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Plant Foods Hum Nutr (2009) 64:141145

DOI 10.1007/s11130-009-0116-1


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 increasing interest on preparing antioxidants from natural
and purification of isohamnetin from marc of sea buckthorn sources, such as herbs, spices, seeds, cereals, fruits, and
was obtained. The antioxidant properties of the pure vegetables. With the function of antioxidants, flavonoids
isolated isorhamnetin were evaluated by the scavenging of play a significant role in natural sources and may be
the diphenylpicrylhydrazyl radical (DPPH), iron (III) to stronger antioxidants than antioxidant vitamins [2]. The
iron (II)reducing, and iron-chelating assays. High purity capacity of flavonoids depends on the number of hydroxyl
isorhamnetin (92.1%) was obtained and the results of groups in the ring A, B, C and the existence of a C2-C3 double
antioxidant assays showed that isorhamnetin performed bond. In other words, the greater the number of hydroxyl
significantly compared with ascorbic acid and BHT, and the groups in the three rings especially in ring B, the greater the
linear correlations were good in these assays. In conclusion, radical scavenging potency of flavonoids [3]. Isorhamnetin
isorhamnetin may have potential as a natural antioxidant to (Fig. 1) is a flavonoid with four hydroxyl groups, the
alternate synthetic substances as food additive with its hydroxyl group at position three enhances its antioxidant
antioxidant activity. activity [4]. Isorhamnetin is highly consumed in the Greek
and Danish traditional diet [5, 6]; it is the main active
Keywords Isorhamnetin . Sea buckthorn . Purification . compound of Pu-huang, a traditional Chinese medicine [7];
Antioxidant activity 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,
Introduction and scavenges the diphenylpicrylhydrazyl radical (DPPH)
[9], prevents endothelial cell injuries, and protects efficiently
During recent years, consumers have been more concerned albumin-bound linoleic acid [10]. Sea buckthorn berries are
about the addition of synthetic additives to food and the squeezed and fermented to made sea buckthorns juice, and
two most commonly used antioxidants, butylated hydrox- the residue, marc of sea buckthorn, in which isorhamnetin
yanisole (BHA) and butylated hydroxytoluene (BHT), have exists is used in low efficiency.
shown DNA damage induction [1]. Therefore, there is an 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
L. Pengfei : D. Tiansheng : H. Xianglin (*) : W. Jianguo relationship between them was observed, however, Hinneburg
State Key Laboratory of Coal Conversion,
et al. [14] found no linear relationship between them.
Institute of Coal Chemistry, Chinese Academy of Sciences,
P.O. Box 165, Taiyuan, Shanxi 030001, Therefore, the aim of this study was to (1) extract and refine
Peoples Republic of China the isorhamnetin from marc of sea buckthorn, (2) investigate
e-mail: the antioxidant activity of the isolated isorhamnetin by the
L. Pengfei : D. Tiansheng
DPPH radical scavenging, iron (III) to iron (II)reducing,
Graduate University of Chinese Academy of Sciences, iron-chelating activities, (3) investigate the correlation between
Beijing 100039, Peoples Republic of China 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
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

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

HPLC Conditions 30
Chromatographic separation was carried out on a Venusil a
ODS column (5.0 m, 150 mm4.6 mm). The mobile 0
phase was methanol-water (60:40, v/v) and the flow rate
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

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

ment by 32 l ethanol (95%) for 60 min each time at room 15
temperature (25 C). The extracts were combined and 2
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 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.
Inhibition (%)


