Food Chemistry 132 (2012) 759–765

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

Antioxidant anthocyanins screening through spectrum–effect relationships

and DPPH-HPLC-DAD analysis on nine cultivars of introduced rabbiteye blueberry
in China
Li-Qiong Sun a,c, Xiao-Ping Ding a, Jin Qi a,c, Hong Yu b,⇑, Shan-an He b, Jian Zhang a,c, Hai-Xia Ge a,d,
Bo-Yang Yu a,c,⇑
a
Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China
b
Institute of Botany, Jiangsu Province & Chinese Academy of Sciences, Jiangsu Province Key Laboratory for Plant Ex Situ Conservation, Nanjing 210014, China
c
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
d
Huzhou Teachers College, Huzhou 313000, China

a r t i c l e i n f o a b s t r a c t

Article history: Blueberries have drawn growing attention in China for various benefits to health. Nine rabbiteye blue-
Received 1 December 2010 berry cultivars which were introduced into China were systematically assessed based on two aspects
Received in revised form 18 August 2011 of chemical profiles and antioxidant activity. The former was achieved by HPLC fingerprints, and the lat-
Accepted 7 November 2011
ter in terms of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging capacity was evaluated with a
Available online 13 November 2011
novel HPLC method. Subsequently, the predominant active anthocyanins were screened from these col-
oured biological samples by two useful methods, respectively. In method I, the spectrum–effect relation-
Keywords:
ships between HPLC fingerprints and the antioxidant activities of blueberry extracts were investigated,
Rabbiteye blueberry
Antioxidant activity
whereas in method II, HPLC analysis followed pre-column reaction of samples with DPPH. The results
DPPH free radical indicated that Gardenblue and Powderblue showed stronger antioxidant activities among all introduced
Spectrum–effect relationship cultivars, and delphinins and anthocyanidin-3-glucosides were the main contributive components to the
HPLC fingerprints antioxidant activities of blueberries. This would be helpful to the quality-oriented cultivation of blue-
berry in China.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Increasing public attention has been paid on blueberries (Vacci-
nium spp.) over the years, due to the fruit’s broad pharmacological
properties, such as antioxidation (Castrejón, Eichholz, Rohn, Kroh,
& Huyskens-Keil, 2008; Dulebohn et al., 2008), anticarcinogenesis
(Seeram, 2008), reduction of coronary heart disease, treatment of
urinary tract disorders (Kalt & Dufour, 1997), and enhancing mem-
ory (Krikorian et al., 2010). Since 1980s, with the improvement of
living standards and the increase in demand for blueberry, blue-
berry cultivation has been introduced in China due to scarce native
species. However, little information has been reported about any
systematic quality evaluation of introduced blueberry cultivars.
DPPH is a well-known antioxidant assessment radical. Colori-
metric method, as a typical off-line detection of DPPH scavenging
⇑ Corresponding authors. Address: Department of Complex Prescription of
Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198,
China. Tel.: +86 25 86185157x8001; fax: +86 25 86185158 (B.-Y. Yu), tel./fax: +86
25 84432966 (H. Yu).
E-mail addresses: hongyu1973_nj@163.com (H. Yu), boyangyu59@163.com
(B.-Y. Yu).
capacity, has been widely used to evaluate the antioxidant activity
(AOA) of blueberry extract due to easy operation (Brambilla, Scal-
zo, Bertolo, & Torreggiani, 2008; Lohachoompol, Mulholland,
Srzednicki, & Craske, 2008; Su & Chien, 2007). However, the weak
changes in the DPPH absorbance could not be detected by the col-
orimetric methods because DPPH and anthocyanins both showed
strong absorption at 500–550 nm. Under this situation, a novel
HPLC method based on the reduction of DPPH peak areas (PAs)
was developed for the determination of DPPH scavenging activity.
It could sensitively discriminate the small changes in the DPPH
absorbance reflected by PAs (Chandrasekar, Madhusudhana,
Ramakrishna, & Diwan, 2006).
Anthocyanins, as the major responsible ingredients, are often
considered as markers in the quality assessment of blueberry prod-
ucts (Gu et al., 2002; Nakajima, Tanaka, Seo, Yamazaki, & Saito,
2004; Prior, Lazarus, Cao, Muccitelli, & Hammerstone, 2001). A
lot of researches on the contribution of individual anthocyanin to
the AOA of blueberry have been reported (Cao, Sofic, & Prior,
1997; Noda, Kaneyuki, Mori, & Packer, 2002; Wang, Cao, & Prior,
1997). Nevertheless, these studies were mainly focused on indirect
screening of compounds purified by phytochemical separation
from the blueberry extracts. In recent years, on-line methods of

