Green coffee methanolic extract and silymarin protect against CCl4-induced hepatotoxicity in albino male rats

The chemicals used in this study were of molecular biology and diagnostic grade. They were purchased from Sigma (St. Louis, MO, USA). Pure virgin olive oil was purchased from a local market. Silymarin was purchased from SEDICO Company, Egypt.

Dried seeds of green Brazilian coffee were purchased from a local market. The green coffee seeds were identified by Prof. Haddad El Rabey (Botanist). A specimen was deposited at the herbarium of Faculty of Science, Botany Department, Mansoura University, Egypt (Accession No. #FSH00134–2019).

The seeds were milled to powder using a mill. A 200 g of the green coffee powder was added to 1 liter methanol (90%), agitated on a shaker at room temperature for 5 days. The methanol was completely evaporated using a rotary evaporator attached to a vacuum pump. For the 20% methanolic extract preparation, 20 g of the extract was mixed with 2 ml of tween 80 (suspending agent), and then the mixture was suspended in 100 ml of distilled water according to the method described by Adebayo et al. [30].

A total of 30 adult Sprague–Dawley strain albino male rats (weighing 159.86 ± 6.96 g) were purchased from the Agricultural Research Center, Giza, Egypt. The rats were housed in polypropylene cages (6/cage). Before the beginning of the experiment, the animals were kept under observation (to be sure that they are healthy), and subjected to acclimatization for the laboratory environment during 1 week. Room temperature and humidity were adjusted at 23 °C and 60%, respectively. Also, the light cycle was fixed at 12 h. The guidelines of the National Institutes of Health (NIH) for the care and use of laboratory animals (NIH Publication, Number 85–23, Revised 1985) were applied to all animal experiments of this study. Also, this study was ethically approved by The Institutional Research Board (IRB) Committee of Mansoura Faculty of Medicine, Mansoura University, Egypt. During the acclimatization period and the whole experiment, food and water were given ad libitum. The acclimatized rats were divided into five groups as follows: negative control group (G1) that received only the vehicle (olive oil), and the other four rat groups received a single dose of CCl4 (1 ml/kg bw) dissolved in olive oil (1:1 volume) in the 1st and 4th day every week of the experiment through an intraperitoneal (IP) injection [6] then they were divided into four groups as follows: untreated positive control group (G2), and three treated groups (G3, G4 and G5) with IP. The third group (G3) was treated with a daily dose of 5 ml/kg of 20% green coffee methanolic extract [7] using oral gavage, the fourth group (G4) was treated with 0.05 g/kg of silymarin [7] and the fifth group (G5) was treated with 0.05 g/kg of silymarin and 5 ml/kg of 20% green coffee methanolic extract, respectively for 30 days. All animal experiments were approved by The Ethical Committee of Mansoura University.

The rats were euthanatized by 2.5% halothane (1:20 halothane vapor: air ratio, flow rate was 70%V/min) at the end of the experiment and blood samples were collected, centrifuged for 5 min at 3000 rpm, and then the serum was transferred into clean tubes for biochemical analysis.

The liver was dissected out, washed with saline solution, divided into two parts, the first part was fixed in 10% formalin for the histological examination and the second part was homogenized in ice-cold phosphate buffer (pH 7.4), and then centrifuged at 4000 rpm for 15 min. Lipid peroxidation (MDA) as a marker of oxidative stress and antioxidant biomarkers were determined in the supernatant.

The activity of all liver function enzymes was estimated in serum using Sigma Aldrich commercial kits from Sigma Aldrich Company (USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were estimated as described by Reichling and Kaplan [31], while alkaline phosphatase activity was estimated as described by Mossner et al. [32]. Total cholesterol (TC), Triglycerides (TAG), high-density lipoprotein (HDL), and low density lipoprotein (LDL) levels were measured according to the manufacturer protocols of bioMerieux kit (France). Serum total protein, bilirubin and albumin were determined according to the protocol of Diamond (Germany). Serum urea, uric acid and creatinine were estimated as described by Kanter [33], Fossati et al. [34], and Bonsens and Taussky [35], respectively using a Human kit (Germany).

Irisin levels were determined in serum using Cono Biotech Co. Ltd. kit (China), interleukin-6 (IL-6) was determined using MyBiosource Kit (San Diego, USA), whereas cytochrome P450 (CYP 450) was determined using Abbexa kit from Cambridge (UK), according to the instructions of the suppliers.

