Improvement on high-cholesterol diet induced atherosclerosis, lipid profile, oxidative stress and genotoxicity in the liver of mice by Echinops spinosissimus Turra subsp. spinosus CURRENT STATUS: UNDER REVIEW

Background Hypercholesterolemia is a major risk factor for the development of atherosclerosis and endothelial dysfunction. Methods The present study investigates the possible mechanism of Echinops spinosissimus Turra subsp. spinosus ( E. s. spinosus ) flower on the high cholesterol diet. Results Our in vitro results demonstrated the richness of E.s. spinosus flower in antioxidant compounds, and its antioxidant activities. The co-administration of E.s. spinosus (100 or 200 mg/kg/day) with high-fat diet attenuated hepatotoxicity as monitored by the improvement of oxidative stress biomarkers and plasma lipid and liver parameters, when compared to the hypercholesterolemic mice. Atherogenic index and body weight were also reduced markedly, compared to control mice. These results were confirmed by the improvement of histological changes and DNA damage. Conclusion These data indicate that E.s. spinosus flower reduces the hypercholesterolemia risk and atherogenic properties of dietary cholesterol. Its hypocholesterolemic effect may be due to its antioxidant activities and its richness in bioactive molecule.


Introduction
Hypercholesterolemia is a dominant risk factor for the development and progression of atherosclerosis, related cardiovascular diseases, and many lipid associated ailments like obesity, liver and kidney failure [1,3]. Epidemiological, clinical, genetic, and experimental studies indicate that a diet high in cholesterol content is a major environmental contributor to an unbalanced lipoprotein [4].
The effect of hypercholesterolemia in vivo has been studied with different animal species. Sparks et al. [5] reported for the first time that hypercholesterolemia affects β-amyloid in rabbits [6,7].
Recently, Granholm and colleagues [8] demonstrated that hypercholesterolemia in rats increase the number of working memory errors. It has been reported that high levels of fat increase fat-mediated oxidative stress and decrease antioxidant enzyme activities [9].
Based on this evidence, many therapeutic agents are available for the management of hypercholesterolemic patients and are employed to promote successful treatment. A number of studies have demonstrated that the use of lipid-lowering drugs can reduce the number of cardiovascular events and mortality from coronary disease [10]. However, due to certain resistances to dietary restriction and financial limitations to use lipid-lowering drugs, many individuals have turned to alternative treatments to control cholesterol levels. Many of these alternative treatments have been used empirically, lacking scientific studies that would allow for more reliable conclusions [11]. In recent years, there has been an increasing interest in antioxidants which play a crucial role in food industry and in providing protection for humans against infections and degenerative diseases [12]. Many studies describe the crucial role of the most important group of secondary metabolites, flavonoids and phenolics compounds, as natural antioxidants in health promotion by reducing the risk of hyperlipidemic diseases and protecting biological systems against the harmful effects of oxidative processes [13].
Echinops spinosissimus (Asteraceae) is a traditional medicinal plant that is abundant in the desert of Egypt and Tunisia [14]. Many researches have subdivided Echinops spinosissimus Turra into two subspecies: Echinops spinosissimus Turra subsp bovei and Echinops spinosissimus Turra subsp.
spinosus (E. s. spinosus) [15]. The latter is drought resistant specie of high medicinal value. E. spinosissimus has been highly reputed as a diuretic, nerve tonic and cough suppressant, and contains a range of sesquiterpene lactones migraine, diarrhea, intestinal worm infestation and hemorrhoids [16]. This specie has high medicinal value due to the presence of functional flavonoids and phenols.
Concerning the genus E. s. spinosus, the ethanolic extract has efficient action on muscular fibers and exhibited a very good anti-inflammatory activity [16]. It was known for its therapeutic effect on health and its extensive anecdotal history in traditional medicines [17]. The Gas chromatography and Mass spectrometry analysis demonstrated that the plant is a potential source of terpenoid compounds and acetylated sterols [17].
The diet-induced hypercholesterolemia animal model has long been used for the assessment of agents with beneficial effects on cholesterol. Therefore, the present investigation was performed to document the effects of E. s. spinosus flower on a high cholesterol diet in mice.

