Oxidative stress influences the progression of liver diseases . By attenuating oxidative stress, cellular antioxidant systems prevent liver diseases . Excessive oxidative stress damages cell membranes, proteins, and DNA. Consequently, it induces toxicity, such as hepatic cancers and muscular dystrophy. Currently available synthetic antioxidants are associated with adverse effects. Therefore, herbal antioxidants are being considered due to their enhanced safety and effectiveness [3,4,5,6].
Zilla spinosa belongs to the family Cruciferae or Brassicaceae, which comprises 3,709 species and 375 genera . Some species of this family are edible. Many species of this family possess anticancer, anti-rheumatic, antidiabetic, antifungal, insecticidal, and antibacterial activities . Z. spinosa is traditionally used for the treatment of kidney and gall bladder stones . It also exerts anti-thyroid effects due to progoitrin and goitrin . It is rich in natural compounds, such as triterpenoids, sterols, progoitrin, flavonoids, goitrin, carbohydrates, and glucosinolates of free sinapine . Owing to these components, different fractions of Z. spinosa exert antiviral, hepatoprotective, antifungal, and antioxidant effects . However, the methanolic and n-hexane fractions have not been evaluated for their hepatoprotective effects. The aqueous ethanol extract of Z. spinosa reduces gamma glutamyl transferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels, indicating the stabilization of plasma membranes of carbon tetrachloride (CCl4)-exposed hepatocytes in animals [11, 12]. The chloroform extract of Z. spinosa is effective against human colon (HCT116) and liver (HEPG2) cancer cells. It induces significant cytotoxicity in HCT116 cells. It also holds significant analgesic and anti-inflammatory potential . Hammada elegans is a shrub or undershrub belonging to the family Amaranthaceae that exerts antidiabetic, antiseptic, and anti-inflammatory effects [13, 14]. Triterpenoids, flavonoids, tannins, alkaloids, glycosides, and saponins are reported to be responsible for its analgesic, anti-inflammatory, hepatoprotective, and antioxidant effects [15,16,17]. The ethanolic extract shows moderate antimicrobial activities against numerous bacterial species, such as Enterococcus faecalis, Escherichia coli, Moraxella lacunata, Proteus mirabilis, Serratia marcescens, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis, Micrococcus luteus, Sarcina ventriculi, and Staphylococcus aureus, and fungi, such as Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus fumigatus, and Penicillium chrysogenum .
The current investigation focuses on the hepatoprotective effectiveness of methanolic and n-hexane fractions of Z. spinosa and H. elegans against CCl4-induced liver injury in rats and their antioxidant potentials.
2 Experimental Procedure
2.1 Plant material
Z. spinosa and H. elegans were collected from Wadi Hafr Al-Batin and Umm oshr Al-Butaihan, Saudi Arabia on 24 February 2016. The specimen vouchers were deposited in the herbarium of the Medicinal Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia with voucher numbers SY282 and SY 270, respectively.
2.2 Extraction and fractionation
Whole plants were air-dried, ground, and soaked in methanol for one week. After filtering, the methanol was evaporated using a Buchi rotary evaporator. The process was repeated three times using methanol. Further, the greenish methanolic crude extract was mixed with distilled water, and partitioned using a separating funnel with solvents of increasing polarity (n-hexane, chloroform, ethyl acetate, n-butanol, and water). Crude methanolic and n-hexane fractions were used for biological evaluations.
2.3 Animals and chemicals
Albino rats (aged two months and weighing 190–202 g) of both sexes were obtained from the College of Pharmacy, Experimental Animal Care Center, King Saud University. The rats were housed in standard polypropylene animal cages at a controlled temperature (22±2°C) and a 12-h light-dark cycle with unrestricted access to water and food (Purina chow). Rats were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, and the experimental protocol was approved by the Institutional Animal Ethics Committee of the College of Pharmacy, King Saud University (clearance number, 05763-891; 6 January, 2014). All solvents used were of analytical grade. Silymarin was purchased from Sigma Aldrich Company (St. Louis, MO, USA).
2.4 Acute toxicity test
The acute oral toxicity of H. elegans methanolic (HEM) and n-hexane (HEH) extracts, and Z. spinosa methanolic (ZSM) and n-hexane extracts (ZSH) was evaluated on rats at several doses (0.1–0.5 mL/kg). The rats were monitored continuously to identify symptoms of toxicity and mortality .
