Natural Product Research

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ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Cytotoxic, hepatoprotective and antioxidant

activities of Silybum marianum variety albiflorum
growing in Egypt

Hayam S. Ahmed, Wafaa R. Mohamed, Abeer S. Moawad, Asmaa I. Owis,

Rasha R. Ahmed & Sameh F. AbouZid

To cite this article: Hayam S. Ahmed, Wafaa R. Mohamed, Abeer S. Moawad, Asmaa I. Owis,
Rasha R. Ahmed & Sameh F. AbouZid (2019): Cytotoxic, hepatoprotective and antioxidant
activities of Silybum�marianum variety albiflorum growing in Egypt, Natural Product Research, DOI:
10.1080/14786419.2019.1582039

To link to this article: https://doi.org/10.1080/14786419.2019.1582039

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Published online: 11 Mar 2019.

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1582039

Cytotoxic, hepatoprotective and antioxidant activities of

Silybum marianum variety albiflorum growing in Egypt
Hayam S. Ahmeda , Wafaa R. Mohamedb, Abeer S. Moawada, Asmaa I. Owisa,
Rasha R. Ahmedc and Sameh F. AbouZida
a
Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt;
b
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef,
Egypt; cCell Biology and Histology Division, Zoology Department, Faculty of Science, Beni-Suef
University, Beni-Suef, Egypt

ABSTRACT ARTICLE HISTORY

Silymarin prepared from the fruits of Silybum marianum (L.) Received 18 November 2018
Gaertn. (Asteraceae) has long been used for the treatment of liver Accepted 8 February 2019
disorders. This study was carried out to evaluate the protective
effect of the fruit extract of white-flowered S. marianum variety KEYWORDS
albiflorum Eig. (WSE) against paracetamol–induced liver toxicity Silybum marianum variety
albiflorum; silyhermin;
in rats. Silyhermin, isosilandrins A/B were identified as the major isosilandrins; cytotoxicity;
flavonolignans in WSE. Cytotoxic activities of WSE and isolated hepatoprotective; oxida-
flavonolignans compared to silymarin were carried out using tive stress
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 mg/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.

1. Introduction
Silymarin in the form of standardized extract of the fruits of Silybum marianum (L.)
Gaertn. (Asteraceae) has long been used to treat liver disorders (Morazzoni and

CONTACT Sameh F. AbouZid sameh.zaid@pharm.bsu.edu.eg

Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1582039.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 HAYAM S. AHMED ET AL.

Bombardelli 1995; Saller et al. 2001; Ferenci 2016). Two varieties of S. marianum grow
in Egypt; variety marianum with purple corollas and variety albiflorum Eig. with white
corollas. The two varieties accumulate flavonolignans in the fruits. Flavonolignans pos-
sess remarkable hepatoprotective activity and considered as the main active constitu-
ents of silymarin. The major flavonolignans isolated from the fruits of S. marianum
variety marianum are silybins A/B, isosilybins A/B, silychristin A, isosilychristin and sily-
dianin. The linear relationships existing between biosynthesis of these flavonolignans
was recently reported (AbouZid et al. 2018). In addition, biotechnological methods
used for enhanced production of these flavonlignans using plant tissue culture techni-
ques were reported (Rahimi et al. 2012, AbouZid 2014). (
)-silandrin, (þ)-silymonin
and 5,7-dihydroxy-chromone were isolated from variety albiflorum (Szilagi et al. 1981).
The structure and stereochemistry of silyhermin and neosilyhermins A and B isolated
from variety albiflorum were described by Fiebig and Wagner (1984).
Paracetamol-induced hepatotoxicity has been used as an experimental model to
test for potential hepatoprotective agents in many studies (Chen et al. 2009; Kiran
et al. 2012). It was proposed that N-acetyl-p-benzoquinone imine is produced from
paracetamol by cytochrome P450. This reactive metabolite is then reduced by
glutathione and excreted as cysteine or mercaptopurine conjugate. In overdosing
paracetamol, glutathione level in the liver is depleted. This causes oxidative stress and
hepatic cell damage (Mahadevan et al. 2006).
The aim of the current work was to investigate the in vitro cytotoxic activity and
in vivo hepatoprotective and antioxidant activities of extract prepared from the fruits
of S. marianum variety albiflorum (WSE) compared to standard silymarin.

