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in vivo International Journal of Experimental and Clinical
Pathophysiology and Drug Research
ISSN (print): 0258-851X, ISSN (online): 1791-7549
Abstract. Background: Silybin is the main component of
silymarin with antioxidant, anti-inflammatory and
cytoprotective actions. Aim: To compare the effect of silybin
used as single substance, silybin–phosphatidylcholine
complex (SilPho), and derivatives of silybin (MannpSil,
GalpSil, GlcpSil, LactpSil) on MKN28 and HepG2 cell
viability and cell death, in vitro, after the induction of
oxidative stress. Materials and Methods: Oxidative stress
was induced by incubating HepG2 and MKN28 cells with
xanthine oxidase in the presence of its substrate xanthine.
Cell viability was determined by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazoliumbromide assay. Determination
of Malondialdehyde in MKN28 cells was performed by High
Performance Liquid Chromatography. Quantitative analysis
of apoptotic cells was carried out using annexin. Results:
SilPho and new silybin glycoconjugates did not affect cell
viability, while silybin induced about 50% cell death in both
MKN28 and in HepG2 cells. The pre-treatment of cells with
silybin and new silybin glycoconjugates (before oxidative
stress induction) did not affect cell viability, while SilPho
had a protective effect. Exposure of MKN28 cells to oxidative
stress caused a twofold increase in cellular MDA
concentration compared to untreated cells. Moreover,
pretreatment with SilPho but not with silybin significantly
prevented oxidative stress-induced increase in cellular
Malondialdehyde. Moreover, silybin induced apoptosis
potentiated by oxidative stress, while SilPho did not induce
any effect. Oxidative stress caused cell death primarily by
necrosis, antagonized by SilPho. Conclusion: The protective
effect of SilPho is partially due to inhibition of radical
oxidative species.
Silybin is the main component of silymarin (famous
antioxidant) with an increasing number of effects (1).
Silibinin is a semipurified, commercially available fraction
of silymarin: Silibinin is an approximately 1:1 mixture of
two diastereoisomeric compounds, silybin A and silybin B
(2). Therefore, purified silybin and silibinin are practically
synonymous (2).
The main effects attributed both in vitro and in vivo to
silybin are related to its antioxidant, anti-inflammatory and
cytoprotective actions (3-5). Silybin is also considered a
chemopreventive and cancer-protective agent because it
modulates a series of mitogenic signaling and cell-cycle
regulators (6, 7), mediating a pro-apoptotic effect (8, 9).
Both bioavailability and therapeutic efficacy of silybin in
vivo are rather limited by low water solubility, low
bioavailability, and poor intestinal absorption (10). To
improve these pharmacological limitations, a silybin
phytosomecomplex (silybin plus phosphatidylcholine;
SilPho) has been co-formulated with vitamin E [Realsil
(RA), Istituto Biochimico Italiano, Lorenzini S.p.a., Italy]
(11, 12). Pharmacokinetic analyses indicated that the
bioavailability of silybinphytosome is much higher than that
of silymarin, and in this pharmaceutical preparation, silybin
is widely distributed in plasma and tissues, which include the
liver, lung, stomach, skin, and prostate (13, 14).
In vivo, silymarin and silybin have been used as
therapeutic herbal products for treatment of acute and
chronic liver diseases: in particular, alcoholic liver disease
and cirrhosis (15-17), nonalcoholic fatty liver disease (18)
and hepatic fibrosis (19, 20). In animals, silybinphytosome
complex reduces oxidative stress, lipid peroxidation,
collagen accumulation and consequently liver damage (19).
1
Correspondence to: A. Federico, MD, Ph.D., Department of
Clinical and Experimental Medicine, Second University of Naples,
Via Pansini 580131 Naples, Italy. Tel: +39 0815666723, Fax: +39
0815666837, e-mail: alessandro.federico@unina2.it
Key Words: Silybin, cell necrosis, oxidative stress.
in vivo 29: xxx-xxx (2015)
Silybin–Phosphatidylcholine Complex Protects Human
Gastric and Liver Cells from Oxidative Stress
ALESSANDRO FEDERICO1, MARCELLO DALLIO1, GIOVANNI DI FABIO2,
ARMANDO ZARRELLI2, SILVIA ZAPPAVIGNA3, PAOLA STIUSO3,
CONCETTA TUCCILLO1, MICHELE CARAGLIA3and CARMELA LOGUERCIO1
Departments of 1Clinical and Experimental Medicine, and 3Biochemistry, Biophysics
and General Pathology, Second University of Naples, Naples, Italy;
2Department of Chemical Sciences, University of Naples Federico II, Naples, Italy
No: 3272-F
Please mark the appropriate
section for this paper
■■Experimental
■■Clinical
■■Epidemiological
0258-851X/2015 $2.00+.40
In men, RA ameliorates some serum and histological
parameters of liver damage and fibrosis (18).
