Hepatoprotective effect of cold-pressed Syzygium aromaticum oil against carbon tetrachloride (CCl₄)-induced hepatotoxicity in rats

Abstract

Contexts: Exposure to environmental pollutants such as carbon tetrachloride (CCl₄) causes liver injuries. There are claims that extracts from Syzygium aromaticum (Linn.) Merrill & L.M.Perry, (Myrtaceae) protects from such injuries.

Objective: This study investigates the protective effects of cold-pressed S. aromaticum oil (CO) against CCl₄-induced liver toxicity in rats.

Materials and methods: CO was orally administered to rats in two doses (100 and 200 mg/kg) along with CCl₄ (1 mL/kg in olive oil) for 8 weeks. Indices of liver and kidney functions, lipid profile, and peroxidation were evaluated in rats’ serum and tissues. Fatty acids and bioactive lipids of CO were analyzed.

Results: High levels of monounsaturated fatty acids (39.7%) and polyunsaturated fatty acids (42.1%) were detected in CO. The oil contained high amounts of tocols and phenolics. The LD₅₀ value at 24 h was approximately 5950 mg/kg. Treatment with 200 mg/kg CO resulted in a decrease of creatinine, urea, and uric acid levels to 0.86, 32.6, and 2.99 mg/dL, respectively. Levels of TL, TC, TAG, LDL-C, and VLDL-C were decreased to 167, 195.3, 584.5, 74.6, and 39.0 mg/L, respectively, after 8 weeks of treatment. Hepatic malondialdehyde levels were reduced and glutathione levels were elevated in CO-treated rats. CO reduced the activities of AST, ALT, and ALP as well as kidney function markers, protein, and lipid profiles, respectively. Histopathological examination of liver indicated that CO treatment reduced fatty degenerations, cytoplasmic vacuolization, and necrosis.

Conclusion: CO possessed a protective effect against CCl₄-induced hepatotoxicity in rats, mediated possibly by the antioxidant properties of the oil.

Key words:

• Antioxidants
• clove
• liver
• metabolic enzyme
• phenolic compounds
• tocols

Introduction

The liver plays an important role in metabolism of endo- and exo-genous substances, wherein the hepatic injury is associated with distortion of these functions. Liver, therefore, is susceptible to diseases including hepatitis, cirrhosis, alcohol-related disorders, and liver cancer (Al-Sayed et al., 2014). A major cause of these disorders is due to exposure to different environmental pollutants and xenobiotics [i.e., carbon tetrachloride (CCl₄), thioacetamide, paracetamol, and alcohol]. These toxicants mainly damage the liver by producing reactive oxygen species (ROS) (Liu et al., 2011). ROS have been known to produce tissue injury through covalent binding and lipid oxidation and have been shown to augment fibrosis as seen from increased collagen synthesis (Adesanoye & Farombi, 2010; Blomhoff, 2005). Scavenging of free radicals by defense systems could reduce the fibrosis process in the tissues (Qian et al., 2008; Shim et al., 2010). Cooperative defense systems that protect body from free radical damage include antioxidants and enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPH)]. The role of protective enzymes is well known and has been studied deeply with in vivo models (Ghassam et al., 2014; Jaydeokar et al., 2014).

CCl₄, an industrial solvent, is extensively used xenobiotic to induce chemical liver injury. CCl₄ is metabolized by hepatic microsomal cytochrome P₄₅₀ to free radical such as trichloromethyl radical (⋅CCl₃) and proxy trichloromethyl radical (⋅OOCCl₃) (Akram et al., 2011; Lee et al., 2004). Trichloromethyl, hepatotoxic metabolite of CCl₄, can react with sulfhydryl groups (e.g., glutathione and protein thiols) and affect enzymes such as CAT and SOD. Over production of trichloromethyl-free radicals initiate a membrane lipid oxidation, eventually leading to various liver pathological processes, such as steatosis, fibrosis, or cirrhosis.

