Food Chemistry 152 (2014) 386390

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Anaytical Methods

Isolation of cyanidin 3-glucoside from blue honeysuckle fruits

by high-speed counter-current chromatography
Liang Chen a, Xiulan Xin a,, Rong Lan a, Qipeng Yuan b, Xiaojie Wang a, Ye Li a
a
b

College of Bioengineering, Beijing Polytechnic, No.1 Shaoyaoju, Beijing 100029, China

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No.15 Beisanhuan East Road, Beijing 100029, China

a r t i c l e

i n f o

Article history:
Received 22 August 2012
Received in revised form 12 November 2013
Accepted 14 November 2013
Available online 22 November 2013
Keywords:
Cyanidin 3-glucoside
Blue honeysuckle fruits
High-speed counter-current
chromatography (HSCCC)

a b s t r a c t
Blue honeysuckle fruits are rich in anthocyanins with many benecial effects such as reduction of the risk
of cardiovascular diseases, diabetes and cancers. High-speed counter-current chromatography (HSCCC)
was used for the separation of anthocyanin on a preparative scale from blue honeysuckle fruit crude
extract with a biphasic solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/
water/triuoroacetic acid (2:2:1:5:0.01, v/v) for the rst time in this paper. Each injection of 100 mg
crude extract yielded 22.8 mg of cyanidin 3-glucoside (C3G) at 98.1% purity. The compound was identied by means of electro-spray ionisation mass (ESI/MS) and 1H and 13C nuclear magnetic resonance
(NMR) spectra.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Blue honeysuckle (Lonicera caerulea L.) is a nutritionally valuable native bush grown in northern temperate zone which can
be widely cultivated in Russia, China, and Japan. Its fruit shape is
oval to long and the avour is similar to bilberry, black currant
and blueberry. Its colour changes from dark navy blue to purple
and the pigment responsible for the attractive colour belongs to
the class of anthocyanin. Blue honeysuckle fruit is anthocyaninrich, whose extract shows signicant antioxidant activity, which
can be correlated to its high content of anthocyanin (Palikova,
Valentova, Oborna & Ulrichova 2009; Palikova et al., 2008; Svarcova et al., 2007). Apart from antioxidant activity, anthocyanin can
promote many positive health benets, such as reduction the risk
of coronary heart diseases and diabetes, anti-cancer, ageing resistance and obesity control (Ghosh & Konishi, 2007; Jankowski,
Jankowska, & Niedworok, 2000; Kwon et al., 2007; Mazza, 2007;
Tsuda, 2008; Vinson, Dabbagh, Serry, & Jang, 1995; Wang & Stoner,
2008).
Irena Palikova et al. (2008) have characterised several kinds of
anthocyanins in blue honeysuckle fruits by HPLC/DAD, and the major anthocyanin was identied as cyanidin 3-glucoside, which
comprised 7988% of total anthocyanin content (Chaovanalikit,
Thompson, & Wrolstad, 2004; Palikova et al., 2008). Cyanidin 3glucoside (C3G), one of the most important anthocyanins, is a potent antioxidant. Hong Wang determined the antioxidant capacity
Corresponding author.
E-mail addresses: Xiulanxin@163.com, leon.c2010@gmail.com (X. Xin).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.11.080