Recently, Zarrelli et al. obtained new 9''–phosphodiester
silybin conjugates with different mono- and di-saccharide
labels through the anomeric hydroxyl group in order to
enhance the biological efficacy of the derivatives by
increasing their in vivo stability, binding affinity, and overall
uptake (21). These silybin derivatives have water solubility
well above that of silybin. Despite a large series of studies
reported in literature, confusion about the different actions
of silybin exists. Therefore the following merit investigation:
i) if the effects of silybin are similar in different cell lines of
different histogenesis; ii) the influence of the concentrations
used in different experimental models; c) the effects of the
different silybin derivatives.
In the present study, we compared the effect of silybin
used as a single agent or as SilPho, and different silybin
derivatives on MKN28 and HepG2 cell death in vitro after
the induction of oxidative stress. We used two cell lines
(MKN28 and HepG2) to verify the results obtained there by
excluding the possibility of interference of the type of cell
on the results.
Materi al s and Methods
Materials. Silybin was a gift from Indena (Milan, Italy). SilPho was
provided by Istituto Biochimico Italiano (G. Lorenzini S.p.A. Milan,
Italy). Silybin derivatives were synthesized according to Zarrelli et
al. (21): in detail, an efficient synthetic procedure leads to new
9''–phosphodiestersilybin conjugates with different mono- and di-
saccharide labels through the anomeric hydroxyl group. In this
approach a suitable 9''-phosphoramidite was used as silybin building
block and 1-OH full protected mono- and di-saccharide derivatives
chosen as sugar starting materials (21). The new silybin conjugates
were: silybin-900-phosphoryl-D-mannopyranoside (MannpSil),
silybin-900-phosphoryl-D-galactopyranoside (GalpSil), silybin-900-
phosphoryl-D-glucopyranoside (GlcpSil), silybin-900-phosphoryl-
D-Lactopyranoside (LactpSil).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide
(MTT) assay was purchased by Sigma (Milan, Italy). Annexin
Apoptosis Detection Kit was obtained from BD Biosciences (San
Diego, CA, USA). Dulbecco’s modified Eagle’s medium
(DMEM):F12, penicillin, streptomycin, fetal bovine serum, L-
glutamine and trypsin/EDTA were obtained from Life Technologies
Inc. (Gaithersburg, MD, USA).
Agent preparation. Pure silybin was dissolved in dimethyl sulfoxide
(DMSO) and used at final concentration of 10, 25, 50, 75, 100 and
200 μM. SilPho was dissolved in DMSO to achieve final
concentrations of silybin similar to those employed for testing
silybin alone (10, 25, 50, 75, 100 and 200 μM). Silybin derivatives
were dissolved in water and used at final concentration of 10, 25,
50, 75, 100 and 200 μM.
Cell culture. HepG2 cells were derived from human hepatocellular
carcinoma (22) and MKN28 cells were derived from a human well-
differentiated gastric tubular adenocarcinoma and showing gastric-
type differentiation (23) (Cell Bank Interlab Cell Line Collection,
IST Genova, Italy). HepG2 and MKN28 cells were grown as
monolayer in DMEM supplemented with 10% fetal calf serum and
1% antibiotic-antimycotic solution (Life Technologies Inc.) at 37˚C
in a humidified atmosphere of 5% CO2 in air. Cytotoxicity
experiments were conducted using serum-free medium.
Induction of oxidative stress. Oxidative stress was induced by
incubating HepG2 and MKN28 cells with xanthine oxidase (XO; 10-
100 mU/ml) in the presence of its substrate xanthine (X; 1 mM) for
periods of up to 3 hours. Exposure of cells in culture to X-XO (1 mM
+ 50 mM) for 2 hours causes significant cell injury (50%) and this
has been demonstrated to be due to generation of radical oxidative
species (ROS) and in particular of OH produced from H2O2 by iron
catalyzed Fenton reaction (22).
We examined the effect of silybin, SilPho, and the new silybin
glycoconjugates (MannpSil, GalpSil, GlcpSil, LactpSil) on X-XO-
induced cell damage. Cells were incubated with serum-free medium
(Control) for 1-48 hours; with serum-free medium for 1-48 hours and
then with X-XO (1 mM + 50 mM) for 2 hours (X-XO control); with
silybin, SilPho, and new silybin glycoconjugates (10-200 μM) for 1-
48 hours and then, after washing, with X-XO (1 mM + 50 mM) for
2 hours.
