Flavonoid C-glycosides from pigeon pea leaves as color and anthocyanin stabilizing agent in blueberry juice

Industrial Crops and Products 58 (2014) 142–147

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Flavonoid C-glycosides from pigeon pea leaves as color and anthocyanin stabilizing agent in blueberry juice You-Zhi Pan a,b,1 , Yue Guan a,b,1 , Zuo-Fu Wei a,b , Xiao Peng a,b , Ting-Ting Li a,b , Xiao-Lin Qi a,b , Yuan-Gang Zu a,b , Yu-Jie Fu a,b,∗ a b

State Engineering Laboratory for Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, PR China Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China

a r t i c l e

i n f o

Article history: Received 27 December 2013 Received in revised form 11 April 2014 Accepted 15 April 2014 Keywords: Anthocyanins Color stabilization Flavonoid C-glycosides Copigment Pigeon pea leaves

a b s t r a c t The influences of flavonoid C-glycoside extracts from pigeon pea leaves (FCGE) and its main components vitexin and orientin on the color and anthocyanins stability of blueberry juice were investigated in the thermal experiments. The color of juice was enhanced by FCGE and its main components in a molar ratio of 1:1 (anthocyanins/copigment). The saturated color of juice with FCGE and its main components could hold for a significantly longer time. Additionally, copigment FCGE, vitexin and orientin significantly enhanced the stability of anthocyanins. Half-life of anthocyanins in juice samples with FCGE (1:1), orientin and vitexin increased 87%, 79%, and 62%, respectively. These results indicated FCGE showed dramatic effect on the color and anthocyanins stability of juice. Furthermore, juice samples with copigments possessed higher total phenolics content and higher DPPH radical-scavenging activity. Therefore, natural, easily available, inexpensive FCGE has potential as color enhancer and anthocyanins stabilizer in food industry. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Anthocyanins are responsible for most blue, red, and related colors in flowers and fruits (Clifford, 2000) and exhibit a very positive effect on some diseases, including potent cardioprotective, neuroprotective, antiinflammatory, and anticarcinogenic properties (Barba et al., 2013). In recent years, the interest in anthocyanins has dramatically increased because of their high antioxidant capacity. Many fruits are rich in anthocyanins, especially berry fruits. Among very popular berry fruits (strawberry, raspberry and blueberry), blueberry exhibits the highest antioxidant capacity, which is related to its highest anthocyanins content (Juranic and Zizak, 2005; Nicoué et al., 2007). Anthocyanins are very susceptible to degradation (Giusti and Wrolstad, 2003). The factors influencing the color and stability of anthocyanins mainly include pH, concentration, light, temperature, presence of copigments, enzymes, sugars, metals, oxygen, and sulphur dioxide (Cavalcanti et al., 2011; Breness et al., 2005;

∗ Corresponding author at: Engineering Research Center of Forest Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China. Tel.: +86 451 82190535; fax: +86 451 82190535. E-mail address: yujie [email protected] (Y.-J. Fu). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.indcrop.2014.04.029 0926-6690/© 2014 Elsevier B.V. All rights reserved.

Jiménez-Aguilar et al., 2011). Low temperature, dark, absence of oxygen and low pH (2–4) are beneficial to the stability of anthocyanins. In addition, the presence of non-anthocyanin polyphenolics significantly increases anthocyanin stability due to the occurrence of copigmentation reactions (Pacheco-Palencia and Talcott, 2010). Copigmentation is a phenomenon in which pigments and other colorless organic compounds, or metallic ions, form molecular or complex associations, resulting in an increase in the color intensity (Boulton, 2001). Some studies (Davies and Mazza, 1993; Mazza and Brouillard, 1990) demonstrated that copigmentation is a primary color-stabilizing mode in plants and food products rich in anthocyanins. Copigmentation generally occurs in the following four interactions: self-association, metal complexation, intramolecular copigmentation and intermolecular interactions. Among the four interactions, intermolecular and intramolecular copigmentation are the most important mechanisms of copigmentation (Sun et al., 2010). Intramolecular copigmentation, in which the central anthocyanin chromophore and aromatic acyl residues covalently linked to their glycosyl moieties (Gómez-Míguez et al., 2006; Figuereido et al., 1999; George et al., 2001), is predominant in flower vacuoles (Sun et al., 2010; Bloor and Falshaw, 2000). Intermolecular copigmentation occurs when colorless compounds are attracted to anthocyanins via weak hydrogen bonds and hydropho˜ et al., 2009; Eiro and Heinonen, bic forces (Castaneda-Ovando 2002).

