Preparation and effects of 2,3-dehydrosilymarin, a promising and potent antioxidant and free radical scavenger

Article abstract of DOI:10.1111/j.2042-7158.2010.01210.x

Objectives Silymarin or silybin has been effectively used for treating liver diseases and acute liver injury partly due to its antioxidant activity. In this study, 2,3-dehydrosilymarin, a compound exhibiting remarkable antiradical/antioxidant activity, wa

Full text of DOI:10.1111/j.2042-7158.2010.01210.x

Research Paper
JPP 2011, 63: 238–244
© 2010 The Authors
JPP © 2010 Royal
Preparation and effects of 2,3-dehydrosilymarin, a promising
Pharmaceutical Society
Received June 17, 2010
Accepted September 24, 2010
DOI
10.1111/j.2042-7158.2010.01210.x
ISSN 0022-3573
and potent antioxidant and free radical scavengerjphp_1210 238..244
Shanshan Tong, Chang Chu, Yuan Wei, Li Wang, Xizhe Gao,
Ximing Xu and Jiangnan Yu
Department of Pharmaceutics, School of Pharmacy, Jiangsu University, Zhenjiang, China
Abstract
Objectives Silymarin or silybin has been effectively used for treating liver diseases and
acute liver injury partly due to its antioxidant activity. In this study, 2,3-dehydrosilymarin, a
compound exhibiting remarkable antiradical/antioxidant activity, was prepared from sily-
marin for the first time. The solubility, radical scavenging capacity and liver protecting
activity of 2,3-dehydrosilymarin were studied and compared with silybin, dehydrosilybin
and silymarin.
Methods The structures of its main components were verified by ultra-performance liquid
chromatography/mass spectrometry (UPLC-MS) and other spectral analysis. In addition, a
rapid screening method, online high-performance liquid chromatography/1,1-dipheny1-2-
picrylhydrazyl (HPLC-DPPH) system, was developed for identifying the individual antioxi-
dants in 2,3-dehydrosilymarin.
Key findings Both in-vitro and in-vivo results markedly proved that dehydrosilymarin has
decent aqueous solubility and remarkable antiradical/antioxidation capacity. Moreover, 2,3-
dehydrosilybin and 2,3-dehydrosilychristin were identified to be the two major active com-
pounds contained in 2,3-dehydrosilymarin.
Conclusions Our results suggest that 2,3-dehydrosilymarin may be a promising and potent
alternative for inhibition of free radical and prevention of oxidation.
Keywords antioxidant; dehydrosilymarin; free radical scavenger; online HPLC-DPPH
Introduction
The flavonolignan silybin is the major bioactive component of the extract from the seeds of
the milk thistle (Silybum arianum (L.) Gaertn.). Silybin is often used in the prevention and
treatment of various liver diseases.[1,2] It acts mainly as an effective radical scavenger
(anti-lipoperoxidant), and also as an antioxidant. Nevertheless, the bioavailability of silybin
is rather limited by its low solubility in water.[1] An oxidized form of silybin (so-called
2,3-dehydrosilybin) was found to exhibit significantly greater antioxidant and anti-cancer
activity than silybin.[3] Unfortunately, dehydrogenization of silybin led to an impaired water
solubility, which considerably compromised the therapeutic efficacy of 2,3-
dehydrosilybin.[2,4]
Silymarin consists of 70–80% of flavanolignans, including silybin, isosilybin, silydianin
and silychristin; the remaining ingredients are mainly polyphenolic compounds.[1] It has
been used in Europe since the 16th century and continues to be used there in the treatment
of liver disease.[5] The therapeutic effects of silymarin suggest that it possesses potent
antiradical and antioxidant activity.
Silybin, 2,3-dehydrosilybin and silymarin have been extensively studied in recent years.