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.11.030
760 L.-Q. Sun et al. / Food Chemistry 132 (2012) 759–765
HPLC combined with chemiluminescence detection were devel-
oped for the simultaneous detection and identification of radical
scavengers in plant extracts (Dapkevicius, van Beek, Niederländer,
& de Groot, 1999; Ding et al., 2009; Zhang, Zhang, & Sun, 2006).
However, these chemiluminescence reaction systems require an
alkaline medium, whereas the HPLC separation for blueberry
should be performed under strong acidic conditions (Brambilla
et al., 2008; Lohachoompol et al., 2008; Prior et al., 2001). Recently,
an on-line HPLC-DPPH assay for screening of antioxidants in com-
plex matrixes has been successfully developed (Bandonienė &
Murkovic, 2002; Koleva, Niederländer, & van Beek, 2000). How-
ever, this method could not be applied to detect blueberry due to
the presence of very high amounts of anthocyanins in this kind
of fruit, which has a strong absorption band centred at 520 nm
around as to DPPH. A simple and low-cost on-line HPLC method
by pre-column reaction of samples with DPPH radical has been
introduced to screen and identify main antioxidants in biological
samples (Tang, Li, Chen, Guo, & Li, 2008). Additionally, investiga-
tion on the spectrum–effect relationships between HPLC finger-
prints and the total biological activities of plant extracts has been
successfully applied to search for principal bioactivity components
of herbal medicines (Kong, Zhao, Shan, Xiao, & Guo, 2008; Kong
et al., 2009).
In this paper, the chemical characteristics and bioactivity of rab-
biteye blueberry cultivars (Vaccinium ashei Reade) in China were
compared with those of wild European species (Vaccinium myrtil-
lus) and local species (Vaccinium uliginosum L.) by HPLC fingerprints
and total AOA analysis. The responsible anthocyanins for strong
AOA of blueberries were screened and identified with two simple
and low-cost methods: method I was based on the investigation
of spectrum–effect relationships between HPLC fingerprints and
total AOA of blueberries, while method II was based on pre-column
reaction of samples with DPPH radical followed by HPLC-DAD
analysis. This study may provide a useful pilot trial in the screening
of active compounds from coloured bioactive samples such as
blueberries.
2. Experimental
2.1. Chemicals and samples
HPLC grade acetonitrile was from Tedia (Fairfield, OH, USA);
analytical grade phosphoric acid was from Nanjing Chemical Plant
(Jiangsu, China); methanol, formic acid, acetic acid were of analyt-
ical grade (Jiangsu Hanbon Sci. & Tech. Co. Ltd., Jiangsu, China). The
water used was purified by a Millipore water purification system
(Millipore, Bedford, MA, USA). L-Ascorbic acid was purchased from
Shanghai Chemical Reagent Company of Chinese Medical Group
(Shanghai, China); DPPH was purchased from Sigma–Aldrich Co.
(St. Louis, MO, USA).
Eleven batches of samples used in this study were taken for the
11 group code numbers S01–S11, respectively. Nine introduced
rabbiteye blueberries cultivars, which are represented by the hexa-
ploid V. ashei Reade that is extremely heterozygous, were provided
by Institute of Botany, Jiangsu Province and Chinese Academy of
Sciences. Their genotypes and codes were listed as follows: Pow-
derblue (S02) (mid to late season) was selected from ‘Tif-
blue’  ‘Menditoo’; Gardenblue (S03) (early to mid-season) was
from ‘Myers’  ‘Clara’; Tifblue (S04) (mid to late season) was se-
lected from the progeny of ‘Ethel’  ‘Clara’; Brightwell (S05) (early
season) were selected from ‘Tifblue’  ‘Menditoo’; Climax (S06)
(early season) resulted from a cross of ‘Callaway’  ‘Ethel’; Premier
(S07) (early season) was from ‘Tifblue’  ‘Homebell’; Choice (S08)
(mid to late season) originated from open-pollinated seeds from
fruit of T-31 (‘Satilla’  ‘Callaway’); Baldwin (S09) (late season)
was from ‘Tifblue’  G-40 (‘Myers’  ‘Black Giant’); Centurion
(S10) (late season) originated from a cross of W-4  ‘Callaway’. V.
myrtillus (S01), which is one of the most important natural re-
sources in Finland (Lätti, Riihinen, & Kainulainen, 2008), was pur-
chased from the market in Finland, and local species V.
uliginosum L. (S11) was collected from Da Xinganling Mountains
in China. All berries were collected in 2009. After harvesting, fresh
berry samples were frozen and stored at 20 °C until transported
to the laboratory. The frozen samples were freeze-dried and
ground into powder. The powders were kept at 70 °C until
analysed.
2.2. Sample/extracts preparation
The freeze-dried powders were extracted as reported previously
(Wu, Gu, Prior, & McKay, 2004). One gram of accurately weighed
sample was transferred into a 15 ml screw-cap tube and vortexed