Preparation Of Extracts
The dried flower heads of E. s. spinosus (500 g) were extracted sequentially by maceration in hexane, dichloromethane, ethyl acetate (EtOAc), and MeOH at room temperature, for 48 hours. After filtration, the solvents were evaporated under vacuum at 50 °C to afford 6.9, 4.9, 7.3 and 19.1 g, respectively.

Determination Of Total Phenolic Content
The amount of total phenolic content was evaluated using a slightly modified colorimetric method described by Singleton and Rossi [18] using the Folin-Ciocalteu reagent. The absorption was measured at 760 nm by spectrophotometer. The estimation of the phenolic compounds was carried out in triplicate and the results was expressed as gallic acid equivalents (GAE, mg gallic acid/per g of extract).

Determination Of Total Flavonoid Content
Total flavonoid contents of flower heads E. s. spinosus were determined using the aluminium trichloride colorimetric method [19]. The total flavonoid content was expressed as mg of quercetin (QE)/g of extract.

Phytochemical Studies
All the plant extracts were subjected to preliminary phytochemical screening following the standard methods [20], to detect the presence of active principles such as sterols, triterpenoïds, tropolone, quinones, alkaloïds, and flavonoïds.
A sample of each extract is dissolved in 2 mL of solvent adequate then will be added to various reagents according to the methods cited below: Test for sterols and triterpenoïds: (Reaction of Liebermann). 1 mL of acetic anhydride and few drops of concentrated sulphuric acid were mixed to the extracts solution and a violet changing to blue-green indicate a positive test for sterols and (or) triterpenoïds.
Test for flavonoïds: (Shinoda test). The stock extract solution (1 mL) was taken in a test tube and added to 1 mL of EtOH, 1 mL of distillated water and a few drops of dilute Hydrochloric acid. A positive reaction was recorded when an intense red violet coloration was appeared which become after blue.
Test for alkaloïds: (Mayer Reaction). Test solution (1 mL) when treated with 0.5 mL of HCl (0.1 N) and 5 drops of Mayer reagent shows immediately a white precipitate showed the presence of alkaloïds.
Test for tropolone: (Reaction of Wiustater). It was manipulated by adding MeOH (0.2 mL), 1 drop of FeCl 3 (0.005 M), water (0.6 mL) and CHCl 3 (0.4 mL) to 1 mL of our test solution. A red colour of the chloroform layer was formed which showed the presence of tropolone.
Test for quinones: (Reaction of Borntraeger). To detect the presence of quinones in our extracts, 2 mL of each solution sample was mixed with 2 mL of NaOH (0.1 M). The changement of coloration in the aqueous phase from red to purple would show a positive result for the presence of quinones.

Antioxidant Capacity Estimation
Three methods of antioxidant assessment, β-carotene bleaching by linoleic acid assay, Diphenylpicrylhydrazyl (DPPH) radical scavenging and ferric-reducing anti-oxidant power were used for investigation of antioxidant activities of E. s. spinosus flower heads extracts. These results were compared to those of α-tocopherol, BHT and gallic acid used as positive control.

Ferric-reducing antioxidant power (FRAP) assay
The FRAP method measure the ability of an antioxidant to donate electron to Fe (III), which could be monitored at 700 nm. This assay was carried out according to the procedure employed by Oyaizu with slight modifications [21]. Absorbance of the reaction mixture was read spectrophotometrically at 700 nm.
FRAP result was expressed as gallic acid equivalents (GAE) in mg/g of tested sample. y = 0.775 x + 0.117; r 2 = 0.906 DPPH free radical scavenging assay The antioxidant activity of DPPH is based on scavenging of DPPH. from antioxidants in the vegetal sample, which produce a spectrophotometric loss in absorbance at 515 nm. The DPPH assay was evaluated as described by [22]. The mixture was prepared in test tubes by dilution of 50 µL of ALE in 735 mL of 100% methanol. 750 mL of 0.1 mM methanolic DPPH reagent was added to the mixture of ALE-methanol. Then, the mixture was incubated at room temperature in a chamber without any light during 30 min. After incubation, the estimation of the scavenging ability was performed by measuring at 517 nm in spectrophotometer (T70 UV-Vis).
The capacity of inhibition percentage (PI) of DPPH radicals was calculated as

