In the present study, the hepatoprotective activity of the unsaponifiable matter (UNSAP) of olive oil, linseed, and sesame oils against CCl4-induced liver toxicity in rats was investigated. In a preliminary antioxidant study, UNSAP showed pronounced DPPH radical scavenging activity (IC50 6.2-10.8 mg/mL). The constituents of UNSAP were determined by GC-MS. The subcutaneous administration of CCl4, caused liver injury. The hepatoprotective effect of UNSAP was comparable to that of α-tocopherol, a standard antioxidant agent. The co-administration of the investigated UNSAP normalized the activities of serum marker enzymes, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Furthermore, the serum alkaline phosphatase (ALP) activity and hepatic malondialdehyde (MDA) level were found to be alleviated by pre-treatment with the UNSAP. A histopathological evaluation showed marked improvement in the liver of UNSAP- and α-Tocopherol-treated animals. The hepatoprotective effect could be attributed to the antioxidant characteristics of UNSAP.
The liver has a wide range of vital functions for the human body. It detoxifies toxic substances that cause oxidative damages. Liver injury induced by carbon tetrachloride (CCl4) is a commonly used model for screening the hepatoprotective activity of natural compounds. The toxicity of CCl4 is a result of its biotransformation to the highly reactive tri-chloro-methyl, and tri-chloro-methyl peroxy free radicals. Both radicals cause the oxidative degradation of lipids, loss in integrity of cell membranes, and damage to hepatic tissue (Zhou
Crude linseed oil, extra virgin olive oil, and crude sesame oil are traditionally edible oils. Feeding piglets a 5% flaxseed oil diet for 3 weeks alleviated liver damage induced by lipopolysaccharide and reduced serum ALT, AST and ALP activities (Wang
Sesame oil unsaponifiables (sesamin, sesamolin, sesamol, and γ-tocopherol) have antioxidant activity (Fukuda
The UNSAP fraction represents a “fingerprint” of each oil. It is present in considerable amounts in crude oils. It contains hydrocarbons, sterols, triterpene alcohols, and tocopherols (Fontanel,
There are limited data in the literature on the hepatoprotective effect of UNSAP. The current study was designed to evaluate the antioxidant activity and hepatoprotective effect of UNSAP from linseed oil, extra virgin olive oil, and crude sesame seed oil in CCl4-intoxicated rats.
Linseed oil, extra virgin olive oil, and sesame oil were obtained from Tanta Oil and Soap Company (Egypt), Agriculture Research Center, Giza, Egypt, and private Mill, Cairo, Egypt, respectively.
Kits used for the determination of ALT, AST, and ALP were obtained from the Spinreact Company (Girona, Spain). Kits used for the determination of superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), and MDA were obtained from the Biodiagnostic Company for Diagnostic and Research Reagents (Cairo, Egypt). α-tocopherol was purchased from Hoffman La Roche (Basel, Switzerland). All other chemicals and reagents were of analytical grade.
Forty-two Sprague-Dawley male rats weighing 170±10 g were obtained from The Holding Company for Biological Products and Vaccines, Egypt. The study was carried out in accordance with ethical procedures approved by the Institutional Animal Care and Use Committee of Cairo University, Giza, Egypt (Approval number CU II F 31 17). All institutional and national guidelines for the care and use of laboratory animals were followed.
Acid value, peroxide value, and UNSAP of the investigated crude oils were determined according to AOCS (
Extraction of UNSAP from the oils was carried out according to AOCS (
The UNSAP were analyzed using the gas chromatograph Thermo Scientific, Trace GC ultra/ISQ, USA, equipped with single quadruple MS, and TG-5MS-fused silica capillary column (30 m, 0.25 mm, 0.1 mm film thickness). The column temperature was programmed from 260 to 300 °C while the injection temperature was set at 280 °C. The helium flow rate was set at 1 mL/min. The identification of peaks was based on the retention time of standard substances and MS spectra of the NIST Mass Spectral Database. The calculation of percent composition of the identified components was based on peak area.
The DPPH radical scavenging activity of the investigated UNSAP was determined according to Ramadan
where A0 is the absorbance of the blank, and As is the absorbance of the sample. All samples were analyzed in three replicates.