2.5 CCl4-induced liver toxicity
Rats of both sexes were randomly divided into eleven groups (n=6)—group 1 (control), group 2 (CCl4 only), group 3 (CCl4+silymarin), group 4 (CCl4+HEM 250 mg/kg), group 5 (CCl4+HEM 500 mg/kg), group 6 (CCl4+HEH 250 mg/kg), group 7 (CCl4+HEH 500 mg/kg), group 8 (CCl4+ZSM 250 mg/kg), group 9 (CCl4+ZSM 500 mg/kg), group 10 (CCl4+ZSH 250 mg/kg), and group 11 (CCl4+ZSH 500 mg/ kg). The test extracts were administered intraperitoneally. Pretreatment with the test extracts and silymarin was initiated three weeks before CCl4 administration, and was continued until the end of the experiment. Blood samples were withdrawn directly into tubes containing disodium EDTA from the retro orbital plexus of all rats under anesthesia 24 h after CCl4 treatment. After centrifuging the clotted blood samples at 2,500 ×g for 10 min, serum samples were separated. The rats were sacrificed after blood withdrawal. Liver tissues were isolated for biochemical and histopathological analyses. They were cleaned with refrigerated normal saline, and 10% (w/v) liver homogenates were prepared in ice cold 0.15 M KCl solution using a motorized Teflon pestle.
2.6 Estimation of hepatotoxicity marker enzymes and bilirubin
2.7 Estimation of lipid profile
To evaluate lipid profile, levels of total cholesterol, triglycerides, very low-density lipoprotein (VLDL), LDL, and high-density lipoprotein (HDL) were measured using commercial diagnostic kits .
2.8 Determination of malondialdehyde (MDA)
Liver tissue homogenates (10% w/v) were prepared in KCl (0.15 M) at 4°C in a Potter–Elvehjem type C homogenizer. After incubating the tissue homogenates (1 mL) at 37°C for 3 h in a metabolic shaker, they were mixed with 10% aqueous trichloroacetic acid (1:1), centrifuged at 800 ×g for 10 min, and the supernatants were separated. Equal volumes (1 mL) of supernatants and thiobarbituric acid solution (0.67% in water) were mixed, and placed in a water bath for 10 min at 100°C. After cooling, the samples were diluted with 1 mL of distilled water, and the optical densities were measured using a spectrophotometer at 535 nm. Sample MDA levels were estimated by comparing with a standard curve of MDA solution .
2.9 Estimation of non-protein sulfhydryls (NP-SH) and total protein
Ice-cold 0.02 mM EDTA was used for the homogenization of liver tissues. Tissue homogenates (5 mL) were mixed with distilled water (4 mL) and 50% trichloroacetic acid (1 mL) in test tubes. After shaking to mix for 10 min, the mixtures were centrifuged at 3,000 rpm, and the supernatants (2 mL) were mixed with Tris buffer (0.4 M, pH 8.9, 4 mL) and 5,5′-dithio-bis (2-nitrobenzoic acid) (0.1 mL) for 5 min; the optical densities were immediately measured at 412 nm against the blank reagent.
A colorimetric method was used to determine total serum protein (g/L); the optical density of the titrated blue/violet product was measured at 546 nm, and sample protein concentrations were calculated based on the following equation .
2.10 Histopathological assessment
An American-made optical rotary microtome was used for producing 5-μm sections of liver tissues. The sections were stained with hematoxylin and eosin, and pathomorphological changes were studied under a light microscope .
2.11 Antioxidant activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to evaluate the antioxidant potential of all four extracts at different concentrations (100, 150, 200, and 2,500 μg/mL). Freshly prepared purple colored DPPH solution turned yellowish after incubating with test samples for 30 min at room temperature, and color changes were measured using a spectrophotometer (UVmini-1240, Shimadzu, Japan) at 514 nm. The following equation was used for the calculation of antioxidant activity :
2.12 Statistical analyses
Results are expressed as mean±standard error (SE) of four samples. The data were statistically analyzed by ANOVA, followed by Dunnett’s multiple comparisons test.
3 Results and Discussion
3.1 Acute toxicity
No mortality or clinical pathologies were observed after treatment with the four extracts even at the highest dose (0.5 mL/kg, p.o.).
3.2 Effects of the extracts on hepatotoxicity marker enzymes and bilirubin in the serum
Results of biochemical parameters indicative of liver function are summarized in Table 1. CCl4 administration caused severe hepatotoxicity in rats as evidenced by significantly elevated serum levels of ALT, AST, ALP, GGT, and bilirubin compared to control. The elevation in these levels was significantly reduced by silymarin pretreatment (10 mg/kg). Pretreatment with HEH and HEM significantly reduced these levels at both doses (250 and 500 mg/kg) compared to control; however, HEH was more effective than HEM. Pretreatment with ZSM and ZSH yielded nonsignificant results at both doses (250 and 500 mg/kg).