of 14 anthocyanins and anthocyanidins. Among them, kuromanin

(cyaniding 3-glucoside) had the highest ORAC (Oxygen Radical
Absorbance Capacity) activity, which was 3.5 times stronger than
Trolox (vitamin E analogue) (Wang, Cao, & Ronald, 1997). Especially, researchers are highlighting on the cancer preventative
activity of C3G in vitro and in vivo recently. Lots of evidences
proved that C3G exhibited the anti-cancer effect through several
key steps: (i) antioxidant effect; (ii) phase II enzyme activation;
(iii) anti-cell proliferation; (iv) induction of apoptosis; (v)
anti-inammatory effects; (vi) anti-angiogenesis; (vii) antiinvasiveness; (viii) induction of differentiation (Chen et al., 2006;
Serano et al., 2004; Shih, Yeh, & Yen, 2007; Wang & Stoner,
2008). Therefore, C3G may be of great value in developing a
potential cancer prevention agent.
High-speed counter-current chromatography (HSCCC) is a support-free liquidliquid partition chromatographic technique. It has
signicant advantages over the conventional liquidsolid chromatography by eliminating all complications caused by the solid support matrix such as reduction of adsorptive loss, deactivation of
samples, tailing of solute peaks and contamination, and the maximum capacity with an excellent sample recovery. Recently, HSCCC
has been popularly used in separation and purication of natural
products (Foucault & Chevolot, 1998; Friesen & Pauli, 2007; Ito,
2005; Marston & Hostettmann, 2006; Oka, Oka, & Ito, 1991).
The purpose of this study was to establish a method to purify
cyanidin 3-glucoside from anthocyanin-rich blue honeysuckle
fruits grown in China using HSCCC. The chemical structure of compound was identied by electro-spray ionisation mass (ESI/MS)
and nuclear magnetic resonance (NMR).

L. Chen et al. / Food Chemistry 152 (2014) 386390

2. Experimental
2.1. Apparatus
The analytical HPLC equipment was an Agilent 1100 system
consisting of a G1311A solvent delivery unit, a G1332A degasser,
a G1315B UVVis photodiode array detector, a sample injection
valve (Model 7725i) with a 100 lL loop, and an Agilent HPLC workstation (Agilent, German). The column applied in this work was a
Zorbax SB-C18 column (250 mm  4.6 mm, I.D., 5 lm, Agilent
Technologies China).
The preparative HSCCC instrument (Shanghai Tauto Biotech Co.,
Ltd., Shanghai, China) used in this study was a TBE-300A highspeed countercurrent chromatograph with three multilayer coil
separation columns connected in series (I.D. of the tubing = 2.6 mm, total volume = 280 ml) and a 20 ml sample loop.
The revolution radius or the distance between the holder axis
and central axis of the centrifuge (R) is 5 cm, and the b values of
the multilayer coil varied from 0.5 at the internal terminal to 0.8
at the external terminal (b = r/R, where r is the distance from the
coil to the holder shaft). An HX 1050 constant-temperature circulating implement (Beijing Boyikang Lab Instrument, Beijing, China)
was used to control the separation temperature. The solvent was
pumped into the column with a model TBP-50A constant ow
pump and continuous monitoring of the efuent was achieved
with a model TBD-23UV detector.
ESI/MS spectra, 1H and 13C NMR spectra were obtained by analysts at the centre of Analysis, Beijing University of Chemical
Technology.

387

during HPLC analysis using a diode array detector. Spectral measurements were made over the wavelength range 200600 nm in
steps of 2 nm.
2.5. Total anthocyanin content determination
The anthocyanin was exhaustively extracted from 10 g blue
honeysuckle fruits, in triplicate, with methanol acidied with
0.1% HCl (v/v). The blue honeysuckle extract sample was analysed
by HPLC using the conditions described in Section 2.4. Commercially available standard of cyanidin 3-glucoside was used as standard stock solution for generating calibration curve. The
calibration curve was Y (peak area) = 2233X (C3G equivalent content) + 13.38 (R2 = 0.999). The content was expressed as mg of
cyanidin 3-glucoside per 100 g of fresh weight.
2.6. Anthocyanin extract of blue honeysuckle fruits
The blue honeysuckle fruits (1 kg) were extracted with methanol acidied with 0.1%HCl at room temperature for 24 h three
times. Routinely, the ratio of the extraction solvent to the fruit
was 10:1. The combined extract was evaporated to syrup. The syrup was dissolved in 500 ml of distilled water acidied with 0.1%
HCl (v/v). The water solution of the syrup was subjected to column
chromatography of AB-8 macro-porous resin orderly eluted with
water and ethanol. The ethanol elution solution was evaporated
to syrup and freeze-dried to yield crude blue honeysuckle anthocyanin extract (CBA).
2.7. Determination of partition coefcients (K)