Based on the comparison between silybin and 2,3-dehydrosilybin, we inferred that 2,3-
dehydrosilymarin might have a better antioxidant activity than silymarin. To the best of our
knowledge, few studies involve 2,3-dehydrosilymarin and no reports on the drug effects of
2,3-dehydrosilymarin were found. Therefore, we prepared 2,3-dehydrosilymarin, and then
studied its antioxidant activity and radical scavenging capacity. 1,1-Dipheny1-2-
picrylhydrazyl free radical (DPPH) is often used for the evaluation of the general radical
scavenging ability of antioxidants.[6,7] The O.D.517 nm absorbance decreases as the radical
is scavenged by antioxidants to form the stable DPPH-H molecule. However, DPPH assay
alone is not able to identify those active ingredients in a mixture of compounds such
as silymarin or dehydrosilymarin. An improved online HPLC-DPPH method combining
Correspondence: Jiangnan Yu
and Ximing Xu, Department of
Pharmaceutics, School of
Pharmacy, Jiangsu University,
Zhenjiang 212013, China.
E-mail: yjn@ujs.edu.cn;
xmxu@ujs.edu.cn
238

2,3-Dehydrosilymarin, potent antioxidant and free radical scavenger
Shanshan Tong et al.
239
Table 1 Composition of the mobile phase used in the UPLC-MS
analysis
separation and activity evaluation of antioxidants was firstly
[8]
reported by Koleva et al.
This method has subsequently
been successfully used to detect radical scavenging com-
Time (min)
Flow rate
(ml/min)
%A
%B
pounds by another two groups.[9–11]
In this study, we investigated the antioxidant activity and
the radical scavenging capacity of 2,3-dehydrosilymarin. We
also identified its active ingredients with the online HPLC-
DPPH method. The method, which combines separation of
antioxidants and activity evaluation, presents a major advan-
tage for such investigations. Our results suggested that 2,3-
dehydrosilymarin had better antiradical and antioxidative
capacity than silybin, 2,3-dehydrosilybin and silymarin. More
interestingly, the solubility of 2,3-dehydrosilymarin was sig-
nificantly improved compared with 2,3-dehydrosilybin, and
could lead to a better bioavailability and therapeutic efficacy.
Hepato-protective effects against CCl4 were observed when
mice were pre-treated with 2,3-dehydrosilymarin (doses
28.4 mg/kg, 142 mg/kg, 284 mg/kg), while no protection was
afforded when mice were pretreated with silybin, 2,3-
dehydrosilybin or silymarin at the same doses. Therefore,
2,3-dehydrosilymarin might be a good candidate for further
development as an antioxidant remedy.
Initial
0.10
10.00
12.00
12.10
0.300
0.300
0.300
0.300
0.300
30.0
30.0
100.0
100.0
30.0
70.0
70.0
0.0
0.0
70.0
A: Methanol; B: 0.2% formic acid aqueous solution.
2,3-Dehydrosilybin and 2,3-dehydrosilycristin were pre-
pared in accordance with the methods stated above, expect
silybin and silycristin were used as initial materials. Their
structures, verified by IR and NMR, were consistent with the
results reported in literature.[12]
UPLC-MS analysis
UPLC/MS/MS were performed on Waters MALDI Synapt
Q-TOF MS (Milford, MA, USA) using an ESI source with
positive ion mode. Chromatography was performed on an
ACQUITY UPLC BEH C-18 column (100 ¥ 2.1 mm, i.d.
1.7 mm particle size; Waters, Milford, MA, USA). The binary
gradient employed methanol (A) and 0.2% formic acid
aqueous solution (B) according to Table 1. The flow rate was
0.3 ml/min. The column temperature was kept at 40°C. UV
spectra were recorded over the range of 200–750 nm. The
injection volume was 5 ml. The ESI source was operated at
100°C in positive mode to produce MH+ ions. The desolva-
tion temperature was set at 250°C, extract voltage was 3.0 V,
desolvation gas and cone gas was set at 500 and 50 l/hr,
respectively. The full-scan mass spectra were acquired over
the range of 50–1000 amu. Capillary voltages were 3.5 kV in
ESI+ and cone voltages were 30 V.
Materials and Methods
Chemicals
The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH.) was pur-
chased from Sigma-Aldrich (St Louis, MO, USA). Silybin
and silymarin were kindly provided by Zhongxing Pharma-
ceutical Co. Ltd (Jiangsu, China). Silycristin was from Tauto
Biotech Co. Ltd (Shanghai, China). All other solvents/
chemicals were purchased from Guoyao Chemical Regent
Co., Ltd (Shanghai, China) except specifically mentioned.
HPLC-grade solvents were used in this study.