3.3 Effect of the extracts on lipid profile
CCl4-exposed rats had significantly elevated levels of triglycerides, cholesterol, VLDL, and LDL compared to normal rats (Table 2). These levels were significantly lower in CCl4-exposed rats pretreated with silymarin than in rats exposed to CCl4 only. HEH and HEM pretreatment at both doses (250 and 500 mg/kg) significantly (p<0.001) lowered these levels compared to CCl4 exposure alone. However, ZSM and ZSH pretreatment at both doses (250 and 500 mg/kg) demonstrated insignificant (p>0.05 and 0.01) improvement in the lipid profile.
3.4 Effect of the extracts on MDA, NP-SH, and total protein in liver tissue homogenates
CCl4 exposure without any treatment significantly (p<0.001) increased MDA, while decreased total protein and NP-SH levels compared to control (Table 3). CCl4-exposed rats pretreated with HEH or HEM at both doses (250 and 500 mg/kg) had significantly (p<0.001) decreased MDA, total protein, and NP-SH levels compared to rats exposed to CCl4 alone. However, ZSM and ZSH pretreatment at both doses (250 and 500 mg/kg) caused insignificant changes (p>0.05 and 0.01).
3.5 Effect of the extracts on liver histopathology
As shown in Figure 1, histopathological findings were consistent with results of biochemical analyses. Severe necrosis and focal hepatic cellular decay of the lobules were observed in the CCl4 only group compared to the control group. Hepatocytes and the central vein were normal in rats pretreated with the plant extracts. In addition, liver tissues of rats treated with silymarin exhibited a normal central vein and sinusoidal hepatocytes. HEH pretreatment at 500 mg/kg was found to be as effective as silymarin with negligible liver toxicity.
3.6 Antioxidant activities of the extracts
The plant extracts exerted antioxidant effects in a dose-dependent manner (Table 4). HEM, HEH and ZSM at 250 μg/mL showed significant antioxidant activities of 38.16, 35.65 and 31.18%, respectively. However, the antioxidant activities of ZSM and ZSH were insignificant. These results further reinforced the significant hepatoprotective potentials of HEM and HEH.
Hepatic diseases remain a worldwide health concern, despite remarkable progress in the field of medicine. Numerous hepatoprotective herbal formulations exist . Plant extracts rich in phenolic compounds are associated with hepatoprotective effects [26,27,28,29]. To our knowledge, this is the first study on the methanolic and n-hexane extracts of the selected plant species. CCl4 becomes toxic after biotransformation by cytochrome P450 to trichloromethyl (CCl3•) and trichloromethylperoxy (CCl3OO•) free radicals, which further initiate lipid peroxidation and disturb the levels of bilirubin and other biomarker enzymes [30,31]. The current study showed that CCl4 damages the structure and functions of the liver, thereby severely disturbing the levels of serum marker enzymes and bilirubin. The results indicated that the tested extracts protect the liver from damage and restore enzyme levels.
Both the extracts showed significant antioxidant as well as hepatoprotective activities; accordingly, a previous study showed that antioxidant effects translate to anti-inflammatory activities . Histopathology results (Figure 1) indicated that HEH at 500 mg/kg shows the best hepatoprotective effects. CCl4 damages hepatocytes as observed by the increased release of various cytosolic enzymes into the blood; therefore, the levels of these enzymes in the blood act as an indicator of the extent of hepatotoxicity . The identification and characterization of pharmacologically active phytoconstituents is crucial for new drug development. Although HEH showed significant therapeutic potential, further studies are required to explore the exact phytoconstituent responsible for its hepatoprotective activity.
It can be concluded that HEM and HEH of H. elegans significantly protect against CCl4-induced hepatotoxicity, and exert antioxidant effects in a dose-dependent manner. However, ZSM and ZSH crude extracts showed poor hepatoprotective and antioxidant effects. This study identifies the association between antioxidant activity and hepatoprotective efficacy. HEH at 500 mg/kg exhibits potent pharmacological activity, and holds significant therapeutic potential.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding support (research group no. RGP-262).
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List of abbreviations
gamma glutamyl transferase
Hammada elegans methanolic extract
Hammada elegans n-hexane extract
very low-density lipoprotein
Zilla spinosa methanolic extract
Zilla spinosa n-hexane extract
About the article
Published Online: 2018-03-02
Ethics approval: Animals were maintained in accordance with the recommendations of the ‘Guide for the Care and Use of Laboratory Animals’ approved by institutional animal ethics committee of College of Pharmacy, King Saud University, Riyadh, Saudi Arabia (Clearance No. 05763–891; January 6, 2014).
Competing interests: The authors declare that they have no competing interests.
Authors’ contributions: All authors contributed equally. All authors read and approved the final manuscript.
Citation Information: Open Chemistry, Volume 16, Issue 1, Pages 133–140, ISSN (Online) 2391-5420, DOI: https://doi.org/10.1515/chem-2018-0021.
© 2018 Riaz Ullah et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0