The rats were housed in individual cages in the animal house of the National Nutrition Institute, Ministry of Health and Population, Egypt. A basal diet was prepared according to AOAC (
G1: Negative control group received olive oil (1 mL/kg b.w, subcutaneous (s.c.)) on days 2, 3, 7, 8.
G2: Positive CCl4 control group received a 1:1 mixture of CCl4, and olive oil (2 mL/kg b.w, s.c.) on days 2, 3, 7, and 8.
G3 and G4: α -tocopherol groups were treated with the standard α -tocopherol, (10 and 50 mg/kg b.w, respectively, oral administration (p.o.)) daily for 10 days and also received the same CCl4–olive oil mixture (1:1, 2 ml/kg b.w, s.c.) on days 2, 3, 7 and 8, after 30 min of administration of α-tocopherol.
The remaining groups (G5, G6 and G7) were administered the investigated UNSAP orally, every day for 10 days at a dose equivalent to the antioxidant activity (DPPH radical scavenging activity, IC50) of 10 mg α–tocopherol.
G5: received 500 mg UNSAP from linseed oil/Kg b.w
G6: received 600 mg UNSAP from olive oil/kg b.w
G7 received 900 mg UNSAP from sesame oil/kg b.w
The rats in these groups (G5, G6 and G7) received the same CCl4–olive oil mixture (1:1, 2 mL/kg b.w, s.c.) on days 2, 3, 7, and 8, after 30 min of administration of the UNSAP.
On day 11, the rats were weighed, blood samples were collected, and diethyl ether anaesthetized animals were scarified by cervical dislocation.
Animals were fasted for 8 h before blood sampling. Blood was collected from the portal hepatic vein in serum and heparin glass tubes. The serum samples were obtained after centrifugation of the coagulated blood at 950 x g, for 15 min at 4 °C. Serum samples were then stored at -20 °C, until analysis. The heparinized whole blood was centrifuged at 950 x g for 15 min at 4 °C; the plasma layer was collected and stored at -20 °C until analysis. The erythrocyte pellets were washed three times with cold physiological saline (0.9% w/v), and stored at -80 °C until analysis.
Immediately after scarifying the rats, the liver organ of each rat was dissected out, washed with cold physiological saline (0.9% w/v), blotted dry, and weighed. Liver tissue samples were homogenized with a cold buffer (50 mM potassium phosphate, pH 7.5), and centrifuged at 950 x g for 15 min. The supernatant was collected, and stored at -80 °C for biochemical analysis.
The determination of blood serum AST, ALT, and ALP activities were carried out using commercial kits according to the manufacturers’ protocols.
Whole blood was used for the determination of GSH. Blood plasma was used for the determination of MDA level and CAT activity while blood erythrocyte lysate was used for the determination of SOD activity. The clear supernatant of liver homogenate was used for the measurement of GSH, SOD, CAT activities, and MDA level. Biochemical determinations were carried out using kits according to the manufacturers’ protocols.
The liver specimens were taken from the rats and fixed with 10% neutral formalin for 72 h before proceeding with the preparation of 5 µm-thick paraffin sections. These sections were sequentially stained with Hematoxylin and Eosin (H & E) for histopathological examination using light microscope according to Bancroft
Results were reported as mean ± S.D. The data were analyzed by one-way ANOVA and Tukey’s test. A bivariate analysis of the data was carried out by Pearson’s correlation test. A value of
The freshness of the investigated oils was confirmed and their acid and peroxide values were compatible with the Codex Alimentarius (
Quality characteristics of oils and GC-MS identified components of unsaponifiable matter
Parameter | Linseed oil | Olive oil | Sesame oil | ||
---|---|---|---|---|---|
Acid value (mg KOH/g Oil) | 0.26±0.01 | 0.57±0.01 | 0.90±0.02 |
||