for 30 s followed by sonication for 5 min with 15 ml of methanol/
water/acetic acid (85:15:0.5; v/v, MeOH/H2O/HOAc). The tube
was shaken twice during sonication to re-suspend the sample.
The tube was then kept at room temperature for 10 min, being vor-
texed for 30 s per 5 min. The tube was then centrifuged at 4550g
for 10 min to collect the supernatant. The residual sample was ex-
tracted again with 10 ml of MeOH/H2O/HOAc following the same
procedure, and the supernatants were pooled and filtered through
a 0.45 lm film prior to anthocyanins analysis.
2.3. Systematic quality assessment of blueberries
2.3.1. Based on chemical profiles
2.3.1.1. Instrumentation. The HPLC equipment used was a Shima-
dzu LC-2010C system consisting of binary pump, autosampler,
thermostated column compartment and DAD, together with LCso-
lution software. A ZORBAX Stablebond Analytical SB-C18 column
(4.6  250 mm, 5 lm, Agilent Technologies, Rising Sun, MD) was
used for all chromatographic separations.
2.3.1.2. Chromatographic conditions. S01, V. myrtillus, was chosen to
test the feasibility and suitability of the experimental method. Five
microlitre of S01 solution was injected into the HPLC system. Mo-
bile phase and detection wavelength were optimised as these two
parameters play a key role in resolution and sensitivity. Conse-
quently, a gradient elution of A (3% aqueous H3PO4) and B (aceto-
nitrile) was used for chromatographic separation. The flow rate
was 1 ml/min, and the detection wavelength was set at 520 nm.
A gradient program used was as follows: 5% B, 0–5 min; 5–10% B,
5–15 min; 10% B, 15–25 min; 10–12% B, 25–35 min; 12–15% B,
35–50 min; 15–18% B, 50–60 min; 18–25% B, 60–80 min; 25–30%
B, 80–90 min. Under the present chromatographic conditions,
higher resolution could be achieved for most of the peaks.
2.3.1.3. Validation of methodology. Method reproducibility and rep-
licability were evaluated by the analysis of five replicate injections
of one sample and the injections of 5 samples prepared indepen-
dently from S01, respectively, and the stability study of the sample
was performed at different intervals of time in a day (0, 2, 4, 8, 16
and 24 h).
2.3.1.4. Evaluation of fingerprints. The HPLC fingerprints were
matched automatically by professional software named Similarity
Evaluation System for Chromatographic Fingerprint of Traditional
Chinese Medicine composed by Chinese Pharmacopoeia Commit-
tee (Version 2004 A). The reference fingerprint was formed by
the system using the Median method from the general comparison
of the chromatograms of 11 blueberry extracts and the similarity
 L.-Q. Sun et al. / Food Chemistry 132 (2012) 759–765
values between the reference fingerprint and the chromatograms
of blueberry extracts were calculated using this software.
2.3.1.5. Hierarchical clustering analysis. The hierarchical clustering
analysis (HCA) of S01–S11 was performed using SPSS statistics
software (SPSS for Windows 17.0, SPSS Inc., USA).
2.3.1.6. HPLC–ESI–MS analysis. The above HPLC system was inter-
faced with an Agilent 1100 LC/MSD Trap XCT ESI (Agilent Technol-
ogies, MA, USA). The HPLC–MS analysis was performed under the
same gradient program with HPLC-DAD using 5% (v/v) aqueous
formic acid (A) and acetonitrile (B). The ESI–MS spectra were ac-
quired in positive ionisation mode recorded on a mass range of
m/z 400–600. Capillary voltage was 3300 V. Drying gas tempera-
ture was set at 350 °C with a flow rate of 9.0 L/min and nebulizing
pressure was of 40 psi. Data were processed by HPLC/MSD Trap
Software 4.2 and Data Analysis 2.2.
2.3.2. Based on antioxidant capacity
Fresh DPPH stock solution was prepared by dissolving
394.32 mg DPPH in 100 ml of methanol, sealed and stored in the
refrigerator at 4 °C, and diluted to the appropriate concentration
before using. Fresh L-ascorbic acid solution was prepared by dis-
solving 10 mg ascorbic acid to 10 ml of MeOH/H2O/HOAc. The ber-
ry extract solutions were diluted to different multiple by MeOH/
H2O/HOAc.
The AOA was determined by HPLC method as reported previ-
ously (Chandrasekar et al., 2006). Briefly, an aliquot of 200 ll berry
extract solutions with different diluted ratios was added to 200 ll
of DPPH solution (at the final concentration of 0.5 mM). The mix-
ture was vortexed for a few seconds and left to stand in the dark
for 20 min at room temperature. Then the sample was filtered
through 0.45 lm filter and 20 ll of the sample was injected to
HPLC. The blank control was prepared by adding 200 ll of
MeOH/H2O/HOAc to 200 ll of DPPH stock solution.
Chromatographic analysis was carried out by using isocratic
elution with 100% acetonitrile at a flow rate of 1.0 ml/min. The
DPPH peaks were monitored at 517 nm. The difference in the
