Pharmaceutical Biology

2007, Vol. 45, No. 10, pp. 735738

Cancer Preventive Agents. 7. Antitumor-Promoting Effects of Seven

Active Flavonolignans from Milk Thistle (Silybum marianum)

Pharmaceutical Biology Downloaded from informahealthcare.com by Monash University on 06/03/13

For personal use only.

on Epstein-Barr Virus Activation

An-Shen Lin1, Makio Shibano1, Kyoko Nakagawa-Goto1, Harukuni Tokuda2, Hideji Itokawa1,
Susan L. Morris-Natschke1, and Kuo-Hsiung Lee1
1

Natural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, North
Carolina, USA; 2Department of Biochemistry and Molecular Biology, Kyoto Prefectural University of Medicine,
Kamigyo-ku, Kyoto, Japan

Abstract
Seven pure flavonolignans were isolated from an extract
of milk thistle [Silybum marianum (L.) Gaertn (Asteraceae)], by semipreparative reverse-phase HPLC, and
identified based on spectroscopic and LC-MS=IT-TOF
data. All seven compounds were screened as potential
antitumor-promoting agents by using the in vitro
short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)induced Epstein-Barr virus early antigen (EBV-EA) activation assay in Raji cells. They showed good inhibitory
activity (87.794.9%) at 1000 mol ratio=TPA. Silychristin A (1) and silychristin B (2) were slightly more potent
than the well-known antitumor promoter b-carotene.
Silychristins A (1) and B (2) and isosilybins A (6) and
B (7) were more active than the clinically proven cancer
prevention components silybins A (4) and B (5).
Keywords: Antitumor promotors, cancer preventiveagents,
flavonolignans, milk thistle, Silybum marianum, silymarin.

Introduction
Milk thistle, Silybum marianum (L.) Gaertn (Asteraceae),
is native to the Mediterranean area and was grown in
southern Europe as a vegetable (Ball & Kowdley,
2005). Extracts of S. marinaum have been used as traditional medicine since the time of ancient Greece (Flora
et al., 1998; Dhiman & Chawla, 2005; Rambaldi et al.,

2005). Silymarin is the collective name of several flavonolignans, including silybinin, silydianin, and silychristin, extracted from S. marianum (Luper, 1998; Crocenzi
& Roma, 2006). For more than 25 years, silymarin has
been used clinically in Europe as an antihepatotoxic
agent (Bhatia et al., 1999), and in recent years, milk thistle has become one of the most popular herbal remedies
used by patients with liver disease and ranked 10th in
U.S. sales among all supplements sold in 2000 (Jacobs
et al., 2002).
Previous studies have related the hepatoprotective
effects of silymarin to its antioxidant and strong free
radical scavenging effects (Luper et al., 1998) and more
recently to anticholestatic properties (Crocenzi & Roma,
2006). In addition, other studies have reported its effects
in the prevention and therapy of cancer as summarized
recently by Aggarwal and Shishodia (2006). The anticancer effect of silymarin has been linked to the main
constituent, silybin, in milk thistle. Silybin consists of
two isomers, silybin A and silybin B, which have shown
antineoplastic activity in several cancer models, and a
phase I clinical study in prostate cancer patients was
reported recently (Flaig et al., 2007). Additional studies
of silybin in cancer prevention were also summarized
(Katiyar, 2005; Singh & Agrawal, 2005). In our continuing investigation of antitumor-promoting agents, an
extract of milk thistle was subjected to high-performance
liquid chromatography (HPLC). Seven flavonolignans

Accepted: July 3, 2007.

Address correspondence to: Kuo-Hsiung Lee, Natural Products Research Laboratories, School of Pharmacy, 315 Beard Hall, Campus
Box #7360, University of North Carolina, Chapel Hill, NC 27599-7360, USA. Tel.: 1-919-962-0066; Fax: 1-919-966-3893; E-mail:
khlee@unc.edu
DOI: 10.1080/13880200701585592

# 2007 Informa Healthcare USA, Inc.

736

A.-S. Lin et al.

were collected and identified based on spectroscopic and

LC-MS=IT-TOF data. All seven compounds were
screened for antitumor-promoting effects. The results
showed that these compounds are potent inhibitors of
skin tumor promotion.

