Plant Foods Hum Nutr (2009) 64:141145
DOI 10.1007/s11130-009-0116-1
ORIGINAL PAPER
Antioxidant Properties of Isolated Isorhamnetin
from the Sea Buckthorn Marc
Liu Pengfei & Deng Tiansheng & Hou Xianglin &
Wang Jianguo
Published online: 15 May 2009
# Springer Science + Business Media, LLC 2009
Abstract In the present study, the process of separation
and purification of isohamnetin from marc of sea buckthorn
was obtained. The antioxidant properties of the pure
isolated isorhamnetin were evaluated by the scavenging of
the diphenylpicrylhydrazyl radical (DPPH), iron (III) to
iron (II)reducing, and iron-chelating assays. High purity
isorhamnetin (92.1%) was obtained and the results of
antioxidant assays showed that isorhamnetin performed
significantly compared with ascorbic acid and BHT, and the
linear correlations were good in these assays. In conclusion,
isorhamnetin may have potential as a natural antioxidant to
alternate synthetic substances as food additive with its
antioxidant activity.
Keywords Isorhamnetin . Sea buckthorn . Purification .
Antioxidant activity
Introduction
During recent years, consumers have been more concerned
about the addition of synthetic additives to food and the
two most commonly used antioxidants, butylated hydrox-
yanisole (BHA) and butylated hydroxytoluene (BHT), have
shown DNA damage induction [1]. Therefore, there is an
L. Pengfei : D. Tiansheng : H. Xianglin (*) : W. Jianguo
State Key Laboratory of Coal Conversion,
Institute of Coal Chemistry, Chinese Academy of Sciences,
P.O. Box 165, Taiyuan, Shanxi 030001,
Peoples Republic of China
e-mail: houxianglin311@126.com
L. Pengfei : D. Tiansheng
Graduate University of Chinese Academy of Sciences,
Beijing 100039, Peoples Republic of China
increasing interest on preparing antioxidants from natural
sources, such as herbs, spices, seeds, cereals, fruits, and
vegetables. With the function of antioxidants, flavonoids
play a significant role in natural sources and may be
stronger antioxidants than antioxidant vitamins [2]. The
capacity of flavonoids depends on the number of hydroxyl
groups in the ring A, B, C and the existence of a C2-C3 double
bond. In other words, the greater the number of hydroxyl
groups in the three rings especially in ring B, the greater the
radical scavenging potency of flavonoids [3]. Isorhamnetin
(Fig. 1) is a flavonoid with four hydroxyl groups, the
hydroxyl group at position three enhances its antioxidant
activity [4]. Isorhamnetin is highly consumed in the Greek
and Danish traditional diet [5, 6]; it is the main active
compound of Pu-huang, a traditional Chinese medicine [7];
and it can be found in the Chinese sea buckthorn juice [8].
According to published work, isorhamnetin shows effect on
HL-60 cell proliferation and cycle distribution, apoptosis,
and scavenges the diphenylpicrylhydrazyl radical (DPPH)
[9], prevents endothelial cell injuries, and protects efficiently
albumin-bound linoleic acid [10]. Sea buckthorn berries are
squeezed and fermented to made sea buckthorns juice, and
the residue, marc of sea buckthorn, in which isorhamnetin
exists is used in low efficiency.
The correlation between the antioxidant activities and
the quantification of total phenols determined is still under
discussion. Some authors [1113] reported that a good linear
relationship between them was observed, however, Hinneburg
et al. [14] found no linear relationship between them.
Therefore, the aim of this study was to (1) extract and refine
the isorhamnetin from marc of sea buckthorn, (2) investigate
the antioxidant activity of the isolated isorhamnetin by the
DPPH radical scavenging, iron (III) to iron (II)reducing,
iron-chelating activities, (3) investigate the correlation between
the amount of isorhamnetin and the antioxidant capacity.
142 Plant Foods Hum Nutr (2009) 64:140145

OCH3 filter of 0.45 m quickly. The filtrate was cooled down to

0 C kept at this temperature for 60 min to get a precipitate.
8 1
O 2 The precipitate was recrystallized in 70% ethanol water
HO B OH solution (30 ml, 60 min, from 70 C to 0 C) to obtain a
7 A pale yellow powder (0.7112 g) after vacuum-dried. The
3 purity of pale yellow powder was 92.1% detected by HPLC
6
5 4 HO and the chromatograms are shown in Fig. 2. This powder
was dissolved in ethanol and used to asses the antioxidant
OH O activity.

