Protective effect of grape seeds proanthocyanidins against
naphthalene-induced hepatotoxicity in rats.
Amany A. E. Ahmed*, Amal J. Fatani
Pharmacology Department, Faculty of Pharmacy, King Saud University, Riyadh,
11495, P.O. Box 22452, Saudi Arabia.
*Corresponding author Telephone: +9661 2913314, Fax: +9661 2064501
E-mail address: aresearch2007@yahoo.com
Abstract
Oxidative stress with subsequent production of reactive oxygen species has been
postulated as one of the mechanisms of naphthalene toxicity. In the present study, the
effect of oral administration of the natural antioxidant and free radical scavenger,
proanthocyanidins present in grape seeds (GSP, 10 or 50 mg kg-1, p.o.), has been
investigated in rats following the concomitant administration of naphthalene (1 g kg-1,
p.o., 15 days) and measurement of selective parameters indicative of liver function and
oxidative stress. Serum aminotransferases (ALT and AST), alkaline phosphatase (AP)
activities, and total bilirubin (T Bil) oncentration were measured, as well as hepatic tissue
lipid peroxidation (MDA), DNA fragmentation, and glutathione (GSH) contents. The
effects of GSP on naphthaline-evoked changes in the above-mentioned parameters were
compared with the known hepatoprotectant agent, silymarin.
Naphthalene hepatotoxicity was evident by the significant elevation of rat serum
activities of ALT, AST, AP, and T Bil concentration. This effect was accompanied with
significant increases in MDA and DNA fragmentation plus the depletion of GSH in
hepatic tissues.
Concurrent administration of GSP significantly attenuated the naphthalene-induced
disturbances in serum liver function enzymes, and markedly antagonized the lipid
peroxidation, DNA fragmentation and GSH depletion induced by naphthalene in hepatic
tissues.
In conclusion, GSP appears to be a potent candidate to ameliorate the oxidative
stress and hepatotoxicity associated with naphthalene in rats.
Key words:
Naphthalene hepatotoxicity, Grape seeds proanthocyanidins, Liver biomarker enzymes,
Oxidative stress, DNA fragmentation, Bromosulphalein dye clearance.
2
1. Introduction
The bioactivated xenobiotic naphthalene is a pervasive environmental contaminant.
It is one of the volatile aromatic hydrocarbons that are widely used commercially. Humans
are exposed to naphthalene from a number of different sources including workplace
exposures as in the aluminum smelting industry or where naphthalene is a starting material
in the production of phthalic anhydride. It is used in the synthesis of dyes, resins, plastics,
plus many pharmaceuticals including veterinary medicine, insect repellents, moth balls
and toilet bowel deodorants (1, 2). In addition, naphthalene is a natural constituent of coal
tar and crude oil and has been identified in cigarette smoke and emission from fossil fuel
combustion (3).
Naphthalene toxicity is highly species, tissue and cell selective (3). Naphthalene
exposure is associated with several toxic manifestations in humans and laboratory animals,
it has been found to cause cataract, hemolytic anemia, and damage of the bronchial
epithelial (Clara) cells and proximal tubules of the kidney (4, 5). Moreover, naphthalene
was involved in hepatocyte injury and liver dysfunction (4, 6, 7).
Toxic manifestations of naphthalene have been attributed to oxidative stress caused
by generation of reactive oxygen species (ROS) (4, 8, 9). These species may be generated
during phase I metabolism by cytochromes P-450 or by the redox cycling of its
metabolites 1,4 & 1,2 naphthoquinone as well as its hydroxylated products 1-naphthol, 2naphthol and 1,2-dihydroxynaphthalene (10).
Tingle et al. (6) reported that human liver is capable of metabolizing naphthalene
rapidly and efficiently into stable protein-reactive and cytotoxic metabolites, but if these
metabolites are not rapidly detoxified by microsomal enzymes it can bind to microsomal
proteins and damage cell membrane and tissue macromolecules including DNA, proteins
3
and lipids. Moreover, intracellular reduced glutathione was proven to provide a major
detoxification process for naphthalene metabolites (11).
