Background. Lipotoxicity is characterized by a metabolic disturbance leading to the development of nonalcoholic fatty liver disease (NAFLD). Some medicinal plant extracts exert hepatoprotective activity by modulating oxidative stress, inflammation, and metabolic disorders. Scolymus hispanicus or the golden thistle can be considered an important natural source of antioxidants. In traditional medicine, the consumption of this plant is recommended for diseases of the liver and intestines. Objective. In this study, we aimed to determine the effects of Scolymus hispanicus on a hyperfatty diet- (HFD-) induced metabolic disorders, oxidative stress, and inflammation. Materials and Methods. Our experiment focused on the administration of an HFD (40%) in Rattus norvegicus for 2 months and treatment with the aqueous extract of Scolymus hispanicus at a rate of 100 mg/kg during the last eight days of experimentation. In this context, several aspects were studied: the evaluation of blood biochemical parameters, liver function such as lipids and glycogen, markers of oxidative stress (TBARS, carbonyl proteins, advanced oxidation proteins, catalase, and SOD) and inflammation (NO and NFkB), morphological study of hepatocytes in primary culture, and histological study of the liver. Results. Lipotoxicity induced metabolic disorders, both serum and tissue. HFD induced an increase in the total lipids and a decrease in glycogen reserve and an alteration in the oxidant-antioxidant balance. HFD induced an increase in markers of liver damage, which resulted in NAFLD, confirmed by histological study and hepatocytes cell culture. Scolymus appears to have lipid-lowering, hypoglycemic, anti-inflammatory and antioxidant properties. It improved glucose tolerance and the condition of fatty liver disease. Conclusion. Golden thistle improves glucose tolerance and hyperlipidemia and ameliorates hepatic steatosis by reducing oxidative stress, inflammation, and lipid accumulation. Its incorporation into a dietary program or as an aliment supplement would prevent hepatic complications associated with an HFD.
Overweight and obesity have become major global public health problems. Increasing consumption of more energy-dense, nutrient-poor foods with high levels of sugar and saturated fats and the increase in the availability of obesogenic ultraprocessed foods combined with reduced physical activity have increased obesity rates threefold or more since 1980 . Overnutrition leads to excess calories, which induce the installation of obesity, indicating an imbalance in the energy balance, which occurs when the calories ingested are greater than those spent by the body. The intake will be higher and the storage lipids will therefore be increased. The increase in the storage of lipids and lipid derivatives leads to the expansion of adipose tissue (hyperplasia and hypertrophy) and the installation of lipotoxicity, which has harmful effects resulting in nonalcoholic fatty liver disease (NAFLD), which is associated with obesity .
NAFLD was recently redefined as metabolic-associated fatty liver disease (MAFLD) to reflect better the pathogenesis . NAFLD is the most common chronic liver disease that affects around 25% of the population. NAFLD encompasses a broad spectrum of diseases that include simple fatty infiltration nonalcoholic steatohepatitis (NASH), which is defined as the presence of fat leading to inflammatory damage to hepatocytes, fibrosis, and finally cirrhosis. The importance of NAFLD lies in the possibility of its gradual progress to advanced fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) [2, 3]. The overall prevalence of NAFLD is growing in parallel with the global epidemic of obesity . The pathophysiology is complex and involves multiple concurrent mechanisms in the context of abnormal metabolic processes that arise mostly in individuals with risk factors. Comorbidities associated with NAFLD include obesity, type 2 diabetes (T2D), arterial hypertension, and dyslipidemia, as traits of metabolic syndrome (MetS) .
MetS is a clinical syndrome that includes obesity, dyslipidemia, arterial hypertension, and T2D . NAFLD is strongly linked with all segments of MetS and it is in fact liver manifestation of MetS. Some authors have suggested that NAFLD could be defined as a fifth component of the MetS . Both conditions were related to insulin resistance (IR), the main pathogenic factor underlying NAFLD and MetS. Abdominal fat overage is a fundamental determinant in NAFLD pathogenesis due to its association with IR and a possible source of free fatty acids (FFA) [3, 6]. Trunk fat was found to be indicative of elevated ALT, supporting the potential involvement of the metabolically active intra-abdominal fat in increased liver injury .