Nowadays, many people are suffering from hepatic damage induced by alcohol, chemicals and infections. Therefore, liver diseases continue to be a serious health problem worldwide. Interest in the role of naturally originated agents for the treatment of liver diseases was considered. Some natural compounds are demonstrated to have a protective role against liver diseases (Pramyothin et al., 2007; Shaker et al., 2010; Wan et al., 2009). Studies revealed that natural bioactive compounds, containing antioxidants, protect the liver against lipid oxidation, and impairment in antioxidant status induced by CCl₄ (Noyan et al., 2006; Sanmugapriya & Venkataraman, 2006; Shaker et al., 2010, 2011). ROS production is linked with oxidative stress, which is defined as an imbalance in the generation of oxidants and the antioxidant defense. ROS have an important role in liver disease, while antioxidants might prevent hepatic damage through scavenger activity and increase the activity of intracellular antioxidant enzymes including SOD, GSH and CAT (Upur et al., 2009). Polyphenolic compounds have marked antioxidant effect and can prevent the liver damage caused by free radicals (Al-Sayed et al., 2014). Studies reported that natural antioxidants and plant extracts are efficacious in preventing oxidative stress-related liver pathologies due to particular interactions and synergism (Vitaglione et al., 2004; Yeh et al., 2013).

There is much evidence indicating that natural bioactive substances, extracts, and oils from vegetable, fruits, oilseeds, and medicinal plants exhibit strong antioxidant potential that could act against hepatic toxicity. One such candidate is clove [Syzygium aromaticum L. (Myrtaceae)] oil, which was chosen in the present study. S. aromaticum essential oil has antimicrobial and antioxidant properties (Gulcin et al., 2012; Huang et al., 2002). Cloves treatment reduced blood sugar and lipid oxidation in streptozotocin-induced diabetic rats by restoring the levels of antioxidant enzymes (Shukri et al., 2010). Clove oil was listed as a ‘‘Generally Regarded As Safe’’ substance by Food and Drug Administration (FDA) when administered at levels under 1500 ppm in food. In addition, the World Health Organization (WHO) Expert Committee on Food Additives has established the acceptable daily human intake of clove oil at 2.5 mg/kg body weight (Kildeaa et al., 2004). Cloves contain a variety of bioactive compounds such as sesquiterpenes, tannins, and triterpenoids. The main aroma constituent of clove buds, eugenol (4-allyl-2-methoxyphenol), was reported to have antifungal activity (Lee & Shibamoto, 2002; Miyazawa & Hisama, 2003). Eugenol was classified by United States Food and Drug Administration (FDA) to be a substance that is generally regarded as safe (Gulcin et al., 2012). Eugenol was reported to inhibit malonaldehyde formation from cod liver oil and the formation of hexanal (Gulcin et al., 2004). Over the last years, interest in cold pressed oils has been increased because cold pressing involves no heat or chemical treatments. Cold pressing also involves no refining process and may contain a high level of lipophilic bioactive phytochemicals including antioxidants (Ramadan et al., 2012). Recently, it was reported that cold-pressed clove oil (CO) is a rich source of essential fatty acids and bioactive compounds including tocols, phospholipids and phenolic compounds. CO was also characterized by its high antiradical potential against free radicals including DPPH and galvinoxyl radicals (Ramadan et al., 2013).

It is hard to find data in the literature on the protective impact of CO on CCl₄-induced oxidative damage in rats. Therefore, in the present study, we investigated the protective impacts of CO against CCl₄-induced oxidative stress and hepatotoxicity in rats. The extent of CCl₄-induced liver injury was monitored by measuring different biochemical and histopathological parameters.

Materials and methods

Chemicals

Cold-pressed CO was obtained from a local market (Zagazig, Egypt). CO has specifications and codex standard approved by Egyptian organization for standardization and quality (approval number 66/92). CCl₄ and standards used for tocols were purchased from Merck (Darmstadt, Germany). Assay kits and other chemicals were of the analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO).