A large number of synthetic and natural antioxidants have been demonstrated to
induce beneficial effects on human health and disease prevention. Grapes and grape
products are good sources of dietary flavonoids, which are powerful antioxidant
compounds. Furthermore, the inedible component of the fruit such as seeds and skin
contain some compounds that are able to scavenge superoxide radicals in living
organisms’ cells (12, 13). Moreover, grape seeds polyphenols (proanthocyanidins) were
found to be highly bioavailable providing significantly greater protection against free
radicals, free radical-induced lipid peroxidation and DNA damage, than that observed with
vitamin C, E and β-carotene, possibly due to its broad spectrum of health benefits (14).
Thus, many recent studies suggest that in-vivo grape seeds proanthocyanidins exposure
may protect multiple organs from a variety of toxic assaults (13, 15).
The commercially available grape seeds proanthocyanidins extract, used in the
present study (GSP, Nulife international, USA) is a standardized water-ethanol extract
from red grape seeds and contains monomeric, dimeric, trimeric and tetrameric
proanthocyanidins, plus other oligomeric proanthocyanidin bioflavonoids. Generally, the
dimer-, trimer-, and tetrameric proanthocyanidins, also referred to as extractable
proanthocyanidins or bioflavonoids, have been shown to be highly bioavailable and
provide excellent health benefits (16).
The liver is the main detoxifying organ in the body, and as such it possesses a high
metabolic rate and it is subjected to many insults potentially causative of oxidative stress.
Consequently, a correct status of the hepatic antioxidant defense system is of major
importance for the maintenance of health. On the other hand, liver is the major organ
involved in the metabolism of absorbed polyphenols (together with the intestinal mucosa
4
and kidneys). Therefore, the potential beneficial effects of food polyphenols would take
place primarily in the liver as well as in blood.
Thus the objective of the present investigation was to evaluate the in-vivo effect of
15-days oral administration of the environmental xenobiotic toxicant, naphthalene, on rat
liver, in an attempt to ascertain the involvement of lipid peroxidation, DNA fragmentation
and glutathione depletion in naphthalene-induced hepatotoxicity. Additionally, this study
examined whether the concomitant administration of the natural antioxidant, grape seeds
proanthocyanidins extract would protect rats from naphthaline-evoked disturbances in the
selected liver biomarkers.
2. Materials and methods
2.1. Animals
Adult male Wistar rats (150-175 g), were provided by the Experimental Animal
Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. The rats
had free access to standard rat chow and water, were housed in cages, 8-10 rats per cage,
and kept in a room maintained at 25 ± 2ºC with a 12 h light/dark cycle. All experiments on
animals were carried out according to the Guidelines of the Animal Care and Use
Committee, King Saud University, Riyadh, Saudi Arabia.
2.2. Drugs and chemicals
Grape seeds proanthocyanidins extract (GSP, 95% purity) was purchased from
Nulife international (USA). Bromosulphalein (BSP, 5% concentration ampoules) were
obtained from Fluka AG (Germany). Diphenylamine was purchased from Koch Light
Laboratories (England). All other chemicals and reagents used were of analytical grade
and were obtained from Sigma- Aldrich Chemical Co. (St. Louis, USA).
5
2.3. Experimental design
Experiments were performed on five groups (n=10 rats). The first group received
naphthalene dissolved in corn oil (1 g kg-1, p.o.) (4) daily for 15 days. The 2nd, 3rd, and 4th
groups were given a saline solution of either GSP (10 or 50 mg kg-1, p.o.) (16) or the
reference hepato-protectant silymarin (100 mg kg-1) (17), respectively, 2 h before
administration of naphthalene (1 g kg-1, p.o.) for 15 days. The fifth normal control group
was administered an equal volume of the vehicle (corn oil) orally for 15 days. At the end
of the experiments, two hours after administration of naphthalene, all animals were killed
by decapitation, and midstream blood samples (4 ml) were collected in heparinized tubes.
