Olive oil polyphenols modify liver polar fatty acid composition and
inhibit CCl4-induced hepatotoxicity in Balb/c mice
Jasminka Giacomettia, Hrvoje Križana, Neven Franjićc and Alena Buretić-Tomljanovićb
University of Rijeka, School of Medicine, aDept. of Chemistry and Biochemistry, bDept. of Biology and Medical Genetics, cStudent of
the School of Medicine, Braće Branchetta 20, HR-51000 Rijeka, Croatia
E-mail: [email protected]
INTRODUCTION
Hepatic injury through carbon tetrachloride (CCl4) induced lipid peroxidation is well known and has been extensively used in the experimental models to understand the
cellular mechanisms behind oxidative damage and further, to evaluate the therapeutic potential of drugs and dietary antioxidants. Many of these methods can be used
often to study the potential preventive effect of dietary antioxidants when considering the measurements in vivo.
The object of this study was to observe the effect of olive oil polyphenols on acute liver injury by determining the changes in the phospholipid (PL FAs) and free fatty
acids (FFAs) composition, as well as the total antioxidant capacity (TEAC) of serum using the carbon tetrachloride (CCl4) induced liver injury on animal model.
EXPERIMENTAL
Male Balb/c mice were divided into 4 groups as follows: Group 1 - control; Group 2 - CCl4 treated; Group 3 - CCl4 and olive oil phenolics extract simultaneously treated;
Group 4 – 3-day olive oil phenolics extract pre-treated and then CCl4 treated. Treated animals were sacrificed 24 hrs after mentioned treatments; the serum and liver
were collected.
The olive oil phenolics extract was previously characterized by qualitative and quantitative evaluations of phenolics analysed with high performance liquid
chromatography (HPLC) using gallic acid as an internal standard.
Serum PL FAs and FFAs fractions were isolated and purified by the solid-phase-extraction (SPE) method using an aminopropylsilica column and subsequently determined
by gas chromatography (GC). Total antioxidant status in the serum was measured as Trolox Equivalent Antioxidant Capacity (TEAC).
Immediately after removal, the liver tissue was fixed in 10% formaldehyde and processed for histological examination according to the conventional method and
staining with hematoxylin and eosin (H&E).
Calculations and Statistical Analysis
DYNAMICS OF EXPERIMENTS
Group 1
Group 2
Group 3
SFAs=Σ% (14:0+16:0+18:0+20:0+22:0+24:0); MUFAs=Σ% (14:1+16:1+18:1+20:1); PUFAs=Σ% (PUFAn-3 + PUFAn-6);
PUFAs n–3 =Σ% (20:5n-3+22:5n-3+22:6n-3);
PUFAs n–6 =Σ% (18:2n-6+18:3n-6+20:2n-6+20:3n-6+20:4n-6+22:4n-6); D9 C18 index =18:1n-9/18:03; D6D
index=[(18:3n-6+20:3n-6)/18:2n-6]1; D5D index=[(20:4n-6/(20:3n-6)]1.
ACL=[(Σ% Total FAn x n)]/100 (n=carbon atom number)2; DBI=Σ % of unsaturated FAs x number of duble bonds of each
unsaturated FAs2;
PI=[(%Monoenoic x 0.025) + (%Dienoic x 1) + (%Trienoic x 2) + (%Tetraenoic x 4) + (%Pentaenoic x 6) + (%Hexaenoic x
8)] 2;
Data are reported as mean ±S.D. Statistical significance was assumed with p<0.05. All statistical analyses were conducted
using the Statistica software package for Windows, StatSoft, Inc. (2005), Version 7.1. The significance of the differences was
analyzed by the Descriptive Statistics by Groups (Breakdown) - Post-hoc Comparisons of Means using Scheffe test.