2. Results and discussion

Fractionation of the WSE by two step column chromatography led to isolation of the
major flavonolignans. These were identified as silyhermin, isosilandrin A and isosilan-
drin B. Chemical structures of the isolated flavonolignans are shown in Figure S1.
Determination of flavonolignans contents in the extracts used in assessing biological
activity was carried out using quantitative nuclear magnetic resonance (qNMR). The
flavonolignans content in WSE were quantified using the doublet signal at 4.90 for iso-
silandrins (367.06 mg/mg extract) and the double doublet signal at 5.38 for silyhermin
(174.18 mg/mg extract). The flavonolignans contents in silymarin were quantified using
broad singlet signal at 2.73 for silydianin (35.08 mg/mg extract), doublet signal at 5.47
for silychristin (143.50 mg/mg extract) and the two double doublets signals around 7.
10 for silibinins and isosilibinins (325.53 mg/mg extract). 1H NMR spectra of WSE,
silyhermin and isosilandrins are shown in Figure S2.
In vitro cytotoxic activity of WSE and isolated flavonolignans was carried out against
normal human cell line (HFB-4) and human cancer cell line (Hep-G2) using sulforhod-
amine B (SRB) assay. Results revealed that WSE and the isolated flavonolignans have
no obvious harmful effects on HFB-4 cells. WSE (IC50 = 78.95 mg/ml) was 4.7 fold safer
than silymarin (IC50 = 16.83 mg/ml) on HFB-4 cells. Silyhermin (IC50 = 84.34 mg/ml)
showed slightly lower cytotoxicity than isosilandrins (IC50 = 72.14 mg/ml) on HFB-4
cells. Cytotoxic activities against Hep-G2 were in the following order: doxorubicin
 NATURAL PRODUCT RESEARCH 3