reduction of peak areas (PAs) for DPPH between the blank and
the sample was used to evaluate radical scavenging activity of
the sample according to the following equation:
PAblank  PAsample
Radical scavenging ð%Þ ¼  100%:
PAblank
IC50 values, the concentrations of samples scavenging 50% DPPH
free radicals, were calculated by nonlinear regression analysis and
expressed by the mean of thrice determination results. L-Ascorbic
acid was used as reference compound for the evaluation of free
radical scavenging activity.
2.4. Predominant antioxidant anthocyanins screening
2.4.1. Method I: spectrum–effect relationships evaluation
Multivariant correlation analysis was used for evaluation of the
spectrum–effect relationships between the values of PAs in HPLC
fingerprints and the total AOA of blueberries by SPSS statistics soft-
ware (SPSS for Windows 17.0, SPSS Inc., USA).
2.4.2. Method II: DPPH-HPLC-DAD analysis
DPPH-HPLC-DAD analysis was performed according to the
method reported previously (Tang et al., 2008). One millilitre of
blueberry extract and 1 ml of DPPH stock solution (at the final con-
centration of 1.25 mM) were mixed to react for 0.5 h in the dark at
room temperature. After filtration through 0.45 lm filter, 10 ll of
sample solution was used for chromatographical analysis with
the proposed gradient elution. One millilitre of methanol was
761
added into the extract as control. The reduction of anthocyanins
PAs between blank and sample was used to evaluate the consum-
ing anthocyanins according to the following equation:
PAblank  PAsample
Consuming anthocyanins ð%Þ ¼  100%
PAblank
3. Results and discussion
3.1. Results of HPLC analysis
3.1.1. HPLC fingerprints
The reproducibility of the same sample solution was in the
range of 0.05–0.09% for retention times (tR) and 0.36–1.98% for
PAs of characteristic peaks. The repeatability of experiment was
below 0.09% for retention times (tR) and 3.17% for PAs of character-
istic peaks. The sample stability precision was below 0.08% for
retention times (tR) and 2.38% for PAs of characteristic peaks. This
indicated that the developed method was robust and suitable for
sample analysis. Fig. 1 showed the HPLC fingerprints and the refer-
ence fingerprint of blueberry extracts from 11 batches of samples.
Fourteen peaks with large areas and good resolution under the
present chromatographic conditions were collectively presented
in the 11 chromatograms and selected as the common characteris-
tic peaks. The relative composition of anthocyanins was similar in
all cultivars and two other species, while PA of each profile was
breed-dependent.
3.1.2. Similarity of fingerprints
China’s State Food and Drug Administration suggested that all
of chromatographic fingerprints should be evaluated by their sim-
ilarity. The relationship within a set of chromatographic finger-
prints could be analysed through comparison in terms of
similarity between the objects and the reference fingerprint. The
similarity values of nine introduced V. ashei Reade cultivars
(S02–S10) were all above 0.9, and they were 0.973, 0.912, 0.985,
0.932, 0.986, 0.937, 0.984, 0.927 and 0.968, respectively. However,
the components in other two Vaccinium species were different
from those in V. ashei Reade and the similarity value was 0.741
for V. myrtillus (S01) and just 0.589 for V. uliginosum L. (S11). This
result indicated that the present method could effectively discrim-
inate different berry species and the content of anthocyanins in the
berries differed from species to species.
3.1.3. Results of hierarchical clustering analysis
HCA, a multivariate analysis technique, is used to discriminate
different samples. The result of HCA was shown in Fig. 2. It was
obvious that the samples could be classified into three clusters:
S06, S08, S02, S04, S10, S05, S07, S09 and S03 in cluster 1, S01 in
cluster 2 and S11 in cluster 3. All of the samples in cluster 1 were
V. ashei Reade cultivars and there were relatively small differences
among them except for S09 (Baldwin) and S03 (Gardenblue).
Whereas, the distance between S01 (V. myrtillus) in cluster 2 and
S11 (V. uliginosum L.) in cluster 3 was considerably large due to
the difference between their varieties. The results were consistent
with those of similarity analysis and showed that HCA could pro-
vide a qualitative comparison of the cultivars and effectively dis-
tinguish the different species of berries.
3.1.4. Peak identification and assignment
Peak identification and assignment in HPLC fingerprints of ber-
ries were based on the comparison of their retention times and mass
spectral data with published data (Lohachoompol et al., 2008; Prior
et al., 2001; Wu & Prior, 2005). The fourteen characteristic peaks
762 L.-Q. Sun et al. / Food Chemistry 132 (2012) 759–765