Ex, 24 hrs
Group 4
Control
group
CCl4 treated
group
CCl4 and
OOP pretreated
group
Control
group
CCl4 treated
group
CCl4 and
OOP pretreated
group
14.87±1.47
17.29±1.11*
16.89±1.08
15.92±2.67
1.48±0.51
0.45±0.28
0.80±0.53
5.04±3.54*
0.00
0.22±0.15*
0.15±0.15*
0.00
0.21±0.08
0.43±0.21
0.51±0.35
0.71±0.27*
20:2n-6
0.14±0.10
0.11±0.07
0.13±0.06
0.14±0.07
0.29±0.11
1.09±0.90
0.99±0.80
0.54±0.19
20:3n-6
1.29±0.31
0.52±0.15*
0.60±0.14*
0.47±0.06*
0.86±0.86
1.10±0.90
0.99±0.80*
2.33±1.36*
20:4n-6
12.31±1.14
7.62±0.63*
8.32±0.82*
6.27±0.56*
6.46±2.65
4.50±2.57*
6.57±5.13*
9.43±3.02*
Σ n-6
28.61 ± 2.66
25.89 ± 1.11
26.11 ± 0.86
18.11 ± 2.09*
22.81 ± 3.09*
9.27 ± 2.86
6.46 ± 2.99
8.87 ± 4.98
20:5n-3
0.69±0.20
0.72±0.30
0.67±0.22
0.29±0.12*
0.05±0.05
0.00
0.11±0.35
0.10±0.12
22:6n-3
17.02±1.38
16.07±1.90
17.42±1.53
11.62±1.08*
0.43±0.25
0.63±0.27
0.37±0.33
2.77±2.42*
Σ n-3
18.14 ± 1.49
17.01 ± 1.91
18.58 ± 1.83
12.21 ± 1.08*
0.49 ± 0.32
0.63 ± 0.27
0.48 ± 0.38
2.90 ± 2.55
Σ PUFA
46.76 ± 3.24
42.99 ± 2.67*
44.69 ± 1.48
35.01 ± 3.50*
9.76 ± 2.96
7.09 ± 3.03
9.35 ± 5.03
21.01 ± 2.04
0.11±0.02
0.11±0.02
0.29±0.14*
a.
50
b.
45
40
25
0.08±0.02
7.29±2.42
2.54±0.37*
4.61±2.96*
2.08±0.87*
28.40±3.16
27.72±1.29
27.06±1.07
29.20±1.75
16.88±4.56
33.79±7.01*
36.10±10.82*
32.63±7.14*
20
18:0
13.05±1.06
17.89±1.14*
17.95±0.72*
19.56±3.07*
17.35±17.25
24.21±4.00
22.31±12.62
22.40±2.72
15
20:0
0.36±0.28
0.05±0.05
0.22±0.22
0.40±0.24
0.00
0.00
0.00
0.00
10
24:0
0.00
0.00
0.00
0.00
0.62±0.57
0.63±0.28
0.50±0.36
5.57±3.15*
Σ SFA
41.89 ± 2.60
45.77 ± 2.26*
45.34 ± 0.86*
49.45 ± 2.29*
42.15 ± 16.05
61.36 ± 5.50*
63.81 ± 13.15*
62.69 ± 6.46*
16:1n-7
1.91±0.43
1.83±0.48
1.61±0.26
2.04±1.10
1.92±1.20
0.69±0.57
1.02±0.69
1.03±1.41
18:1n-9
9.34±0.65
9.38±0.96
8.28±0.78
13.49±2.64*
36.22±18.57
24.65±7.19
19.90±10.41*
12.93±6.30*
20:1n-9
0.00
0
0.03±0.03
0.00
0.14±0.13
0.00*
0.03±0.03*
0.00*
Σ MUFA
11.35 ± 0.92
11.24 ± 1.37
9.97 ± 1.14
15.53 ± 3.44*
48.09 ± 15.93
31.55 ± 7.21*
26.84 ± 11.97*
16.30 ± 6.72*
32.23±4.99
28.69±5.00
30.06±8.30
32.52±11.34
Table 2. Differences in liver PL characteristics based on FA profiles
determined in control and test groups
PL
Control
group
CCl4 and OOP
treated
group
0.99±0.05*
CCl4 and OOP
pre-treated
group
0.71±0.09*
3.89±0.64
4.54±0.60
2.39±0.68*
0.44±0.06*
0.49±0.07*
0.40±0.07*
2.11±0.13*
2.10±0.24*
1.86±0.23*
0.64±0.07
0.66±0.08
0.71±0.09
0.54±0.09
D9 C18
0.73±0.10
0.53±0.07*
0.47±0.05*
0.70±0.14
D6D
0.09±0.02
0.04±0.01*
0.04±0.02*
0.03±0.01*
D5D
10.13±2.71
15.28±3.23*
14.62±3.88*
13.41±1.88
ACL
18.38±0.14
18.25±0.12
18.33±0.09
17.99±0.07*
DBI
202.71±13.22
180.96±13.62*
189.31±9.98
146.82±8.60*
PI
211.00±15.61
185.15±18.41*
197.37±15.17
138.96±10.34*
PUFA/SFA
1.12±0.15
PUFA/MUFA
4.16±0.59
20:4/18:2
0.83±0.06
22:6/20:4
1.39±0.11
n-3/n-6
CCl4 treated
group
0.94±0.10*
Values are area per cent (mean±SD); *significantly different from the control
determined by the Descriptive Statistics by Groups (Breakdown) - Post-hoc
Comparisons of Means using Scheffe test (p<0.05).
