Separation and elucidation of anthocyanins in the fruit of mockstrawberry (Duchesnea indica Focke)

Article abstract of DOI:10.1080/14786410802496903

Anthocyanin pigments in the fruit of mockstrawberry (Duchesnea indica Focke), were extracted with 0.1% HCl in ethanol, and the crude anthocyanin extract was purified by a C₁₈ Sep-Pak cartridge open-column chromatography. High-performance liquid

Full text of DOI:10.1080/14786410802496903

This article was downloaded by: [Laurentian University]
On: 07 December 2014, At: 15:58
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly
Natural Product Letters
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gnpl20
Separation and elucidation of
anthocyanins in the fruit of
mockstrawberry (Duchesnea indica
Focke)
Chuanguang Qin a , Yang Li a , Ruijie Zhang a , Weining Niu a & Yan
Ding a
a Faculty of Life Science , Northwestern Polytechnical University ,
Xi’an 710072, Shaanxi, China
Published online: 22 Sep 2010.
To cite this article: Chuanguang Qin , Yang Li , Ruijie Zhang , Weining Niu & Yan Ding (2009)
Separation and elucidation of anthocyanins in the fruit of mockstrawberry (Duchesnea indica
Focke), Natural Product Research: Formerly Natural Product Letters, 23:17, 1589-1598, DOI:
10.1080/14786410802496903
To link to this article: http://dx.doi.org/10.1080/14786410802496903
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Natural Product Research
Vol. 23, No. 17, 20 November 2009, 1589–1598
Separation and elucidation of anthocyanins in the fruit of mockstrawberry
(Duchesnea indica Focke)
*
Chuanguang Qin , Yang Li, Ruijie Zhang, Weining Niu and Yan Ding
Faculty of Life Science, Northwestern Polytechnical University, Xi’an 710072, Shaanxi, China
(Received 18 June 2008; final version received 18 September 2008)
Anthocyanin pigments in the fruit of mockstrawberry (Duchesnea indica Focke),
were extracted with 0.1% HCl in ethanol, and the crude anthocyanin extract was
purified by a C18 Sep-Pak cartridge open-column chromatography. High-
performance liquid chromatography (HPLC) with photodiode array detection
and mass spectrometry (MS) was applied for the isolation and composition
analysis of anthocyanins in the fruit of mockstrawberry and their aglycones
from acid hydrolysis. Three anthocyanins were found in the fruit of mock-
strawberry and they were identified as cyanidin 3-O-rutinoside (61%), peonidin
3-O-rutinoside (34%), and petunidin 3-O-rutinoside (5%), respectively, by
spectroscopic methods (UV-vis and MS). The two major anthocyanins were
isolated by preparative HPLC, and their chemical structures were further
characterised by H 1 NMR. On the basis of chromatographic data, the total
anthocyanin content was 205 mg gꢀ1 of the fresh fruit of mockstrawberry.
Keywords: anthocyanins; mockstrawberry; Duchesnea indica Focke; natural
colourants; composition analysis
1. Introduction
Anthocyanins are a group of over 500 compounds that are largely responsible for the
many blue, purple, red, and orange colours exhibited by plants (Andersen & Jordheim,
2006). Chemically, they are classified as flavonoids and have a molecular structure based
on the 2-phenylbenzopyrylium (or flavylium) cation. Although anthocyanins have been
widely studied because of their central role in determining the colour of plant organs, they
also have biochemical properties that are believed to be beneficial to human health. Like
other flavonoids, anthocyanins are polyphenolic compounds and exhibit substantial
in vitro antioxidant capacity (Kahkonen & Heinonen, 2003; Konga, Chiaa, Goha, Chiaa,
& Brouillardb 2003). Additionally, anthocyanins have been shown to have a range of
potential therapeutic properties such as anti-inflammatory, cancer chemoprevention,
antiobesity, and vasoprotection (Chen et al., 2006; Cooke, Steward, Gescher, & Marczylo,
2005; Hou, 2003; Jayaprakasam, Vareed, Olson, & Nair, 2005; Katsube, Iwashita,
Tsushida, Yamaki, & Kobori, 2003; Tsuda, Horio, Uchida, Aoki, & Osawa, 2003; Xu,
Ikeda, & Yamori, 2005). Since anthocyanins accumulate to high concentrations in some
foods, for example, berry fruit, consumption ranging up to several 100 mg per serving can
*Corresponding author. Email: qinchg@nwpu.edu.cn
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410802496903
http://www.informaworld.com