Cell viability. Cell viability was determined by the MTT assay.
Briefly, 10 μl of MTT (5mg/ml saline) were added to each well, and
treated cells were incubated for 90 min at 37˚C and centrifuged for
five minutes. After aspiration of supernatant, cells were lysed and
solubilised by addition of 100 μl of 0.04N HCl containing
isopropanol. The absorbance of each sample was analyzed at 540
nm. Cell viability (%) was calculated by dividing the absorbance of
samples obtained from cells incubated with test drugs by the
absorbance of samples obtained from cells incubated with tissue
culture medium only (control) and multiplying this ratio by 100.
Data are presented as the mean±standard deviation (SD) of three
experiments run in duplicate.
Determination of lipid peroxidation. Malondialdehyde (MDA) is
considered a presumptive biomarker for lipid peroxidation in live
organisms and cultured cells (24). Determination of MDA in
MKN28 cells was performed by High Performance Liquid
Chromatography (HPLC) with fluorimetric detection, according to
the method of Bergamo and colleagues (25). Cellular pellets were
extracted with 250 ml of Milli-Q water in an ultrasonic bath for 30
minutes after the addition of 250 ml of cold 10% tricloracetic acid
(TCA). Samples were vigorously mixed (three minutes) and
centrifuged (5 minutes, 10000 × g). The supernatant was added to
700 ml of thiobarbituric acid prepared using thiobarbituric acid in 2
M acetate buffer at pH 3, degassing by a vacuum pump (5 minutes),
and flushing the final solution with nitrogen for 10 minutes. The
mixtures were degassed and then incubated for 30 minutes at 90˚C.
At the end of the incubation period, samples were cooled,
centrifuged (5 minutes, 10000 × g) to remove particulate material
and, finally, sample aliquots (20 ml) were analyzed by HPLC.
Quantification of MDA was obtained from a calibration curve
constructed by injecting increasing amounts of standard MDA.
MDA concentration was expressed as mg/106cells.
Quantitative analysis of apoptotic cells by flow cytometry.
Quantitative analysis of apoptotic cells with and without treatment
in vivo 29: xxx-xxx (2015)
2
of silybin was carried out using the Annexin Apoptotic Detection
Kit II (BD Biosciences).
Briefly, MKN-28 cells were treated with silybin alone or SilPho
at the doses previously described for 24 h with or without X-XO-
induced cell damage. Cells were harvested, washed twice with cold
Phosphate Buffered Saline (PBS) and then resuspended in 1X
binding buffer at a density of 1×106 cells/ml. Cellular pellets were
subjected to annexin and propidium iodide staining at room
Federico et al: Silybin–Phosphatidylcholine Complex Combats Andoxidative Stress
3
Figure 1. Evaluation of cell viability in MKN28 (A) and HepG2 (B) cultured cells after incubation with silybin, silybin–phosphatidylcholine complex
(SilPho), silybin-900-phosphoryl-D-mannopyranoside (MannpSil), silybin-900-phosphoryl-D-galactopyranoside (GalpSil), silybin-900-phosphoryl-
D-glucopyranoside (GlcpSil), silybin-900-phosphoryl-D-Lactopyranoside (LactpSil), before and after induction of oxidative stress with xanthine-
xanthine oxidase (X-XO). The data are reported as means±SD of three experiments. The concentration of agents utilized was 50 μg and the time of
observation was 24 hours. *p<0.01 vs. Control and SilPho alone.
temperature for 15 minutes in the dark and analyzed by flow
cytometry within 1 hour after The addition of 400 μl of 1X binding
buffer. Apoptotic cells, stained with annexin and propidium iodide,
were analyzed by fluorescence activated cell sorting using a Cell
Quest 3.4 software (FACS Calibur; BD Biosciences, San Jose, CA,
USA). The apoptotic cells stained with annexin exhibited green
fluorescence, whereas the cells stained with propidium iodide
exhibited red and green fluorescence.
Experiments were conducted as it follows: I: Evaluation of
silybin, SilPho, MannpSil, GalpSil, GlcpSil and LactpSil toxicity in
MKN28 and HepG2 cultured cells under basal conditions; II:
evaluation of cell viability after the induction of oxidative stress;
III: evaluation of cell viability in MKN28 and HepG2 cultured cells
after incubation with silybin, SilPho, MannpSil, GalpSil, GlcpSil
and LactpSil and subsequent induction of oxidative stress; IV:
determination of MDA as a marker showing the induction of
oxidative stress in cultured cells after incubation with silybin alone
and with SilPho; V: quantitative analysis of apoptotic cells with and
without treatment of silybin and SilPho.