Gas chromatography (GC) of fatty acids methyl esters (FAME) in CO

Fatty acids in CO were transesterified into fatty acid methyl esters (FAME) by N-trimethylsulfoniumhydroxide (Macherey-Nagel, Germany) according to Arens et al. (1994). FAME were detected on a Shimadzu GC-14A equipped with flame ionization detector (FID) and C-R4AX chromatopac integrator (Shimadzu Inc., Kyoto, Japan). The flow rate of the carrier gas helium was 0.6 mL/min and the split value with a ratio of 1:40. A sample of 1 μL was injected on a 30 m × 0.25 mm × 0.2 μm film thickness Supelco SPTM-2380 (Bellefonte, PA) capillary column. The injector and the FID temperature were set at 250 °C. The initial column temperature was 100 °C programmed by 5 °C/min until 175 °C and kept 10 min at 175 °C, then 8 °C/min until 220 °C, and kept 10 min at 220 °C. A comparison between the retention times of the samples with those of an authentic standard mixture (Sigma, St. Louis, MO; 99% purity specific for GLC), run on the same column under the same conditions, was made to facilitate identification.

HPLC of tocols in CO

A solution of 250 mg of CO in 25 mL n-heptane was directly used for the HPLC (Ramadan, 2013). The HPLC analysis was conducted using a Merck Hitachi low-pressure gradient system, fitted with an L-6000 pump, a Merck-Hitachi F-1000 Fluorescence Spectrophotometer (the detector wavelength was set at 295 nm for excitation, and at 330 nm for emission) and a D-2500 integration system; 20 μL of the samples were injected by a Merck 655-A40 Autosampler onto a Diol phase HPLC column 25 cm × 94.6 mm ID (Merck, Darmstadt, Germany) using a flow rate of 1.3 mL/min. The mobile phase used was n-heptane/tert-butyl methyl ether (99:1, v/v).

Extraction and quantification of phenolic compounds in CO

CO (1 g) was dissolved in n-hexane (5 mL) and mixed with 10 mL methanol–water (80:20, v/v) in a glass tube for 2 min in a vortex (Ramadan et al., 2010). After centrifugation at 3000 rpm for 10 min, the hydroalcoholic extracts were separated from the lipid phase by using a Pasteur pipette then combined and concentrated in vacuo at 30 °C until a syrup consistency was reached. The lipidic residue was redissolved in 10 mL methanol-water (80:20, v/v) and the extraction was repeated twice. Hydroalcoholic extracts were redissolved in acetonitrile (15 mL) and the mixture was washed three times with n-hexane (15 mL each). Purified phenols in acetonitrile were concentrated in vacuo at 30 °C then dissolved in methanol for further analysis. Aliquots of phenolic extracts were evaporated to dryness under nitrogen. The residue was re-dissolved in 0.2 mL water and diluted (1:30) Folin–Ciocalteu’s phenol reagent (1 mL) was added. After 3 min, 7.5% sodium carbonate (0.8 mL) was added. After 30 min, the absorbance was measured at 765 nm using a UV-260 visible recording spectrophotometer (Shimadzu, Kyoto, Japan). Gallic acid was used for the calibration and the results of triplicate analyses are expressed as parts per million of gallic acid.

Experimental animals

Experimental procedures involving the animals and their care were conducted in conformity with the institutional guidelines and the Guidelines for Care and Use of Laboratory Animals in Biomedical Research as adopted and promulgated by the World Health Organization. Healthy adult male albino rats (Wister Strain) of approximately same age, weighing approximately 120–140 g, were purchased from Organization of Biological Products and Vaccines (Helwan Farm, Helwan, Egypt). Animals were housed under ambient temperature (25 ± 2 °C) with 50 ± 5% relative humidity and a 12 h light-dark cycle. Rats received standard pellet diet comprised of 20% crude protein, 5% fat, 4.5% crude fiber, 8% ash, 2% calcium, 0.6% phosphorus, 3.4% glucose, 2% vitamins, and water ad libitum.