1590
C. Qin et al.
be achieved. However, despite the identified in vitro therapeutic properties of anthocyanins
and their widespread presence in the diet, studies to date indicate that the apparent
bioavailability is very low compared with other polyphenolics and flavonoids (Manach,
Williamson, Morand, Scalbert, & Remesy, 2005; McGhie, Ainge, Barnett, Cooney, &
Jensen, 2003; Sykerget, et al., 2005), which suggest that anthocyanins might not exhibit
a therapeutic effect when consumed as part of the diet. As a class of compounds,
anthocyanins have a diverse range of molecular structures that are likely to play important
roles in determining their biological activity and associated factors such as bioavailability
and metabolism. Berry fruits, such as raspberry, blackberry, blueberry, mulberry,
strawberry, bayberry, and black raspberry, in addition to hybrid berries like marionberry,
boysenberry, and tayberry, were recently reported to contribute a range of anthocyanins,
and each type of berry fruit has a particular spectrum of anthocyanins (Chaovanalikit,
Thompson, & Wrolstad, 2004; Longo & Vasapollo, 2005a, 2006; McGhie, Rowan, &
Edwards, 2006; Mullen, Lean, & Crozier, 2002; Tian, Giusti, Stoner, & Schwartz, 2001,
2006). We are particularly interested in the anthocyanin composition in the fruit of the
mockstrawberry (Duchesnea indica Focke), which is a kind of traditional Chinese herbal
medicine and has been used in folk remedies for the treatment of pains, carbuncles,
inflammations, and cancer for a long time. In this article, the profile of anthocyanins in the
fruit of mockstrawberry was reported for their potential application as natural colourants
and antioxidant agents by food, pharmaceutical, and cosmetic industries.
2. Materials and methods
2.1. Chemicals and standards
HPLC grade solvents, such as methanol, acetonitrile, isopropanol and chloroform etc.,
were purchased from Fisher Scientific (NJ, USA). Trifluoroacetic acid (TFA) and
tetrahydrofuran of HPLC grade were purchased from Tedia Co. Inc. (Fairfield, OH,
USA). d-Trifluoroacetic acid (CF3COOD, 99.8%), d4-methanol (CD3OD, 99.8%) and
trimethylsilicane (TMS) was purchased from Sigma Chemical Co. (St. Louis, MO, USA),
and cyanidin 3-O-rutinoside was from Extrasynthese (Genay, France). Deionised water
was used to prepare all solutions.
2.2. Sample collection
Wild growing mockstrawberry fruits (Figure 1(a)) were hand-picked in Xi’an, China,
during May 2007, placed in polyethylene bags, and stored at ꢀ20ꢁC until use. The plant
was classified at the Department of Biology, Northwest University, China, as Duchesnea
indica Focke.
2.3. Extraction of anthocyanins
The milled fresh mockstrawberry fruits (500 g) were extracted in the dark by stirring with
1000 mL of 0.1% HCl (v/v) in ethanol for 20 h at room temperature. The samples were
filtered on a Buchner funnel, and the solid residue was washed with an additional 50 mL of
0.1% HCl (v/v) in ethanol. Filtrates were combined and dried using a rotary evaporator at
30ꢁC. The remaining solid was dissolved in 0.01% HCl (v/v) in deionised water and
successively purified.

Natural Product Research
1591
(a)
(b)
Figure 1. Pigment from the fruit of mockstrawberry (Duchesnea indica (Andr.) Focke).
2.4. Purification of anthocyanins
The anthocyanin aqueous solution obtained from the extraction procedure described
earlier was passed through a 5 g sorbent weight C-18 Sep-Pak cartridge (Waters Corp.,
Milford, MA), previously activated with methanol followed by 0.01% aqueous HCl (v/v).