Fig. 1. The HPLC fingerprints of the extracts of various blueberries and the reference atlas from the 11 chromatograms. The 14 characteristic peaks were tentatively
identified: peak 1, delphinidin 3-galactoside; peak 2, delphinidin 3-glucoside; peak 3, cyanidin 3-galactoside; peak 4, delphinidin 3-arabinoside; peak 5, cyanidin 3-glucoside;
peak 6, petunidin 3-galactoside; peak 7, cyanidin 3-arabinoside; peak 8, petunidin 3-glucoside; peak 9, peonidin 3-galactoside; peak 10, petunidin 3-arabinoside; peak 11,
peonidin 3-glucoside; peak 12, malvidin 3-galactoside; peak 13, malvidin 3-glucoside and peak 14, malvidin 3-arabinoside.

Fig. 2. Hierarchical clustering analysis of blueberry samples.

were tentatively identified as delphinidin 3-galactoside (peak 1, tR
(min) 22.1, [M+] (m/z) 465, MS/MS (m/z) 303), delphinidin 3-gluco-
side (peak 2, tR (min) 24.7, [M+] (m/z) 465, MS/MS (m/z) 303), cyani-
din 3-galactoside (peak 3, tR (min) 26.9, [M+] (m/z) 449, MS/MS (m/z)
287), delphinidin 3-arabinoside (peak 4, tR (min) 28.6, [M+] (m/z)
435, MS/MS (m/z) 303), cyanidin 3-glucoside (peak 5, tR (min)
31.8, [M+] (m/z) 449, MS/MS (m/z) 287), petunidin 3-galactoside
(peak 6, tR (min) 33.5, [M+] (m/z) 479, MS/MS (m/z) 317), cyanidin
3-arabinoside (peak 7, tR (min) 36.6, [M+] (m/z) 419, MS/MS (m/z)
287), petunidin 3-glucoside (peak 8, tR (min) 39.2, [M+] (m/z) 479,
MS/MS (m/z) 317), peonidin 3-galactoside (peak 9, tR (min) 41.3,
[M+] (m/z) 463, MS/MS (m/z) 301), petunidin 3-arabinoside (peak
10, tR (min) 43.5, [M+] (m/z) 449, MS/MS (m/z) 317), peonidin 3-glu-
coside (peak 11, tR (min) 46.6, [M+] (m/z) 463, MS/MS (m/z) 301),
malvidin 3-galactoside (peak 12, tR (min) 49.6, [M+] (m/z) 493, MS/
MS (m/z) 331), malvidin 3-glucoside (peak 13, tR (min) 51.1, [M+]
(m/z) 493, MS/MS (m/z) 331) and malvidin 3-arabinoside (peak 14,
tR (min) 54.5, [M+] (m/z) 463, MS/MS (m/z) 331), respectively.
3.2. Total antioxidant capacity of the blueberry extracts
The chromatograms of DPPH with or without biological sample
treatment were monitored at 517 nm, and the obvious decrease of
DPPH PAs was observed antioxidant reaction. The AOA of L-ascor-
 L.-Q. Sun et al. / Food Chemistry 132 (2012) 759–765
Table 1
Antioxidant capacities of the blueberry extracts measured in DPPH-HPLC assay
(n = 3).
Sample Antioxidant activity (mg Sample Antioxidant activity (mg
No. AE g1 dry weight) No. AE g1 dry weight)
S01 189.85 ± 2.85 S07 35.31 ± 0.54
S02 233.49 ± 4.55 S08 63.40 ± 1.10
S03 244.92 ± 4.82 S09 52.78 ± 1.03
S04 33.19 ± 0.59 S10 12.69 ± 0.23
S05 11.55 ± 0.18 S11 52.76 ± 0.63
S06 28.88 ± 0.29 Ascorbic 1000
acid
bic acid measured in this method was consistent with the previous
report (Tang et al., 2008), indicating the data from the experiments
was scientific and reliable. The AOA of different berry extracts were
compared with that of L-ascorbic acid by HPLC assay, and
were expressed as mg of L-ascorbic acid equivalent per 1 g of dry
weight. The results (Table 1) ranged from a high of 244.92 mg L-
ascorbic acid equivalents (AE) g1 dry weight for Gardenblue
(S03) to a low of 11.55 mg AE g1 dry weight for Climax (S06),
reflecting a 21-fold difference in scavenging DPPH radical among
the samples. It indicated that all of berry extracts possessed a good
free radical scavenging capacity, and the AOA of various berry ex-
tracts were clearly different. Among these berries, introduced Gar-
denblue and Powderblue showed stronger antioxidative effect than
other cultivars. This information would provide some references
for the introduction and domestication of blueberry in China.
3.3. Predominant antioxidant anthocyanins screening
3.3.1. Results of method I
The multivariant correlation analysis between AOA and PAs of
14 common characteristic peaks in the HPLC fingerprints were
achieved by SPSS statistics software (Table 2). The results showed
that peak 1, 2, 4, 5, 8 and 13 in HPLC fingerprints possessed a close
correlation on the AOA of berry extracts, and these peaks might be
the main antioxidant components. The predominant peak 11 dis-
played a weak influence on the total AOA, while peak 8 with small
PA revealed a prominent contribution. Consequently, the strong
