Roder JD. Acetaminophen. Veterinary Toxicology. USA:
measurement with the Folin phenol reagent. J Biol Chem Butterworth Heinemann; 2001. p. 65–9.
1951;193:265–75. Salmi M, Adams D, Jalkanen S. IV. Lymphocyte trafficking in
Luck H. Catalase. In: Bergmeyer H, editor. Methods of the intestine and liver. Am J Physiol Gastrointest Liver
enzymatic analysis. New York: Academic Press; 1965. Physiol 1998;274:1–6.
p. 855–94. Savides MC, Oehme FW. Acetaminophen and its toxicity.
MacNaughton SM. Acetaminophen toxicosis in a Dalmatian. J Appl Toxicol 1983;3:96–111.
Can Vet J 2003;44:142–4. Sener G, Sehirli O, Cetinel S, Yegen BG, Gedik N, Ayanoglu-
Melli M, Kayaalp O. Parasetamol. In: Kayaalp O, editor. Dulger G. Protective effects of MESNA (2-mercaptoethane
Rasyonel Tedavi Yönünden Tibbi Farmakoloji. Hacettepe- sulphonate) against acetaminophen-induced hepatorenal
Tas-; 2006. p. 849–51. oxidative damage in mice. J Appl Toxicol 2005;25:20–9.
Meotti FC, Rosa JM, Brocardo PS, Balz D, Waltrick AP, Seo KW, Park M, Song YJ, Kim SJ, Yoon KR. The protective
Bagio A, et al. Protective effect of crude extract from effects of propolis on hepatic injury and its mechanism.
Wedelia paludosa (Asteraceae) on the hepatotoxicity Phytother Res 2003;17:250–3.
induced by paracetamol in mice. J Pharm Pharmacol Sun Y, Oberley LW, Li Y. Simple method for clinical assay of
2006;58:137–42. superoxide dismutase. Clin Chem 1988;34:497–500.
Merino N, González R, González A, Remirez D. Histopatho- Tanaka M, Nakata K, Takase K, Mita S. The protective effect
logical evaluation on the effect of red propolis on liver of (4R) hexahydro-7,7-dimethyl-6-oxo-1,2,5-dithiazocine-
damage induced by CCl4 in rats. Arch Med Res 1996;27: 4-carboxylic acid (SA3443), a novel cyclic disulfide, on
285–9. acetaminophen-induced liver injury. Nippon Yakurigaku
Meyer DJ, Harvey JW. Hematologic changes associated with Zasshi 1991;97:191–8.
serum and hepatic iron alterations in dogs with congenital Tee LBG, Davies DS, Seddon CE, Boobis AR. Species
portosystemic vascular anomalies. J Vet Intern Med 1994;8: differences in the hepatotoxicity of paracetamol are due
55–6. to differences in the rate of conversion to its cytotoxic
Mirochnitchenko O, Weisbrot-Lefkowitz M, Reuhl K, Chen metabolite. Biochem Pharmacol 1987;36:1041–52.
L, Yang C, Inouye M. Acetaminophen toxicity. Opposite Therond P, Bonnefont-Rousselot D, Davit-Spraul A, Conti
effects of two forms of glutathione peroxidase. J Biol Chem M, Legrand A. Biomarkers of oxidative stress: an analytical
1999;274:10349–55. approach. Curr Opin Clin Nutr Metab Care 2000;3:373–84.
Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB. Tirmenstein MA, Nelson SD. Acetaminophen-induced oxida-
Acetaminophen-induced hepatic necrosis. IV. Protective tion of protein thiols: contributions of impaired thiol-
role of glutathione. J Pharm Exp Ther 1973;187:211–7. metabolising enzymes and the breakdown of adenosine
Nagai T, Inoue R. Preparation and the functional properties nucleotides. J Biol Chem 1990;265:3059–65.
of water exctract and alkaline extract of royal jelly. Food Towsend GF, Morgan JF, Hazlett B. Activity of 10-hydro-
Chem 2004;84:181–6. xydecenoic acid from royal jelly against experimental
Ozcan S, Senyuva HZ. Improved and simplified liquid leuaemia and ascitic tumours. Nature 1959;183:1270–1.
chromatography/atmospheric pressure chemical ionization Turgut K. Klinik enzimoloji. Veteriner klinik laboratuar tes-his
mass spectrometry method for the analysis of underivatized kitabı. Konya; 2000. p. 179–201.
132 M. Kanbur et al. / Experimental and Toxicologic Pathology 61 (2009) 123–132

Uzbekova D, Chugunova L, Makarova V, Ryabkov A, Yoshioka T, Kawada K, Shimada T, Mori M. Lipid

Mirgorodskaya L. Efficacy of royal jelly and lactulose on peroxidation in maternal and blood and protective
thyroxin-induced liver damage in rats. Hepatology 2006;28:157. mechanism against activated-oxygen toxicity in the blood.
Yamashina S, Ikejima K, Enomoto N, Takei Y, Yato N. Am J Obstet Gynecol 1979;135:372–6.
Glycine as a therapeutic immuno-nutrient for alcoholic Zimmermann A. Liver regeneration: the emergence of new
liver disease. Alcohol Clin Exp Res 2005;29:162–5. pathways. Med Sci Monit 2002;8:53–63.