Cytotoxic, hepatoprotective and antioxidant activities of Silybum marianum variety albiflorum growing in Egypt

Silymarin prepared from the fruits of Silybum marianum (L.) Gaertn. (Asteraceae) has long been used for the treatment of liver disorders. This study was carried out to evaluate the protective effect of the fruit extract of white-flowered S. marianum variety albiflorum Eig. (WSE) against paracetamol-induced liver toxicity in rats. Silyhermin, isosilandrins A/B were identified as the major flavonolignans in WSE. Cytotoxic activities of WSE and isolated flavonolignans compared to silymarin were carried out using sulforhodamine B assay. WSE, silyhermin and isosilandrins had no obvious harmful effect on normal human cell line compared to silymarin with IC50 values 78.95, 84.34, 72.14 and 16.83 µg/ml, respectively. The hepatoprotective activity of WSE at dose 50 mg/kg was comparable to silymarin (100 mg/kg). These data were supplemented with histopathological studies on liver sections. The hepatoprotective effects of WSE on oxidative stress induced by administration of paracetamol are probably associated with its antioxidant properties.

3 Agilent column (9.4 x 250 mm, 5 µm), USA. The NMR spectrometer used was Bruker model AVANCE III HD (Fälladen, Switzerland) operating at the basic frequency of 400.13 MHz (O1). The solvents used in this study were of analytical grade. Methanol used for HPLC preparative separation of isolated compounds was of HPLC grade (Sigma-Aldrich, Steinheim, Germany). Standard silymarin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Plant material
S. marianum variety albiflorum Eig. fruits were collected from Assiut city, Upper Egypt on 15 April 2015. Botanical authentication was carried out by Dr. Abdel Halim Mohamed, Flora and Phytotaxonomy Department, Agricultural Research Center, Cairo, Egypt. The fruits were manually separated from the heads and freed from their pappus.

Isolation of major flavonolignans from S. marianum variety albiflorum
The fruits S. marinaum variety albiflorum (150 g) were extracted with methanol according to a reported method (AbouZid et al. 2016). The pericarp of the fruits (67.1 g) was extracted with methanol (x3, 100 ml) to yield 5.8 g crude extract. The extract was monitored by TLC using methylene chloride : acetone : formic acid 7.5:1.6:0.9, under short ultraviolet light and sprayed with 10% sulfuric acid spray reagent. The crude extract (5.8 g) was loaded on silica gel (10 g), dried and applied onto a silica gel column (200 g).
The column was developed using methylene chloride (0.1% formic acid) as a mobile phase and increasing the polarity by acetone (0-50%). Twenty three fractions (50 ml) were collected, concentrated under reduced pressure and monitored by TLC. Fractions 16 and 17 eluted from the previous column with 40% acetone in methylene chloride (0.1% formic acid) were pooled, evaporated to dryness (0.5 g) and further purified on sephadex LH-20 column (50 g) using methanol. Fifteen sub-fractions were collected, concentrated and monitored by TLC, examined under short ultraviolet light short wavelength and sprayed with 10% sulfuric acid spray reagent. Sephadex LH-20 column fractions 8 and 9 showed one spot at R f value 0.6. The two fractions were pooled, evaporated to dryness (184.4 mg), analyzed by 1 H NMR and identified as silyhermin (Biedermann et al. 2016).
Fractions 13-16 from the first silica gel column were pooled, evaporated to dryness (2.9 g) and further purified on sephadex LH-20 (50 g) column using methanol as a mobile phase. Fourteen sub-fractions (40 ml) were collected, concentrated under reduced pressure, and monitored by TLC, examined under short ultraviolet light short wavelength and sprayed with 10% sulfuric acid spray reagent. Sub-fractions 7-10 showed one spot.
These fractions were pooled and evaporated to dryness (199.7 mg). The pooled fractions were further purified by preparative HPLC using methanol : water containing 0.1% formic acid 55:45, at 3 ml/min. Two compounds were isolated, analyzed by 1 H NMR spectroscopy and identified as isosilandrin A (R t = 54.18 min) and isosilandrin B (R t = 57.46 min). Signals of γ1-H and γ2-H were obscured by water signal.