Obesity is associated with an increase in adipose tissue lipolysis, secretion of inflammatory, and fibrotic mediators, which can reach the liver. The accumulation of inflammatory/immune cells and the modification of the activities of these cells in the adipose tissue contributed to chronic low-grade inflammation during obesity [2, 3]. This sustained inflammation mediates IR and provides a contributing link between its development and NAFLD [2, 3]. The accumulation of hepatic diacylglycerol and the activation of inflammatory pathways are promoted. Diacylglycerols activate protein kinase ε and inhibit insulin signaling, leading to hepatic IR [2, 3]. The dysregulation of insulin-mediated control of hepatic production of glucose and lipids appears to be the main event in the development of NAFLD . Normally, insulin impairs gluconeogenesis while promoting lipogenesis. There is a paradoxical situation in NAFLD, especially in the context of T2D. IR results in a reduced ability to inhibit gluconeogenesis but insulin-driven lipogenesis still occurs and is even enhanced .
Varieties of natural products have been proposed as a pharmacological treatment of MetS and T2D. Scolymus hispanicus, the golden thistle species, is food source and can be considered an important natural source of antioxidants. The golden thistle (Scolymus hispanicus), locally known as “Guernina” or “Thaghadiwth,” is one of the most popular plants in Algeria, Spain, and other Mediterranean countries . In Algeria, we eat the petioles (“stems” of the leaf, or more exactly the main vein) cooked in the broth (red sauce with meat) that accompanies couscous.
Scolymus hispanicus has been linked to many medicinal properties such as diuretic, depurative, digestive, choleretic, and lithiuretic properties . Moreover, in traditional medicine, consuming this plant in the green or cooked state is recommended for liver and intestines diseases . The flaky stems are used for digestive tract care, bronchitis, and cold and have emmenagogic and antidiarrhoeal properties . The roots in decoction are recommended as an antidiabetic. Consumption of the ribs (main veins) of this plant fresh or cooked is recommended for liver and intestinal diseases . Other uses in the traditional medicine of golden thistle have been reported, such as in Malta fever and eye infection ; it can also be used as an appetizer and as a hemostatic agent . The antioxidant activity of Scolymus has been reported [10, 12].
Phytochemical analysis has demonstrated that the plant contains many biologically active compounds and a high content of α-tocopherol and identified 3 flavonoids (catechin, rutin, and tannic acid) and 13 phenolic acids, such as gallic acid, pyrogallol, chlorogenic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, salicylic acid, and rosmarinic acid resveratrol .
In this context, the present study aims to evaluate the effect of the aqueous extract of Scolymus hispanicus on HFD-related metabolic disorders, steatosis hepatic, inflammation, and stress markers.
2. Materials and Methods
2.1. Preparation of Aqueous Extract from Scolymus hispanicus
The aerial part of Scolymus hispanicus or the golden thistle was harvested in Algiers in February 2019. The voucher specimen (INA/P/No 54) has been preserved in the herbarium of the Botany Department, National Institute of Agronomy (INA), Algiers, Algeria. The stems and leaves of Scolymus were washed and separated from the roots, cut into small slices, dried, then added to 1000 mL of water, and left to boil for 50 min on a thermostated stirrer. After the cooling, the extract was filtered through muslin. The filtrate was centrifuged at 1500 rpm for 5 min and a second time at 2000 rpm for 10 min to obtain a homogeneous liquid. After the centrifugation, all samples were filtered through filter paper (Whatman with a pore size of 11 μm). The collected aqueous extract was then lyophilized (Cryodos 80, −75°C, 5 m³/h) to find an extract yield of 4.3%. The extract was stored in sealed glass vials at ± 4°C before being tested and analyzed.