Experimental design

Twenty-four rats were used to study the protective role of CO on the CCl₄-induced hepatotoxicity in rats. CCl₄ was mixed with olive oil in the ratio of 1:1 (w/w) and used for hepatoprotective potential against CCl_4-induced liver damage. Animals were divided randomly into four equal groups (six rats each) and treated as follows:

Group 1 (negative control group): Rats were fed on standard synthetic diet for 8 weeks.

Group 2 (positive control group): Rats were fed on standard diet and received with equal mixture of CCl₄ and olive oil orally (three time a week) by gastric gavages at a dose of 1 mL/kg (b.w.) for 8 weeks.

Group 3: Rats were fed on standard diet and received CO orally (three times a week) by gastric gavages at a dose of 100 mg/kg, simultaneously with equal mixture of CCl₄ and olive oil by gastric gavages at dose of 1 mL/kg during the last 4 weeks.

Group 4: Rats were fed on standard diet and received CO orally (three times a week) by gastric gavages at a dose of 200 mg/kg, simultaneously with equal mixture of CCl₄ and olive oil by gastric gavages at dose of 1 mL/kg during the last 4 weeks.

Blood sampling and biochemical evaluation

Blood samples were collected at the end of experiment and obtained from the retro-orbital plexus veins from the individual rat by means of fine capillary heparinized tubes. Blood samples was collected and allowed to clot, serum was separated by centrifugation at 3000 rpm for 15 min. Serum was used to investigate the biochemical parameters including liver and kidney function tests and serum lipid profile.

Activities of liver enzymes including alanine transaminase (ALT) and aspartate transaminase (AST), alkaline phosphatase (ALP) as well as the serum total bilirubin (TB), direct bilirubin (DB), total protein and serum albumin were determined according to the methods of Reitman and Frankel (1957), Tietz et al. (1983), Doumas et al. (1973), and Doumas et al. (1971), respectively. The globulin was calculated by subtracting the albumin from serum total protein. Kidney function parameters including urea, uric acid, and creatinine were measured by using the method of Amer et al. (2007). Lipid profile including total lipids (TL), triglycerides (TAG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) was determined according to the methods of Ramadan et al. (2008).

Antioxidant markers

Liver samples were washed immediately with ice-cold saline to remove excess blood. Liver tissue was homogenized in cold 0.1M potassium phosphate saline (pH = 7.4) at a concentration of 10% (w/v). The homogenate was then centrifuged at 5000 rpm for 10 min at 4 °C to obtain the supernatant, which was used to investigate antioxidant markers. Lipid peroxides (malondialdehyde, MDA) were measured according to Mihara and Uchiyama (1978). Reduced glutathione (GSH) content in liver was measured spectrophotometrically using Ellman’s reagent (DTNB) following the method described by Moron et al. (1979). The absorbance was read at 412 nm and the standard graph was drawn using different concentrations of a standard GSH solution and GSH contents were calculated as nM per mg of tissue protein.

Histopathological examination

Small tissue specimens were collected from fresh liver tissue of the rats and rapidly fixed in 10% neutral buffered formalin. After proper fixation, thin paraffin sections were routinely prepared and stained with hematoxylin and eosin stain (H&E) for the histopathological lesions in hepatic and renal tissues. The liver sections were graded numerically to assess the degree of histopathological features of acute hepatic injury.

Statistical analysis

All data were analyzed by one-way ANOVA. Duncan’s new multiple-range test was used to resolve the difference among treatment means. All statistical analyses were performed using the statistical software SPSS 11.0 (SPSS Ltd., Surrey, UK). A p < 0.05 was considered statistically significant. Ratio values were not arcsin transformed before statistical analysis.

