Protective Effect of Chrysanthemum Pacificum Against Ccl4-Induced Injury on the Human Hepatoma Cell Line (Huh7)

Nermeen F. Farag et al /J. Pharm. Sci. & Res. Vol. 7(9), 2015, 633-639 Protective Effect of Chrysanthemum Pacificum Against CCl4-Induced Injury on the Human Hepatoma Cell Line (Huh7) Nermeen F. Farag, Enas H. Abdelrahman*, Shadia M. Azzam and El-Sayeda A. El-Kashoury Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, P.B. 11562, Cairo, Egypt Abstract Aims: Evaluation of hepatoprotective, free radical scavenging potentials of the plant parts and isolation, characterization of major active compounds. Methods: An in-vitro assay using human hepatoma cell line (Huh7) was used to evaluate the effect of methanol extract of C.pacificum Nakai parts (flowers, aerial parts, roots) and fractions of flowers (10,100,1000 µg/ml) on enzymes activities before and after exposure of the cells to carbon tetrachloride (CCl4). Results: Among the tested parts, flowers ethanol extract (100 µg/ml) exhibited the highest significant increase in superoxide dismutase (SOD) activity (237.18±6.5U/ml) and liver glutathione (GSH) level (25.42±1.74 mg/dl). Furthermore it showed the highest free radical scavenging activity (IC50, 1.2 mg/ml) even more potent than silymarin (IC50, 1.84 mg/ml). Ethyl acetate (EtOAc) and butanol (n-BuOH) fractions of flowers (100 µg/ml) exhibited higher potency than other fractions and silymarin in normalizing alanine transaminase (ALT) (11.42±0.43 & 13.68±0.87 U/ml respectively) and SOD (276.7±1.31&276.7±2.19 U/ml respectively) activities. Moreover, butanol fraction was more effective in neutralizing GSH level (18.4 ±0.10 mg/dl) compared to silymarin (16.73±1.33 mg/dl). Bioassay guided fractionation led to the isolation of luteolin, luteolin-7-O- glucuronide, luteolin-7-O-β-rutinoside (scolymoside) and 1,5-di-O-caffeoylquinic acid (cynarin), the latter compounds are active hepatoprotectives in artichoke. Conclusion: This is the first report for the protective and radical scavenging potentials of C. pacificum which may partly attributed to the presence of active phenolics as cynarin. Keywords: Antioxidant, Chrysanthemum pacificum, hepatoprotective, Huh7 INTRODUCTION was used for recording UV spectra and Chrom Tech model Oxidative stress is considered a main mechanism in (CT-2400) for determination of total phenolic and contributing to the initiation and progression of hepatic flavonoid content. NMR analyses were run on Varian damage in a variety of liver disorders (1-3). Many plant VNMRS 600 NMR spectrometer (Bruker Daltonics, phenolics have potent antioxidant and hepatoprotective Bremen, Germany) 1H-NMR, 600 MHz, 13C-NMR, 125 effects (4-7). Genus Chrysanthemum has been reported to MHz. or Bruker NMR-spectrophotometer (Microanalytical contain signiﬁcant amounts of ﬂavonoids and Unit, Faculty of Pharmacy, Cairo university), 1H-NMR, 400 hydroxycinnamoyl-quinic acids which demonstrate radical- MHz, 13C-NMR, 100 MHz, Japan relative to TMS in scavenging and anti-hepatotoxic activities(5-7,8-12). Our CD3OD or DMSO. For column chromatography, Sephadex previous study on C. pacificum Nakai family LH-20 (Pharmacia, Uppsala, Sweden), Silica gel 60 H for Asteraceae(13) ascertained the metabolites profiling in the VLC (E-Merck, Darmstadt, Germany), silica gel 60 for plant on an organ basis using UPLC-MS. Hydroxycinnamic column chromatography (Fluka,70–230 mesh, acid derivatives and flavonoids were detected as the most Germany)and silica gel 100 C18–reversed phase (70–230 abundant phytoconstituents. Herein we report on for the mesh, Fluka) were used. Pre-coated silica gel 60 F254 plates first time an in-vitro assay using the human hepatoma cell (Merck, Germany) were used for TLC. line (Huh7) to evaluate the possible hepatoprotective, Plant material antioxidant potentials of C. pacificum organs against CCl4- Samples of the flowers capitulum, aerial parts and roots induced injury. Furthermore, this study correlates the were collected during 2012-2013 from the plants cultivated activity among organs with their phenolics and flavonoids in the Experimental Station of Medicinal plants, content. Pharmacognosy Department, Faculty of Pharmacy, Cairo University. The plant was kindly identified by Mrs. MATERIALS AND METHODS Therease Labib, taxonomy specialist, Orman Botanical General techniques Garden, Giza, Egypt. A voucher specimen (27.2.2014) was The antioxidant activity was determined using Jenway deposited at the herbarium of Pharmacognosy Department, double beam spectrometer (UK). Shimadzu UV-1650 PC Faculty of Pharmacy, Cairo University. 