2.2. Preparation of the Hyperfatty Diet
The hyperfatty diet at 40% was prepared in the cellular and molecular physiopathology team/BPO Laboratory/USTHB. According to the recommended nutritional intake, fats should not exceed 30% of the total daily energy intake to avoid unhealthy weight gain. In our study, we used a rate of 40% of lipids to confirm the installation of obesity with metabolic dysfunctions in the rats. The hyperfatty diet is based on cooked sheep fat; the cooking increases saturated fatty acids. The lipid intake in these rats is represented by 40 g of cooked sheep fat equivalent of 360 kcal; this fat is added to 60 g of the standard laboratory food equivalent of 186 kcal to constitute 100 g of food equivalent of 546 kcal. A daily diet of 20 g of hyperfatty food provides 109.2 kcal/day.
This study was carried out on 28 female rats of the Rattus norvegicus with average weights of 111.33 ± 27.66 g, which were reared at the animal facility of the Faculty of Biological Sciences, USTHB, with controlled temperature (22 ± 1°C), lighting (12-hour dark/light cycle), and free access to food and water.
The animals were divided into 4 groups:(1)Control batch: seven control rats subjected to a standard laboratory diet for 2 months of experimentation. The feed was provided by the National Animal Feed Office; the calories intake contained in 20 g of food is 62 calories.(2)Control batch treated with the aqueous extract of Scolymus hispanicus at a rate of 100 mg/kg of body weight/day during the last eight days of experimentation by intraperitoneal injection (7 animals).(3)Batch subjected to a hyperfatty diet (HFD) at 40% for two months with a daily intake of 20 g per rat. The calorie intake contained in 20 g of food was 109.2 calories.(4)Batch subjected to an HFD and treated with the aqueous extract of Scolymus hispanicus at a rate of 100 mg/kg of body weight/day during the last eight days of experimentation by intraperitoneal injection while maintaining the hyperfatty diet (7 animals).
2.4.1. Chemical Study
(1) Total Phenolic Content. The content of total polyphenols in the aqueous extract of Scolymus hispanicus was determined using the Folin–Ciocalteu reagent according to the method of Singleton et al., using gallic acid as a reference . An aliquot of the aqueous (0.2 mL) extract contains 1000 μg of Scolymus mixed with 46 mL of distilled water and 1 mL of Folin–Ciocalteu reagent in a volumetric flask. The mixture was incubated for 3 min in the dark. After that, 3 mL of sodium carbonate solution (7.5%) was added to the mixture. After 2 hours of incubation in the dark, the absorbance was measured at 740 nm in a spectrophotometer (Shimadzu 1800, Mulgrave, Victoria, Australia). The total phenolic content was evaluated from a standard calibration curve of gallic acid, and the results were expressed as micrograms of gallic acid (GA) equivalents (E) per milligram of extract (µg GAE/mg).
(2) Determination of Total Flavonoids. The total flavonoids were determined according to the modified method described by Lebreton et al. using quercetin as a reference . Four milliliters (4 mL) of dilution solution was mixed with 4 mL of aluminum trichloride solution (2% in methanol). After 15 min of incubation, the absorbance was measured at 415 nm. Quercetin (Q) was used as a reference compound to produce the standard curve. The results were expressed as μg QE/mg.
(3) Antioxidant Activity: Scavenging Effect on DPPH Radical. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was carried out as described by Brand-Williams et al. . It is based on the degradation of the DPPH radical dissolved in an 80% methanol/water mixture. An antioxidant will have the ability to donate an electron to the synthetic radical DPPH (purple coloration) to reduce it to nonradical DPPH (yellow-green coloration). The aqueous extract was dissolved in methanol. A sample of 25 μL of each concentration (100, 200, 400, 600, 800, and 1000 μg/mL) was added to the DPPH methanol solution (60 μM, 975 μL). After 30 min of incubation at 25°C, the absorbance at 517 nm was measured by UV spectrophotometer (Jasco, V-530). Ascorbic acid and α-tocopherol were used as compounds reference. The radical scavenging activity was then calculated using the following equation: % of radical scavenging activity = ((Abs control−Abs sample)/Abs control) × 100, where Abs control is the absorption of the blank sample and Abs sample is the absorbance of the tested extract.