Cytotoxic activity
In vitro cytotoxic activity was carried out using the sulphorhodamine B assay.
Doxorubicin was used as a positive control. Normal human cell line (HFB-4) and human cancer cell line (Hep-G2) were seeded in 96 well microtiter plates at a concentration of 1,000-2,000 cells/well, 100 µl/well. After 24 h, cells were incubated for 72 h with various concentrations of tested samples. For each concentration, three wells were used.
The plates were incubated for 72 h. The medium was discarded. The cells were fixed with 150 μl cold trichloroacetic acid for 1 h at 4°C. The plates were washed with distilled water using a Tecan automatic washer (Crailsheim, Germany) and stained with 50 μl 0.4% SRB dissolved in 1% acetic acid for 30 min at room temperature in the dark. The 5 plates were washed with 1% acetic acid to remove unbound dye and air-dried for 24 h.
The dye was solubilized with 150 µl/well of 10 mM tris base (pH 7.4) for 5 min on a shaker at 1,600 rpm. The optical density (OD) of each well was measured spectrophotometrically at 490 nm with an ELISA microplate reader. The percentage of cell survival was calculated as follows: Surviving fraction = O.D. (treated cells)/O.D.

Ethics Statement
All procedures in the present study were approved by the ethics committee of the Faculty of Pharmacy, Beni-Suef University. The guidelines declared by U.S.A. national institute of health for care and use of laboratory animals were followed in all experimental procedures.

Animals
Wistar male rats weighing 150-180 g were obtained from the animal house of Nahda University in Beni-Suef (NUB), Beni-Suef, Egypt. Animals were kept under the following conditions: temperature (25 ± 2°C), humidity (60 ± 10%), a 12/12 h light-dark cycle and allowed to access water and food freely.

Experimental design
Fifty six rats were divided into seven groups (eight rats each). First group in which the rats received vehicle only for 7 days (Normal). Second group in which the rats received WSE (200 mg/kg, p.o.) for 7 days (W200). Third group in which the rats received vehicle for 7 days and paracetamol (600 mg/kg, p.o.) at the 7 th day of the experiment (APAP).

Blood and tissue preparation
At the end of the experiment blood samples were withdrawn and serum was prepared by centrifugation at 3000 r.p.m. for 15 min at 4°C. The serum was used for assessment of ALT, AST, albumin, direct and total bilirubin levels. The animals were sacrificed and livers were dissected out immediately and washed with cold saline. One part of the liver was immersed in 10% formal saline for histopathological examination. Another part was homogenized in 4 volumes of phosphate buffered saline, centrifuged at 1000 g for 15 min at 4°C and the supernatants were then stored at -80°C till analysis. Liver homogenates were used for determination of reduced GSH and MDA contents as well as SOD activity.

Biochemical tests
ALT and AST estimation was carried out according to the procedures of the assay kits (Randox, UK). The assay depends on colorimetric measurement of the reaction at 546 nm. Serum albumin and bilirubin were determined colorimetrically using test reagent kits. Liver tissue homogenates were used for the determination of reduced GSH content using BlueGene Biotech assay kit (BlueGene Biotech, China). Hepatic content of MDA was estimated according to the kit instruction (Lifespan Bioscience, USA). Liver SOD activity was determined by superoxide dismutase kit (MyBio-source, USA).

Histopathological Examination
The isolated kidneys were washed using normal saline, fixed in 10% formal saline, dehydrated in ascending grades of alcohol and embedded in paraffin wax. Sections were taken at thickness of 5 m. Staining was done by hematoxylin and eosin (H&E). The section fields were examined under light microscope by a histopathologist (Bancroft et al. 1996).

Statistical analysis
The present results are illustrated as mean ± SEM. All the statistical analyses were performed using one way analysis of variance (ANOVA) test with Tukey's post hoc comparison test. Significance was based on P value < 0.05. Data analysis was done using Prism 5 (GraphPad Software, USA).

Table S1
Effects of oral administration of white-flowered Silybum marianum fruit extract on liver functions and oxidative stress biomarkers in paracetamol-induced hepatotoxicity in rats (n = 8 rats per group). Each bar represents the mean ± SEM of the group.