Anthocyanins and other polyphenolics were adsorbed onto the Sep-Pak column, while
sugars, acids, and other water soluble compounds were removed by washing the cartridge
with 2 volumes of 0.01% aqueous HCl (v/v). Less polar polyphenolics were subsequently
eluted with 2 volumes of ethyl acetate. Anthocyanins were then eluted with methanol
containing 0.01% HCl (v/v). The acidified methanol solution was evaporated using
a rotary evaporator at 30ꢁC. The remaining solid was dissolved in 0.01% HCl (v/v)
aqueous solution to have a known concentration solution (2 mg mLꢀ1) and immediately
analysed. This solution (Figure 1(b)) was stored at ꢀ20ꢁC until used for successive acid
and alkaline hydrolyses.
2.5. Acid hydrolysis of anthocyanins
First, 5 mL of 2 N HCl was added to 1 mL of the purified anthocyanin solution
(2 mg mLꢀ1) in a screw-cap test tube, flushed with nitrogen, and capped. The pigments
were hydrolysed for 2 h at 100ꢁC; then, the solution was immediately cooled in an ice bath
(Ordaz-Galindo, Wesche-Ebeling, Wrolstad, Rodriguez-Saona, & Argaiz-Jamet, 1999).
The hydrolysate was purified by using a 0.5 g sorbent weight C-18 Sep-Pak cartridge
(Waters) as previously described.
2.6. HPLC–PAD–MS analysis
The high-performance liquid chromatography (HPLC)–photodiode array detection
(PAD)–mass spectrometry (MS) analyses were performed using a Waters 2696 separation

1592
C. Qin et al.
module equipped with a 996 photodiode array detector (PAD) coupled to a mass
spectrometer (quadrupole analyser) equipped with an electrospray ionisation interface.
Chromatographic separation was carried out using a 250 ꢂ 4.6 mm i.d., 5 mm Kromasil
C18 column (No.: NC-2546-06251151) with a 4 ꢂ 3 mm i.d. Phenomenex C18 guard
cartridge, both thermostated at 32ꢁC. The mobile phase was composed of 0.1% TFA in
water (solvent A) and 0.1% TFA in acetonitrile (solvent B) at a flow rate of 1 mL minꢀ1
.
The following gradient was utilised: 0 min, 10% B; 0–2 min, 10% B; 2–35 min, 10–90% B;
35–40 min, 90–100% B; 40–60 min, 100% B. Absorbance spectra were recorded every 2 s,
between 200 and 600 nm, with a bandwidth of 4 nm, and chromatograms were acquired at
520, 440, 310, and 280 nm. MS parameters were as follows: capillary voltage, 4000 V;
fragmentor, 160 V; drying gas temperature, 350ꢁC; gas flow (N2), 10 L minꢀ1; nebuliser
pressure, 50 psig. The instrument was operated in positive ion mode scanning from m/z
100 to 800 at a scan rate of 1.43 s per cycle. The wavelength used for quantification was
520 nm (De Pascual-Teresa, Santos-Buelga, & Rivas-Gonzalo, 2002; Hong & Wrolstad,
1990). The calibration curve was produced by the integration of absorption peaks
generated from the analysis of dilution series of cyanidin 3-O-rutinoside.
2.7. Preparative HPLC
The major anthocyanins were isolated by a preparative HPLC system consisting of
a Shimadzu model LC-8A pump, a Shimadzu SCL-10A VP system controller, a manual
injector fitted with a 1 mL sample loop, and a Shimadzu model SPD-10A UV/vis detector
equipped with a preparative flow cell. A 250 ꢂ 21.2 mm i.d., 7 mm Zorbax Stable Bond-C18
preparative column, coupled to a 50 ꢂ 21.2 mm i.d., 5 mm Zorbax Stable Bond-C18 guard
column, was used for the separation and isolation of anthocyanins. The mobile phase was
formic acid/water/methanol (10 : 75 : 15 v/v). The isocratic flow rate was 20 mL minꢀ1, and
the detector was set at 520 nm. The anthocyanin fractions of interest were isolated, and
their purity was checked by analytical HPLC–PAD–MS analysis, as previously described.