Abbreviations: D9 C18-delta 9 desaturase index of the 18:0; D6D- delta 6
desaturase index; D5D-delta 5 desaturase index; ACL-average chain length;
DBI-double bond index; PI-peroxidizability index.
Acknowledgements
This work was supported by the Croatian Ministry of Science,
Project No. 062-0000000-0221 and 062-0982522-0369,
DIOKI,d.d. Zagreb and The university of Rijeka foundation.
*
30
16:0
Values are area per cent (mean±SD); *significantly different from the control determined by the Descriptive Statistics by Groups
(Breakdown) - Post-hoc Comparisons of Means using Scheffe test (p<0.05).
Abbreviations: PUFA-polyunsaturated fatty acid; MUFA-monounsaturated fatty acid; SFA-saturated fatty acid.
#
+
35
14:0
Plasma
total
(μM)
Liver and serum
collection
3 days OOP pre-treated
OOP=10 mg/kg olive oil phenolics extracts (saline solution, i.p.)
CCl4 and OOP
treated group
18:3n-6
18:2n-6
Ex, 24 hrs
CCl 4=2 ml/kg (1:1, v/v, Olive oil:CCl4) (i.p.)
FFAs
CCl4 and
OOP treated
group
4 th day
CCl 4 treated (i.p.)
Group 1 - control; Group 2 - CCl4 treated; Group 3 - CCl4 and olive oil phenolics extract
simultaneously treated; Group 4 – 3-days olive oil phenolics extract pre-treated and then
CCl 4 treated
mM Trolox
PL FAs
Liver and serum
collection
START
Table 1. Differences in liver PL FAs and FFAs profiles between the control and test (treated) groups
Fatty
acids
START
1
2
3
4
Gro u p
Figure 1. a. Histological analysis of the liver from male Balb/c mice. H&Estained liver sections from: A-normal, B-CCl4 treated, C-CCl4 and OOP
simultaneously treated and D- 3-days OOP pre-treated and then CCl4 treated
mice, magnification = 400X;
b. Serum total antioxidative capacity (TAC) * significantly different from the
control and group 2, + from the group 2 and 3, # from the group 2 and 4,
determined by the Descriptive Statistics by Groups (Breakdown) - Post-hoc
Comparisons of Means using Scheffe test (p<0.05).
CONCLUSIONS
The concentrations of PL FAs and FFAs were altered by CCl4 administration, as well as by the simultaneous
use of olive oil phenolics with CCl4.
Significant differences in PL FAs were noted in the liver tissue after the administration of CCl4. The percent
contribution of liver PL FAs in group 2 as compared to the control group was significantly higher for 18:0,
18:2n-6, 18:3n-6 and saturated fatty acids (SFA), and lower for 20:3n-6, 20:4n-6 and polyunsaturated
fatty acids (PUFAs). In the liver FFAs fraction, percent contribution was significantly lower as compared to
the control group for 14:0, 20:4n-6, 20:1n-9 and monounsaturated fatty acids (MUFAs), while significantly
increased for 16:0 and saturated fatty acids (SFAs). Compared to the CCl4 treated group, olive oil
antioxidants group showed a reduction in the 18:1n-9.
Our experimental study showed that pre-treatment with olive oil phenolics resulted in more fatty acid
changes in the liver. Compared to CCl4 treated group, significant changes in 14:0, 18:1n-9, 20:4n-6 in the
PL FAs fraction, and 14:0 in the FFAs fraction were found. Major changes were noted in both lipid liver
fractions compared to group 3 and 4. So, comparison between pre-treated and treated olive oil phenolics
group showed significant differences in 14:0, 16:0, 18:1n-9, 18:3n-6, 20:3n-6, 20:4n-6, 20:5n-3 and
22:6n-6 in the PL FAs fraction and 14:0, 18:2, 20:3n-6, 24:0 and 22:6n-3 in the FFAs fraction.
CCl4-treated groups showed that total antioxidant capacity was higher compared to the control. Liver
histopathology indicated that olive oil polyphenols reduced the injury score of fatty degeneration incidence
and liver lesions induced by CCl4 in mice.
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