Animals
All the mice used were adult males, 17–22 g, purchased from
the animal distribution center ofYangzhou University (Jiangsu,
China). The mice were kept on an artificial 12-h light–dark
cycle and given free access to standard laboratory diet and
water according to the regulations for the administration of
affairs concerning experimental animal care (State Council of
the China, 1988). All the research protocols were approved by
the Research Ethics Committee of Jiangsu University.
Solubility studies
The experiments were carried out essentially according to
the reported method[13] with slight modifications. To prepare
saturated solutions, excess amount (20 mg) of silybin,
2,3-dehydrosilybin, silymarin, 2,3-dehydrosilymarin were
dissolved in 10 ml double-distilled H2O and sonicated for 1 h
by a sonicator (KQ-500DE, Kunshan, China). After sonica-
tion, those samples were shaken in a vibrator with homother-
mal air bath (25°C) for 60 h, and then centrifuged at 15 000g
for 10 min. The supernatants were filtered through 0.45 mm
cellulose acetate membrane filters to remove undissolved
compounds. Once saturated solutions were prepared, they
were diluted for UV absorbance analysis. The OD 288 nm
absorbances of silybin and silymarin solutions, and the OD
255 nm absorbances of 2,3-dehydrosilybin and 2,3-
dehydrosilymarin solutions were determined by a UV–Vis
spectrophotometer (UV-2401PC, Shimadzu, Japan).
Preparation of 2,3-dehydrosilymarin,
2,3-dehydrosilybin and 2,3-dehydrosilycristin
Silymarin(6 g) was dissolved in 400 ml pyridine and heated to
90°C under reflux for 77 h with stirring. After reaction, pyri-
dine was removed using a rotary evaporator under 45 mbar at
60°C. To remove the residual pyridine, 50 ml toluene was
added and evaporated in vacuum at 80°C. The remaining pellet
was dissolved in ethyl acetate, loaded onto a silica gel column,
and then eluted with hot acetone. After these procedures,
acetone was removed from the products by distillation. The
leftover pellet was re-dissolved in hot ethanol and filtered
through a Double-ring #102 filter paper (Xinhua Paper Indus-
try Co. Ltd, Hangzhou, China). The pass-through was air dried
until ~4 g brown 2,3-dehydrosilymarin pellet was obtained.
To quantify the concentrations of the saturated solutions,
serial ethanol solutions of silybin, 2,3-dehydrosilybin, sily-
marin, and 2,3-dehydrosilymarin were prepared. The standard
curves were plotted according to their OD 255 nm or 288 nm

240
Journal of Pharmacy and Pharmacology 2011; 63: 238–244
UV
detector
(288/255
nm)
HPLC
UV
detector
(517nm)
pump
(mobile
phase)
C18 column
mixer
Rection coil
HPLC
pump
(DPPH)
Sample
introduction
Figure 1 Instrumental setup for the online RP-HPLC-DPPH measurement of radical scavenging compounds.
Table 2 The malondialdehyde values of fresh liver tissue samples from 10 tested groups
Group
Treatment
MDA (nmol/mg protein)
Group I
Blank
CCl4
1.62 Ϯ 1.86
Group II
Group III
Group IV
Group V
Group VI
Group VII
Group VIII
Group IX
Group X
5.5912 Ϯ 1.9471a
5.0742 Ϯ 1.5732a
4.0071 Ϯ 2.0734
4.5482 Ϯ 0.8705a
4.0259 Ϯ 2.2688
4.6483 Ϯ 1.1161a
2.8854 Ϯ 0.8841b
2.5416 Ϯ 0.6359b
2.7666 Ϯ 0.8815b
Silybin (142 mg/kg) + CCl4
2,3-Dehydrosilybin (28.4 mg/kg) + CCl4
2,3-Dehydrosilybin (142 mg/kg) + CCl4
2,3-Dehydrosilybin (284 mg/kg) + CCl4
Silymarin (142 mg/kg) + CCl4
2,3-Dehydrosilymarin (28.4 mg/kg) + CCl4
2,3-Dehydrosilymarin (142 mg/kg) + CCl4
2,3-Dehydrosilymarin (284 mg/kg) + CCl4
MDA, malondialdehyde. The experiments were repeated 10 times for each sample (n = 10). Data are shown as mean Ϯ SD. The differences across all
10 groups were significant (Kruskal–Wallis test, H = 43.433, d.f. = 9, P < 0.001). aSignificantly higher than the MDA values in Group I (Dunn’s test
subsequent to the Kruskal–Wallis test, P < 0.05). bSignificantly lower than the MDA values in Group II (Dunn’s test subsequent to the Kruskal–Wallis
test, P < 0.05).