Afterwards, serum were quickly separated by centrifugation for 15 min at 3000 G, and
kept at -80 Cº until used for enzyme assays. Livers were also immediately removed,
washed with chilled saline and kept until assayed at -80 Cº.
2.4. Estimation of liver injury markers
2.4.1. Assay of serum enzymes and components
Serum alanine aminotransferase (ALT) and asparatic acid aminotransferase (AST)
activities were estimated according to the method of Ritman and Frankel (18), while
alkaline phosphatase (AP) activity was assayed depending on the method of Bowers and
Mc Comb (19). Commercial kits (Spinreact, Spain) were used and the activities of these
enzymes were expressed as international units (U L-1). Moreover, serum total bilirubin
concentration (T Bil), was assayed depending on the method of Martinek (20).
Commercial kits (Spinreact, Spain) were used and concentrations were expressed as mg
dL-1.
6
2.4.2. Estimation of liver lipid peroxidation (MDA) and glutathione (GSH) contents
Lipid peroxidation was assayed according to the method described by Ohkawa
et al. (21), where the levels of thiobarbituric acid reactive substances (TBARs), a
commonly utilized specific marker used to measure the degree of lipid peroxidation, were
measured in 20% of liver homogenates, using thiobarbituric acid reaction and the results
were expressed as malondialdehyde (MDA) (nmol g-1 wet weight).
Glutathione (GSH) content in the liver homogenates was determined according to
the method described by Ellman (22), depending on the reaction of Ellman’s reagent (5, 5dithiobis (2-nitrobenzoic acid), (DTNB), with the thiol group of GSH at pH 8.0 to give
yellow color of 5-thiol-2-nitrobenzoate anion. This was measured spectrophotometrically
at 412 nm and the results were expressed as µmol g-1 wet weight.
2.5. Quantitative DNA fragmentation assay
Quantitative estimation of DNA fragmentation was determined by colorimetric
diphenylamine assay as described by Burton (23). Liver samples from different groups
were homogenized in chilled lysis buffer (10 mmol TRIS-HCl, 20 mmol EDTA, 0.5%
Triton X-100, pH 8.0). Homogenates (1 ml) were then centrifuged at 27,000 g for 20 min
to separate intact DNA in the pellet from fragmented/damaged DNA in the supernatant
fractions. Perchloric acid (to reach a final concentration of 0.5 M) was added separately to
both the pellets and supernatant samples. Samples were heated at 90 ºC for 15 min then
centrifuged at 1500 g for 10 min to remove proteins. Resulting supernatants, whether
containing whole or fragmented DNA, were left to react with diphenylamine (0.088 M) for
16-20 h at room temperature, afterwards absorbance was measured at 600 nm. DNA
fragmentation was expressed as a percentage of total to fragmented DNA. Treatment
effects were reported as percentage of fragmentation observed in control group.
7
2.6. Measurement of hepatic Bromosulphalein (BSP) clearance
Rats were divided into five groups each of six animals. One group served as
control and was given only the vehicle (corn oil). Another group was administered
naphthalene dissolved in corn oil (1 g kg-1, p.o.) for 15 days. Three groups were given
saline solutions of either GSP (10 or 50 mg kg-1, p.o.) or silymarin (100 mg kg-1, p.o.) two
hours before administration of naphthalene (1 g kg-1, p.o.) for 15 days. After 18 h from the
last dose of naphthalene, bromosulphalein (BSP) dye (100 mg kg-1) was injected
intravenously to all animals. Midstream blood from decapitated animals (4 ml) were
collected in heparinized tubes after exactly 30 min from the dye injection. Afterwards,
blood samples were centrifuged for 15 min at 3000 G and the dye concentration in plasma
was estimated spectrophotometrically at 518 nm. Data were presented as both BSP
retention (percentage retained of originally injected amount, 100 mg), as well as excretory
capacity (%) calculated as (Rt – Rn/Rc – Rn) x 100, where R= BSP retention, while t, n, c,
represent treatment, naphthalene & control, respectively (24).