Each fraction was evaporated using a rotary evaporator at 30ꢁC to completely remove the
methanol and then loaded on a 5 g sorbent weight C-18 Sep-Pak cartridge (Waters)
previously activated with methanol followed by 0.01% aqueous HCl (v/v). The cartridge
was rinsed with 5 volumes of 0.01% aqueous HCl (v/v), and the adsorbed anthocyanin was
eluted with 0.1% HCl (v/v) in methanol. The eluent solution was evaporated to dryness
using a rotary evaporator at 30ꢁC, and the resulting solid was resolubilised with the
suitable solvent for NMR analysis.
2.8. NMR analysis
1H NMR spectral data were recorded on an INOVA-400 instrument (Varian Inc., Palo
Alto, CA, USA) operating at 400 MHz in solvent CD3OD–CF3COOD (9 : 1) with TMS as
internal standard. Sample temperature was stabilised at 25ꢁC (Longo & Vasapollo, 2005b;
Longo, Vasapollo, & Rescio, 2005).
3. Results and discussion
The anthocyanin composition of the fruit of mockstrawberry was determined by means of
HPLC–PAD–MS analysis. The HPLC profile recorded at 520 nm (Figure 2(a)) indicated

Natural Product Research
1593
(a)
0.50
0.40
0.30
0.20
0.10
0.00
5.00
10.00
15.00
(b) 0.20
0.15
1
0.10
3
2
0.05
0.00
6.00
8.00
10.00
12.00
14.00
(c)
4
0.04
6
0.02
0.00
5
8.00
10.00
12.00
14.00
16.00
18.00
Figure 2. HPLC–PAD chromatogram recorded at 520 nm corresponding to (a) the crude extract
from the fruit of mockstrawberry; (b) the purified anthocyanins from the fruit of mockstrawberry;
(c) the purified anthocyanidins from the mockstrawberry anthocyanins after acid hydrolysis.
that there were six components in crude extract from the fruit of mockstrawberry at
retention time range of 8–11 min, which absorb in this region of the visible region. After
purification through the C-18 column of Sep-Pak cartridge (Waters Corp., Milford, MA),
the chromatogram of the purified anthocyanin extract from the fruit of mockstrawberry,
recorded at 520 nm, was shown in Figure 2(b). As can be seen, there are only three peaks in
the chromatogram, indicating the presence of three different anthocyanins in the fruit of
mockstrawberry. The chromatogram of the purified product after acid hydrolysis of the
anthocyanin extract, recorded at 520 nm (Figure 2(c)), showed that three different
aglycones could be obtained from the three anthocyanins in the mockstrawberry fruit.
These three anthocyanins and their corresponding aglycones, the structures of which were
shown in Figure 3, were identified by comparison of HPLC retention times, elution order,
photodiode array UV/vis spectroscopic, and ESI–MS spectrometric data (Table 1).
The two major anthocyanins corresponding to peaks 1 and 3 (Figure 2(b)) represented
about 61 and 34%, respectively, of the total peak area revealed at 520 nm. Peak 1 was
identified as cyanidin 3-O-rutinoside on the basis of its ꢀmax of 516 nm and a mass
spectrum comprising a Mþ at m/z 595 and two fragment ions at m/z 449 and 287 resulting
from the loss of the deoxyglucose (Mþ ꢀ142) and the rutinose (Mþ ꢀ308), respectively.

1594
C. Qin et al.
R
5′
2
R2
5′
OH
6′
2′″
OH
R1
4′
3′
OH 4′″
8
1
B
6′
HO
O
2
O
4′
HO
7
8
1
OH
CH
B
3′″
5′″
1′
R1
O
2
O
O
HO
7
H
C
2′
1"
A
3
1′
3′
3
5″
3″
1′″
2′
OH
6
A
C
6′″
3
O
6″
5
4
6
OH
2″
HO
5
4
OH
4″
HO
OH
1 R =OH,
R =H
4 R1= OH,
5 R1= OH,
6 R1= OCH3, R2= H
R2= H
R2= OCH3
1
2
2 R =OH,
R =OCH3
1
2
3 R =OCH , R =H
1
3
2
Figure 3. Chemical structures of the anthocyanins identified in the fruit of mockstrawberry
and their aglycones after acid hydrolysis: 1, cyanidin 3-O-rutinoside (Cy-3-Rut); 2, petunidin
3-O-rutinoside (Pt-3-Rut); 3, peonidin 3-O-rutinoside (Pn-3-Rut); 4, cyanidin (Cy); 5, petunidin (Pt);
6, peonidin (Pn).