absorbances. The concentrations of those samples were cal- UV–Vis spectrophotometer at 517 nm. The radical inhibition
culated from the corresponding standard curves.
efficacy was calculated from the absorbance change. The
radical scavenging activity of silybin, 2,3-dehydrosilybin,
silymarin and 2,3-dehydrosilycristin was also measured and
compared with that of 2,3-dehydrosilymarin. Their DPPH
scavenging efficacies were compared by IC50 values (the
concentration of tested compound which inhibited 50% radi-
cals), calculated from the mean values of triplicates at differ-
ent concentrations.
Online HPLC-DPPH analysis
The samples were dissolved in methanol at 100 mg/l concen-
tration for on-line HPLC-DPPH analysis. The instrumental
setup was applied according to the reported method
(Figure 1).[8,9,11] The separation of antioxidative components
was performed on a 150 ¥ 4.6 mm i.d., 5 mm particle size,
shimpak RP-18 column (Shimadzu, Japan). The mobile phase
was methanol and water (6 : 4) and the flow rate was 0.3 ml/
min. The signals were detected with a shimadzu SPD-10Avp
photo diode array at 288 nm wavelength for silymarin and
255 nm for 2,3-dehydrosilymarin. For online DPPH radical-
scavenging analysis, the flow reagent (2 mg/l DPPH in metha-
nol) was set to be 0.3 ml/min, and the induced bleaching was
detected photometrically as a negative peak at 517 nm. The
length of the capillary used for the postcolumn reaction was
adjusted to 465 cm (to achieve a reaction time of 90 s).
In-vivo study on the liver protection effects of
2,3-dehydrosilymarin
One-hundred mice were obtained and randomly divided into
10 groups (n = 10 in each group). Group I (blank group) mice
were orally administered with a 0.5% aqueous solution of
sodium carboxymethylcellulose (CMC-Na, 20 ml/kg dose)
daily for 15 days, and sesame oil (5 ml/kg dose) on day 16.
Group II (positive control group) were orally treated with
0.5% CMC-Na daily (20 ml/kg dose) for 15 days and then
challenged with CCl4 in sesame oil (0.3%, dose = 5 ml/kg) on
day 16. The protective effects of silybin, 2,3-dehydrosilybin,
Quatification of DPPH radical scavenging activity
The superoxide radical scavenging capacity of 2,3- silymarin and 2,3-dehydrosilymarin were tested in Groups
dehydrosilymarin was examined by free radical scavenging III–X (Table 2). Group III were pre-treated orally with silybin
(DPPH, superoxide) assay.[6] A 1-ml volume of ethanol solu- (7.1 mg/ml in 0.5% CMC-Na, 20 ml/kg dose) daily for 15
tion of DPPH (19.8 ¥ 10-4 mm) was added into 3 ml of 2,3- days and CCl4 in sesame oil (0.3%, 5 ml/kg body weight) on
dehydrosilymarin ethanol solutions at serial concentrations day 16. Group IV–VI mice were orally administered with
(1.8, 3.6, 5.4, 9, 27, 54 and 200 mg/l). After 30 min, the 2,3-dehydrosilybin (1.42 mg/ml, 7.1 mg/ml or 14.2 mg/ml in
absorbance changes of mixed samples were measured with a 0.5% CMC-Na, 20 ml/kg dose, respectively) daily for 15 days

2,3-Dehydrosilymarin, potent antioxidant and free radical scavenger
Shanshan Tong et al.