Liver homogenates were used for the determination of catalase (CAT), superoxide dismutase (SOD), and glutathione transferase (GST) activities, as well as total antioxidant capacity (TAC), and reduced glutathione (GSH) and malondialdehyde (MDA) levels using Biodiagnostic Kit (Egypt), according to the method described by the supplier.

The liver tissue was immediately fixed in 10% formalin, dehydrated in ethanol (70, 80 and 90%), cleared in xylene, embedded in paraffin, and then sectioned by a microtome. The sections were then stained with hematoxylin and eosin (H&E) as described by Bancroft and Stevens [36].

The obtained data were expressed as means ± standard deviation and they were analyzed by one-way analysis of variance (ANOVA statistical measure). Duncan’s test was used as a post hoc test for comparison of significance between groups using the statistical package program (SPSS) version 17.0 [37]. The SigmaPlot software (AUTOSIGNAL V1.7) was used in plotting graphics.

Table 1 showed that serum AST, ALT, and ALP activities, as well as bilirubin, creatinine, uric acid, urea, cholesterol, triacylglycerol, and low-density lipoprotein levels were significantly increased, whereas albumin, total protein and high-density lipoproteins levels were significantly decreased in the positive control group (G2) compared to the negative control group (G1). However these parameters were partially restored to their normal values approaching the normal values in the treated groups (G3, 4 and 5) compared to G2. The combination of green coffee methanolic extract and silymarin in G5 was more efficient in reducing the activities of all liver function enzymes compared to the other groups (G3 and G4).

Figure 1(a,b,c,d,e), (and supplementary Table 1) showed that CCl4 toxicity in the positive control group (G2) significantly decreased the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST), total antioxidant capacity (TAC), and the level of reduced glutathione (GSH) in the liver tissue homogenate. However, these markers were increased in rats treated with green coffee methanolic extract, silymarin and their synergic combination (G1, G2 and G3, respectively) compared to the positive control group in G2

.

Figure 1f (and supplementary Table 1) also showed that; CCl₄ toxicity in the positive control group (G2) significantly elevated the amount of lipid peroxidation (MDA) when compared to the negative control in G1. Whereas treating with green coffee methanolic extract, silymarin and their combination in G3, G4 and G5, respectively significantly reduced this high peroxidation level, although still significantly higher than that of the untreated negative control group (G1). Also, the combination of green coffee methanolic extract and silymarin in G5 was more efficient in reducing lipid peroxidation and increasing antioxidants compared to other groups (G3 and G4).

Figure 2 (a,b,c) (and supplementary Table 2) showed similar findings in G2. A significant decrease in the levels of both irisin and CYP450 (p < 0.05) and an increase in IL-6 level (P < 0.05) in comparison to the negative control group (G1) was observed in G2. Rats treated with green coffee methanolic extract, silymarin and their combination (G3, G4 and G5, respectively) showed a significant increase in both irisin and CYP450 levels (p < 0.05), in comparison with those in G2. Moreover, treating with the mixture of green coffee-methanolic extract and silymarin in G5 was more efficient in increasing both irisin and CYP450 and decreasing IL-6

Furthermore, the possible correlations among the studied parameters were evaluated (See supplementary Table 3). A positive correlation was observed between antioxidants and each other. A positive correlation was also observed between antioxidants, CYP 450 and irisin. In contrast, a significant negative correlation was found between MDA and antioxidants. Furthermore, a positive and highly significant correlation was observed between CYP 450 and irisin. Also, a highly negative correlation was observed between IL-6 and both CYP 450 and irisin (Supplementary Table 3).

Figure 3 (a,b,c,d,e) showed the histopathological examinations of rats under study. Figure 3a showed the normal hepatic tissues of the untreated negative control group (G1). The CCl₄ toxicity (Fig. 3b) severely damaged the liver tissue in G2. The hepatic tissues of G2 showed fatty infiltrated and degenerated hepatocytes, congested hepatic vein, and sinusoid degenerated biliary duct and bridging necrotic cells. However, rats treated with green coffee methanolic extract, silymarin and their combination in G3 (Fig. 3c), G4 (Fig. 3d) and G5 (Fig. 3e), respectively showed ameliorated hepatic tissues, particularly in G5 that showed nearly normal hepatocytes without any congestion

More studies are needed to determine the mechanism(s) of the hepatoprotective effect of green coffee methanolic extract and its bioactive compounds. More than one mechanism underlying this effect may be involved because of the mixture of bioactive components present in green coffee.