Chemicals
Vitamin D purchased from Novartis Company. Eugenol purchased from Sigma-Aldrich Company.
Experimental animals
Forty young healthy male Westar rats weighing about 200 gm were obtained from Animal House from the company for biological products and vaccines (VACSERA), Cairo, Egypt. Animals were housed in plastic cages under controlled temperature (23±28
oC) and maintained in groups of five per cage in a light-dark cycle. They were given free access to a commercial pellet diet and tap water and allowed to acclimatize for three days before initiation of the experiment. They were divided into four groups (10 animals each) and will be treated as follows:
Group I: Control group (-ve) each animal orally given saline (100 mg/kg b/w) for 2 months.
Group IV: Each animal orally given vitamin D (100 mg/kg b/w) then orally given eugenol (100 mg/kg b/w) for 2 months. By the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, 8th edition, all protocols and animal handling at the Department of Zoology, Faculty of Science, Helwan University were approved by the Committee on Research Ethics for Laboratory Animal Care (approval number: HU/Z/010-19).
Total phenolic content
Total phenolic compound content of
G. eugenol extract was assayed by the Folin-Ciocalteu method as described previously
(Chaieb et al., 2007). Briefly, 0.1 mL of the sample’s extract was mixed with 2.5 mL of distilled water in a test tube and then 0.1 mL of undiluted Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) was added. The solution was mixed well and then allowed to stand for 6 min before adding 0.5 mL of 20% sodium carbonate solution. The color developed for 30 min at room (20
oC) temperature and the absorbance was measured at 760 nm using a spectrophotometer (PD 303 UV spectrophotometer, Apel Co., Limited, Saitama, Japan). A blank sample was prepared using 0.1 mL of methanol instead of the extract. The measurement was compared to a calibration curve of gallic acid solution and expressed as milligram (mg) equivalent (eq.) of gallic acid per gram (g) of dry weight extract.
Total flavonoids
The aluminium chloride colorimetric method was used to determine the total flavonoid content of eugenol extract as described previously
(Baba and Malik, 2015). Briefly, in a test tube, 50 mL of the extract was mixed with 4 mL of distilled water, 0.3 mL of 5% NaNO
2 solution and 0.3 mL of 10% AlCl3. 6H
2O. The mixture was allowed to stand for 6 min and then 2 mL of 1 mol/L NaOH solution was added; distilled water was subsequently added to bring the final volume to 10 mL. The mixture was allowed to stand for another 15 min and the absorbance was measured at 510 nm. The total flavonoid content was calculated from a calibration curve and the result is expressed as mg eq. rutin/g dry weight.
DPPH (2,2–diphenyl-1-picrylhydrazyl) scavenging activity radical
The power of the eugenol extract to scavenge DPPH radicals was assayed as described previously
(Akillioglu et al., 2010). A fresh solution of 0.08 mM DPPH radical in methanol was prepared. Next, 950 mL of DPPH solution was mixed with 50 mL extract and incubated for 5 min. Exactly 5 min later, the absorbance of the mixture was measured at 515 nm (PD 303 UV spectrophotometer, Apel Co., Limited). Antioxidant activity (AA) is expressed as percentage inhibition of DPPH radical using the equation below:
where,
A sample = Absorbance of the sample at time.
t= 5 min.
A control = Absorbance of the control.
ABTS [2,4,6-tri(2-pyridyl)-s-triazine] radical scavenging activity
The ABTS assay was used to determine the DPPH radical scavenging activity according to the method of
Gouveia and Castilho (2011). The ABTS+ radical solution was prepared by reacting 50 mL of 2 mM ABTS solution with 200 mL of 70 mM potassium persulfate solution. This mixture was stored in the dark for 16 h at room temperature and it was stable in this form for two days. For each analysis, the ABTS+ solution was diluted with pH 7.4 phosphate buffered saline (PBS) solution to an initial absorbance of 0.700±0.021 at 734 nm.
This solution was freshly prepared for each set of analysis. To determine the antiradical scavenging activity, an aliquot of 100 mL methanolic solution was mixed with 1.8 mL of ABTS+ solution and the decrease in absorbance at 734 nm (PD 303 UV spectrophotometer, Apel Co., Limited, Saitama, Japan) was recorded during 6 min. The results are expressed as µmol Trolox equivalent per g of dried extract (mmol eq. Trolox/g), based on the Trolox calibration curve.
Ferric reducing antioxidant power (FRAP)
Ferric reducing antioxidant power (FRAP) was performed as described previously
(Benzie and Strain, 1996).
Real time PCR
The total RNA was isolated from the liver tissue using an RN easy plus Minikit (Qiagen, Valencia, CA). One microgram of the total RNA and random primers were used for cDNA synthesis using the Revert Aid H minus Reverse Transcriptase (Fermentas, Thermo Fisher Scientific Inc., Canada). For real time PCR analysis, the cDNA samples were run in triplicate and GAPDH was used as a reference gene. Each PCR amplification included non-template controls and all reagents except for the cDNA. Real time PCR reactions were performed using Power SYBR Green (Life Technologies, CA) and were conducted on the Applied Biosystems 7500 Instrument. The typical thermal profile is 95
oC for 3 min, followed by 40 cycles of 95
oC for 15s and 56
oC for 30s. After PCR amplification, the 1Ct was calculated by subtracting the GAPDH Ct from each sample Ct. The method of Pfaffl was used for the data analysis
(Pfaffl, 2001). The PCR primers for iNOS, IL-2 and TNF-α genes were synthesized by Jena Bioscience GmbH (Jena, Germany). Primers were designed using the Primer-Blast program from NCBI. For a reference gene, GAPDH was used.
The primer sets used were as the following:
GAPDH: Sense: 5'-GCATCTTCTTGTGCAGTGCC-3',
Antisense: 5'-GATGGTGATGGGTTTCCCGT-3';
iNOS: Sense:5'-GTTCCTCAGGCTTGGGTCTT-3',
Antisense:5'- TGGGGGAACACAGTAATGGC-3';
IL-2: Sense: 5'-CTGCAGCGTGTGTTGGATTT-3',
Antisense: 5'-GGCTCATCATCGAATTGGCAC-3';
TNF-α: Sense: 5'-AGAACTCAGCGAGGACACCAA-3',
Antisense: 5'-GCTTGGTGGTTTGCTACGAC-3'
Histopathological studies
Liver tissue samples were fixed in 10% neutral formalin for 24 h and paraffin blocks were routinely processed for light microscopy. Slices of 4-5 mm were obtained from the prepared blocks and stained with hematoxylin and eosin as well as Masson’s trichrome for hepatic fibrosis. The preparations obtained were visualized using a Nikon microscope.
Statistical analysis
Results were expressed as Mean±SE (standard error). Data for multiple variable comparisons were analysed by one-way analysis of variance (ANOVA). For the comparison of significance between groups, Duncan’s test was used as a post hoc test according to the statistical package program (SPSS version 22).