Statistical analysis. Data are expressed as the mean±SD.
Significance of differences was assessed by one-way analysis of
variance (ANOVA) and, when the F value was significant, by
Tukey-Kramer test for multiple comparisons or by Student’s t-test
for comparison between two means. Differences were considered to
be significantly different if p<0.05.
Results
Effect of oxidative stress on MKN 28 and HepG2 cell
viability. Oxidative stress was induced by incubating MKN28
and HepG2 cells with XO (10-100 mU/ml) in the presence
of its substrate (1 mM) for periods of up to 3 hours. Two-
hour incubation with X-XO (1 mM and 10-100 mU/ml)
caused a dose-dependent and significant reduction in cell
viability, as assessed by the MTT assay (Figure 1). For the
subsequent experiments, a concentration of X-XO of 1 mM
plus 50 mU/ml was selected that led to a decrease in cell
viability close to 60%.
Effect of silybin, SilPho, and new silybin glycoconjugates on
X-XO induced cell damage. Underbasal conditions, the
incubation of MKN28 and HepG2 cells with silybin,SilPho,
and new silybin glycoconjugates led to two different results
(Figure 1). SilPho and new silybin glycoconjugates did not
affect cell viability, while silybin induced cell death of about
50%, even at the lower dose used, both of MKN28 and
HepG2 cells. The pre-treatment of cells with silybin and new
silybin glycoconjugates (before X-XO incubation) did not
affect cell viability, while SilPho had a protective effect
(Figure 1). In Figure 1, the concentration of molecules
utilized and the time of observation reported are 50 μg and
24 hours, respectively. With the exception of the SilPho (see
later), the same results have been verified at 10, 25, 50, 75,
100, 200 μM and at 1-48 hours of observation (data not
shown).
As the oxidative damage induced by X-XO and the effect
of pre-treatment were similar in MKN28 and HepG2 cells
we decided to perform the following experiments only in
MKN28 cell line.
Effect of silybin and of SilPho on X-XO induced lipid
peroxidation in MKN28 cells. ROS-induced cell damage is
associated with cell membrane disruption due to lipid
in vivo 29: xxx-xxx (2015)
4
Figure 2. Effect of silybin (A) and silybin–phosphatidylcholine complex
(SilPho) (B) on Malondialdehyde (MDA) in MKN28 (A) and HepG2 (B)
cell before and after induction of oxidative stress with xanthine-xanthine
oxidase (X-XO). The data are reported as means±SD of three
experiments. *p<0.05 and **p<0.01 vs. 0 μg/ml.
peroxidation. Therefore, we hypothesized that silybin and
SilPho might prevent lipid peroxidation caused by ROS
generated by X-XO. In this light, we evaluated whether
pretreatment with silybin and SilPho was able to counteract
X-XO-increased cellular MDA, a marker of lipid
peroxidation. Exposure of MKN28 cells to X-XO caused
an approximately two-fold increase in cellular MDA
concentration compared with untreated cells (Figure 2).
Moreover, pretreatment with SilPho (Figure 2B) (25-100
μg) but not with silybin (10-50 μg) (Figure 2A)
significantly prevented X-XO-induced increase of cell
MDA. These results suggest that the protective effect of
SilPho was partially due to inhibition of ROS-induced lipid
peroxidation.
Data regarding new silybin glycoconjugates are not
reported because experimental results were similar to those
obtained with silybin.
Effect of silybin and SilPho on MKN28 cell apoptosis and
necrosis before and after induction of oxidative stress with
X-XO. The pre-incubation with SilPho showed a dose-
dependent protective effect (Table I). These effects are,
almost in part, explained by the results obtained by flow
cytometry (FACS). In fact, as reported in Table II, we
found that the two assessed substances differently affected
cell vitality. Under basal conditions, silybin induced
apoptosis and SilPho did not induce any effect. Oxidative
stress caused cell death primarily by inducing cell
necrosis. The concomitant presence of silybin and
oxidative stress enhanced the ability of the latterto induce
apoptosis. SilPho had no effects on apoptosis, but
significantly counteracted cell necrosis. The increase of
silybin and SilPho concentrations up to 100 μg did not
modify these results.
Discussion
Several reports have been published on silymarin and its
flavonolignan obtained from the seeds of milk thistle (Silybum
marianum) constituents regarding their liver-protective,
antioxidant, and free-radical scavenging activities (1, 3-5).