633 Nermeen F. Farag et al /J. Pharm. Sci. & Res. Vol. 7(9), 2015, 633-639 Chemicals and standards the absorbance was measured spectrophotometrically Silymarin, carbon tetrachloride (CCl4) and dimethyl against a blank at 517 nm. Trolox [(±)-6-hydroxy-2,5,7,8- sulfoxide were purchased from Sigma–Aldrich chemicals tetramethylchromane-2-carboxylic acid] and silymarin Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s were used as positive controls at a concentration of (0.05, medium, fetal bovine serum, trypsin 0.25%, penicillin G, 0.07, 0.09, 0.11, 0.13 mg/ml), and (0.5, 1, 1.5, 2, 2.5, 3 streptomycin and phosphate-buffered saline were obtained mg/ml), respectively. Free radical scavenging activity is from Gibco Invitrogen (Carlsbad, CA, USA). Kits for expressed as the concentration of extract inhibiting DPPH measurement of AST, ALT, SOD activities and GSH level formation by 50% relative to methanol (IC50 value). were purchased from Biodiagnostic for diagnostic reagents (Dokki, Giza, Egypt). Determination of polyphenolic content 2, 2-Diphenyl-1-picrylhydrazyl (DPPH reagent), trolox and Aliquots (2 ml, each) of 50% methanolic extracts and gallic acid (Sigma-Aldrich, St. Louis, MO, USA) for free standard solution of gallic acid (0.008, 0.016, 0.024, 0.032, radical scavenging estimation. Folin-Ciocalteu reagent 0.04, 0.048, and 0.056 mg/mL) was added to 1.5 mL of used for determination of total polyphenolic content Folin-Ciocalteu reagent and 4 ml of 20% sodium carbonate obtained from Loba-Chemie (Mumbai, india). solution, then the solution was diluted to 25 ml with distilled water. The absorbance of the resulting solution Cell culture was measured, after 30 min at λmax 765 nm, against a blank Human hepatoma cancer cells (Huh-7) obtained from prepared at the same time, using 2 ml of distilled water, VACSERA (Dokki, Giza, Egypt). Cells were maintained in instead of the tested solution. For each concentration, DMEM medium supplemented with 100 mg/ml triplicate experiments were carried out. The results were streptomycin, 100units/ml penicillin and 10% heat- expressed as µg of gallic acid equivalents/ mg of extract inactivated fetal bovine serum in a humidified, 5% CO2 (GAE) atmosphere at 37 C. Determination of total flavonoids content Hepatoprotective potential estimation Aliquots (0.5 ml, each) of 80% methanol extracts and The dried powders of flowers, aerial parts and roots were standard solution of quercetin (0.01, 0.02, 0.04, 0.08 and macerated in 70% ethanol till exhaustion and dried under 0.1 mg/mL) was added to 0.1 Ml of 10% aluminium vacuum. The fresh flowers of C. pacificum Nakai was chloride, 0.1 mL of 1M potassium acetate aqueous solution extracted with 70% ethanol at room temperature till and 2.8 mL of distilled water. The solution was well mixed exhaustion, the extract, in each case, was dried under and the absorbance was measured spectrophotometrically at vacuum till dryness. Dry ethanol extract was re-suspended 415 nm against blank. Total flavonoids content was in H2O and successively extracted with PE, CHCl3, EtOAc expressed as mg quercetin equivalent (QE)/g plant extract. and n-BuOH saturated with water till exhaustion. Each Each measurement was performed in triplicate. extract was concentrated under reduced pressure till dryness. The extracts of the different organs and fractions Extraction and isolation of flowers extract were used for the hepatoprotective assay. Fresh flowers (3.5 kg) was macerated in 70% ethanol till To assess the hepatoprotective effect of the tested samples, exhaustion. The ethanolic extract was evaporated under exponentially growing cells were collected using 0.25% vacuum to yield (95 g). The residue was suspended in trypsin-EDTA and plated in 6-well plates at 105cells/well. water and partitioned successively with petroleum ether, Cells were treated with various concentrations of the plant chloroform followed by ethyl acetate and n-butanol extracts (10, 100, 1000 μg/ml in phosphate-buffered saline) saturated with water. The solvents were then evaporated for one hour, followed by the addition of 40 mM CCl4 in under reduced pressure to give different fractions. The 0.05% dimethyl sulfoxide and subsequent incubation for EtOAc residue (10.5 g) was subjected to silica gel CC and two hours. The supernatant was collected and assayed for eluted by gradient addition of CHCl3, EtOAc and MeOH AST, ALT, SOD activities and GSH level. Silymarin was to yield fractions I & ΙΙ. Fr. I (350 mg) was purified by used as a reference agent. All variables were measured in sephadex LH-20 using MeOH-H2O (1:1 v/v) to yield triplicates and the experiment was repeated three times. compound 1. Fraction II was purified by sephadex LH-20 Results were expressed as mean ± standard deviation using MeOH-H2O (1:1 v/v) and further purified by (S.D.), calculated from n = 3. Data were analyzed using rechromatography on a silica gel column (13 cm L×1 cm one-way analysis of variance (ANOVA), followed by D). Gradient elution was performed using CHCl3-MeOH Turkey-Kramer’s post hoc test. Statistical significance was (95:5 v/v) with increasing MeOH percentage by 1% till accepted at a level of p<0.001. reaching 10% MeOH to yield compound 2. n-BuOH fraction was subjected to VLC column eluted with CHCl3 Free radical scavenging activity estimation and increasing the polarity by 5% increments with EtOAc, Lyophilised 70% methanol extracts of C.

Load more