Table 1. Chromatographic, spectroscopic and spectrometric characteristics of the anthocyanins
found in the fruit of mockstrawberry and their aglycones after acid hydrolysis.
Peak no. tR
(Figure 2) (min)
ꢀmax
(nm)
A(440nm)/A
M
Mþ
(calcd.) (found)
Mþ-Rut
(m/z)
Peak
assignment
ðꢀmax
Þ
1
2
3
4
5
6
9.6
9.8
10.6
12.5
12.8
14.1
518 (281)
526 (281)
516 (280)
525 (270)
525 (255)
524 (268)
30.3
33.1
32.2
25.8
26.7
30.5
595.17
625.18
609.18
287.1
317.1
301.1
595.22
625.24
609.25
287.2
317.3
301.2
287.20
317.35
301.3
Cy-3-Rut
Pt-3-Rut
Pn-3-Rut
Cyanidin (Cy)
Petunidin (Pt)
Peonidin (Pn)
–
–
–
The aglycone corresponded to the molecular ion of cyanidin (4). The ESI–MS profiles
of the peak 3 presented the molecular ions Mþ at m/z 609 and the fragment ions at m/z 467
and 301 resulting from the loss of the deoxyglucose (Mþ ꢀ 142) and rutinose (Mþ ꢀ 308),
respectively. The aglycone corresponded to the molecular ion of peonidin (6). Peak 3 was
therefore identified as peonidin 3-O-rutinoside. The minor anthocyanin (peak 2, around
5%) had a ꢀmax of 526 nm and a mass spectrum consisting of a Mþ at m/z 625 and
fragment ions at m/z 483 and 317 resulting from the loss of the deoxyglucose (Mþ ꢀ 142)
and rutinose (Mþ ꢀ 308), respectively. The aglycone corresponded to the molecular ion of
petunidin (5). Anthocyanin (2) was therefore identified as petunidin 3-O-rutinoside. The
UV/vis absorbance spectra of these compounds (Figure 4) confirmed their identity.
The Abs440/Absꢀmax ratio values calculated for each anthocyanin, ranging from 31 to
32%, indicated a substitution in the C-3 position of the flavylium ring (Giusti, Rodrıguez-
Saona, & Wrolstad, 1999). It is well known that anthocyanins with glycosidic substitutions
at position 3 exhibit a ratio of the absorbance at 400–440 nm to the absorbance at the
visible maximum wavelength (520 nm) that is almost twice as large as for anthocyanins
with glycosidic substitution at position 5 or both 3 and 5 (Harborne, 1976). In addition,
the obtained Abs280/Absꢀmax (67–100%) and Abs310/Absꢀmax (13–22%) ratios
confirmed that mockstrawberry anthocyanins were simple anthocyanin molecules without
acylation of glycoside with aromatic acids (Mozetic, Trebse, & Hribar, 2002; Woodall &
Stewart, 1998).

Natural Product Research
1595
0.28
0.26
0.24
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.50
0.40
0.30
0.20
0.10
0.00
1
2
250.00 300.00 350.00 400.00 450.00 500.00 550.00
250.00 300.00 350.00 400.00 450.00 500.00 550.00
0.070
4
3
0.80
0.60
0.40
0.20
0.00
0.060
0.050
0.040
0.030
0.020
0.010
0.000
250.00 300.00 350.00 400.00 450.00 500.00 550.00
250.00 300.00 350.00 400.00 450.00 500.00 550.00
0.090
0.080
0.070
0.060
0.050
0.040
0.030
0.020
0.010
0.000
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
5
6
250.00 300.00 350.00 400.00 450.00 500.00 550.00
250.00 300.00 350.00 400.00 450.00 500.00 550.00
Figure 4. UV–visible spectrum of peaks 1–6 recorded with a PAD from 200 to 600 nm: 1. peak 1; 2.
peak 2; 3. peak 3; 4. peak 4; 5. peak 5; 6. peak 6.