Peroxide value (mEq O2/kg oil) | 3.25±0.04 | 6.50±0.05 | 2.80±0.15 | ||
Unsaponifiable matter % | 0.80±0.03 | 1.70±0.05 | 2.50±0.01 | ||
30.90 | Phytol | 296 | 2.66 | ND |
4.02 |
41.67 | Tricosane | 324 | ND | ND | 0.66 |
43.73 | Tetracosane | 338 | ND | ND | 0.77 |
50.12 | Tetratriacontane | 478 | 36.63 | ND | ND |
51.38 | Squalene | 410 | ND | 96.14 | 0.88 |
51.86 | Cycloeucalenol | 426 | ND | 0.58 | ND |
52.12 | Pentatriacontane | 492 | 1.43 | ND | ND |
53.10 | Triacontane | 422 | 2.10 | ND | ND |
54.61 | Tritriacontane | 464 | 24.92 | ND | ND |
55.56 | γ-Tocopherol | 416 | ND | 5.43 | 18.44 |
56.80 | α-Tocopherol | 430 | ND | 0.80 | ND |
57.56 | Rutin | 610 | ND | ND | 1.05 |
57.63 | Tetracontane | 562 | 10.30 | ND | ND |
57.76 | Sesamin | 354 | ND | ND | 28.95 |
58.46 | Campesterol | 400 | 5.84 | ND | 12.99 |
58.83 | Stigmasterol | 412 | 1.87 | ND | 3.85 |
59.82 | β- Sitosterol | 414 | 6.20 | 0.79 | 27.02 |
60.00 | Cycloartenol | 426 | ND | ND | 1.38 |
60.03 | Dotriacontane | 450 | 2.62 | ND | ND |
62.04 | 24-methylenecycloartanol | 440 | ND | 0.77 | ND |
63.36 | 6-[3-[3-[3-(5,11 dioxoindeno [1,2-c]isoquinolin-6-yl) propylamino] propylamino] propyl] indeno [1,2-c] isoquinoline-5,11-dione | 648 | ND | 0.91 | ND |
Mean ± Standard Deviation (n=3).
RT: Retention time in minutes.
MW: Molecular weight.
ND: Not detected.
Gas chromatographic analysis data of the extracted UNSAP are shown in
The scavenging activity of the investigated UNSAP against DPPH radicals is shown in
DPPH radical scavenging activity of the investigated unsaponifiable matter
Unsaponifiable matter | IC50 |
---|---|
Linseed oil | 6.20±0.08 |
Olive oil | 7.30± 0.16 |
Sesame oil | 10.80± 0.35 |
IC50 value denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals. IC50 value of α-tocopherol was found to be 121.32 ± 0.48 (μg/mL).
Mean ± Standard Deviation (n=3).
In the present study, the antioxidant activity of the UNSAP from linseed oil (IC50 = 6.2 mg/mL) was close to that of the UNSAP from olive oil (7.3 mg/mL). The radical scavenging activity of the UNSAP from sesame oil (IC50 = 10.8 mg/mL) represented less than 50% of the activity of the other investigated UNSAP. Farhoosh
The activities of the AST, ALT, and ALP enzymes in the serum of the rats are presented in
Liver function and oxidative stress biomarkers in the blood of CCL4 - intoxicated rats treated with unsaponifiable matter and α-tocopherol
Parameter Group | AST (U/L) | ALT (U/L) | ALP (U/L) | GSH (mg/dL) | SOD (U/mL) | CAT (U/L) | MDA (nmol/mL) |
---|---|---|---|---|---|---|---|
Control | 82.33 ± 3.06c | 65.00 ±4.36b | 119.00 ±11.79d | 26.85 ± 0.98a | 365.38 ±4.81a | 435.30 ±29.50b | 9.23 ±1.44bc |
CCl4 | 239.30 ±35.20a | 160.67 ±17.01a | 283.67 ±3.21a | 17.52 ±1.70c | 328.80 ±27.10b | 286.30 ±19.50c | 19.56 ±0.75a |
α-Tocopherol (10 mg/Kg b.w) + CCl4 | 157.67 ±15.28b | 69.30 ±18.60b | 237.33 ±25.80ab | 19.04 ±0.86bc | 355.30 ± 4.24ab | 275.00 ±47.30c | 7.77 ±2.74c |
α-Tocopherol (50 mg/Kg b.w) + CCl4 | 158.33 ±2.52b | 55.67 ±10.97b | 230.70 ±31.90bc | 22.88 ±0.67ab | 358.11 ± 4.77ab | 434.70 ±34.20b | 7.93 ±1.42c |
Linseed oil UNSAP (500 mg/Kg b.w) + CCl4 | 125.67 ±15.53bc | 52.33 ±14.98b | 215.67 ±4.93bc | 19.38 ±1.97bc | 328.13 ±5.39b | 513.00 ±39.10a | 13.42 ±0.94b |
Olive oil UNSAP (600 mg/Kg b.w) + CCl4 | 121.00 ±4.36bc | 61.00 ±7.21b | 187.67 ±10.02c | 24.27 ±2.17ab | 350.33 ±2.47ab | 308.36 ±15.57c | 13.90 ±3.25b |
Sesame oil UNSAP (900 mg/Kg b.w) + CCl4 | 127.3 ±19.30bc | 69.67 ±6.66b | 123.00 ±9.64d | 23.46 ±3.38ab | 364.31 ±7.54a | 320.90 ±23.40b | 13.08 ±1.62bc |
Data are expressed as Mean ± Standard Deviation (n=6). Significant differences among the means were determined by analysis of variance and Tukey’s test. Means with the same letter in the same column are not significantly different at 0.05 level of significance.