2.7. Statistical analysis
Statistical analysis was performed using one way analysis of variance (ANOVA)
followed by Tukey Kramer post hoc multiple comparisons test. The results were expressed
as mean ± SEM of 10 values in each group, and the level of significance was considered at
p <0.05.
3. Results
3.1. Effect of treatments on serum levels of liver enzyme markers
Administration of naphthalene for 15 days caused a significant (p<0.001) increase
in the activities of serum ALT (150%), AST (152%), AP (178%) and T Bil concentration
8
(214%) as compared to control values (Fig. 1, 2, 3 and 4), indicating that naphthalene
induced significant liver injury with cholestasis.
Oral administration of GSP in rats (10 or 50 mg kg-1, 2 hrs before naphthalene for
15 days), significantly protected the liver and attenuated the naphthalene-induced changes
in activities of the estimated liver enzymes ALT (76% and 73%), AST (67% and 71%),
AP (79% and 73%) and concentration of T Bil (67% and 59%), respectively, as compared
to the responses seen with naphthalene alone. In general, the effects of GSP (10 and 15 mg
kg-1) were comparable to that of the standard silymarin, appearing to offer more
pronounced protection against naphthalene-evoked changes in AST activity and T Bil
concentration (Fig. 1, 2, 3 and 4).
3.2. Effect of treatments on hepatic lipid peroxidation and glutathione contents
The present study showed that oral administration of naphthalene for 15 days
produced a significant (p<0.001) increase in hepatic lipid peroxides, as indicated by the
rise in the levels of the MDA (224%) as well as a significant (p<0.001) depletion of
hepatic GSH contents (300%) as compared with values in control group (Fig. 5 and 6).
Concurrent administration of GSP (10 or 50 mg kg-1) protected the liver and
significantly attenuated the extent of naphthalene-elicited hepatic lipid peroxidation (60%
and 53%, respectively) as compared to results seen in rats given naphthalene alone. Values
in GSP-treated groups were not significantly different than that of control. Once again the
protective effects of GSP were comparable to that of silymarin, with the higher dose
appearing to be more effective (Fig. 5).
Moreover, GSH was restored to approximately control tissue levels upon coadministration of GSP (10 or 50 mg kg-1) and naphthalene, whereas values reached
300% and 357%, respectively, of those seen in group administered naphthalene alone.
Silymarin administration also preserved the liver GSH, but to a lesser extent than GSP,
9
indicating that GSP maybe more potent than silymarin in replenishing liver GSH contents.
Surprisingly, in the group given the higher dose of GSP, levels of hepatic GSH were even
higher than that seen in the control group (Fig. 6).
3.3. Effect of treatments on hepatic DNA fragmentation
Naphthalene-induced DNA fragmentation in rat hepatic tissues is illustrated in Fig.
7. The comparative protective abilities of silymarin (100 mg kg-1) and GSP (10 or 50 mg
kg-1) are also presented. The results showed that 15 days oral administration of
naphthalene induced an increase in DNA fragmentation in the rat hepatic tissues reaching
approximately 140% of control values. Concomitant administration of GSP (10 or 50 mg
kg-1) significantly decreased naphthalene-induced hepatic DNA fragmentation where the
values reached 84% and 79%, respectively, of values seen in group given naphthalene
alone. The results in GSP-treated groups were not significantly different than that of
control. Additionally, their effects were comparable to that of silymarin, and indicated the
protective ability of GSP against naphthalene-induced DNA fragmentation in rat hepatic
tissues.