Acid hydrolysis of the purified anthocyanins produced three peaks (4, 5 and 6), as
shown in the chromatogram of Figure 2(c). The ESI–MS profiles of these compounds
presented their molecular ions Mþ at m/z 287 (4), 317 (5) and 301 (6), corresponding to
the molecular ions of cyanidin, petunidin, and peonidin, respectively (Pridham, 1964).
The absorbance spectra of these compounds confirmed their identity (see Figure 4).

1596
C. Qin et al.
Table 2. 1H NMR spectroscopic data of two anthocyanins (1 and 3) isolated
from the extract of mockstrawberry fruit (ꢁ in CD3OD–CF3COOD (9 : 1) at
25ꢁC).
Anthocyanin
1 ꢁ (J in Hz)
3 ꢁ (J in Hz)
Aglycone
H-4
H-6
9.01 s
6.65 d (2.1)
6.89 brs
8.06 d (2.4)
7.05 d (8.5)
8.29 dd (2.5, 8.8)
8.95 s
6.69 d (1.8)
6.90 brs
8.05 d (2.5)
7.03 d (8.7)
8.30 dd (2.4, 8.5)
3.92 s
H-8
H-20
H-50
H-60
O–CH3
3-Glucosyl
H-100
H-200, H-300, H-400, H-500, H-600
5.31 d (7.8)
3.20–3.55 m
5.30 d (7.8)
3.18–3.52 m
Among the three pigments detected in the fruit of mockstrawberry, the two major
anthocyanins (compounds 1 and 3, Figure 2(b)) were isolated by preparative HPLC
and their identity and structure were confirmed on the basis of 1H NMR
spectroscopic data. The chemical shifts (ꢁ) obtained from the 1H NMR analysis of
anthocyanins 1 and 3 were reported in Table 2. Signals in the downfield of the
spectra between ꢁ 6.7 and 9.0 ppm were clearly attributable to the aromatic protons
(A and B rings) of the aglycone molecule as previously reported (Frøytlog, Slimestad,
& Andersen, 1998; Kuskoski et al., 2003) for these compounds. The signal doublets at
ꢁ 5.3 corresponded to the protons on the anomeric carbon from the glucose residues,
confirming that they were in position C-3 as also indicated by the Abs440/Absꢀmax
ratio values. The ꢂ-configuration of this moiety was confirmed from the magnitude
1
00 00
(J ¼ 7.6 Hz) of the J1 2 coupling constant in the H NMR spectra (Gonnett & Fenet,
2000; Pale, Nacro, Vanhaelen, & Vanhaelen-Fastre, 1997). The spectrum of the
compound presented a doublet at ꢁ 5.29 (d, J ¼ 7.6 Hz, H-1 glucose) confirming the
presence of glucose as sugar moiety (Fossen, Øvstedal, Slimestad, & Andersen, 2003;
Frøytlog et al., 1998). It was not possible to confirm by means of NMR analysis the
identity of the anthocyanins (2) (Figure 2(b)), which were characterised as rutinoside
derivative of petunidin by means of HPLC–PAD–MS analysis; their low concentration
in the mockstrawberry extract did not enable us to preparatively isolate this minor
anthocyanin for their NMR characterisation.
4. Conclusion
The total amount of anthocyanins in the fruit of mockstrawberry, determined on the
cyanidin 3-O-rutinoside basis, was 205 mg gꢀ1 of stoned fresh fruit of mockstrawberry.
Cyanidin 3-O-rutinoside was the most predominant anthocyanin (125 mg gꢀ1), followed by
peonidin 3-O-rutinoside (70 mg gꢀ1). The amount of petunidin 3-O-rutinoside was
10 mg gꢀ1. To our knowledge, this is the first time that the anthocyanin composition of
mockstrawberry fruit has been described.

Natural Product Research
1597
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China
(20672086), China Postdoctoral Science Foundation (20070411144) and the Scientific Research
Foundation for Returned Overseas Chinese Scholars, State Personnel Ministry of China (2007).
