African Journal of Agricultural Research Vol. 7(26), pp.

3772-3780, 10 July, 2012

Available online at http://www.academicjournals.org/AJAR
DOI: 10.5897/ AJAR11.1386
ISSN 1991-637X 2012 Academic Journals

Full Length Research Paper

Isolation and determination of major anthocyanin

pigments in the pericarp of P. communis L. cv. Red Du
Comices and their association with antioxidant activity
Wen-jiang Huang1,2, Shao-ling Zhang1, Gai-hua Qin1, Le Wenquan3 and Jun Wu1*
1
College of Horticultural Science, Nanjing Agricultural University, Nanjing 210095, China.
2
College of life Science, Anhui Normal University, Wuhu 241000, China.
3
Changli institute of Pomology, Changli Hebei 06600, China.
Accepted 29 February, 2012

The anthocyanins in the fruit skin of Pyrus communis cv. Red Du Comice were isolated, identified and
quantified using high-performance liquid chromatography/diode array detection (HPLC/DAD) and
HPLC/electrospray ionization/mass spectrometry (HPLC/ESI/MS). The individual anthocyanins were
identified by comparing their mass spectral data and retention times with those of standards and
published data. Cyanidin-3-galactoside was the major compound, taking up to 93% of the total
-1
anthocyanin content. The content of Cyanidin-3-galactoside reached 23.7 3.2 mg 100 g fresh weight,
followed by the eighth peak (2.14%) and Cyanidin-3-glucoside (1.23%). Pelargonidin-3-rutinoside (pg-3-
rutinoside) was identified for the first time in P. communis L. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
ferric reducing antioxidant power (FRAP) assays showed that the extract from the fruit peel of Red Du
Comice contained anthocyanins and possessed high antioxidant capacity.

Key words: Pyrus communis, anthocyanin, high-performance liquid chromatography-electrospray ionization-

mass spectrometry, antioxidant activity.

INTRODUCTION

Color is an important factor determining fruit outer quality traditionally been used for the identification of antho-
which has been used successfully in the characterization cyanins (Aneersen, 1985; Matysik and Benesz, 1991;
of fruits (Gautier-Hion et al., 1985). This property has an Sherma, 2000; Timberlake and Bridle, 1976) but with the
important effect on overall acceptability to the consumer advance of analytical technology, analysis and
(Gamble et al., 2006). Anthocyanins are responsible for identification of anthocyanins developed by using High
most of the red, blue and purple colors of fruits, performance liquid chromatography (HPLC) with mass
vegetables, flowers and other plant tissues or products spectrometry (MS) or with tandem MS (Bakker et al.,
(Gould et al., 2002; Manetas, 2006; Steyn et al., 2002; 1986; Hong and Wrolstad, 1990). In recent years, HPLC
Stintzing and Carle, 2004). The isolation and identification coupled with MS has been becoming the standard
of anthocyanins are difficult as a result of their ability to method for identification and separation in most labora-
undergo structural transformations and complex reac- tories (Desai et al., 2010; Huang et al., 2009; Li et al.,
tionary. In addition, they are difficult to measure indepen- 2009; Zheng et al., 2010). HPLC can provide a faster and
dently from other flavonoids as they have similar reaction improved separation of complex compounds and allows
characteristics. Paper and thin-layer chromatography have their tentative identication based on their retentive
characteristics. It is still difficult to obtain each reference
compound for the same spectrum that represents the
anthocyanins. Therefore, HPLC coupled with MS, ESI/MS
*
Corresponding author. E-mail: [email protected]. Tel: are possibly the most powerful methods for identifying the
+86 25 84396485. Fax: +86 25 84396485. structure of anthocyanins. These methods allow the
 Huang et al. 3773