Results

Fatty acids and bioactive lipids of CO

Nine fatty acids were found in CO. The percentages of C10:0, C12:0, C14:0, C16:0, C16:1, C18:0, C18:1n-9, C18:2n-6, and C18:3 were 2.76, 0.07, 0.06, 8.57, 0.36, 6.59, 39.4, 40.2, and 1.99%, respectively. Linoleic and oleic acids were the main fatty acids comprising together almost 80% of total fatty acids. CO contained significant levels of monounsaturated fatty acids (MUFA, 39.7 g/100 g total fatty acids) and had a polyunsaturated fatty acids (PUFA) content of 42.1 g/100 g of total fatty acids. Palmitic and stearic were the major saturated fatty acids (SFA), comprising together about 15% of total identified FAME. CO was characterized by high levels of unsaponifiables (25.3 g/kg). Levels of α-, β-, γ- and δ-tocopherols were 14 890, 56, 4184, and 186 mg/kg CO, respectively. In addition, amounts of α-, β-, γ-, and δ-tocotrienols were 1110, 55, 85, and 9498 mg/kg CO, respectively. α-Tocopherol constituted about 49.5% of the total tocols followed by δ-tocotrienol (31.6%) and γ-tocopherol (13.9%). Other tocols were measured in lower levels. CO was also characterized by high level of phenolics (4.6 mg GAE/g).

Effect of CO on liver enzymes, kidney function indicators, protein profile, lipids profile, and antioxidant markers in CCl₄-induced liver injury rats

The LD₅₀ value of CO was 5950 mg/kg based on a 24 h oral toxicity study. In a long-term toxicity study of CO (100, 200, and 400 mg/kg, p.o.), only 400 mg/kg induced a decrease in body weight. The effect of two doses of CO on serum liver enzymes in CCl₄-treated rats was studied and the results are given in Table 1. After injection of CCl₄, activities of AST, ALT, and ALP enzymes in the CCl₄-treated group (groups 2) were significantly increased (p <0.05), as compared with the negative control (group 1). Administration of CO attenuated the increased levels of AST, ALT, and ALP enzymes induced by CCl₄ and caused a subsequent recovery toward normalization comparable with the negative control (group 1). Treatment of animals with both doses of CO significantly reduced the activities of serum AST, ALT, and ALP enzymes as compared with the positive group of CCl₄-treated alone. In addition, levels of TB and DB were also reduced significantly upon treatment with CO (groups 3 and 4).

Table 1. Changes in liver enzymes in CCl₄-induced oxidative damage in rat liver.

Group	Treatment	ALT (U/mL)	AST (U/mL)	ALP (U/L)	TB (mg/dL)	DB (mg/dL)
1	Negative control (Normal)	28.33 ± 0.99^c	29.00 ± 0.59^b	95.11 ± 0.66^c	0.75 ± 0.046^b	0.123 ± 0.011^b
2	Positive control (CCl4)	138.33 ± 1.55^a	171.6 ± 1.44^a	293.3 ± 2.00^a	1.65 ± 0.13	0.623 ± 0.015^a
3	Clove oil (100 mg/kg) + CCl4	36.66 ± 1.66^b	36.67 ± 1.33^b	125.6 ± 1.25^b	0.74 ± 0.026^b	0.146 ± 0.013^b
4	Clove oil (200 mg/kg) + CCl4	30.00 ± 1.82^bc	35.66 ± 1.10^b	83.36 ± 1.29^d	0.85 ± 0.034^b	0.156 ± 0.009^b

^{a, b, c}There is no significant difference (p>0.05) between any two means with the same letter in each column.

Table 2 presents the impact of CO on kidney function indicators and protein profile of CCl₄-treated rats. Levels of creatinine, urea and uric acid in negative control (group 1) after 8 weeks of experiments were 0.716, 25.3, and 3.63 mg/dL, respectively. On one hand, it could be seen that treatment with CCl₄ (group 2) resulted in increase in the levels of creatinine, urea and uric acid levels (1.85, 56.3, and 7.93 mg/dL, respectively). On the other hand, treatment with CO resulted in decrease of creatinine, urea, and uric acid levels. Concerning protein profile (total protein, albumin, globulin, and A/G ratio), results revealed that treatment with CCl₄ (group 2) reduced the protein parameters in general (Table 2), while treatment with both doses of CO increased protein profile to levels resemble negative control (group 1).