References
Andersen, O.M.,
& Jordheim, M. (2006). The anthocyanins. In O.M. Andersen &
K.R. Markham (Eds.), Flavonoids chemistry, biochemistry and applications (pp. 471–551).
Boca Raton, FL: CRC Press, Taylor and Francis.
Chaovanalikit, A., Thompson, M.M., & Wrolstad, R.E. (2004). Characterization and quantification
of anthocyanins and polyphenolics in blue honeysuckle (Lonicera caerulea L.). Journal of
Agricultural and Food Chemistry, 52, 848–852.
Chen, P.N., Chu, S.C., Chiou, H.L., Kuo, W.H., Chiang, C.L., & Hsieh, Y.S. (2006).
Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-rutinoside, exhibited an
inhibitory effect on the migration and invasion of a human lung cancer cell line. Cancer
Letters, 235, 248–259.
Cooke, D., Steward, P.W., Gescher, J.A., & Marczylo, T. (2005). Anthocyans from fruits and
vegetables – Does bright colour signal cancer chemopreventive activity? European Journal of
Cancer, 41, 1931–1940.
De Pascual-Teresa, S., Santos-Buelga, C., & Rivas-Gonzalo, J.C. (2002). LC-MS analysis of
anthocyanins from purple corn cob. Journal of the Science of Food and Agriculture, 82,
1003–1006.
Fossen, T., Øvstedal, D.O., Slimestad, R., & Andersen, Ø.M. (2003). Anthocyanins from a
Norwegian potato cultivar. Food Chemistry, 81, 433–437.
Frøytlog, C., Slimestad, R., & Andersen, Ø.M. (1998). Combination of chromatographic techniques
for the preparative isolation of anthocyanins – Applied on blackcurrant (Ribes nigrum) fruits.
Journal of Chromatography A, 825, 89–95.
Gonnett, J.F., & Fenet, B. (2000). Cyclamen red colors based on a macrocyclic anthocyanin in
Carnation flowers. Journal of Agricultural and Food Chemistry, 48, 22–26.
Giusti, M.M., Rodrıguez-Saona, L.E., & Wrolstad, R.E. (1999). Molar absorptivity and color
characteristics of acylated and nonacylated peonidin-based anthocyanins. Journal of
Agricultural and Food Chemistry, 47, 4631–4637.
Harborne, J.B. (1976). Comparative biochemistry of the flavonoids. New York: Academic Press.
Hong, V., & Wrolstad, R.E. (1990). Use of HPLC separation/photodiode array detection for
characterization of anthocyanins. Journal of Agricultural and Food Chemistry, 38, 708–715.
Hou, D.X. (2003). Potential mechanisms of cancer chemoprevention by anthocyanins. Current
Molecular Medicine, 3, 149–159.
Jayaprakasam, B., Vareed, S.K., Olson, L.K., & Nair, M.G. (2005). Insulin secretion by bioactive
anthocyanins and anthocyanidins present in fruits. Journal of Agricultural and Food
Chemistry, 53, 28–31.
Kahkonen, M.P., & Heinonen, M. (2003). Antioxidant activity of anthocyanins and their aglycons.
Journal of Agricultural and Food Chemistry, 51, 628–633.
Katsube, N., Iwashita, K., Tsushida, T., Yamaki, K., & Kobori, M. (2003). Induction of apoptosis
in cancer cells by bilberry (Vaccinium myrtillus) and the anthocyanins. Journal of Agricultural
and Food Chemistry, 51, 68–75.
Konga, J.M., Chiaa, L.S., Goha, N.K., Chiaa, T.F., & Brouillardb, R. (2003). Analysis and
biological activities of anthocyanins. Phytochemistry, 64, 923–933.
Kuskoski, E.M., Vega, J.M., Rios, J.J., Fett, R., Troncoso, A.M., & Asuero, A.G. (2003).
Characterization of anthocyanins from the fruits of baguacuu (Eugenia umbelliflora Berg).
Journal of Agricultural and Food Chemistry, 51, 5450–5454.

1598
C. Qin et al.
Longo, L., & Vasapollo, G. (2005a). Determination of anthocyanins in Ruscus aculeatus L. berries.