241
and CCl4 in sesame oil (0.3%, 5 ml/kg) on day 16. Group VII those of corresponding pure standards. Interestingly, the
mice were given silymarin (7.1 mg/ml in 0.5% CMC-Na, tR = 6.37 min peak (Figure 2) had similar prominent
20 ml/kg dose) orally for 15 days and then CCl4 in sesame oil fragments to those of 2,3-dehydrosilybin and 2,3-
(0.3%, 5 ml/kg body weight) on day 16. Group VIII–X mice dehydroisosilybin in m/z 400 to 200 region. This finding
were given 2,3-dehydrosilymarin orally at different concen- suggested that this peak could be a 2,3-dehydro-derivative of
trations (1.42 mg/ml, 7.1 mg/ml or 14.2 mg/ml in 0.5% CMC- isosilybin.
Na, 20 ml/kg dose, respectively) for 15 days and treated with
CCl4 in sesame oil (0.3%, 5 ml/kg) on day 16. All the mice
Solubility of 2,3-dehydrosilymarin
were sacrificed 24 h after the CCl4 challenge. Fresh liver
The aqueous solubility of 2,3-dehydrosilymarin was studied
samples were then dissected and the acute toxicity was tested
on the room temperature (25°C). The solubilities of silybin,
with malondialdehyde (MDA) studies.
2,3-dehydrosilybin and silymarin were also tested for com-
parison. The results are shown in Table 3. Among all samples,
Liver malondialdehyde determination
the solubility of 2,3-dehydrosilybin was the lowest, though
Fresh liver samples were immediately washed with ice-cold
2,3-dehydrosilybin has been frequently reported to have
saline. 0.3 g liver tissue was added into 3.0 ml ice-cold saline,
significant antioxidant activity. The poor solubility of
homogenized with a Fluker homogenizer (Fluker, Gemany)
for 15 s, and then centrifuged at 785g for 10 min to remove
2,3-dehydrosilybin in water was probably due to the dehydro-
genation between C2-C3. A flavonoid plane structure, which
debris. MDA concentrations in the supernatants were deter-
led to a substantial decrease of solubility, was formed
mined with a kit from Jiancheng Co. Ltd (Nanjing, China)
along with the conjugated double bond at C2-C3 in 2,3-
according to its instructions. The protein concentrations of the
dehydrosilybin. The application of 2,3-dehydrosilybin has
been substantially restricted by its poor solubility.[3] However,
in our results, the solubility of 2,3-dehydrosilymarin was dis-
tinctly higher than that of silybin and 2,3-dehydrosilybin.
Therefore, 2,3-dehydrosilymarin, with better aqueous solubil-
ity, could be a more promising antioxidant drug, having
improved bioavailability.
supernatants were determined with Coomassie Brilliant Blue
assay. The final liver MDA values were normalized by the
protein concentrations.
Statistical analysis
Kruskal–Wallis one-way analysis of variance on ranks fol-
lowed by Dunn’s test was used for the solubility analysis and
the MDA assay. Analysis was made using SigmaStat Software
(Version 3.5, Jandel Scientific Software, Corte Madern, CA,
USA)
DPPH radical scavenging activity of
2,3-dehydrosilymarin
The ability of various antioxidants – both pure compounds
and plant extracts – to terminate radical chain processes was
indirectly evaluated by different methods using various sub-
strates.[14] Conventional procedures for the screening and
identification of antioxidants in complex matrices require the
separation and purification of chemical compounds, which is
Results and Discussion
Preparation and composition analysis of
2,3-dehydrosilymarin
2,3-Dehydrosilymarin was prepared essentially according to a tedious and time-consuming. On-line HPLC radical scaveng-
reported method, which was originally designed for the prepa- ing activity measurement makes it possible to directly identify
ration of 2,3-dehydrosilybin.[3] Before the composition analy- active constituents in complex matrices.[15,16] In this study, an
sis of the 2,3-dehydrosilymarin, we prepared pure standards online DPPH-HPLC assay was used to identify antioxidant
of 2,3-dehydrosilybin and 2,3-dehydrosilycristin. The infra- constituents in silymarin and 2,3-dehydrosilymarin sepa-
red spectra of 2,3-dehydrosilybin and 2,3-dehydrosilycristin rately. Combined UV (positive signals) and DPPH quenching
were recorded using KBr pellets on a NICOLET AVATAR- (negative signals) chromatograms, fractions from 2,3-
1
370 spectrometer. The H NMR spectra were obtained on dehydrosilymarin are presented in Figure 4. With online
Bruker AVANCE DPX 200 and AMX 400 WB spectrometers. HPLC-DPPH analysis of silymarin, negative (DPPH quench-
DMSO-d6 was used as solvent, in which the observation of ing) peaks were hardly observed in any of the fractions. But
OH signals is possible. The solid-state infrared spectrum and for 2,3-dehydrosilymarin, two fractions were detected as posi-
1H NMR (400.13 MHz, 296 K) data of 2,3-dehydrosilybin tive peaks on the UV detector (255 nm), which showed
and 2,3-dehydrosilycristin were consistent with the results hydrogen-donating ability (negative peaks, with 90 s lag time
reported in literature.[12]
for reaction) toward the DPPH radicals. These two fractions
The composition of our 2,3-dehydrosilymarin was studied (tR = 16.0 min and tR = 50.7 min) were identified as 2,3-
using UPLC-MS analysis. typical profile of 2,3- dehydrosilychristin and 2,3-dehydrosilybin by comparing
dehydrosilymarin is shown in Figure 2. The retention time of their retention time with standard compounds.