Table 2. Impact of CO on kidney function indicators and protein profile in CCl₄-induced injury in rat.

Group	Treatment	Creatinine (mg/dL)	Urea (mg/dL)	Uric acid (mg/dL)	T-protein (g/dL)	Albumin (g/dL)	Globulin (g/dL)	A/G ratio
1	Negative control (Normal)	0.71 ± 0.026^b	25.33 ± 1.33^c	3.63 ± 0.33^b	6.50 ± 0.17^a	3.78 ± 0.05^b	2.72 ± 0.24^a	1.40 ± 0.55^ab
2	Positive control (CCl4)	1.85 ± 0.036^a	56.33 ± 1.21^a	7.93 ± 0.26^a	5.40 ± 0.37^b	3.01 ± 0.06^d	2.38 ± 0.33^a	1.312 ± 0.16^ab
3	Clove oil (100 mg/kg) + CCl4	0.57 ± 0.051^c	31.33 ± 1.16^b	2.41 ± 0.72^c	6.53 ± 0.61^a	3.65 ± 0.02^c	2.88 ± 0.41^a	1.27 ± 0.17^b
4	Clove oil (200 mg/kg) + CCl4	0.68 ± 0.013^b	32.66 ± 1.10^b	2.99 ± 0.62^bc	6.53 ± 0.14^a	4.14 ± 0.07^a	2.39 ± 0.26^a	1.74 ± 0.66^a

^{a, b, c}There is no significant difference (p>0.05) between any two means with the same letter in each column.

Impact of CO on lipid profile of CCl₄-induced liver injury rats is presented in Figure 1

Figure 1. Changes in lipid profiles (mg/L) in CCl₄-induced injury in rat. Error bars show the variations of three determinations in terms of standard deviation.

. Generally, treatment with CCl₄ resulted in significant increase in all lipids parameters (TL, TC, TAG, LDL-C, and VLDL-C) except of HDL-C. After 8 weeks of experiment levels of TL, TC, TAG, LDL-C, and VLDL-C in positive control (group 2) were 213.5, 292.6, 691.6, 119.5, and 58.5 mg/L, respectively. After the same period (8 weeks) with 200 mg/kg CO treatment, levels of TL, TC, TAG, LDL-C, and VLDL-C were significantly decreased to 167, 195.3, 584.5, 74.6, and 39.0 mg/L, respectively. Concerning HDL-C, treatment with CCl₄ resulted in decrease the level of HDL-C after 8 weeks of experiment while treatment with both doses of CO increased HDL-C levels.

As shown in Table 3, the MDA (nmol/mg) level was markedly higher in CCl₄-treated rats (group 2) after 8 weeks. Expected increases lipid oxidative indices in the CCl₄-treated model confirmed that oxidative damage has been induced. Negative control (group 1) and groups (3 and 4) that treated with CO were characterized by lower levels of MDA. On one hand, it could be concluded that CO inhibited the elevation of MDA levels upon CCl₄ administration. On the other hand, GSH level was markedly lower in CCl₄-treated rats (group 2). However, the GSH level was significantly increased by CO treatment (groups 3 and 4) when compared with the positive control group. Administration of CO (groups 3 and 4) did significantly increase (p >0.05) the activity of GSH as compared to the CCl₄-treated group.

Table 3. Changes in antioxidant markers in CCl₄-induced injury in rats.

Group	Treatment	MDA (nmol/mg)	GSH (mmol/g)
1	Negative control (normal)	3.350 ± 0.14^d	1.487 ± 0.066^a
2	Positive control (CCl4)	9.252 ± 0.26^a	0.835 ± 0.087^b
3	Clove oil (100 mg/kg) + CCl4	5.976 ± 0.35^b	1.320 ± 0.069^a
4	Clove oil (200 mg/kg) + CCl4	4.735 ± 0.17^c	1.380 ± 0.074^a

^{a, b, c}There is no significant difference (p>0.05) between any two means with the same letter in each column.