Journal of Agricultural and Food Chemistry, 53, 475–479.
Longo, L., & Vasapollo, G. (2005b). Anthocyanins from bay (Laurus nobilis L.) berries. Journal of
Agricultural and Food Chemistry, 53, 8063–8067.
Longo, L., & Vasapollo, G. (2006). Extraction and identification of anthocyanins from Smilax
aspera L. berries. Food Chemistry, 94, 226–231.
Longo, L., Vasapollo, G., & Rescio, L. (2005). Identification of anthocyanins in Rhamnus alaternus
L. berries. Journal of Agricultural and Food Chemistry, 53, 1723–1727.
Manach, C., Williamson, G., Morand, C., Scalbert, A., & Remesy, C. (2005). Bioavailability and
bioefficacy of polyphenols in humans. 1. Review of 97 bioavailability studies. American
Journal of Clinical Nutrition, 81, 230S–242S.
McGhie, T.K., Ainge, G.D., Barnett, L.E., Cooney, J.M., & Jensen, D.J. (2003). Anthocyanin
glycosides from berry fruit are absorbed and excreted unmetabolised by both humans and
rats. Journal of Agricultural and Food Chemistry, 51, 4539–4548.
McGhie, T.K., Rowan, D.R., & Edwards, P.J. (2006). Structural identification of two major
anthocyanin components of boysenberry by NMR Spectroscopy. Journal of Agricultural and
Food Chemistry, 54, 8756–8761.
Mozetic, B., Trebse, P., & Hribar, J. (2002). Determination and quantification of anthocyanins and
hydroxycinnamic acids in different cultivars of sweet cherries (Prunusa Vium L.) from Nova
Gorica Region (Slovenia). Food Technology and Biotechnology, 40, 207–212.
Mullen, W., Lean, M.E.J., & Crozier, A. (2002). Rapid characterization of anthocyanins in red
raspberry fruit by high-performance liquid chromatography coupled to single quadrupole
mass spectrometry. Journal of Chromatography A, 966, 63–70.
Ordaz-Galindo, A., Wesche-Ebeling, P., Wrolstad, R.E., Rodriguez-Saona, L., & Argaiz-Jamet, A.
(1999). Purification and identification of Capulin (Prunus serotina Ehrh) anthocyanins.
Food Chemistry, 65, 201–206.
Pale, E., Nacro, M., Vanhaelen, M., & Vanhaelen-Fastre, R. (1997). Anthocyanins from bambara
groundnut (Vigna subterranea). Journal of Agricultural and Food Chemistry, 45, 3359–3361.
Pridham, J.B. (1964). Paper electrophoresis of phenolic compounds. Methods of polyphenol chemistry.
New York: Pergamon Press.
Sykerget, M., Kotnik, P., Hadolin, M., Hras, A.R., Simonic, M., & Knez, Z. (2005). Phenols,
ˇ ˇ
proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant
activities. Food Chemistry, 89, 191–198.
Tian, Q.G., Giusti, M.M., Stoner, D.G., & Schwartz, J.S. (2001). Identification of anthocyanins in
berries by narrow-bore high-performance liquid chromatography with electrospray ionization
detection. Journal of Agricultural and Food Chemistry, 49, 3987–3992.
Tian, Q.G., Giusti, M.M., Stoner, D.G., & Schwartz, J.S. (2006). Characterization of a new
anthocyanin in black raspberries (Rubus occidentalis) by liquid chromatography electrospray
ionization tandem mass spectrometry. Food Chemistry, 94, 465–468.
Tsuda, T., Horio, F., Uchida, K., Aoki, H., & Osawa, T. (2003). Dietary cyanidin 3-O-beta-D-
rutinoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice.
Journal of Nutrition, 133, 2125–2130.
Woodall, G.S., & Stewart, G.R. (1998). Do anthocyanins play a role in UV protection of the red
juvenile leaves of Syzygium? Journal of Experimental Botany, 49, 1447–1450.
Xu, J.W., Ikeda, K., & Yamori, Y. (2005). Cyandin-3-rutinoside regulates phosphorylation of
endothelial nitric oxide synthase. FEBS Letters, 574, 176–180.