Materials and Methods

Pharmaceutical Biology Downloaded from informahealthcare.com by Monash University on 06/03/13

For personal use only.

Chemistry
Milk thistle extract was dissolved in methanol and separated on a semipreparative HPLC system (Shimadzu
SLC 10A system controller, Shimadzu LC-20A pump)
with a photodiode array detector (Shimadzu SPDM10A detector). The separation was performed on a
Alltech Alltima C18-5 22 i.d.
250 mm column. Seven
major peaks were collected at UV absorption 254 nm
with an isocratic methanol-water (52:48) solvent system.
At a flow rate of 4.5 mL=min, the retention times for
each compound were 34.8, 38.8, 44.5, 87.9, 97.6, 126.2,
and 135.0 min, respectively. From UV, 1H NMR, 13C
NMR, and LC-MS=IT-TOF data, these compounds
were identified as silychristin A (1), silychristin B (2),
silydianin (3), silybin A (4), silybin B (5), isosilybin A
(6) and isosilybin B (7) (Fig. 1), and these assignments

Figure 1. Structures of compounds 17.

were confirmed by comparing their spectroscopic data

with prior literature (Lee & Liu, 2003; Smith et al., 2005).

In vitro EBV-EA activation assay

Epstein-Barr virus early antigen (EBV-EA) positive
serum from a patient with nasopharyngeal cancer
(NPC) was provided by Professor H. Hattori (Department of Otorhinolaryngology, Kobe University). The
EBV genome-carrying human lymphoblastoid cell (Rahi
cells derived from Burkitt lymphoma) were cultivated in
10% fetal bovine serum (FBS) RPMI 1640 medium
(Sigma R8758; Sigma, St. Louis, MO, USA). Spontaneous activation of EBV-EA in a subline of Raji cells
was less than 0.1%. The inhibition of EBV-EA activation
was assayed using Raji cells (virus nonproducer type)
incubated at 37C for 48 h in 1 mL of medium containing
n-butyric acid (4 mM, inducer), TPA (32 pM 20 ng in
2 mL DMSO), and various amounts of the test compound
(17) dissolved in 5 mL DMSO. Smears were made from
the cell suspension. The EBV-EA inducing cells were
stained with high-titer EBV-EA positive serum from
NPC patients and detected by an indirect immunofluorescence technique. In each assay, 500 cells were counted,
and the number of stained cells (positive cells) was

Pharmaceutical Biology Downloaded from informahealthcare.com by Monash University on 06/03/13

For personal use only.

Antitumor-promoting flavonolignans
recorded. Triplicate assays were performed for each
data point. The average EBV-EA induction of the test
compound was expressed as a ratio relative to the
control experiment (100%), which was carried out with
n-butyric acid (4 mM) plus TPA (32 pM). EBV induction was ordinarily around 35%, and the viability of
treated Raji cells was assayed by Trypan blue staining
method. The cell viability of the TPA positive control
was greater than 80%. Only compounds that induced
less than 80% (% of control) of the EBV-active cells
(those with a cell viability of more than 60%) were considered able to inhibit the activation caused by promoter substances.

Cytotoxicity determination
For the determination of the cytotoxicity or cell viability
of surviving cells, the Trypan blue staining method was
used. After EBV-EA activating assays, 0.1 mL of treated
cells (suspended in PBS) was stained with 0.1 mL of
0.25% Trypan blue solution. Dead cells were stained
blue. Viable unstained cells were counted.