Histopathology of the liver

The results of hepatic histopathological examination are shown in Figure 2(A–D

Figure 2. Photomicrograph of liver from (A) control rats showing normal histobasoligical structure (H&E × 300), (B) rats received CCl₄ (100 mg/kg) showing severe fatty change in the hepatocytes (H&E × 300), (C) rats received CCl₄ (100 mg/kg) and CO (100 mg/kg) showing degenerative changes in hepatocytes in the form of vaculation with focal mononuclear aggregation (H&E × 800), (D) rats received CCl₄ 100 mg/kg and CO (200 mg/kg) showing congestion of the central vein and hepatic sinusoids (H&E × 300).

). The histological observations supported the obtained biochemical results. Liver sections from negative control rats showed normal lobular architecture, hepatic cells with a well-preserved cytoplasm and well-defined nucleus and nucleoli (Figure 2A). When compared with the normal liver tissues of controls, liver tissue in the rats treated with CCl₄ revealed extensive liver injuries characterized by moderate to severe hepatocellular degeneration and necrosis around the central vein, lipidosis, hepatic fibrosis, and cholangiocyte hyperplasia (Figure 2B). The liver of rats received CCl₄ showing severe fatty changes in the hepatocytes, the cytoplasme of hepatocytes showing clear vacuoles which squeezing their nuclei in one side giving signet ring shape (Figure 2B). The histopathological hepatic lesions induced by administration of CCl₄ were only remarkably ameliorated in central lobular necrosis, hepatic lipidosis, and hepatic fibrosis by treatment with CO (Figure 2C and D). In group 3, which received CO (100 mg/kg), only mild degenerative changes in the hepatocytes in the form of vacuolar degeneration and activation of von-Kupffer cells together with focal mononuclear aggregations in the portal areas were shown (Figure 2C). The liver of group 4 which received a 200 mg/kg dose of CO, showed congestion of the central vein and mild congestion of the hepatic sinusoids (Figure 2D).

Discussion

The liver, as a key organ of metabolism and excretion, is constantly endowed with the task of detoxification. Hepatotoxicants, including viruses, fungal products, bacterial metabolites, minerals, environmental pollutants and chemotherapeutic agents, can induce various disorders of the organ (Ha et al., 2005). Among the various health problems, liver diseases, including hepatocellular carcinoma, fibrosis, cirrhosis and hepatitis appear to be the most serious. Hepatotoxins, such as ethanol, acetaminophen and CCl₄, induced liver injury, which is characterized by varying degrees of hepatocyte degeneration and cell death (Yeh et al., 2013). Vitaglione et al. (2004) suggested that ROS including superoxide and hydroxyl radicals have been proved to be associated with the intoxication by CCl₄. Evidence suggested that CCl₄ has been commonly used as a hepatotoxin in experimental hepatopathy (Hsu et al., 2008). Covalent binding of the metabolites of CCl₄, trichloromethyl-free radicals, to cell proteins is considered to be the initial step in a chain of events that eventually lead to membrane lipid oxidation and finally to cell death.

In recent years, there has been a substantial increase in the use of so-called complementary dietary phenolics and alternative therapies to treat patients with liver disease. Epidemiological studies suggested an inverse relation between the consumption of phenol-rich foods and the risk of degenerative diseases, particularly cancers and cardiovascular diseases (Arts & Hollman, 2005). In this regard, there has been a great deal of interest in the screening and characterization of novel potentially therapeutic of phenolics, phenol-rich foods and medicinal plants (Murcia et al., 2001; Seeram et al., 2005). The antioxidant properties of phenolic compounds and their ability to modulate the activity of enzymes have been demonstrated in vitro studies and are believed to be a primary mechanism for their biological impacts. However, the question remains of whether these properties demonstrated in in vitro studies are relevant to protect against oxidative damage in vivo, where polyphenolic compounds are found at a very low concentrations depending on bioavailability and metabolism.