2.2. Reagents
Methanol and formic acid used for HPLC analysis were of chromatography grade and purchased from Beijing Chemical Factory
(Beijing, China). The standard of cyanidin 3-glucoside was purchased from Mansite biological technology Co., Ltd. (Chengdu,
China). All organic solvents used for sample preparation and
HSCCC were of analytical grade and purchased from Beijing
Chemical Factory (Beijing, China). Triuroacetic acid (TFA) used
for HSCCC analysis was of chromatography grade and purchased
from Merck Co. (Hohenbrunn, Germany). AB-8 macro-porous resin
was purchased from Boen adsorbent material Technology Co., Ltd.
(Cangzhou, China).

The partition coefcients (K) were determined as follows: a

small amount of CBA was dropped into a 10 ml test tube to which
2.0 ml of each phase of the equilibrated two-phase solvent system
was added. The tube was shaken vigorously for 1 min to thoroughly equilibrate the sample between the two phases. Then
1 ml of each phase was separated in a ask, and the solvents were
evaporated in a rotary evaporator. The residue of each phase was
dissolved with methanol acidied with 0.1% HCl equally, and then
the resulting solution was injected to HPLC, using the conditions
described in Section 2.4. The K value was calculated by the ratio between the peak areas.
2.8. Preparation of two-phase solvent system and sample solution

2.3. Fruit sample

Blue honeysuckle (L. caerulea L.) fruits were provided by Jilin
agricultural university, Changchun China, which were cultivated
on the Changbai mountains area in northern China. The sample
of fully mature fruits was harvested by hand from several different
plants of the same variety during the period of July to November in
2011 and stored in plastic containers at 40 C for chemical
analysis.
2.4. HPLC analysis of anthocyanins
HPLC conditions of all the samples in this paper were the same.
The solvent system was consisted of methanol as uid phase A and
5% (v/v) formic acid in water as uid phase B. Gradient conditions:
02 min, 5 A in B; 210 min, 520% A in B; 1015 min, 2025% A in
B; 1525 min 2530% A in B; 3035 min, 45% A in B. This was followed by a 10 min re-equilibration. The column oven temperature
was set at 30 C. The ow rate was 1.0 ml/min, and 10 lL aliquots
were injected into the column. Anthocyanin was detected at 280
and 520 nm. UVVis absorption spectra were recorded on-line

The solvents were mixed in a separatory funnel according to the

selected volume ratio and thoroughly equilibrated by vigorous
shaking at room temperature. The upper phase and the lower
phase were separated and degassed by sonication for 20 min before used. The sample solution for HSCCC was prepared by dissolving 100 mg of CBA into 10 ml two-phase solvent system.
2.9. HSCCC separation
Headtail elution mode was carried out, with the upper organic
phase as stationary phase. The multiplayer coiled column was rst
entirely lled with the upper phase. The lower phase was then
pumped into the headed of the column at a ow rate of 2.0 ml/
min, while the apparatus was run at a revolution speed of
850 rpm. After hydrodynamic equilibrium was established
throughout the coil, the sample solution was injected into the separation column. During the separation the column temperature
was controlled at 25 C. The UV detector was set at 280 nm. Each
peak fraction was collected according to the chromatogram. The
retention of the stationary phase was computed from the volume

388

L. Chen et al. / Food Chemistry 152 (2014) 386390

100 g and the major anthocyanin in blue honeysuckle was cyanidin

3-glucoside (84.77%). Although cyanidin 3-glucoside is common in
many coloured fruits, the cyanidin 3-glucoside content in blue
honeysuckle is higher than that in others, such as blackberry, raspberry and blueberry. Therefore, blue honeysuckle is a cyanidin 3glucoside-rich fruit.