Results and Discussion

The biological screening was carried out by using a shortterm in vitro synergistic assay on EBV-EA activation
induced by TPA (Henle & Henle, 1966; Takasaki et al.,
1990). In this assay, we used three different reference
compounds, b-carotene, curcumin, and glyzyrrhizin,
which are all widely studied natural products and known
to be active in this widely used test for cancer prevention
using animal models (Suzuki et al., 2006; Wang et al.,
2006). In this test system, all seven compounds showed
potent inhibition of EBV activation (Table 1), which
was comparable or better than that of the reference
compounds. In addition, all of the test compounds
showed low cytotoxicity toward Raji cells (IC50 398
482 mM). The results in Table 1 show that the isomeric
compounds 1 and 2 had significant EBV-EA inhibition
effects (92.7% and 64.1% for 1; 94.9% and 66.7% for
2) at test concentrations of 1000 and 500 mol ratio=TPA,
TPA, respectively, and were the two most potent compounds among all test samples. Compounds 6 and 7
showed similar inhibition effects to those of b-carotene
(91.4% inhibition at 1000 mol ratio=TPA, 65.8% at
500 mol ratio=TPA). Moreover, at concentrations of
100 and 10 mol ratio=TPA, 6 and 7 displayed greater
inhibition than b-carotene. Compounds 3, 4, and 5 also
showed good inhibition effects at concentrations of
1000, 500, and 100 mol ratio=TPA.
Silybins A and B (4 and 5) are the major components
in the active extract of milk thistle (silymarin) and have
been linked previously to skin cancer prevention effects

737

Table 1. Inhibitory effects of compounds 17 on TPAmediated EBV-EA induction.

Concentration (mol ratio=TPAa)
1000
Compound
1
2
3
4
5
6
7
b-Carotenec
Curcuminc
Glyzyrrhizinc

500

100

10

IC50

Percentage of EBV-EA induction (%)

7.3
5.1
11.9
12.3
10.0
7.6
9.1
8.6
0
27.4

(60)b
(60)
(60)
(60)
(60)
(60)
(60)
(70)
(60)
(70)

35.9
33.3
41.6
46.1
43.8
36.4
42.7
34.2
22.8
63.3

76.4
75.1
77.3
79.0
77.2
75.9
77.9
82.1
81.7
83.3

96.1
93.5
97.2
100
97.0
96.7
97.2
100
100
100

398
347
425
482
447
400
437
397
292
572

TPA, 12-O-tetradecanoylphorbol-13-acetate; concentration is

20 ng=mL (32 pmol=mL).
b
Values in parentheses are viability percentages of Raji cells.
c
Positive control.

via anti-inflammatory, antioxidant, and immunomodulatory mechanisms (Katiyar, 2005). Our study results
now reveal that all seven major compounds in silymarin
show good EBV-EA inhibition activities. In our testing
model, silychristins A and B (1 and 2) and isosilybins
A and B (6 and 7) had better inhibition effects compared
with b-carotene, and silybins A and B (4 and 5) showed
lower activity compared with the other flavonolignans in
silymarin. Further in vivo testing on mouse skin papillomas is ongoing.
In conclusion, seven pure compounds silychristin A
(1), silychristin B (2), silydianin (3), silybin A (4), silybin
B (5), isosilybin A (6), and isosilybin B (7) were isolated
from an extract of milk thistle. Evaluation with an in vitro
EBV-EA activation assay showed that silychristin B (2)
was the most active compound with 94.9% EBV-EA
inhibition at 1000 mol ratio=TPA. As it also showed
low cytotoxicity, 2 could be valuable as an antitumor
promoter or as a lead compound for new cancer
preventive drug development.

Acknowledgments
This investigation was supported in part by grant
CA-17625 from the National Cancer Institute and
GM-076152 from National Institute of General Medial
Sciences, NIH, awarded to K.H.L. This study was also
supported in part by a grant from the Ministry of
Education, Sciences, Sports and Culture, and the Ministry of Health and Welfare, Japan (H.T.). Thanks are due
to Dr. Takashi Tatsuzaki of Tokiwa Phytochemical Co.,
Ltd, for the gift of Silybum marianum extract.

738

A.-S. Lin et al.

Pharmaceutical Biology Downloaded from informahealthcare.com by Monash University on 06/03/13

For personal use only.