3.4. Effect of treatments on hepatic Bromosulphalein (BSP) clearance
As shown in table 1, BSP retention after 30 minutes from its injection into
naphthalene-treated rats was significantly elevated (p < 0.001) when compared to values
seen in normal rats. However, in animals treated with GSP (10 or 50 mg kg-1), the
naphthalene-evoked increase in BSP retention was significantly reduced to 71% (p<0.05)
and 56% (p<0.001),
respectively when
compared to
values in animals
given
naphthalene alone. This lead to a significant reduction in the liver excreting capacity for
BSP dye reaching 51.2% and 77.8%, respectively, as compared to naphthalene-
10
treated group. In general, the protective effects of GSP were comparable to that seen with
the hepato-protectant agent, silymarin, and the values in the group treated with the lower
dose of GSP were not significantly different than that of control.
4. Discussion
In the present investigation, rats intoxicated with naphthalene (1 g kg-1, p.o.) for 15
days, showed signs of liver injury with cholestasis as observed from the significant and
dramatic increase in activities of liver enzymes (ALT, AST and AP) and T Bil
concentration.
Elevation in serum AST and ALT is a reflection of radical-mediated lipid
peroxidation of liver cell membrane (25). It has been reported that AP is membrane bound
and its alteration is likely to affect the membrane permeability and produce derangement
in the transport of metabolites. Furthermore, AP is well documented to act as an indicator
of cholestatic changes and both AP activity and T Bil concentration are indices of biliary
damage (26). Several studies have reported changes in the activities of liver function
enzymes which may be accompanied with cholestasis and elevation in serum T Bil
concentrations during naphthalene or naphthalene derivatives intoxication. Similarly,
naphthalene was reported to cause hepatocytes injury and liver dysfunction in different
animal models (6, 27, 28).
Xenobiotics and hepatotoxic agents were proven to induce hepatotoxicity via
various mechanisms such as activation of cytochrome P450 (29), generation of ROS (8,
30), or through the effects of the active toxic metabolites on protective actions of GSH
(31). Naphthalene-induced hepatotoxicity in the present study was also accompanied by an
increase in hepatic lipid peroxidation (MDA) and a decrease in GSH contents, indicating a
11
clear association between oxidative stress plus lipid peroxidation with the development of
naphthalene-elicited hepatotoxicity.
Lipid peroxidation, an important indicator of oxidative damage of biological
tissues, was found to be induced in rats exposed to naphthalene (8). Similarly, oxidative
stress was induced by naphthalene in different animal models, whereas its administration
resulted in the elevation of the levels of serum and liver lipid peroxides in mice (28, 32),
and in rats (33). In addition, naphthalene has been shown to induce oxidative stress as
evidenced by hepatic and brain lipid peroxidation, glutathione depletion, DNA-single
strand breaks and excretion of urinary lipid metabolites in rats (34) as well as in cultured
macrophage J774A.1 cells (35).
In the present study, liver glutathione levels of rats intoxicated with naphthalene
were reduced leading to liver injury which was reflected by impairment in BSP dye
excretion rate. GSH is a well known protectant of liver cells against oxidative stress,
through non-enzymatic and enzymatic reactions (36). The depletion of liver GSH contents
is thought to result from inhibition of GSH efflux across the hepatocyte’s membrane (27).
Moreover, reduced glutathione has been reported to form either nucleophil-forming
conjugates with the active metabolites or act as a reductant for peroxides and free radicals
(37), which might explain its depletion. The resultant reduction in GSH level may thus
increase susceptibility of the tissue to oxidative damage including lipid peroxidation and
DNA fragmentation.
In accordance with the results of the present study, hepatic DNA fragmentation has
been reported in many models of hepatotoxicity following treatment with certain drugs or
chemicals (25, 38), indicating crucial associations between the generation of ROS,
oxidative damage to membrane lipids plus DNA molecules, lipid peroxidation and DNA
fragmentation (38, 39). Moreover, naphthalene, through its active metabolites, has been
12
reported to cause DNA damage and lipid peroxidation in cells (35), which is also
consistent with the concept of the involvement of ROS as a mechanism of its toxicity. It
has also been reported that GSH may provide a major detoxification process for
naphthalene metabolites (11), and its depletion may cause their accumulation.