DPPH radicals (PI) = [( -)/A b ] × 100
Where refers to the absorbance of control (without plant extract) and to the absorbance of sample (with plant extract).
BHT was used as standard at the same concentrations of plant extracts.
Antioxidant assay using the β-carotene bleaching method The antioxidant activity of the E. s. spinosus flower was evaluated using β-carotene bleaching method as described by Chevolleau et al. [23] with some modification. All determinations were carried out in triplicate.
The antioxidant activity coefficient (AAC) was calculated according to the following equation: Absorbance of control (0min) -Absorbance of control (120min)

Animal Diet And Tissue Preparation
This study was performed in accordance with the Institute Ethical Committee for the Care and Use of Laboratory Animals guidelines [24] and Sciences Faculty of Sfax (n°1204) [25]. Adult mice (30 ± 5 g) were randomly divided into four groups of 12 animals each; group received either no treatment

Protein Quantification
Protein content in the liver was assayed following the methods previously described by Lowry et al [26].

Determination of Oxidative Stress Markers
We determined the amount of malondialdehyde (MDA) in the liver using 1,1,3,3-tetraethoxypropane and following the methods described by Draper and Hadley [27]. The values are expressed as nmol MDA/mg protein.
Advanced oxidation protein products (AOPPs) were assayed using the method described by Witko [28]. The level of AOPP in liver tissue was calculated using an extinction coefficient of 261 and expressed as µmol/mg protein.
Protein carbonyls (PCO) were measured using the method of Reznick and Packer [29]. The absorbance of the sample was measured at 370 nm. The carbonyl content was calculated based on the molar extinction coefficient of DNPH (5 2.2 3 104 cm/M) and expressed as nmoles/mg protein.
Superoxide dismutase (SOD) enzyme activity was measured using the method described by Beauchamp and Fridovich [30]. One unit of SOD activity was defined as the amount of enzyme required to cause a 50% inhibition of nitro blue tetrazolium photoreduction. SOD activity in cerebellar tissue is expressed as units/mg protein.
Glutathione peroxidase (GPx) enzyme activity was measured using the method described by Flohe and Gunzler [31]. We calculated the modification in absorbance at 340 nm. GPx enzyme activity is expressed as nmol GSH/min/mg protein.
Liver reduced glutathione (GSH) contents were determined by Ellman's method [32], and modified by Jollow et al. [33], based on the development of a yellow color when 5,5-dithiobis-2 nitro benzoic acid was added to compounds containing sulfhydryl groups. The absorbance was measured at 412 nm after 10 min. Total reduced glutathione content was expressed as nmol/mg of protein.

Molecular Analysis
DNA samples required for the DNA fragmentation analysis of normal and experimental mice were isolated from liver tissues by the method described previously by Kanno et al. [35]. The DNA fragmentation assay was performed by electrophoresis on genomic DNA samples using agarose/EtBr gel following the procedure described by Sellins and Cohen [36].

Histopathological Examination
Some liver tissue collected from each group was randomly selected for light microscopy. Samples were fixed in formalin solution and embedded in paraffin. The liver was then sectioned in the sagittal plane and stained with hematoxylin-eosin [37]. The nonalcoholic steatohepatitis (NAS) calculation system was applied to evaluate the steatosis, inflammation, and ballooning [38].

Statistical Analysis
All experiences and statistical analyses were made in triplicate. All results are expressed as the mean ± standard deviation. Statistical analysis was performed with SPSS 17.0 statistical package for Windows (SPSS, Inc., Chicago, IL). A two-way ANOVA followed by Tukey's post-hoc test was performed to compare treatment and control groups. Statistical significance was set at α = 0.05.