Silybin acts, both in vitro and in vivo, as a radical scavenger by
increasing the levels of superoxide dismutase and glutathione
peroxidase and by reducing MDA and 4-hydroxynonenal (26),
markers of lipid peroxidation. Similarly, vitamin E and
phospholipids are well-known antioxidants and the
conjugation of these three substances without any alteration in
their stability enhances antioxidant action (27, 28). The
conjugation of silybin with phospholipids was performed in
order to modify its solubility and absorption in vivo. In fact,
while silybin has very low solubility in water, its conjugation
with other substances allowed its intravenous administration
and enhanced its oral bioavailability (11). In vivo, the complex
Federico et al: Silybin–Phosphatidylcholine Complex Combats Andoxidative Stress
5
Table I. Cell viability on treatment with silybin–phosphatidylcholine complex (SilPho) and xanthine-xanthine oxidase (X-XO). SilPho protects against
X-XO-induced damage in MKN28 and HepG2 cells.
Cell viability (%)
0 μg/ml 10 μg/ml 25 μg/ml 50 μg/ml 75 μg/ml 100 μg/ml 200 μg/ml
MKN28
Control 100 - - - - - -
X-XO 60 - - - - - -
SilPho - 85 80 83 84 83 80
SilPho+X-XO - 61 64 70 67 68 66
HepG2
Control 100 - - - - - -
X-XO 60 - - - - - -
SilPho - 88 85 89 85 88 84
SilPho+X-XO - 60 63 71 68 66 65
Table II. Cell deathwith pre-incubation of cells with silybin and
silybin–phosphatidylcholine complex (SilPho) under basal conditions
and after the induction of oxidative stress.
Apoptosis (%) Necrosis (%)
Basal 2.99 1.65
Silybin, 25 μg/ml 7.52* 6.38*
SilPho, 25 μg/ml 2.89 2.22
Oxidative stress 5.50* 12.97*
Oxidative stress + silybin, 25 μg/ml 22.05* 6.40*
Oxidative stress+ SilPho, 25 μg/ml 3.69 8.41*
*p<0.05 vs. basal.
of silybin with phospholids and vitamin E (RA) is rapidly
absorbed, with a blood peak concentration at 2 hours and a
large inter-organ distribution (14).
The new silybin derivatives obtained by Zarrelli et al. have
a higher water solubility than that of silybin, with enhanced
biological efficacy, binding affinity, and overall uptake (21).
In the present study, we assessed the effects of all these
compounds on cell viability and evaluated whether silybin or
SilPho pre-treatments were able to counteract X-XO-induced
increase of intracellular MDA. Exposure of MKN-28 cells to
X-XO caused an approximately two-fold increase in MDA
level as compared to untreated cells. Moreover, pretreatment
with SilPho and silybin prevented X-XO-induced
intracellular MDA increase. This suggests that the protective
and antioxidant effect of SilPho and silybin is, at least in
part, due to inhibition of ROS-mediated lipid peroxidation.
In vitro studies revealed that flavonoids can have
considerable antioxidant activity in a wide range of chemical
oxidation systems (29, 30). In our study, silybin and SilPho
exhibited powerful spontaneous antioxidant capacity in
human gastric and liver cells. Moreover, we evaluated the
protective effect on X-XO induced injury in MKN-28 cells
line measuring cell viability, and we found that only SilPho
had a dose-dependent protective effect. It is likely that
phospholipids have a protective effect against X-XO-induced
cell death by stabilizing plasma membranes.
In our experimental system, cell death induced by
oxidative stress followed two different patterns. The first led
to necrosis, a typical consequence of acute metabolic
perturbation, and the second to apoptosis, the consequence
of programmed death (31). Silybin enhanced X-XO-induced
apoptosis and reduced X-XO-mediated necrosis, whereas
SilPho significantly counteracted only cell necrosis.
Previously, we demonstrated that RA induced a
normalization of circulating lipids in patients with non-
alcoholic steatohepatitis, probably by improving liver
function (32).
In conclusion, our results show that both silybin and
SilPho act as antioxidants in an in vitro cell system, reducing
MDA levels induced by oxidative stress. Moreover, SilPho
protects MKN-28 cells from X-XO-induced cell death, being
more active than silybinin protecting cells from oxidative
stress.
Con fli ct of Interest S tatement
All of authors have declared no personal or family conflicts of
interest in regard to this study. This study was not funded.
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Received May 15, 2015
Revised July 6, 2015
Accepted July 8, 2015
Federico et al: Silybin–Phosphatidylcholine Complex Combats Andoxidative Stress
7
July 8, 2015
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