sequential fragmentation of a given molecular ion and 1 g peel powder. Anthocyanins were extracted at 4C for 60 min in
provide information on the identication of anthocyanins dark environment; this procedure was repeated three times to
collect the extract solution. The extraction was concentrated under
based on their ion fragment patterns (Wilson et al., 2005). vacuum at 30C using a rotary evaporator until dryness. The dry
Several studies have been reported on the structural extraction was dissolved in 5 ml of 1% formic acid in distilled water.
elucidation of anthocyanins by means of HPLC-DAD-MS. About 1 ml of extracted solution was passed through a 0.45 m
Using the HPLC-DAD-ESI-MS method, Lopes-da-Silva et millipore lter for analysis.
al. (2002) discovered that strawberry extract contained
three major anthocyanins, Pelargonidin-3-rutinoside (pg-
HPLC-MS analysis
3-rut), Pelargonidin-3-glucoside (pg-3-gluc), Cyanidin-3-
glucoside (cy-3-gluc) and 12 minor anthocyanins, A Waters LC-MSD 1525 series, equipped with a UV detector and
although identity could be only assigned to ve of them Agilent Zorbax SB-C18 column was used. The solvents were (A)
as pg-3-acetylglucoside, cy-3-rutinoside, pg-3- aqueous 2% formic acid, and (B) acetonitrile: water (1:1 v/v)
malylglucoside, pg-diglucoside, and cy-3- containing 2% formic acid. The gradient was from 6 to 10% B for 4
min, from 10 to 25% B for 8 min, isocratic 25% B for 1 min, from 25
malonylglycosyl-l-5-glucoside, the latter three were a
to 40% for 7 min, from 40 to 60% for 15 min, from 60 to 100% for 5
novel discovery in strawberry. So this technique has been min, from 100 to 6% for 5 min, at a flow rate of 1.0 ml/min. Injection
shown to be appropriate for isolation and identification of volumes were 15 l, and the detection wavelength was 516 nm. MS
complex mixtures. Many studies focused on the conditions were as follows: ESI interface, positive ion model, 35 psi
composition of anthocyanins in apple (DuPont et al., nebulizer pressure, 10 L/min dry gas flow rate, 350C dry gas
2002; Shoji et al., 2006), strawberry (Aaby et al., 2007; temperature and scans at m/z 150 to 1000. All analyses were
duplicated.
Gil et al., 1997; Wu et al., 2006), blueberry (Gu et al.,
2002; Prior et al., 2001), grape (Garca-Beneytez et al.,
2003; Huang et al., 2009; Oliveira et al., 2010) and other Antioxidant capacity determined by FRAP
fruits (Wu et al., 2006). Pear is one of the most important
temperate fruits. Fruit skin color is a significant factor that This procedure involved the reduction of ferric ion (Fe 3+) to the
affects the quality and customer acceptance. There is a ferrous ion (Fe2+) to a blue colored complex Fe2+/TPTZ in the
presence of bioactive compounds (antioxidants) which was
few studies that have been conducted on pear phenolic revealed as the increase of absorption at 593 nm (Benzie and
compounds studies using liquid chromatography (LC) or Strain, 1999). Extracts was added to the FRAP reagent. The FRAP
liquid chromatography with mass spectrometric detection reagent contained 10 mM TPTZ in 40 mM HCl, 20 mM FeCl 3 and
(LC-MS) have reported such polyphenols in pear 300 mM acetate buffer (pH3.6) that was freshly prepared in the ratio
materials (Francis, 1970; Muchuweti and Chikwambi, of 1:1:10 and warmed 30 min at 37.8C before and after adding of
sample extract. The FeSO47H2O compound was used to prepare
2008; Oleszek et al., 1994).
the increasing concentrations of standard solutions. Results were
This study aimed to isolate and identify the anthocyanin expressed as ascorbic acid equivalent antioxidant capacity (mg
molecules responsible for fruit skin coloration in P. ascorbic acid/100 g fresh weight).
communis L. cv. Red Du Comices.
Antioxidant capacity determined by DPPH-radical scavenging
MATERIALS AND METHODS activity (DPPH assay)

Plant samples The ability to scavenge DPPH free radical was determined based
on the method of Brand Williams (Brand-Williams et al., 1995), with
The fruit samples were picked at commercial maturity from Changli minor modification. Briefly, reaction mixtures containing 25 l
Institute of Pomology(Hebei, China), and transported to the extracts and 2 ml 6.25 10-5 M DPPH solution were prepared,
laboratory immediately. Fruit free of defects and without evidence of mixed, and then reacted in the dark for 30 min. A control sample
mechanical damage were selected. Fruit skins were peeled and containing the same volume of solvent in place of extract was used
frozen in liquid nitrogen and kept at -70C until being analysed. to measure the maximum 2,2-diphenyl-1-picrylhydrazyl (DPPH)
absorption. The absorbance at 517 nm was recorded to determine
the concentration of the remaining DPPH. Results were expressed
Chemicals as ascorbic acid equivalent antioxidant capacity (mg ascorbic
acid/100 g fresh weight).
HPLC-grade methanol and acetonitrile were obtained from Merck
KGaA, Darmany, Germany. Formic acid, cyanidin-3-glucoside,
cyanidin-3-galactoside, cyanidin-3-arabinoside, peonidin-3- RESULTS AND DISCUSSION
glucoside, peonidin-3-galactoside and DPPH, 2,4,6-tripyridyl-s-
triazine (TPTZ) were obtained from Sigma Chemical Co. All other Test for anthocyanins
chemicals used in this study were analytical grade.
The red, purple and blue colors found in many plants are
due to two classes of water soluble pigments:
Extraction of anthocyanins
anthocyanins and betacyanins. The anthocyanins are
The extraction of anthocyanins was performed according to Wu flavonoids, a class of phenolic molecules that are
(Wu et al., 2006), with some modifications. 20 ml methanol with 1% synthesized through the Shikimic acid pathway and are
(v/v) formic acid was added into 100 ml Erlenmeyer ask containing widespread in the plant kingdom. Betalains, a group of
3774 Afr. J. Agric. Res.