AOA of S01, S02 and S03 (Table 1) could attributed to higher con-
tents of delphinidin 3-galactoside, delphinidin 3-glucoside and del-
phinidin 3-arabinoside (peak 1, 2 and 4) (Fig. 1). The three
compounds were delphinins according to their aglycon, while del-
phinidin 3-glucoside, cyanidin 3-glucoside, petunidin 3-glucoside
and malvidin 3-glucoside (peak 2, 5, 8 and 13) were glucosides
in terms of their glycosyl. This illustrated that delphinins and anth-
ocyanidin-3-glucosides might be responsible for the remarkable
AOA of blueberries. Curiously, the total AOA value of S01 was smal-
ler than those of S02 and S03 (Table 1), while the PAs of most of
peaks in S01, including delphinidin 3-galactoside, delphinidin
3-glucoside and delphinidin 3-arabinoside, were much larger than
those in S02 and S03 (Fig. 1). Some possible explanations were
listed as follows: (1) S02 and S03 might possess other strong anti-
oxidant components, which cannot be monitored at 520 nm; (2)
the therapeutic effect of herbal medicines is often based on the
synergic effect of their mass constituents, and the concentration
proportion of mass constituents in the herbal medicines might
have impact on the therapeutic effect (Li et al., 2007).
3.3.2. Results of method II
Recently, Tang et al. (2008) developed an efficient approach,
which was based on pre-column reaction of samples with DPPH
radical followed by HPLC-DAD analysis, for identification of major
antioxidants in biological samples. The hypothesis was that the
763
Table 2
The correlation coefficients between antioxidant activity and common characteristic
peaks.
Peak No. Correlation coefficient Peak No. Correlation coefficient
1 0.545 8 0.627
2 0.627 9 0.173
3 0.436 10 0.509
4 0.527 11 0.164
5 0.555 12 0.136
6 0.391 13 0.518
7 0.418 14 0.064
conjugated system in the molecular structure would be destroyed
due to the reaction of compounds with DPPH, so that the PAs of
compounds with potential antioxidant effect in the HPLC chro-
matograms would be significantly reduced or disappeared. Fig. 3
showed the chromatograms of S01 with and without DPPH (at
the final concentration of 5 mM) treatment monitored at 520 nm,
and PAs for most of peaks obviously decreased after spiking the
DPPH solution. In this test, the concentration of DPPH solution
was the key factor. If the overdose of DPPH was used, the differ-
ences of free radical scavenging capacity between individual
anthocyanin profile and others could not be measured. On the con-
trary, the consuming anthocyanins could not be inspected due to
the insufficient DPPH.
The overall AOA and PAs of most peaks for S10 were smaller than
those of other samples, so it was selected for the determination of
the concentration of DPPH solution. It was found that the PAs of
three peaks (1, 2 and 4) were reduced more than a half when the final
concentration of DPPH was 1.25 mM. The decreases of PAs ranged
from a high of 78.05% for peak 4 to a low of 2.76% for peak 13, reflect-
ing a 28-fold difference in scavenging DPPH radical capacity among
the peaks (Table 3). While the other samples were treated by DPPH
solution (at the final concentration of 1.25 mM), the decrease ten-
dencies of PAs for most peaks in various samples were unanimous
(Table 3). The results showed that delphinidin 3-galactoside, del-
phinidin 3-glucoside and delphinidin 3-arabinoside (peak 1, 2 and
4) scavenging effect on DPPH radical were rather stronger, or their
reaction speeds with DPPH radical were faster than other peaks.
Cao et al. (1997) proved that, in general, compounds with more hy-
droxyl substitutions on the B ring might show stronger AOA. Accord-
ingly, the higher AOA of delphinins could be attributed to three
hydroxyl substitutions on their B ring, and this was identical with
the result of multivariant correlation analysis and published re-
searches (Noda et al., 2002). However, PAs of the other peaks slightly
Fig. 3. HPLC-DAD chromatograms of S01 (V. myrtillus) extract monitored at 520 nm.
(A) Before reaction with DPPH free radicals, (B) after reaction with DPPH free
radicals (DPPH final concentration 5 mM).
764 L.-Q. Sun et al. / Food Chemistry 132 (2012) 759–765