Results
In vitro antioxidant activity of E.s.spinosus flower

Amounts Of Polyphenols And Flavonoids
The obtained values for total phenolic (TPC) and flavonoid contents are summarized in Table 1. The methanolic extract was found to have a high amount of phenolic content (895.14 mg gallic acid/g of extract) and the ethyl acetate extract has been found to be rich in flavonoids (215.36 mg quercetin/g of extract).

FRAP assay
The reducing power assay is often used to evaluate the ability of an antioxidant to donate an electron. In this assay, the ability of E.s.spinosus extracts to reduce Fe 3+ to Fe 2+ was determined.
Reducing power increased with the concentration of the extracts. All analyzed samples demonstrated significant antioxidant capacities with FRAP test except the hexane and dichloromethane extracts of this plant. In fact, the methanolic extract showed the highest ability to reduce Fe 3+ (Fig. 2).
-Carotene Bleaching Assay The antioxidant activity through β-carotene-linoleate system of the four extracts of E.s. spinosus flower is measured by the ability of a compound to minimize the loss of -carotene during the oxidation of linoleic acid in an emulsified aqueous system. The antioxidant activity coefficient (AAC) was compared with butylated hydroxyanisole and it is presented in Table 3. Results presented in Fig. 3 indicated that body weights of hypercholesterolemic mice were significantly increased, while body weight of the group co-treated with E.s.spinosus flower methanolic extract was similar to that of control group.

Effect of E.s.spinosus flower on lipid peroxidation, and protein oxidation in the liver
Our findings revealed changes in the levels of lipid peroxidation products in the experimental groups.
In the hypercholesterolemic mice, MDA level, index of lipid peroxidation showed a significant increase (P = 0.003). Similarly, a remarkable rise in AOPP and PCO levels in the liver was also evident in the hypercholesterolemic group, when compared with controls (P < 0.001; P = 0.076) ( Table 4).
Supplementation of E.s.spinosus flower methanolic extract to the hypercholesterolemic group ameliorated all parameters cited above.

Effect of E.s.spinosus flower on enzymatic and non-enzymatic antioxidants in the liver
Our results showed the effect of Echinops spinosus methanolic extract on enzymatic and nonenzymatic antioxidants of control and experimental animals. ANOVA demonstrated a significant rise (p < 0.001) in the activities of enzymatic antioxidants (SOD and GPx) (  Effect of E.s.spinosus flower on some plasma biomarkers of liver toxicity Bilirubin level as well as AST and ALT activities were significantly increased (P < 0.001), respectively, in the hypercholesterolemic group, when compared with the controls (Table 6). Co-administration of Echinops spinosus methanolic extract to the hypercholesterolemic group significantly decreased plasma bilirubin, AST, and ALT to near normal values, as compared with the hypercholesterolemic mice.

Liver histopathological analysis
Histopathological examination of control group (Fig. 4A) showed unremarkable changes with normal architecture and appearance of the central vein with a radiating pattern of cell plates that were normal in shape and size. The hypercholesterolemic regime induced degenerative changes, such as the hepatic steatosis, leucocytes infiltration, and hepatocyte vacuolization (Fig. 4B). The administration of E.s.spinosus flower to the hypercholesterolemic provoked a marked improvement in the hepatocyte structure ( Fig. 4 (C,D).