Fig. 1 Structure of isorhamnetin (C16H12O7, wt 316.26) Antioxidant Assays

Materials and Methods
Materials
DPPH radical, linoleic acid and ferrozine were purchased
from Sigma Chemical Co. (St. Louis, MO, USA).
Standard isorhamnetin was from the National Institute for
the Control of Pharmaceutical and Biological Products
(NICPBP, Beijing, China). Marc of sea buckthorn was from
the Shan-di-yang-guang group (Shanxi, China). BHT
(food-grade) was from Hua-yu food additives Co. Ltd
(Zhengzhou, China). HPLC system was LC-10ATVp
(Shimadzu, Japan). Ultrasonic equipment (As2060 B) was
from Autoscience Instrument Co. Ltd. (Tianjin, China). The
rest of the other reagents were of analytical grade and the
water was deionized.
DPPH Radical-Scavenging
The ability of isorhamnetin to scavenge DPPH free radicals
was determined as previously described [12, 15]. Briefly, a
given volume of the isolated isorhamnetin (0.02444 mg/ml),
ascorbic acid (0.1136 mg/ml), or BHT (0.378 mg/ml) was put
in a test tube, and then 2.0 ml of the DPPH-ethanol solution
(0.1260 mg/ml) was added, after that, the content in the test
tube was further diluted to 5.0 ml with ethanol. The controls
contained all the reaction reagents except isorhamnetin or
positive control substance. After 20 min incubation in
darkness and at room temperature, the resultant UV
absorbance was recorded at 517 nm. The percentage
inhibition values were calculated from the absorbance of the
control (Ac) and of the sample (As) using Eq. (1). Three
1
Voltage (mv)

40
HPLC Conditions 30
20
Chromatographic separation was carried out on a Venusil a
10
ODS column (5.0 m, 150 mm4.6 mm). The mobile 0
phase was methanol-water (60:40, v/v) and the flow rate
1
was 1.0 ml/min. The column temperature was maintained at 10
Voltage (mv)

32 C and the detection wavelength was set at 360 nm. 8

6
4 b
Samples Preparation 2
2
0
Marc of sea buckthorn was dried and ground into powder. 1
25
Dried powder (590 g) was extracted by ultrasonic equip-
Voltage (mv)