reduced after mixed with DPPH solution, which were not tentatively

23.92 ± 0.43
23.60 ± 0.52

24.75 ± 0.47
0.55 ± 0.01

1.33 ± 0.03
1.59 ± 0.04
2.49 ± 0.08
1.62 ± 0.05
0.00 ± 0.01
1.60 ± 0.02
1.65 ± 0.03
0.01 ± 0.01
1.73 ± 0.03
1.52 ± 0.03
considered as efficient radical scavengers in blueberries.

S11
3.3.3. Comparative study on two methods
The representations above showed that the results of spectrum–
effect analysis and DPPH-HPLC-DAD analysis were grossly consis-
77.21 ± 0.88
75.32 ± 0.97
6.08 ± 0.17
78.05 ± 0.79
5.67 ± 0.13
23.53 ± 0.37
6.08 ± 0.17
20.12 ± 0.41
5.79 ± 0.11
22.66 ± 0.32

2.76 ± 0.12
4.54 ± 0.15
2.88 ± 0.07
3.73 ± 0.09
tent. For spectrum–effect analysis, it needs to get the AOA of each
sample, and the profiles of biological components of samples should
S10

be basically the same. As a consequence, the comprehensive infor-

mation was obtained, including not only the chemical characteris-
tics and bioactivity of various cultivated rabbiteye blueberries but
23.75 ± 0.63
22.81 ± 0.45

23.89 ± 0.33

4.27 ± 0.14
1.93 ± 0.04

2.79 ± 0.09

1.39 ± 0.03
2.57 ± 0.06
0.03 ± 0.01
4.44 ± 0.09
0.80 ± 0.02
2.37 ± 0.07
0.76 ± 0.01
0.78 ± 0.03
also the contribution of individual anthocyanin profile to the total
AOA of blueberry. Gilbert and Alves (2003) proved that the actions
of medicines derived from plants depend not only on main active
S09

principles but also on adjuvant substances in the plant which could

enhance the activity of the components. Consequently, whether an
28.24 ± 0.51
27.19 ± 0.47

28.43 ± 0.48

4.58 ± 0.14
0.81 ± 0.01

1.99 ± 0.07

1.41 ± 0.03
4.36 ± 0.09
0.01 ± 0.02
4.54 ± 0.09
0.53 ± 0.01
0.02 ± 0.01
0.39 ± 0.01
0.80 ± 0.02

anthocyanin was the main antioxidant or the substance which could

synergistically enhance the AOA of other anthocyanins, its value of
correlation coefficient with total AOA of berries should be large.
S08

Therefore, it might be used to explain that in method I, 3-glucosides

of anthocyanidin (peak 2, 5, 8 and 13) showed better correlation
16.03 ± 0.14
15.31 ± 0.26

15.82 ± 0.22

4.55 ± 0.13

2.71 ± 0.12

0.12 ± 0.11
-0.03 ± 0.01

0.47 ± 0.01
2.17 ± 0.06
1.27 ± 0.03

0.04 ± 0.07

0.17 ± 0.03
0.42 ± 0.04
1.71 ± 0.08

with total AOA of blueberry than the anthocyanins with other glyco-
syl, whereas their free radical scavenging capacities were not obvi-
ously superior to others in method II. In DPPH-HPLC-DAD analysis,
S07

except for that the amount of DPPH must be strictly controlled, the
number of batches for tested samples was not restricted and single
24.42 ± 0.23
23.88 ± 0.41

23.49 ± 0.28

4.10 ± 0.12

3.99 ± 0.12

3.11 ± 0.13
0.76 ± 0.01

0.07 ± 0.02

0.33 ± 0.08

3.26 ± 0.07
4.39 ± 0.07
0.57 ± 0.02

0.54 ± 0.02
0.21 ± 0.01

sample was just enough for investigation; it also could be used to

discriminate different species groups. The obtained information
embodied the free radical scavenging capacity or reactive speed of
S06

respective anthocyanin.
Overall, both method I and method II could be used to screen
68.16 ± 0.57
67.50 ± 0.44
14.32 ± 0.13
68.26 ± 0.49
8.53 ± 0.15
29.40 ± 0.45
16.61 ± 0.28