Figure 1. HPLC chromatograms of extract of Red Du Comice pear peel of fruit (516 nm).

Table 1. HPLC/DAD and HPLC/ESI -MS of anthocyanins in fruit skin of Red Du Comice.

+ 2
Peak number tR(min) [M ] m/z MS m/z Compounds
1 28.82 449 287 Cyanidin-3-galactoside
2 32.42 449 287 Cyanidin-3-glucoside
3 34.50 419 287 Cyanidin-3-arabinoside
4 35.23 595 449/287 Cyanidin-3-rutinoside
5 37.64 579 433/271 Pelargonidin-3-rutinoside
6 38.51 463 301 Peonidin-3- galactoside
7 40.17 463 301 Peonidin-3- glucoside

Cyanidin-3-methylmalonylgluside?
8 44.52 549 287 Cyanidin-3-succinylglucoside?
Cyanidin-malylrhamnoside?

Standard 28.76 449 287 Cyanidin-3-galactoside

Standard 32.33 449 287 Cyanidin-3-glucoside
Standard 34.30 419 287 Cyanidin-3-arabinoside
Standard 38.43 463 301 Peonidin-3- galactoside
Standard 40.1 463 301 Peonidin-3- glucoside

pigments that includes the betacyanins are indole-derived from fruit skin of Red Du Comice using the HPLC-DAD
alkaloids and contain nitrogen. Pear peel extracts (100% chromatograms at 516 nm. A total of eight anthocyanin
methanol with 1% HCl) were tested for the presence of compounds were identified by their elution order and by
anthocyanins by observing pigment color under acidic or comparing the m/z of each anthocyanin molecule and its
alkaline conditions by adding HCl or sodium hydroxide. 3 fragmentation to prepared standards and previous reports
ml of extract and 3 ml HCl were mixed in a test-tube and (Table 1). Five peaks (peaks 1, 2, 3, 4 and 8) showed
2
then placed in boiling water bath for 5 min. The mixture fragment ions at m/z 287 in MS analyses that could be
was stable and did not lose color when boiled. A blue- tentatively identified as cyanidin derivatives. Peaks 1 and
green colour was observed when adding sodium 2 showed identical molecular ions at m/z 499 and
hydroxide to the extract, which indicated the presence of fragmentation patterns, but their retention time in the
anthocyanins in the extracts. HPLC system were 28 and 32 min for peaks 1 and 2,
respectively. Their elution order and MS characteristics
suggest that they could be cyanidin-3-galactoside (Figure
Identification of pear anthocyanins 2) and cyanidin-3-glucoside, respectively, which concurs
with that found in previous studies (Cerezo et al., 2010)
Figure 1 showed the anthocyanin profile of the extract and coincides with the available standard used for
 Huang et al. 3775

M+
Intensity

MS2
Intensity

Figure 2. MS data of cyanidin -3-galactoside.

confirmation purposes (Table 2). Peaks 3 and 8 showed methyl derivative of cyanidin-3-malonylglucoside.
the same major cyanidin fragment ions at m/z 287, but However, other structures that also match the molecular
different molecular ions at m/z 419 and 549, respectively. ion of peak 8 are cyanidin-malylrhamnoside and cyanidin-
Peak 3 produced an MS pattern corresponding to 3-succinylglucoside (Da Silva et al., 2007). Unfortunately,
cyanidin-3- arabinoside, which coincides with the no information could be obtained to confirm suggestions
confirmation marker. Peak 8 might correspond to the about its structure.
3776 Afr. J. Agric. Res.

Table 2. Anthocyanins content and percentage in the extract of Red Du Comice

peel.

a b
Compounds Content %
Cyanidin-3-galactoside 23.73.2 93.71
Cyanidin-3-glucoside 0.310.05 1.23
Cyanidin-3-arabinoside 0.030.00 0.12
Cyanidin-3-rutinoside 0.020.00 0.08
Pelargonidin-3-rutinoside 0.280.02 1.11
Peonidin-3- galactoside 0.190.03 0.75
Peonidin-3- glucoside 0.220.02 0.87
Peak 8 0.54 0.03 2.14
a
Individual anthocyanins were analyzed using HPLC-ESI-MS. Each anthocyanin was
quanitified in cyanidin equivalents (mg/100 g fresh weight). b Percentage of the total
content.