The administration of CCl4 induced hepatic injury as evidenced by a significant (
The UNSAP doses were sufficient to overcome the destructive effect of CCl4 on liver enzymes. The antioxidant activity of each UNSAP could be attributed to the synergistic effect of its constituents.
The administration of α-tocopherol (a positive control) at both investigated doses in CCl4-treated rats significantly (
The antioxidant effects of α-tocopherol and UNSAP on the CCl4-intoxicated rats are shown in
MDA is one of the main lipid peroxidation products. Pre-treatments of CCl4 intoxicated rats with the investigated UNSAP or α-tocopherol at both investigated doses significantly reduced (
The effects of the investigated treatments on the activities of SOD and CAT in the liver and the levels of GSH and MDA are shown in
Oxidative stress biomarkers in the liver tissue of CCL4-intoxicated rats treated with unsaponifiable matter and α-tocopherol
Group | GSH (mg/g tissue) | SOD (U/mg tissue) | CAT (U/g tissue) | MDA (nmol/g tissue) |
---|---|---|---|---|
Control | 115.54 ±2.28a | 24.42 ±0.90a | 155.55 ±6.73ab | 23.82 ±0.85d |
CCl4 | 71.97 ±7.83c | 8.32 ±1.01d | 129.37 ±2.45c | 115.21 ±10.56a |
α-Tocopherol (10 mg/Kg b.w) + CCl4 | 80.30 ±1.23bc | 19.14 ± 3.12bc | 143.66 ±11.49bc | 44.18 ±9.17bc |
α-Tocopherol (50 mg/Kg b.w) + CCl4 | 93.13 ±5.12b | 22.33 ± 1.20ab | 171.44 ±5.10a | 27.53 ±0.92cd |
Linseed oil UNSAP (500 mg/Kg b.w) + CCl4 | 118.60 ±9.08a | 18.78 ±0.59bc | 132.13 ±4.56c | 28.35 ±3.43cd |
Olive oil UNSAP (600 mg/Kg b.w) + CCl4 | 76.21 ±10.26bc | 20.10 ±2.78abc | 156.85 ±3.43ab | 47.09 ±3.95b |
Sesame oil UNSAP (900 mg/Kg b.w) + CCl4 | 115.17 ±8.94a | 15.58 ±0.78c | 144.20 ±9.32bc | 51.96 ±7.62b |
Data are expressed as Mean ± Standard Deviation (n=6). Significant differences among the means were determined by analysis of variance and Tukey’s test. Means with the same letter in the same column are not significantly different at 0.05 level of significance.
The activities of liver SOD, and CAT in the CCl4-treated group were significantly (
The pre-treatment with α-tocopherol at 50 mg/Kg b.w significantly increased (
On the other hand, the administration of α-tocopherol at 50 mg/Kg b.w or UNSAP of olive oil in CCl4-treated rats significantly (
In the present study, the MDA level of liver tissue was significantly reduced after the administration of α-tocopherol or the investigated UNSAP. The administration of α-tocopherol at a high dose as well as the UNSAP of linseed oil in CCl4-treated rats significantly (
The data showed that all UNSAP studied contained several compounds at varied concentrations. The antioxidant effect of these UNSAP is, actuality, the accumulative effect of its constituents and could not be directly attributed to one single compound in the mixture.