Results and Discussion

Ascorbic acid
Sample Preparations

As shown in Fig. 2, isorhamnetin and quercetin are the

0.00 0.04 0.08 main compounds in the marc, and the concentrations are
Concentration (mg/ml) 0.47% and 0.12%, respectively. The results of HPLC
quantification demonstrated that a remarkable purification
Fig. 3 DPPH radical scavenging activity of isorhamnetin, ascorbic of isorhamnetin was achieved, and this can be evidenced
acid, and BHT
further by comparison between b and c. Quercetin was
almost eliminated through the process. The purity of the
experiments were performed and the average result was pale yellow powder obtained was quantified by HLPC,
adopted. 92.1% with the yield of 25.6%.
Ac  As
Inhibition%  100 1 DPPH Radical-Scavenging
The effect of the antioxidants on DPPH radical scavenging
Iron (III) to Iron (II)Reducing Activity is thought to be due to their hydrogen donating ability.
DPPH is a stable free radical and accepts an electron or
The ability of isorhamnetin to reduce iron (III) was assessed hydrogen radical to form a stable diamagnetic molecule.
by the method of previous paper [14]. Briefly, a given The reduction capability on DPPH radicals was determined
volume of isorhamnetin (0.02444 mg/ml), ascorbic acid by the decrease of its absorbance at 517 nm. It was visually
(0.1522 mg/ml), or BHT (0.1890 mg/ml) was mixed with noticeable a discolouration from purple to yellow. BHT and
2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of a ascorbic acid were tested as references.
1% aqueous potassium hexacyanoferrate (K3Fe(CN)6) The results of the DPPH the method are presented in Fig. 3.
solution in a 10.0 ml test tube, added some ethanol to In the present study, all samples discolor the DPPH. The free
8.0 ml, and mixed well. After 30 min incubation at 50 C, radical scavenging potentials of the sample and controls were
2.5 ml of 10% trichloroacetic acid (Cl3CCOOH) were found in the order of isorhamnetin > ascorbic acid > BHT.
added to stop the reaction, and the mixture was centrifuged The median inhibitory concentration (IC50) values for the
(2,500 rmp) for 10 min. Then took 2.5 ml of the upper isorhamnetin, ascorbic acid and BHT were 5.0 g/ml,
layer, well mixed with 2.5 ml water and 0.5 ml of 0.1% 5.7 g/ml and 18.0 g/ml, respectively. Similarly, a literature
aqueous FeCl3. After 10 min incubation, the absorbance [17] reported that isrohamnetin exhibited a high scavenging
was measured at 700 nm. Three experiments were efficiency toward DPPH radicals. The linear correlation
performed and the average result was adopted. coefficient (R2) of the first four data of isorhamnetin was
0.9841. The last datum of isorhamnetin of the inhibition
Iron-Chelating Activity reached 94.2% and was a little deviated of the linear.
These results indicated that isorhamnetin had a notice-
Chelating of iron (II) ions by isorhamnetin was carried out able effect on scavenging free radicals. Free radical
as described by previous work [16]. Briefly, a given volume scavenging activity increased correspondingly with the
of the isorhamnetin (0.1222 mg/ml), ascorbic acid increasing concentration of isorhamnetin.
(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 Iron (III) to Iron (II)Reducing Activity
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 Fe (III) reduction is often used as an indicator of electron-
10 min of equilibrium, the absorbance at 562 nm was donating activity, which is an important mechanism of
recorded. The controls contained all reaction reagents phenolic antioxidant action [14, 18]. Ascorbic acid and
144 Plant Foods Hum Nutr (2009) 64:140145

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

Inhibition (%)
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 Isorhamnetin
Ascorbic acid
terminate the free radical chain reaction. A good correla- BHT
tion, of the linear coefficients (R2) of isorhamnetin is
0.9891, was found in this assay, 0.9891. 0.00 0.07 0.14
Concentration (mg/ml)
Iron-Chelating Activity
Fig. 5 Iron-chelating activity of isorhamnetin, ascorbic acid, and
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 best and the other two samples did nearly the same but worse
chelations of ferrous ions by isorhamnetin, ascorbic acid than that of isorhamnetin. The median inhibitory concentra-
and BHT as controls were estimated. In the presence of tion (IC50) values for the isorhamnetin, ascorbic acid and
other chelating agents, however, the complex formation is BHT were 24.5 g/ml, 50.0 g/ml and 47.1 g/ml,
disrupted, resulting in a decrease in the red color. respectively. The linear correlation coefficient (R2) of
Measurement of the rate of color reduction, therefore, isorhamnetin in this assay was 0.9227. This activity has
allows estimation of the chelating activity of the coexisting significant value in the pharmacological investigation for
chelator. Chelating agents, which form -bonds with a the treatment of Alzheimers and Parkinsons diseases [19].
metal, are effective as secondary antioxidants because they
reduce the redox potential and then stabilize the oxidized Correlation Between Amount of Antioxidants
form of the metal ion. and Antioxidant Activity
As shown in Fig. 5, the efficiencies of Fe2+-ferrozine
complex increase with the increasing concentration of the The correlation between the antioxidant activities and the
three antioxidants. In this assay, isorhamnetin performed 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-
0.8 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
Absorbance at 700nm

might exhibit opposite activities, moreover, some might

have a strong activity on DPPH radical scavenging but a
0.4 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
Ascorbic acid will result in inevitable deviation when the extract is
considered as single component. In order to eliminate the
0.0 interferences of the impure materials, we used a certain
0.00 0.03 0.06 0.09
component, isorhamnetin. As expected, isorhamnetins
Concentration (mg/ml) activities increased with the increasing concentration and
Fig. 4 Iron (III) to iron (II)reducing activity of isorhamnetin, good linear correlation coefficients (R2) were observed in
ascorbic acid, and BHT this paper.
Plant Foods Hum Nutr (2009) 64:140145 145

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