An experiment has been designed on rat model to evaluate the protective effects of the CO against the toxicity caused by CCl₄ in rats. CCl₄ increased lipid oxidation, and affects different blood biochemical parameters including liver enzymes, kidney function indicators, protein profile, lipid profile, and antioxidant markers. CO treatment prevented these effects, indicating that CO was able to attenuate the lipid oxidation induced by CCl₄. For example, the protective role of CO was accompanied with a partial prevention of GSH depletion in the liver tissue. It is considered that hepatic GSH represents an enzyme reserve of the liver, which is responsible for reducing the hepatotoxicity induced by the active metabolites of CCl₄. As GSH is also a crucial determinant of tissue susceptibility to oxidative damage, the partial protection of CO on GSH reserves provide an additional action not only to remove the active metabolites of CCl₄ but also to scavenge free radicals, which are involved in lipid oxidation. The effect of CO was further confirmed by histopathological examinations. Hepatocyte necrosis, fatty change, hyaline degeneration, ballooning degeneration, and infiltration of Kupffer cells and lymphocytes were prominent in the histological findings. CO offers a hepatoprotection impact in central lobular necrosis, hepatic lipidosis, and cholangiocyte hyperplasia in rats.

It has been hypothesized that one of the principal causes of CCl₄-induced liver injury is the formation of lipid peroxides by free radical derivatives of CCl₄. Thus, the antioxidant potential or the inhibition of the generation of free radicals is important in protection against CCl₄-induced hepatopathy. The body has an effective defense mechanism to prevent and neutralize the free radical induced damage by a set of endogenous antioxidant enzymes such as SOD and CAT. These enzymes constitute a mutually supportive defense team against ROS. Lipid peroxidation, a ROS-mediated mechanism, has been implicated in the pathogenesis of various liver injuries and subsequent liver fibrogenesis in experimental animals. The significant non-dose-dependent decrease in the hepatic lipid hydroperoxide confirmed that treatment with CO could effectively protect against the hepatic lipid oxidation induced by CCl₄. Hence, it is possible that the mechanism of hepatoprotection of CO may be due to its antioxidant activity.

CO contains high-level health-promoting fatty acids such as MUFA and PUFA. From the health point of view, MUFA have been shown to lower “bad” LDL-C and retain “good” HDL-C. This is the major benefit of olive oil over the highly polyunsaturated oils, wherein PUFA reduce both the “bad” as well as the “good” serum cholesterol levels in blood (Ramadan et al., 2010). On one hand, clove oil also contains high levels unsaponifiables, tocols, and phenolic compounds. Oils rich in phenolics may play an important role in reducing the risk of many diseases. On the other hand, phenolics bioavailability was found to be higher when phenolic compounds are soluble in a lipid matrix than in an aqueous media (Ramadan, 2013). The antioxidant potential of phenolics is mainly due to their redox properties and is the result of various mechanisms: antiradical activity, transition-metal-chelating activity, and/or singlet-oxygen-quenching capacity (Bettaieb et al., 2010). Phenolics exhibit favorable hepatoprotective effects and could prevent chemical induced dyslipidemia, inflammatory response, and mitochondrial oxidative damage of rat hepatocytes. Therefore, bioactive compounds with remarkably high antioxidant activity, superior free radical-scavenging ability, and inhibition of lipid oxidation are contributed to the hepatoprotective effect in rats against liver damage induced by CCl₄ (Hu et al., 2012; Liu et al., 2012; Tang et al., 2012; Yeh et al., 2013; Yousef et al., 2010). Because the phytochemicals, analysis of CO showed the presence of tocols and phenolic compounds, and hepatoprotective activities of such constituents are well known, there has been speculation that these constituents might be responsible for the observed hepatoprotective roles. From the overall results, it was concluded that CO exhibits antiradical and antioxidant activities, which could have a beneficial effect against oxidative liver damage induced by CCl₄.

Conclusion

Cold-pressed clove oil is a rich source of health-promoting antioxidants and possessed a hepatoprotective effect against CCl₄-induced toxicity in rats.

Declaration of interest

The authors report that they have no conflicts of interest.