2.4.2. Biological Study
(1) Analytical Techniques. The animals were bled from the retroorbital venous plexus; this technique eliminates using anesthetic agents affecting measurements of biochemical parameters. Blood, which was collected in dried tubes, was centrifuged at 3000 rpm for 10 min and the sera were stored at −20°C. Blood glucose, triglycerides, cholesterol, and transaminase were measured by enzymatic colorimetric method using a test kit of Biosystem. Blood insulin was determined by radioimmunoassay using a CIS test kit (ORIS INDUS). The evaluation of the redox status was performed in the sera and erythrocytes by assaying the thiobarbituric acid reactive substances (TBARs) and catalase.
(2) Oral Glucose Tolerance Test (OGGT). The oral glucose tolerance test (OGTT) measures the clearance of glucose from the body after its absorption from the intestinal tract. All rats were weighed one day before the test for the calculation of the glucose solution to be administered. The glucose solution (40%) was administered by intraperitoneal injection. Rats received 2 mg of glucose/g of body weight [17, 18].
The rats were fasted for a period of 14 to 16 hours with free access to water. A blood sample was taken from a small incision in the tail using a scalpel to measure basal blood glucose level (=time point 0) with the glucometer vital check [17, 18]. Once basal glucose concentrations were measured in all rats, the glucose solution was given to each animal by intraperitoneal injection. The timer was immediately started after the first administration of glucose to all rats. After 30 min, the blood glucose was measured using a glucometer of each rat in the same order as they were injected. This operation was repeated in 60, 90, and 120 min after glucose administration [19, 20].
(3) Organs Harvesting. At the end of the experiment, animals were sacrificed after anesthesia by intraperitoneal injection of urethane. The liver removed was divided into five fragments, and each fragment was weighed. They were intended for different assays, including total lipids where the fragment is immersed directly in Folch solution. Another fragment was bound in paraformaldehyde at 10%, and the other three fragments were frozen directly in liquid nitrogen to evaluate redox status, inflammatory markers, and hepatic glycogen. Two animals from each batch were kept for the initiation of hepatocyte cell culture.
(4) Histology of the Liver. After fixation in paraformaldehyde at 10% for 24 h, the specimens of liver were dehydrated and embedded in paraffin and cut at 5 μm. The sections were stained with Masson’s trichrome .
(5) Hepatic Glycogen. The principle of the method consists in hydrolyzing the glycogen extracted from the liver of rats into glucose with an acid and determining the amount of the formed glucose using the Folin and Wu method . Concentrations were deduced from a standard curve prepared with standard glucose solution and the amount of glycogen was expressed per 100 g of liver.
(6) Total Lipids. The extraction was carried out according to the method of Folch et al. . The lipids were extracted using chloroform/methanol (2 : 1 ). The total lipids were estimated in mg/100 g of liver.
(7) Oxidant and Antioxidant Activity.(i)Catalase Activity Assay. The enzymatic activity of catalase was determined using the method of Claiborne . The principle was based on the disappearance of H2O2 in the presence of the enzyme source at 25°C. Catalase was evaluated in sera, erythrocytes, and liver of all animal groups. Absorbance was estimated at 240 nm in two time points, t0 and after two min. Erythrocytes and liver were lysed, before all assays, in a lysis buffer .(ii)Superoxide Dismutase (SOD) Activity Assay. The evaluation of the SOD activity was performed according to the method of Giannopolitis and Ries .(iii)Thiobarbituric Acid Reactive Substances Assay (TBARs). After the reaction with thiobarbituric acid (TBA) (Sigma) , the TBARs were measured in sera, erythrocytes, and liver. The MDA contained in the supernatant in the presence of 10% trichloroacetic acid reacted with TBA and caused the formation of a red complex estimated at 532 nm.(iv)Protein Carbonyl Assay. Protein carbonyls (PC) were measured in the liver of all animal groups according to the procedure described by Reznick and Packer  using dinitrophenylhydrazine (DNPH) reagent and spectrophotometric method. The absorbance was measured at 370 nm. The results were expressed as nanomoles of carbonyl groups per milligram of protein using a molar extinction coefficient of 22 000M⁻¹ cm⁻¹.(v)Advanced Protein Oxidation Products Assay. The determination of advanced protein oxidation products (AOPP) levels was performed in the liver by modifying the Witko-Sarsat method . The absorbance of the reaction mixture was immediately estimated at 340 nm. AOPP concentrations were expressed as micromoles/L of chloramine-T equivalents .