Discussion
are prevalent in modern societies [39]. Hyperlipidemia is commonly associated with atherosclerotic vascular diseases and is present as a risk factor for many diseases [10,40] [42,43]. Where, HDL-C is a free radical scavenger and prevents peroxidation of beta lipoproteins [44]. The reduction in HDL following cholesterol feeding may be due, also, to contributed acceleration of apoA-I clearance from the plasma based on cholesterol-enriched diets [45]. The high level of LDL-cholesterol found in hypercholesterolemic rats may be attributed to a down regulation in LDL receptors by cholesterol and saturated fatty acids included in the diet [46]. The hypercholesterolemic mice exhibited a profound increase in atherogenic index as compared to normal ones. This atherogenicity thought to be due to the atherogenic lipoprotein subclasses commonly associated with hyperlipidemia [47]. Our result indicated that, treatment using E.s.spinosus flower exhibited significant decrease of AI, as compared to hypercholesterolemic mice that might be ascribed to their plasma lipid-lowering activity. The E.s. spinosus showed high anti-hyperlipidemic activity in mice. This could be explained by the richness of the plant in polysaccharides and β-carotene. In fact, Godard [43] has reported that polysaccharides are strongly liable with the observed hypolipidemic effects. Shaish et al. [48] demonstrate that βcarotene is responsible for an increase in HDL-cholesterol.
Other biomarkers of liver toxicity like AST and ALT [49] were also studied in the present work. Plasma AST and ALT activities were significantly high in high-cholesterol fed diet than in normal mice; these results agreed with those of Sudhahar et al. [50]. The increase of these activities suggested their leakage from the liver to the plasma [51]. The biochemical alterations were correlated with a marked increase in liver lipid and protein oxidation. The lipid peroxidation, measured as thiobarbituric acid reactive species, is a free-radical mediated propagation of oxidative insult to polyunsaturated fatty acids involving several types of free radicals [52]. The hypercholesterolemic mice of the present study exhibited high significant elevation of hepatic MDA, AOPP and PCO concentrations as compared to normal control group. An increase of lipid peroxidation, in animals fed with a high cholesterol diet has been previously reported [53,54]. There was a positive correlation between plasma total cholesterol and triacylglycerol concentrations and free radicals generation [55]. An abnormal rise in lipid and protein oxidation was reduced with plant administration, due to their antioxidant activities and this antioxidant compounds, emphasized through in vitro experiments. However, the obtained results could be attributed to the reported antioxidant effects of E.s.spinosus flower which in turn lead to decreased free radical generation and decreased oxidative damage of the liver, the main organ involved in cholesterol biosynthesis. Indeed in the current study, oxidative stress was obvious in cholesterol rich diet fed-mice as evidenced by the increase in plasma lipid peroxide level coupled with decreased SOD, GPx and catalase activities. Similar results were previously reported [56].
Supplementation with E.s.spinosus flower was found to increase the activity of the antioxidant enzymes respectively, as compared with the hypercholesterolemic mice. A reduction of these enzymes activities is associated with the accumulation of highly reactive free radicals, leading to deleterious effects such as loss of integrity and function of cell membranes. When they are present in high concentrations, free radicals are able to interact with the enzymes and inactivate them.
Apart from enzymatic antioxidants, non-enzymatic antioxidants and hepatic reduced glutathione play a vital role in protecting cells from oxidative damage. GSH in hypercholesterolemic mice decreased significantly when compared with the normal ones. These reductions may be due to the increased utilization of these anti-oxidants for quenching enormous free radicals produced during hypercholesterolemic condition. Thus, the increased GSH content in hypercholesterolemic group may be attributed to the ability of E.s.spinosus flower to improve the defensive nature of liver against free radicals. Free radicals also attack DNA bases, therefore causing mutagenic lesions. In fact, hypercholesterolemic diet resulted in a significant DNA fragmentation with a subsequent formation of a DNA smear on agarose gel.
The damage induced by hypercholesterolemic diet in the liver of adult mice was confirmed by histological changes, including a marked leucocyte infiltration, steatosis, and apoptosis. In fact, hypercholesterolemic diet caused several liver histological injuries such as distortion in tissue histoarchitecture, congestion of the central vein, sinusoidal dilatation, generalized congestion, hemorrhage, and degenerative changes. Co-treatment of mice with E.s.spinosus flower could prevent DNA and histological damages in hypercholesterolemic mice due to its powerful antioxidant capacity.
In conclusion, the results obtained herein indicate that Echinops spinosissimus Turra subsp. spinosus a protective effect on high-cholesterol diet induced liver damage in mice, possibly through its antioxidant properties. We therefore propose that Echinops spinosissimus may be beneficial in reducing hypercholesterolemia in patients, but further studies are required to determine the optimal doses of this compound.

Conflicts of interest
The authors declare that there are no conflicts of interest.