20
ment by 32 l ethanol (95%) for 60 min each time at room 15
temperature (25 C). The extracts were combined and 2
10
filtered with a membrane filter of 0.45 m. The filtered 5 c
extract (1.0 ml was used to detect the contents of the marc) 0
was dried by the rotary evaporation method obtaining 0 2 4 6 8 10
2.8671 g of a solid substance; then washed with 100 ml Time (min)
water, 100 ml 20% ethanol water (v/v) solution, 100 ml
40% ethanol water solution, 200 ml 70% ethanol water Fig. 2 HPLC chromatograms of the compounds at 280 nm: a HPLC
chromatogram of standard isorhamnetin; b HPLC chromatogram of
solution, successively. Fraction 4 (70% ethanol water
pale yellow powder obtained in our lab; c HPLC chromatogram of
solution, 200 ml) was concentrated to 30 ml with rotary extract of the marc. Peak 1 was isorhamnetin and peak 2 was quercetin
evaporation at 70 C and then filtered with a membrane (identified by standard quercetin)
Plant Foods Hum Nutr (2009) 64:140145
100
Inhibition (%)
50
Isorhamnetin
Ascorbic acid
BHT
0
0.00 0.04
Concentration (mg/ml)
Fig. 3 DPPH radical scavenging activity of isorhamnetin, ascorbic
acid, and BHT
experiments were performed and the average result was
adopted.
Ac
As
Inhibition%  100
Ac
Iron (III) to Iron (II)Reducing Activity
The ability of isorhamnetin to reduce iron (III) was assessed
by the method of previous paper [14]. Briefly, a given
volume of isorhamnetin (0.02444 mg/ml), ascorbic acid
(0.1522 mg/ml), or BHT (0.1890 mg/ml) was mixed with
2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of a
1% aqueous potassium hexacyanoferrate (K3Fe(CN)6)
solution in a 10.0 ml test tube, added some ethanol to
8.0 ml, and mixed well. After 30 min incubation at 50 C,
2.5 ml of 10% trichloroacetic acid (Cl3CCOOH) were
added to stop the reaction, and the mixture was centrifuged
(2,500 rmp) for 10 min. Then took 2.5 ml of the upper
layer, well mixed with 2.5 ml water and 0.5 ml of 0.1%
aqueous FeCl3. After 10 min incubation, the absorbance
was measured at 700 nm. Three experiments were
performed and the average result was adopted.
Iron-Chelating Activity
Chelating of iron (II) ions by isorhamnetin was carried out
as described by previous work [16]. Briefly, a given volume
of the isorhamnetin (0.1222 mg/ml), ascorbic acid
(0.1564 mg/ml), or BHT (0.1890 mg/ml) was added to
50 l of 2.0 mM aqueous FeSO4 in 5.0 ml test tube, then
ethanol was added to 4.0 ml. After 5 min incubation, the
reaction was initiated by 1.0 ml of 5.0 mM ferrozine. After
10 min of equilibrium, the absorbance at 562 nm was
recorded. The controls contained all reaction reagents
143
except isorhamnetin or positive control substance. The
iron-chelating activities were calculated from the absor-
bance of the control (Ac) and of the sample (As) using
Eq. (1). Three experiments were performed and the average
result was adopted.
Results and Discussion
Sample Preparations
As shown in Fig. 2, isorhamnetin and quercetin are the
0.08 main compounds in the marc, and the concentrations are
0.47% and 0.12%, respectively. The results of HPLC
quantification demonstrated that a remarkable purification
of isorhamnetin was achieved, and this can be evidenced
further by comparison between b and c. Quercetin was
almost eliminated through the process. The purity of the
pale yellow powder obtained was quantified by HLPC,
92.1% with the yield of 25.6%.
1 DPPH Radical-Scavenging
The effect of the antioxidants on DPPH radical scavenging
is thought to be due to their hydrogen donating ability.
DPPH is a stable free radical and accepts an electron or
hydrogen radical to form a stable diamagnetic molecule.
The reduction capability on DPPH radicals was determined
by the decrease of its absorbance at 517 nm. It was visually
noticeable a discolouration from purple to yellow. BHT and
ascorbic acid were tested as references.
The results of the DPPH the method are presented in Fig. 3.
In the present study, all samples discolor the DPPH. The free
radical scavenging potentials of the sample and controls were
found in the order of isorhamnetin > ascorbic acid > BHT.
The median inhibitory concentration (IC50) values for the
isorhamnetin, ascorbic acid and BHT were 5.0 g/ml,
5.7 g/ml and 18.0 g/ml, respectively. Similarly, a literature
[17] reported that isrohamnetin exhibited a high scavenging
efficiency toward DPPH radicals. The linear correlation
coefficient (R2) of the first four data of isorhamnetin was
0.9841. The last datum of isorhamnetin of the inhibition
reached 94.2% and was a little deviated of the linear.
These results indicated that isorhamnetin had a notice-
able effect on scavenging free radicals. Free radical
scavenging activity increased correspondingly with the