A
peak 1 was 4.567 min with lmax at 257 nm and 373 nm,
Our results showed that the online HPLC-DPPH method
while the retention time of peak 2 was 6.088 min with lmax could be applied for a quick screening of antioxidant com-
at 254 nm and 369 nm. The mass spectrum of both peaks pounds in silymarin and 2,3-dehydrosilymarin. With the
showed correct mass: m/z 481.1 (MH)+. Peaks 1 and 2 were on-line HPLC-DPPH assay, we demonstrated that 2,3-
identified as 2,3-dehydrosilychristin and 2,3-dehydrosilybin dehydrosilybin and 2,3-dehydrosilychristin were the two
(Figure 3), respectively, by comparing their tR values, absor- major active antioxidants in 2,3-dehydrosilymarin. DPPH
bances from 200 nm to 600 nm, and mass spectra with free radical was also used to further quantify the free

242
Journal of Pharmacy and Pharmacology 2011; 63: 238–244
2
6.06
1
4.58
6.37
%
4.41
4.83
0
Time
10.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
451.1
8.00
9.00
100
Peak 1: 2,3-dehydrosilychristin
463.1
463.1
431.1
%
463.2
327.1
153.0
137.1
301.0
327.1
339.1
464.1
481.1
301.1
403.1
345.1
385.1
357.1
277.1
273.1
237.1
221.1
403.1
481.1
481.2
315.1
297.1
161.1
413.1
122.0
m/z
0
50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650
463.1
100
Peak 2: 2,3-dehydrosilybin
481.1
481.1
%
301.0
481.2
431.1
451.1
273.0
273.0
339.0
339.1
482.1
483.1
302.0
357.1
303.1
303.1
162.1
147.0
287.1
431.0
403.1
245.0
217.1
357.1
358.1
130.0
103.1
163.1
257.0
0
50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650
m/z
Figure 2 LC/MS analysis of 2,3-dehydrosilymarin, 1 = 2,3-dehydrosilychristin (4.58), 2 = 2,3-dehydrosilybin (6.06).

2,3-Dehydrosilymarin, potent antioxidant and free radical scavenger
Shanshan Tong et al.
243
Table 3 The tested aqueous solubilities of silybin, 2,3-dehydrosilybin, silymarin and 2,3-dehydrosilymarin samples at 25°C
Silybin
2,3-Dehydrosilybin
Silymarin
2,3-Dehydrosilymarin
Solubility (mg/ml)
55.28 Ϯ 0.23
3.45 Ϯ 0.39
452.12 Ϯ 13.74a,b
406.39 Ϯ 10.79a,b
The experiments were repeated 6 times for each sample. Data are shown as mean Ϯ SD. The differences across all four groups were significant
(Kruskal–Wallis test, H = 21.609, d.f. = 3, P < 0.001). aSignificantly higher than the solubility values of silybin (Dunn’s test subsequent to the
Kruskal–Wallis test, P < 0.05). bSignificantly higher than the solubility values of 2,3-dehydrosilybin (Dunn’s test subsequent to the Kruskal–Wallis test,
P < 0.05).