The correlation between the investigated parameters using Pearson’s Correlation is shown in
Pearson’s correlation coefficient (r) between liver function enzymes and the oxidative stress biomarkers in the different groups studied
Parameter | AST | ALT | ALP | Blood MDA | Liver MDA | Blood GSH | Liver GSH | Blood SOD | Liver SOD | Blood CAT |
---|---|---|---|---|---|---|---|---|---|---|
ALT | 0.746 |
|||||||||
ALP | 0.794 |
0.529 |
||||||||
Blood MDA | 0.499 |
0.629 |
0.241 | |||||||
Liver MDA | 0.779 |
0.933 |
0.530 |
0.721 |
||||||
Blood GSH | -0.705 |
-0.515 |
-0.748 |
-0.302 | -0.538 |
|||||
Liver GSH | -0.662 |
-0.491 |
-0.633 |
-0.287 | -0.556 |
0.386 | ||||
Blood SOD | -0.579 |
-0.381 | -0.602 |
-0.529 |
-0.382 | 0.672 |
0.279 | |||
Liver SOD | -0.774 |
-0.811 |
-0.493 |
-0.709 |
-0.888 |
0.602 |
0.355 | 0.499 |
||
Blood CAT | -0.433 |
-0.469 |
-0.207 | -0.205 | -0.615 |
0.215 | 0.643 |
-0.042 | 0.498 |
|
Liver CAT | -0.412 | -0.529 |
-0.313 | -0.585 |
-0.549 |
0.619 |
0.042 | 0.605 |
0.706 |
0.158 |
Significant at
Significant at
The results revealed significant (
A microscopical examination of the hepatic tissues of the control group revealed normal liver structure with a normal lobular pattern. The hepatocytes showed well-defined central nuclei and abundant cytoplasm. Magnification at 200x enabled the observation of the normality of the central vein and portal venous channels with normal-sized sinusoids. Bile ducts were lined with normal cuboidal epithelium. (
Photomicrographs of liver sections of CCl4-treated rats administered with Unsaponifiable matter and α-tocopherol (a-g). (a) Control group showing normal hepatocytes (H), normal sized sinusoids (S) and central vein (CV). (b) CCl4-intoxicated rats, showing steatotic hepatocytes (arrowhead), and congested portal vein (arrow). (c) CCl4-intoxicated rats treated with α-tocopherol (10 mg/kg b.w) showing hyaline body (arrow), apoptotic hepatocytes (arrowhead) and restricted portal area (PA). (d) CCl4-intoxicated rats treated with α-tocopherol (50 mg/ kg b.w) showing proliferated bile duct (arrow), periportal inflammation (double arrow) and dilated congested portal vein (PV). (e) CCl4-intoxicated rats treated with the unsaponifiable matter of linseed oil, showing inflammatory cell aggregates (arrow). (f) CCl4-intoxicated rats treated with the unsaponifiable matter of olive oil, showing improvement of most hepatocytes, and lacerated wall of central vein (arrow). (g) CCl4-intoxicated rats treated with the unsaponifiable matter of sesame oil, showing dilated, congested blood vessel (arrow), where most hepatocytes display normal (arrowhead). (Haematoxylin & Eosin×200)
A histopathological examination of the hepatic tissues of the CCl4 group showed a weaker architecture, with edema. Most of the hepatic vasculature revealed congestion, dilatation, and inflammation. Hepatocytes showed steatosis and severe cytoplasmic vacuolation (
The treatment of intoxicated rats with α-tocopherol at 10 mg/Kg b.w revealed a mild degree of improvement, where disturbed architecture with expanded portal areas was observed. A hyaline body with a fragmented nucleus and apoptotic hepatocytes were also seen (
A histological picture of the hepatocytes of UNSAP of the linseed oil group indicated mild to moderate curative effects. The examination revealed areas of intact hepatocytes alternated with areas of hepatocytes with vacuolated cytoplasm or fatty degeneration (
The investigated UNSAP were found to be effectively hepatoprotective, as evidenced by biochemical, and histopathological studies. The efficiency of those UNSAP could be due to an interaction between its components which far exceeds its antioxidant activity.
The authors declare no conflict of interest.