increasing concentration of isorhamnetin.
Iron (III) to Iron (II)Reducing Activity
Fe (III) reduction is often used as an indicator of electron-
donating activity, which is an important mechanism of
phenolic antioxidant action [14, 18]. Ascorbic acid and
144
BHT were used as controls. Higher absorbance of the
reaction mixture indicates greater reducing power. As shown
in Fig. 4, the activities of the three samples are in the order
of isorhamnetin > ascorbic acid > BHT. The activity of
isorhamnetin was better than that of ascorbic acid, while
BHT performed worst in this assay. It was slightly different
from the results of DPPH assay. These results suggested
that isorhamnetin and the other two controls had remark-
able potency to donate electrons to reactive free radicals, to
convert them into more stable non-reactive species and to
terminate the free radical chain reaction. A good correla-
tion, of the linear coefficients (R2) of isorhamnetin is
0.9891, was found in this assay, 0.9891.
Iron-Chelating Activity
Iron-chelating capacity is important since it reduces the
concentration of the catalyzing transition metal in lipid
peroxidation via the fenton reaction. Ferrozine can quanti-
tatively form complexes with Fe2+. In this study, the
chelations of ferrous ions by isorhamnetin, ascorbic acid
and BHT as controls were estimated. In the presence of
other chelating agents, however, the complex formation is
disrupted, resulting in a decrease in the red color.
Measurement of the rate of color reduction, therefore,
allows estimation of the chelating activity of the coexisting
chelator. Chelating agents, which form -bonds with a
metal, are effective as secondary antioxidants because they
reduce the redox potential and then stabilize the oxidized
form of the metal ion.
As shown in Fig. 5, the efficiencies of Fe2+-ferrozine
complex increase with the increasing concentration of the
three antioxidants. In this assay, isorhamnetin performed
0.8
Absorbance at 700nm
0.4
Isorhamnetin
Ascorbic acid
BHT
0.0
0.00 0.03 0.06
Concentration (mg/ml)
Fig. 4 Iron (III) to iron (II)reducing activity of isorhamnetin,
ascorbic acid, and BHT
Plant Foods Hum Nutr (2009) 64:140145
100
Inhibition (%)
50
Isorhamnetin
Ascorbic acid
BHT
0
0.00 0.07 0.14
Concentration (mg/ml)
Fig. 5 Iron-chelating activity of isorhamnetin, ascorbic acid, and
BHT
best and the other two samples did nearly the same but worse
than that of isorhamnetin. The median inhibitory concentra-
tion (IC50) values for the isorhamnetin, ascorbic acid and
BHT were 24.5 g/ml, 50.0 g/ml and 47.1 g/ml,
respectively. The linear correlation coefficient (R2) of
isorhamnetin in this assay was 0.9227. This activity has
significant value in the pharmacological investigation for
the treatment of Alzheimers and Parkinsons diseases [19].
Correlation Between Amount of Antioxidants
and Antioxidant Activity
The correlation between the antioxidant activities and the
quantity of the flavonoids is still under discussion, a good
linear relationship was observed in some published works
[1113]; however, Hinneburg [14] found no linear rela-
tionship between them. The controversy might be contrib-
uted to the complexity of plant materials used by them.
There were massive components in the extract and these
components might produce various results. Some of them
might not perform antioxidant activities, some others
might exhibit opposite activities, moreover, some might
have a strong activity on DPPH radical scavenging but a
weak activity on iron-chelating capacity. The target
products in most works were determined by Folin-
Ciocalteu method, which was used to determine the total
phenols in the extract, but not of certain components. That
will result in inevitable deviation when the extract is
considered as single component. In order to eliminate the
interferences of the impure materials, we used a certain
0.09
component, isorhamnetin. As expected, isorhamnetins
activities increased with the increasing concentration and
good linear correlation coefficients (R2) were observed in
this paper.
Plant Foods Hum Nutr (2009) 64:140145
Conclusion
High purity (92.1%) isorhamnetin was obtained and the
yield was 25.6%, therefore, the process to obtain isorham-
netin from marc of sea buckthorn is feasible. From the
results of the antioxidant activities, we could find that
isorhamnetin had strong antioxidant activities in all of the
three assays. Good linear correlation coefficients (R2)
between amount of substance and antioxidant activity were
found in these assays. Based on the results, isorhamnetin
may have potential as a natural antioxidant to alternate
synthetic substances as food additives.
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