(IC50 ¼ 0.50 mg/ml), WSE (IC50 = 17.18 mg/ml), silymarin (IC50 = 22.75 mg/ml), isosilan-
drins (IC50 = 61.39 mg/ml) and silyhermin (IC50 = 68.19 mg/ml). WSE had comparable
cytotoxic activity to silymarin on Hep-G2 cells. WSE had higher cytotoxic activity on
Hep-G2 cells than the isolated flavonolignans silyhermin and isosilandrins. This result
indicates a possible synergistic effect of the flavonolignans in WSE or the cytotoxic
activity may be attributed to other compounds. Cytotoxicity assay charts are shown
in Figure S3.
Few studies are available on in vitro hepatoprotective and antioxidant activities of fla-
vonolignans isolated from the S. marianum variety albiflorum. For example, silyhermin
showed prominent antihepatotoxic action at the dose of 1.0 mg/ml in carbon tetrachlor-
ide-treated primary cultured rat hepatocytes (Hikino et al. 1984). Silyhermin showed
strongest activity at the dose of 0.1 mg/ml and weaker activity at higher dose (1 mg/ml)
in D-galactosamine-treated primary cultured rat hepatocytes. (
)-silandrin and (
)-isosi-
landrin showed higher activity than (þ)-silybins as inhibitors of oxidative burst of human
polymorphonuclear leukocytes stimulated by phorobol-myristate-acetate (Samu et al.
2004). (
)-isosilandrin A exhibited anti-inflammatory effect in lipopolysaccharide-
stimulated RAW264.7 macrophages (Cho et al. 2014). The underlying molecular mechan-
ism was described through suppressing the production of pro-inflammatory mediators
(NO, iNOS, MCP-1, TNF-a, IL-6, and IL1b) and inhibition of NF-jB activation.
Literature lacks in vivo animal studies on white-flowered S. marianum constituents.
Therefore, we evaluated the in vivo antioxidant and hepatoprotective activities against
paracetamol-induced liver toxicity for WSE on male Wistar rats. WSE at a dose of
200 mg/kg did not significantly affect liver functions and contents of reduced GSH,
MDA or SOD activity as compared to normal control group indicating its safety to liver
tissues. The activities of ALT and AST as well as the contents of direct and total biliru-
bin were significantly increased (p < 0.05) in paracetamol-treated group compared to
the control group (Table S1). Administration of WSE (50, 100 and 200 mg/kg) signifi-
cantly reduced paracetamol-induced hepatotoxicity in rats as illustrated by significant
reduction in serum ALT, AST, direct and total bilirubin as compared to the paracetamol
control group (Table S1). The effect of WSE (50 mg/kg) was similar to that obtained by
administration of the standard silymarin (100 mg/kg) indicating its higher hepatopro-
tective effect. This may be attributed to the higher bioavailability of the lower polarity
flavonolignans contained in the WSE than those contained in standard silymarin.
Administration of WSE (50, 100 and 200 mg/kg) significantly reduced paracetamol-
induced oxidative stress in rats liver homogenates as illustrated by significant elevation
in hepatic reduced GSH content and SOD activity together with significant reduction in
hepatic MDA content as compared to the paracetamol-administered group (Table S1).
The effect of WSE (50 mg/kg) was similar to that obtained by administration of silymarin
(100 mg/kg). Interestingly, WSE (100 and 200 mg/kg) significantly increased hepatic
reduced GSH content and SOD activity while significantly reduced liver MDA content as
compared to standard silymarin (100 mg/kg) indicating higher activity (Table S1).
Figure S4 A–H shows the results of liver histological examination of control and
treated groups. Normal liver parenchyma arranged around the central vein were
observed in the control group rats (Figure S4A). Administration of WSE (200 mg/kg,
p.o.) did not affect normal structure of hepatocytes but showed few degenerating cells
4 HAYAM S. AHMED ET AL.

with activated Kupffer cells (Figure S4 B). Oral administration of paracetamol (600 mg/
kg) produced severe liver damage as evidenced by centrilobular necrosis of hepato-
cytes associated with periportal infiltration of inflammatory cells and degenerative
changes of scattered hepatocytes (Figures S4 C and D). Liver samples from rats admin-
istered paracetamol with silymarin (100 mg/kg) showed apparent normal hepatocytes
with scattered binucleated cells and mild sinusoidal dilatation (Figure S4 E). Oral
administration of WSE (50 mg/kg) with paracetamol produced mild central vein
congestion, mild infiltration of inflammatory cells and mild proliferation of bile ducts
(Figure S4 F). Oral administration of WSE (100 mg/kg) greatly improved abrasions
induced by paracetamol as evidenced by apparent intact hepatocytes with vesicular
nuclei, activated Kupffer cells and moderate sinusoidal dilatation (Figure S4 G). Oral
administration of WSE (200 mg/kg) showed apparent intact many hepatocytes with
mild congestion of hepatic blood vessels and activated Kupffer cells (Figure S4 H).
These effects were comparable to that obtained by oral administration of standard
silymarin (100 mg/kg).

3. Experimental
Experimental details are available online as a supplementary material.

4. Conclusion
The protective effect of WSE on the liver tissues induced by administration of paraceta-
mol is probably associated with its antioxidant properties. This is supported by previous
in vitro studies. In addition, the present data proves that the hepatoprotective effects
produced by WSE are better than silymarin at the same dose level. To the best of our
knowledge this is the first report about in vivo biological effect of extract prepared from
the fruits of S. marianum variety albiflorum. Further studies are needed to identify
the key molecular interactions that govern the regulation of antioxidant and hepato-
protective activities of the extract in response to paracetamol–induced liver injury.

Disclosure statement
No potential conflict of interest was reported by the authors.

ORCID
Hayam S. Ahmed http://orcid.org/0000-0002-7379-3078
Sameh F. AbouZid http://orcid.org/0000-0002-0789-767X

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