Table 1
Partition coefcients (K) of cyanidin 3-glucoside in HSCCC solvent system.
Solvent system

Ratio (v/v)

K of cyandin
3-glucoside

tert-Butyl methyl ether/n-butanol/

acetonitrile/water/triuoroacetic acid

0.5:3.5:1:5:0.01
1:3:1:5:0.01
1.5:2.5:1:5:0.01
2:2:1:5:0.01
2.5:1.5:1:5:0.01
3:1:1:5:0.01

2.400
2.037
1.581
1.028
0.675
0.242

3.2. Selection of two-phase solvent system

of the stationary phase collected from the column after the separation was completed.
2.10. Structure elucidation
The preparative compound was identied by MS, 1H NMR and
C NMR spectrometry. A Micro-mass 70-VSE mass spectrometer
was used with an ion source temperature of 200 C and a probe
temperature of 25 C. The spectrum was scanned at 70 eV from
m/z 200 to 1000. NMR spectra were performed in CD3OD/CF3COOD
(95:5 v/v) using a Bruker high-resolution AV500NMR spectrometer
at 500 MHz.

13

3. Results and discussion

3.1. Total anthocyanin content determination
Quantication of blue honeysuckle anthocyanin was investigated by HPLC. The total anthocyanin content was 681.07 mg/

The successful separation by HSCCC largely relies on the selection of a suitable two-phase solvent system. Essentially, the nonaqueous solvent system is favourable for the isolation of non-polar
target compounds and the organic-aqueous solvent system is
favourable for the polar compounds. In order to achieve an efcient
separation of anthocyanin, which is hydrophilic, organic-aqueous
solvent system was examined in the present work. The partition
coefcient (K), generally within the range of 0.52.0, is an important parameter in HSCCC separation. A smaller K value elutes the
solute closer to the solvent front with lower resolution while a
larger K value tends to give better resolution but broader, more
dilute peaks due to a longer elution time (Ito, 2005).
According to a lot of literatures (Aoki, Kuze, Kato, & Gen, 2002;
Degenhardt, Knapp, & Winterhalter, 2000; Du, Zheng, & Xu, 2008;
Osorio et al., 2010; Schwarz, Hillebrand, Habben, Degenhardt, &
Winterhalter, 2003; Zanatta, Cuevas, Bobbio, Winterhalter, & Mercadante, 2005), tert-butyl methyl ether/n-butanol/acetonitrile/
water/ triuoroacetic acid was a suitable two-phase solvent system for separation of anthocyanin. Since the volume ratio of nbutanol had greater inuence on the polarity of the two phases,
the optimal solvent system was selected through changing the

mAU

OH
OH

300
250

+
O

O O

200
150
100
50

OH

H
OH
OH

0
0

10

20

30

40

min

10

20

30

40

min

mAU
200
150
100
50
0
0

mAU
200

mAU
150

150

100

100

50
0

50

300

400

500

nm

0
0

10

20

30

40

min

Fig. 1. HPLC chromatograms of (A) standard substance cyanidin 3-glucoside, (B) crude blue honeysuckle anthocyanin extract (CBA), and (C) cyanidin 3-glucoside puried
from blue honeysuckle fruits by HSCCC (UV spectrum).