O
O
O
O
OH
OH
HO
HO
OMe
OH
OMe
OH
O
O
O
O
OH
OH
OH
OH
Silybin
OH
Dehydrosilybin
OH
OH
OH
O
O
OMe
OMe
HO
HO
O
O
O
OH
OH
OH
OH
Silychristin
Figure 3 Structures of silybin, 2,3-dehydrosilybin, silychristin and 2,3-dehydrosilychristin.
O
OH
OH
Dehydrosilychristin
100
90
80
70
60
50
40
30
20
10
0
0.005
0.000
500
400
300
200
100
1
–0.005
–0.010
–0.015
–0.020
–0.025
–0.030
–0.035
–0.040
–0.045
2
0
–100
–200
–300
–400
–500
0
50
100
150
200
250
0
5 10 15 20 25 30 35 40 45 50 55 60 65 70
Minutes
Concentration (mg/ml)
Figure 5 Scavenging of the DPPH radical by silybin (᭺), 2,3-
dehydrosilybin (᭹), silymarin (᭝), 2,3-dehydrosilymarin (ꢀ) and 2,3-
dehydrosilycristin (᭛). Results are means Ϯ SD (n = 3).
Figure 4 UV and DPPH radical quenching chromatograms of 2,3-
dehydrosilymarin, 1 = 2,3-dehydrosilychristin, 2 = 2,3-dehydrosilybin.
radical-scavenging activity of 2,3-dehydrosilymarin, sily-
marin, silybin, 2,3-dehydrosilybin and 2,3-dehydrosilycristin.
Their DPPH radical scavenging capacities were shown to be
dose-dependent (Figure 5); the IC50 values were calculated
for each sample. The tested IC50 values of silybin, 2,3-
dehydrosilybin, silymarin, 2,3-dehydrosilymarin and 2,3-
dehydrosilymarin was apparently better than that of silymarin,
as indicated by its lower IC50 value (16.30 mg/ml vs
24.67 mg/ml).
Effect of 2,3-dehydrosilymarin in carbon
tetrachloride-induced hepatotoxicity
dehydrosilycristin
were
803.93 mg/ml,
23.28 mg/ml,
24.67 mg/ml, 16.30 mg/ml and 18.43 mg/ml, respectively. Before the studies on their liver protecting effect, the acute
These results indicated that 2,3-dehydrosilybin, silymarin, toxicity of silybin, 2,3-dehydrosilybin, silymarin and 2,3-
2,3-dehydrosilymarin and 2,3-dehydrosilycristin had compa- dehydrosilymarin was studied in mice. No acute toxicity was
rable DPPH radical scavenging activity, greatly higher than observed in the mice given any of the drugs at oral doses up to
that of silybin. The DPPH radical scavenging activity of 2,3- 320 mg/kg. The liver protecting effects of those drugs were

244
Journal of Pharmacy and Pharmacology 2011; 63: 238–244
studied with a CCl4-intoxicated mouse model. MDA analysis
was used to characterize the extent of liver injury in CCl4-
intoxicated mice. The liver protecting effects of silybin, 2,3-
dehydrosilybin, silymarin and 2,3-dehydrosilymarin in mice
are presented in Table 2. The results suggest that silybin,
2,3-dehydrosilybin or silymarin have little or no significant
effects against liver damage at tested doses. However, 2,3-
dehydrosilymarin showed clear protective effects against
CCl4-induced liver injury. The liver protection of 2,3-
dehydrosilymarin was observed in the mice treated with a oral
dose as low as 28.4 mg/kg. More interestingly, the liver pro-
tecting effects of 2,3-dehydrosilymarin seemed to be indepen-
dent of dose (Group VIII–X).
When compared with silymarin, 2,3-dehydrosilymarin had
slightly improved IC50 and comparable solubility. This fact
suggested that the remarkable in-vivo liver protecting effects
of 2,3-dehydrosilymarin could partly arise from its good
radical scavenging activity and solubility. From online DPPH-
HPLC analysis, we found that 2,3-dehydrosilychristin was
Acknowledgements
The authors are grateful to Mr Tao Guan-Jing, the State Key
Laboratory of Food Science and Technology at Jiangnan Uni-
versity, for LC-MS analyses.
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Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest
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Funding
This work was supported by the National Nature Science
Foundation of China (No.30973677) and the Nature Science
Fund for colleges and universities in Jiangsu Province, China
(No.08KJB360001).