15.15 ± 0.22
28.35 ± 0.47
12.72 ± 0.17

12.69 ± 0.14
-0.05 ± 0.02

12.59 ± 0.20
0.08 ± 0.01

the main active compounds in plants, and were especially suitable

for coloured biological samples. In method I, the predominant
components screened in terms of their correlation values with
S05

the bioactivity of mixtures, did not only include the strong active
compounds but also the adjuvant substances which could exert
32.51 ± 0.33
31.60 ± 0.42
0.36 ± 0.11
32.12 ± 0.29

6.47 ± 0.19

3.92 ± 0.13

6.73 ± 0.17
0.52 ± 0.15
1.53 ± 0.08

1.47 ± 0.08

3.12 ± 0.08

-0.02 ± 0.01
0.28 ± 0.06
0.50 ± 0.07

synergetic effect. In method II, the bioactivity of each HPLC profile

could be provided directly without any special data processing
software. The combination of these two methods could provide
S04

more comprehensive information about the integral and individual

characteristics. This study would provide a meaningful mode for
20.09 ± 0.36
19.58 ± 0.21

1.69 ± 0.14
4.63 ± 0.19

4.90 ± 0.11
1.53 ± 0.24
5.13 ± 0.12
2.08 ± 0.15
11.41 ± 0.23

1.91 ± 0.11
1.67 ± 0.03
19.69 ± 0.09

0.00 ± 0.08

2.00 ± 0.09

choosing a reasonable method according to researcher’s different

perspectives.
S03

4. Conclusions
Consuming anthocyanins after reaction with DPPH free radical (n = 3).

31.14 ± 0.27
30.36 ± 0.46
7.38 ± 0.28
31.18 ± 0.53
7.58 ± 0.14
11.34 ± 0.23
7.49 ± 0.22
11.63 ± 0.17

12.15 ± 0.13
6.76 ± 0.24
7.00 ± 0.32

6.88 ± 0.17
0.00 ± 0.01

7.21 ± 0.03

Blueberry, as a burgeoning fruit which combines nutrition with

health care, has potential to be used in a wide range of applica-
S02

tions. It is suitable for a variety of people, and the focuses people

concern about have shifted from taste and production to chemical
quality, and even bioactivity. In this study, HPLC was firstly used to
11.43 ± 0.15
11.13 ± 0.17

11.80 ± 0.22

0.10 ± 0.12

0.94 ± 0.29
1.09 ± 0.11

1.36 ± 0.44
1.70 ± 0.03

1.62 ± 0.04
2.05 ± 0.01
1.23 ± 0.02
1.96 ± 0.05

2.18 ± 0.04

1.68 ± 0.08

establish the chromatography fingerprints of various rabbiteye

blueberry cultivars in China, and their AOA were evaluated by a no-
S01

vel HPLC method. It was found that the AOA of introduced Garden-
blue and Powderblue were stronger than other cultivars and native
species (V. uliginosum L.), indicating that introducing superior vari-
Consuming anthocyanins (%)

eties from abroad is of great significance and is also very necessary.

Delphinidin 3-arabinoside
Delphinidin 3-galactoside

Petunidin 3-arabinoside
Delphinidin 3-glucoside

This should be beneficial to directive breeding and industrialised

Petunidin 3-galactoside

Malvidin 3-arabinoside
Cyanidin 3-arabinoside

Malvidin 3-galactoside
Peonidin 3-galactoside
cyanidin 3-galactoside

Petunidin 3-glucoside

Malvidin 3-glucoside
Cyanidin 3-glucoside

Peonidin 3-glucoside

planting of blueberry in China.

In this paper, two efficiency and low-cost methods were applied
to the screening of the main antioxidant anthocyanins in blueber-
ries, respectively. In method I, the correlation between individual
anthocyanin profile and the total AOA of blueberry was estimated
Table 3

by spectrum–effect relationships analysis. The results suggested

that delphinins and anthocyanidin-3-glucosides were closely cor-
 L.-Q. Sun et al. / Food Chemistry 132 (2012) 759–765
related to the high AOA of blueberries. In method II, DPPH-HPLC-
DAD analysis was applied to rapid evaluation of the AOA of antho-
cyanins profiles in blueberry, and delphinins were found to be the
major antioxidant anthocyanins. It was clear that these two meth-
ods both could be widely used in the search for principal bioactive
components in complex mixtures, and particularly in studies of the
biological activity of coloured samples.
Acknowledgements
This research was financially supported by the opening fund of
Jiangsu Province Key Laboratory for Plant Ex Situ Conservation
(KF08001), a Grant from the Ministry of Agriculture of the People’s
Republic of China (nyhyzx07-028, 2006-G25), a Grant from the
Specialized Research Fund for the Doctoral Program of Higher Edu-
cation of China (for the youth scholars) (No. 20090096120008) and
a Grant from science and technology protects of Huzhou
(2010GG17).
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