Willems et al. (3) reported that, in rats, approximately all of the naphthalene that is
absorbed is metabolized. In addition, naphthalene was found to undergo bioactivation to a
reactive and cytotoxic protein metabolite, thought to be the intermediate naphthalene 1, 2epoxide. It has also been reported to be the principal toxic metabolite in rat hepatocytes
(11). Moreover, previous studies have shown that, 1-naphthol is metabolized to 1, 2naphthoquinone and 1, 4-naphthoquinone (10, 40). These compounds are cytotoxic, to
hepatocytes, especially in the absence of a functional metabolizing system and in the
presence of depleted intracellular glutathione (40, 41). In addition, Tingle et al. (6)
reported that various human P450 enzymes have been implicated in the bioactivation of
naphthalene, including CYP1A2, 3A4 and 2E1. Induction of human liver CYP2E1
resulted in preferential bioactivation of naphthalene to cytotoxic species which are
probably quinines derived from further metabolism of its stable metabolite, 1-naphthol.
Other suggested mechanisms of naphthalene–induced hepatotoxicity have been put
forth. Amin and Hamza (38) suggested a positive correlation between hepatotoxicity and
DNA fragmentation. They implicated that calcium may play a role in drug-induced
oxidative stress associated with hepatotoxicity. Similarly, Aktay et al. (32) concluded that
the reactive metabolites of naphthalene could result in depletion of glutathione through
binding covalently to tissue macromolecules leading to loss of thiol groups and
disturbances in calcium sequestration activity of the subcellular compartment.
The present investigation demonstrated that administration of grape seed
proanthocyanidins extract greatly attenuated the naphthalene-elicited changes in the levels
13
of liver biomarkers (ALT, AST, AP and T Bil) and significantly reduced lipid
peroxidation, DNA fragmentation and the depletion of glutathione contents in hepatic
tissues. These effects were comparable to those seen with the reference drug silymarin.
Silymarin, a hepato-protectant, was shown to guard the livers of experimental
animals against several hepatotoxic substances. It acts by preventing or inhibiting lipid
peroxide formation that can be induced by different hepato-toxicants. This effect may be
mediated through membrane-stabilization or free radical–quenching activity (17).
The beneficial effects of plants polyphenols or antioxidants against some
xenobiotic hydrocarbons induced- hepatotoxicity has been previously studied. Miyagawa
et al. (42) reported a hepato-protective effect of green tea extract and its phenolic
compounds against 1, 4-naphthoquinone-induced hepatotoxicity in rats. Moreover, the
antioxidant, vitamin E succinate, was reported to protect against naphthalene-evoked
changes in biomarkers of oxidative injury in rats (8). Similarly, garlic extract significantly
protected the liver of mice against naphthalene toxicity (28).
Many investigators have demonstrated the efficacy of GSP as an inhibitor of lipid
peroxidation and as a powerful free radical scavenger in vitro as well as in vivo (25, 35).
Similarly, GSP was found to reduce the oxidation of polyunsaturated fatty acids in mouse
liver microsomes (12) and provide protection against lipid peroxidation and DNA
fragmentation in mice (13, 35). Moreover, in rats exposed to either a vitamin E-deficient
diet or a high cholesterol diet, supplementation of polymeric grape seeds tannins to their
diets normalized the diet-depleted levels of liver glutathione and reduced the elevated
plasma lipid peroxides. In addition, a significant decrease in acetaminophen-induced
serum aminotransferases elevation, hepatic DNA damage, and mortality rate in rats
administered GSP has been reported (25, 43, 44).