References
Aggarwal BB, Shishodia S (2006): Molecular targets of
dietary agents for prevention and therapy of cancer.
Biochem Pharmacol 71: 13971421.
Ball KR, Kowdley KV (2005): A review of Silybum marianum (milk thistle) as a treatment for alcoholic liver
disease. J Clin Gastroenterol 39: 520528.
Bhatia N, Zhao J, Wolf DM, Agarwal R (1999): Inhibition
of human carcinoma cell growth and DNA synthesis by
silibinin, an active constituent of milk thistle: Comparison with silymarin. Cancer Lett 147: 7784.
Crocenzi FA, Roma MG (2006): Silymarin as a new hepatoprotective agent in experimental cholestasis: New possibilities for an ancient medication. Curr Med Chem 13:
10551074.
Dhiman RK, Chawla YK (2005): Herbal medicines for liver
diseases. Dig Dis Sci 50: 18071812.
Flaig TW, Gustafson DL, Su L-J, Zirrolli JA, Crighton F,
Harrison GS, Pierson AS, Agarwal R, Glode LM
(2007): A phase I and pharmacokinetic study of
silybin-phytosome in prostate cancer patients. Invest
New Drugs 25: 139146.
Flora K, Hahn M, Rosen H, Benner K (1998): Milk thistle
(Silybum marianum) for the therapy of liver disease.
Am J Gastroenterol 93: 140143.
Henle G, Henle W (1966): Immunofluorescence in cells derived
from Burkitts lymphoma. J Bacteriol 91: 12481256.
Jacobs BP, Dennehy C, Ramirez G, Sapp J, Lawrence V
(2002): Milk thistle for the treatment of liver disease:
A systematic review and meta-analysis. Am J Med
113: 506515.
Katiyar S (2005): Silymarin and skin cancer prevention:
Anti-inflammatory, antioxidant and immunomodulatory effects (review). Int J Oncol 26: 169176.

Lee DY-W, Liu Y (2003): Molecular structure and

stereochemistry of silybin A, silybin B, isosilybin A,
and isosilybin B, isolated from Silybum marianum (milk
thistle). J Nat Prod 66: 11711174.
Luper S (1998): A review of plants used in the treatment of
liver disease: Part 1. Altern Med Rev 3: 410421.
Rambaldi A, Jacobs BP, Iaquinto G, Gluud C (2005): Milk
thistle for alcoholic and=or hepatitis B or C liver
diseasesa systematic Cochrane hepato-biliary group
review with meta-analyses of randomized clinical trials.
Am J Gastroenterol 100: 25832591.
Singh RP, Agarwal R (2005): Mechanisms and preclinical
efficacy of silibinin in preventing skin cancer. Eur J
Cancer 41: 19691979.
Smith WA, Lauren DR, Burgrss EJ, Perry NB, Martin RJ
(2005): A silychristin isomer and variation of flavonolignan levels in milk thistle (Silybum marianum) fruits.
Planta Med 71: 877880.
Suzuki M, Nakagawa-Goto K, Nakamura S, Tokuda H,
Morris-Natschke SL, Kozuka M, Nishino H, Lee K-H
(2006): Cancer preventive agents. Part 5. Anti-tumorpromoting effects of coumarins and related compounds
on Epstein-Barr virus activation and two-stage mouse
skin carcinogenesis. Pharm Biol 44: 178182.
Takasaki M, Konoshima T, Fujitani K, Yoshida A,
Nishimura H, Tokuda H, Nishino H, Iwashima A,
Kozuka M (1990): Inhibitors of skin-tumor promotion.
VIII. Inhibitory effects of euglobals and their related
compounds on Epstein-Barr virus activation. (1). Chem
Pharm Bull 38: 27372739.
Wang X, Nakagawa-Goto K, Kozuka M, Tokuda H,
Nishino H, Lee K-H (2006): Cancer preventive agents.
Part 6: Chemopreventive potential of furanocoumarins
and related compounds. Pharm Biol 44: 116120.