+ MS2
M
Intensity

Intensity

Figure 3. MS data of cyanidin-3-rutinoside.

Peak 4 produced a fragment pattern matching cyanidin- calibration curve determined using cyanidin-3-glucoside.
3-rutinoside (Figure 3). Peaks 6 and 7 were assigned as Other identified anthocyanins were quantitated through
Peonidin-3-galactoside (Figure 5) and Peonidin-3- the calibration curves of corresponding reference
glucoside, both with the mass spectrometric characteris- compounds. Table 2 shows the individual concentration of
tics and elution orders matching the reference com- the anthocyanins in the extracts of Red Du Comice peel.
pounds and those that had previously been reported in Cyanidin-3-galactoside, the major compound, accounts
+
grape (Huang et al., 2009). Peak 5 showed an [M] at for 93.7% of total anthocyanin which confirms in previous
2
579, which gave MS fragments that were at m/z 271 and studies (Sanchez et al., 2003). Percentages of 94.2% for
433 and was identified as Pelargonidin-3-rutinoside Cyanidin-3-galactoside and 4.5% for Cyanidin-3- rutoside
(Figure 4). Due to the lack of corresponding reference were found by Sanchez et al. (2003) in RedDAnjou. It is
compounds for Cyanidin-3-rutinoside, Pelargonidin-3- followed by the compounds representing peak 8 (2.14%),
rutinoside and the compound representing peak 8, the Cyanidin-3-glucoside (1.23%), Pelargonidin-3-rutinoside
identified anthocyanins were quantitated through a (1.11%), Peonidin-3-glucoside (0.87%) and Peonidin-3-
 Huang et al. 3777

M+
Intensity

MS2
Intensity

Figure 4. MS data of pelargonidin-3-rutinoside.

galactoside (0.75%). Muchuweti et al. (2008) considered infrared-spectra. Cyanidin-3-o-galactoside, Quercetin-3-

that only one anthocyanidin present the pear peel o-glucoside and Cyanidin-3-o-arabinoside were identified
according to paper chromatography and Uv-vis and only in the Max Red Bartlett fruit skin using HPLC-MS by
3778 Afr. J. Agric. Res.

M+
Intensity

MS2
Intensity

Figure 5. MS data of peonidin-3-galactoside.

Luca et al. (2010). According to Tamuras works (1995), strawberry (Ana et al., 2010). Pelargonidin-3-rutinoside
most of anthocyanin contain Pelargonidin (Pg) as has also been previously reported in Concord grape
aglycone and Pg-3-rutinoside is commonly found in (Baublis et al., 1994), cranberry (Heinonen, 2007),
 Huang et al. 3779

strawberry (Da Silva et al., 2007b), blackberry (Wrolstad, glucoside was the major anthocyanin in strawberry
1993), sweet cherry (Gao and Mazza, 1995) and black (Fragaria ananassa) (Cerezo et al., 2010). Delphinidin,
plum (Wu et al., 2006); but not in pear (Lin and Harnly, petunidin and malvidin were the major contributors to
2008; Sanchez et al., 2003). This study is tentatively total anthocyanin content but the proportions of each
proposing, for the first time, that Pelargonidin-3-rutinoside compound were cultivar-dependent. High bush has more
is present in pear. polar anthocyanins than rabbit eye cultivars
(Lohachoompol et al., 2008). The anthocyanin differences
in fruit peels constitute a biochemical marker, so the
Antioxidant capacity gathered information in this study helps towards
characterizing pear cultivars and their mutants. This will
The antioxidant capacity of the total anthocyanin obtained assist in breeding good sensory quality attributes and
from fruit peel of Red Du Comice was evaluated with the defense against ultraviolet light or aggression by
DPPH and FRAP test. The free radical scavenging pathogens (Hamauzu et al., 2005).
activity determined by DPPH was 165.23 11.6 mg/100
-1
g FW and the value of total antioxidant capacity
-1
determined by FRAP was 87.88 9.8 mg/100 g FW ACKNOWLEDGMENT
(ascorbic acid equivalent antioxidant capacity). The
extract possessed higher antioxidant capacity than that This work was supported by the Natural Science Fund of
found in previous studies. This difference might be due to China (300900974). The authors would like to thank Dr.
the differing maturation rate of individual fruit and different Huping Zhang for helpful discussions on antioxidant
methods of extraction and analysis. measurements and Mr. Xu (State key laboratory of Crop
Genetics and Germplasm Enhancement) for technical
assistance with HPLC-MS.
Conclusion

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