14
The protective effects of GSP on GSH changes has been documented (15, 44) and
was confirmed in the present study, where GSP was capable of restoring naphthaleneevoked depletion of GSH levels. This restoration of hepatic GSH contents in the liver of
rats treated with GSP appeared to be effective in providing protection against naphthaleneaffected rat livers, since a significant reduction in lipid peroxidation and DNA
fragmentation were observed in the present study. This effect was also accompanied by a
significant improvement in activities of the liver function enzymes and was reflected by
the increased ability of the rat liver to excrete BSP dye. BSP clearance represents a simple
and sensitive test for the functional integrity of the liver (23, 45). The results of the present
study indicated that GSP, in the doses utilized, significantly improved the capacity of the
damaged liver cells to excrete BSP dye, since the plasma levels of the dye were lower in
the GSP-treated rats than that of rats treated with naphthalene alone.
Plants proanthocyanidins have been demonstrated to inhibit oxidative stress
through modulation of metabolic functions, enhancement of detoxification pathways,
and/or prevention of the interaction of xenobiotics with biological molecules (15). In
addition to the previously-mentioned mechanisms, proanthocyanidins appear capable of
attenuating naphthalene bioactivation in vivo. Tingle et al. (6) and Ray et al. (46) linked
the protective abilities of GSP against naphthalene and acetaminophen-induced
hepatotoxicity through antioxidant effects and through inhibition of CYP liver enzymes,
responsible for metabolism of naphthalene and acetaminophen in mice and rats.
Therefore, It can be suggested that, concomitant presence of GSP, as a powerful
antioxidant, during naphthalene metabolism facilitates the detoxification process, thus
minimizing naphthalene- dependent production of toxic radical species along with
drastically limiting the ongoing lipid peroxidation and DNA fragmentation (6). This effect
15
would result in a significant reduction of serum levels of hepatic enzyme markers and
improve BSP dye excretion capacity of hepatic cells as was seen in this study.
In conclusion, it is plausible to suggest that naphthalene metabolism triggered
production of ROS, coupled with impaired oxidant/ antioxidant balance, leading to a state
of oxidative stress that could have been partially responsible for the resultant DNA
fragmentation, hepatotoxicity, and the disturbance in the level of hepatic enzymes seen in
this study. Restoration of hepatic cells antioxidant capacity appeared to provide protection
against naphthalene-induced oxidative stress. The extract GSP exhibited a beneficial effect
as a natural hepatoprotectant and seemed to abate the oxidative insult in liver tissue,
restore the altered naphthalene-sensitive hepatic biochemical markers to an appreciable
extent, improve the hepatic GSH plus lipid peroxidation contents, and attenuate DNA
fragmentation of the liver cells.
Achnowledgements
This study was supported by a Research Grant provided by King Abdel Aziz City
for Science and Technology (KACST), Saudi Arabia.
16
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21
Table.1.Effect of concomitant oral administration of grape seeds proanthocyanidins (GSP,
10 or 50 mg kg-1) or silymarin (100 mg kg-1) with naphthalene (1 g kg-1)
for 15 days on hepatic bromosulphalein dye (BSP, 100 mg kg-1, i.v.)
clearance in adult male rats.
______________________________________________________________________
Treatments
Dose (mg kg-1)
BSP retention (mg%)
Excretory capacity (%)
_______________________________________________________________________
Control
(corn oil)
_
0.602 ± 0.018
_
Naphthalene
1000
1.399 ± 0.147##
_
100
0.889 ± 0.058
10
0.991 ± 0.068 #
50
0.779 ± 0.079
Naphthalene
+Silymarin
**
64
Naphthalene
+GSP
*
51
Naphthalene
+GSP
***
78
______________________________________________________________________
- Values represent the mean ± SEM of six rats in each group.
- BSP retention, percentage retained of originally injected amount
- Excretory capacity (%) = (Rt – Rn/Rc – Rn) x 100; R: BSP retention; t, n & c: treatment,
naphthalene & control, respectively.
- At #, significantly different than control (#, p<0.05; ##, p<0.001)
- At *, significantly different than naphthalene-treated group (*, p<0.05; **, p<0.01; ***,
p<0.001)
22
Legends for Figures
Fig. 1.