389

L. Chen et al. / Food Chemistry 152 (2014) 386390

volume of n-butanol, and the K values of cyanidin 3-glucoside were

summarised in Table 1.
Han et al. (2008) established a simple mathematic equation,
based on some published and determined data of the K value and
solvent ratios in some successful cases of CCC separation. A simple
mathematic method was developed to predict the solvent ratio
that could give expected K values for these compounds. This method was satisfactorily applied in determining the two-phase solvent
system for the isolation of pseudolaric acid B (PAB) from the roots
of the Chinese medicinal plant Pseudolarix kaempferi Gordon
(Pinaceae).
In Table 1, six different compositions of the two-phase solvent
system of tert-butyl methyl ether/n-butanol/acetonitrile/water/triuoroacetic acid were listed for the respective K values of the target compound. The result demonstrated that the solvent system in
a ratio of 4-X:X:1:5:0.01 displayed their relationship generated by
the linear equation. The regression curve was Y = 0.881X 0.657
(R2 = 0.996). In equation, Y represented the K value and X was the
volume of n-butanol in two-phase solvent system. This kind of
mathematic method could more clearly indicate the relationship
between the composition of the two-phase solvent system and
the K value, which made the HSCCC experiment much simpler
and more efcient. More importantly, it could help to better design
the simultaneous HSCCC separation of several target compounds
from other fruits by more effectively calculating the partition coefcients and the resolutions among different anthocyanins.
After the comparison, the two-phase solvent system of tertbutyl methyl ether/n-butanol/acetonitrile/water/triuoroacetic
acid at a volume ratio of 2:2:1:5:0.01 was found to be satisfactory
for the separation of target compound from the hydrophilic impurities, which had small K value in CBA.
3.3. Preparative HSCCC separation
The HPLC analysis of CBA indicated that it contained several
compounds as shown in Fig. 1B and target compound relative
content in CBA is 29.6%.
Fig.2 showed the result obtained from 100 mg of the crude sample by preparative HSCCC. The shaded part of the peak was cut and
concentrated. This separation yielded 22.8 mg of cyanidin
3-glucoside at 98.1% purity based on HPLC analysis. The mass
recovery was more than 90%.
3.4. Conrmation of cyanidin 3-glucoside
To further conrm the chemical structure of target compound,
the fraction from preparative HSCCC was submitted to the MS
and NMR analysis. A clear mass spectrum was obtained where an

Table 2
C and 1H spectral data (ppm) for cyanidin 3-glucoside.

13

Cyanidin 3-glucoside
Aglycone
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
Glucose
100
200
300
400
500
6A00
6B00

13

C (ppm)

164.30
145.63
136.97
159.47
103.81
170.57
95.16
157.73
113.39
121.25
118.46
147.41
155.80
117.43
128.27
103.41
74.82
78.16
71.13
78.82
62.41

H (ppm)

8.97d5.0
6.63t1.0
6.84 s (broad)

8.00d2.5

6.97dd10.0,5.0
8.20dd3.5,2.0
5.26d8.0
3.68 m
3.65 m
3.56 m
3.90dd12.0,2.0
3.94 s
3.71 m

ion at M+H m/z 449 accounted for the cyanidin 3-glucoside as

previously reported (Chaovanalikit et al., 2004). MS experimentation detected one mass fragment at m/z 287 related to the hexose
([M162]+) molecule, which furnished the cyanidin alycone with
m/z 287. HPLC analysis showed a single peak (Fig. 1B) and the
retention time corresponded to standard substance (Fig. 1A). In
addition, 1H and 13C NMR spectral data (ppm) were shown in
Table 2. Compared with previous publication (Du et al., 2008),
the fraction was also identied as cyanidin 3-glucoside.
4. Conclusions
Blue honeysuckle fruit is rich in cyanidin 3-glucoside predominating 84.77% of the total anthocyanin content in this present
work. Blue honeysuckle is an excellent source for cyanidin 3-glucoside that has a potential for cancer prevention.
The overall results indicated that HSCCC was successfully used
for separation and purication of cyanidin 3-glucoside from blue
honeysuckle. The purication methodology yielded highly puried
compound that was well suited for further biochemical and toxicological research. HSCCC has been demonstrated to be a powerful
tool in separation and purication of bioactive components from
the natural medicines.
Acknowledgements
The authors acknowledge the Special Fund for Agro-scientic
Research in the Public Interest (201103037), the Funding Project
of Competence Development Program for Beijing VET Teachers,
the Scientic Research Base Fund for Science and Technology Innovation and Transformation Program, PHR (IHLB), Beijing Famous
Teacher Program for nancial support, Project supported by the
Science-Technology Foundation of Beijing Municipal Commission
of Education (KM201410858002) and Project supported by the
Youth elite project of Higher Education of Beijing (YETP1801).
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