(8) Measurement of Inflammation Markers.(i)Nuclear Factor-Kappa B (NFκB). The assessment was determined by immunoenzymatic assay. Invitrogen ELISA kits were used for measuring the levels of the NF-kB p65 in the liver of all groups. The estimation was made by Elisa reader at 450 nm (BioTek Instruments).(ii)Nitrogen Monoxide Assay (NO). The determination of nitrite and nitrate was evaluated from supernatants of the liver of different groups. The nitrite bearing in all samples, which were deproteinized and regenerated, was quantified after addition of Griess reagent (0.1% N-(1naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 5% phosphoric acid). Absorbance was measured at 543 nm .
(9) Perfusion and Isolation of Hepatocytes. This technique was carried out using the modified method of Severgnini et al. and Edwards et al. [32, 33]. All steps were performed under sterile conditions.
After anesthesia of the animals by intraperitoneal injection with urethane, insert the cannula in the portal vein and start the perfusion using a peristaltic pump containing phosphate-buffered saline (PBS) at pH 7.2. As soon as the infusion starts, immediately cut the hepatic vein to allow perfusate to run as waste.
The flow was maintained at 5 mL/min for 15 to 20 min thanks to the peristaltic pump to remove the blood completely in each lobe. A second solution at pH 7.4 containing trypsin replaces PBS for tissue digestion. At this stage, the hepatic tissue was rapidly disaggregated.
The liver was collected with a curved spatula and was transported in a sterile Petri dish containing DMEM + fetal calf serum (FCS), where we proceeded with the disruption of the tissue proceeded using a scalpel. This step should be fast in order to avoid damage to hepatocytes.
The medium containing the cells was recovered, followed by centrifugation at 600 rpm for 5 min at room temperature. The supernatant was subsequently removed and the cells were suspended a second time in 30 mL of Percoll cushion at 37.5% for recovering viable cells. Another centrifugation was effectuated for 3 minutes at 1000 rpm at room temperature.(i)Hepatocyte Culture and Microscopy. The viable cells recovered were suspended again in 2 mL of DMEM; the hepatocytes are observed with an inverted microscope after staining with trypan blue. The cells were distributed in flasks, which were adjusted to 5 mL of DMEM supplemented with FCS, L-glutamine, and antibiotics, and they are incubated in a CO2 incubator (Memmert) (5% CO2, 95% air) for the start of the primary culture. After 48 h of incubation, we noted the confluence of the cells. Trypsinization was necessary to perform the first passage [32, 33].
2.4.3. Statistical Analysis
Data were analyzed with ANOVA using STATISTICA version 6 and completed with HSD Tukey’s test. The results were expressed as the mean ± standard deviation. The differences at were considered to be statistically significant.
3.1. Phytochemical Study of Scolymus hispanicus
The aqueous extract of Scolymus hispanicus showed a high content of total polyphenols and flavonoids (Table 1). The antioxidant activity of the aqueous extract of Scolymus hispanicus was evaluated using the DPPH free radical scavenging test. Our extract showed a very important antifree radical activity with an IC50 value of 0.0038 µg/ml, which was extremely higher than the reference values BHA and BHT (21.18 ± 0.12 µg/mL and 12.66 ± 0.18 µg/mL, respectively) (Table 1).
Total phenolic content (µg GAE/ mg)
Total flavonoids (ug QE/mg)
DPPH (IC50) (µg/mL)
270.321 ± 25.44
164.94 ± 9.45
21.18 ± 0.12
12.66 ± 0.18
Each value was expressed as means ± standard deviations for triplicate experiments. n.a.: not applied. Q: quercetin; QE: quercetin equivalents; GA: gallic acid; GAE: gallic acid equivalents; BHA: butylhydroxyanisole; BHT: butylhydroxytoluene.