Effect of 15 days concomitant oral administration of grape seeds proanthocyanidins
(GSP, 10 or 50 mg kg-1) or silymarin (Sily, 100 mg kg-1) with naphthalene (Naph, 1 g kg-1)
on serum alanine aminotransferase (ALT) activity (U l-1) in adult male rats.
Data expressed as the mean ± SEM (n=10 rats). At #, values significantly different than
control (p<0.001). At *, values significantly different than naphthalene-treated group (*,
p<0.05; **, p<0.01).
Fig. 2.
Effect of 15 days concomitant oral administration of grape seeds proanthocyanidins
(GSP, 10 or 50 mg kg-1) or silymarin (Sily, 100 mg kg-1) with naphthalene (Naph, 1 g kg-1)
on serum asparatic acid aminotransferase (AST) activity (U l-1) in adult male rats.
Data expressed as the mean ± SEM (n=10 rats). At #, values significantly different than
control (#, p<0.001). At *, values significantly different than naphthalene-treated group (*,
p<0.05; **, p<0.001).
Fig. 3.
Effect of 15 days concomitant oral administration of grape seeds proanthocyanidins
(GSP, 10 or 50 mg kg-1) or silymarin (Sily, 100 mg kg-1) with naphthalene (Naph, 1 g kg-1)
on serum alkaline phosphatase (AP) activity (U l-1) in adult male rats.
Data expressed as the mean ± SEM (n=10 rats). At #, values significantly different than
control (#, p<0.001). At *, values significantly different than naphthalene-treated group (*,
p<0.05; **, p<0.01).
23
Fig. 4.
Effect of 15 days concomitant oral administration of grape seeds proanthocyanidins
(GSP, 10 or 50 mg kg-1) or silymarin (Sily, 100 mg kg-1) with naphthalene (Naph, 1 g kg-1)
on serum total bilirubin (T Bil) concentration (mg dl-1) in adult male rats.
Data expressed as the mean ± SEM (n=10 rats). At #, values significantly different than
control (#, p<0.01; ##, p<0.001). At *, values significantly different than naphthalenetreated group (*, p<0.05; **, p<0.01). At +, values significantly different than silymarintreated group (+, p<0.05).
Fig. 5.
Effect of 15 days concomitant oral administration of grape seeds proanthocyanidins
(GSP, 10 or 50 mg kg-1) or silymarin (Sily, 100 mg kg-1) with naphthalene (Naph, 1 g kg-1)
on hepatic lipid peroxidation, estimated as MDA contents (nmol g-1 wet weight), in adult
male rats.
Data expressed as the mean ± SEM (n=10 rats). At #, values significantly different than
control (#, p<0.001). At *, values significantly different than naphthalene-treated group (*,
p<0.01; **, p<0.001).
Fig. 6.
Effect of 15 days concomitant oral administration of grape seeds proanthocyanidins
(GSP, 10 or 50 mg kg-1) or silymarin (Sily, 100 mg kg-1) with naphthalene (Naph, 1 g kg-1)
on hepatic reduced glutathione contents ( μmol g-1 wet weight) in adult male rats.
Data expressed as the mean ± SEM (n=10 rats). At #, values significantly different than
control (#, p<0.001). At *, values significantly different than naphthalene-treated group (*,
p<0.05; **, p<0.01; ***, p<0.001).
24
Fig. 7.
Effect of 15 days concomitant oral administration of grape seeds proanthocyanidins
(GSP, 10 or 50 mg kg-1) or silymarin (Sily, 100 mg kg-1) with naphthalene (Naph, 1 g kg-1)
on hepatic DNA fragmentation (% of control) in adult male rats.
Data expressed as the mean ± SEM (n=10 rats). At #, values significantly different than
control (#, p<0.05; ##, p<0.01; ###, p<0.001). At *, values significantly different than
naphthalene-treated group (*, p<0.01).
25