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Many compounds may be copigments, such as flavonoids, alkaloids, amino acids, organic acids, nucleotides, polysaccharides, metals, and anthocyanins themselves (Mazza and Brouillard, 1990). Previous researches indicated the color and stability of anthocyanins could be enhanced by polyphenolic copigments, particularly phenolic acids (ferulic, caffeic acids and rosmarinic acids) (Eiro and Heinonen, 2002). Meanwhile, chlorogenic acid (Brouillard et al., 1989), quercetin-5 -sulphonic acid (Sweeny et al., 1981), morin (Baranac et al., 1997b), rutin (Baranac et al., 1996), quercetin (Baranac et al., 1997a) have also been identified as good copigments. These copigments are expensive or synthetic substances and their natural cheap equivalents are the focus of current studies. The flavonoid C-glycoside from pigeon pea leaves seems to be one of such equivalents. The pigeon pea leaves are rich in flavonoid Cglycosides, mainly including vitexin and orientin (Wu et al., 2009). Pigeon pea is one of the major grain legume crops in the tropics and subtropics. It ranks sixth in areas and production, its leaves can be consumed as vegetable (Wei et al., 2013). Therefore, FCGE may be a natural and abundant copigment. To the best of our knowledge, there has been not the study on the pigeon pea leaves as copigments yet. The objective of this study was to evaluate the influence of FCGE and its main components on the color and anthocyanins stability of blueberry juice, meanwhile, the possible mechanism for the copigmentation reaction was elucidated. 2. Materials and methods 2.1. Chemicals and reagents Vitexin, orientin, rutin, Folin–Ciocalteu reagent and DPPH (2,2diphenyl-1-picrylhydrazyl) were purchased from Sigma–Aldrich (Steinheim, Germany). FCGE were obtained in our laboratory. The pigeon pea leaves was extracted with 80% ethanol at room temperature for one week. The filtered solution was collected and concentrated to dryness under reduced pressure. The resulting extract was diluted in distilled water and further extracted with ethyl acetate. Then, the ethyl acetate fractionation was evaporated to dryness under reduced pressure and submitted to a preliminary chromatographic separation over an ADS-5 macroporous resin column, then eluted with ethanol of 20%, 40%, 60% and 100%, successively. The 40% ethanol fraction was concentrated to dryness under reduced pressure to obtain yellow powder, its main components were confirmed as flavonoid C-glycoside vitexin (55.8%) and orientin (28.3%) by HPLC analysis as described in our previous report (Wu et al., 2009). The yield of flavonoid C-glycoside extracts from pigeon pea leaves (FCGE) was 5.28 mg/g pigeon pea leaves. 2.2. Sample preparation Blueberry fruits were obtained from Greater Khingan Range area in China. Pigeon pea leaves were collected in autumn from Hainan Province, China. Highly colored blueberry fruits were selected and washed, destemmed and squeezed with a certain amount of water (fruit/water = 1:6, w/w), then filtered to yield juice. Vitexin, orientin and rutin were added in juice (30 mL) with an anthocyanins/copigment molar ratio of 1:1, while FCGE was added in juice with three anthocyanins/copigment molar ratio of 1:0.1, 1:0.5 and 1:1 (FCGE calculated as vitexin and orientin). The control sample was the juice without copigment, the positive control sample was the juice with rutin. All the prepared samples were stored in the same transparent glass bottles with stopper and kept at constant temperature (40 ◦ C), the data were acquired at the second, fifth, seventh, tenth and twentieth day, respectively. All experiments were repeated in triplicate.

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2.3. HPLC–UV-MS analyses of anthocyanins Anthocyanins in blueberry juice were analyzed using an Agilent 1200 HPLC system. Chromatographic separation was carried out on a HIQ Sil reversed phase column (C18, 250 mm × 4.6 mm, 5 ␮m). The sample was filtered through 0.45 ␮m nylon membranes prior to HPLC analysis. The mobile phase consisted of aqueous 2% formic acid (A) and acetonitrile containing 2% formic acid (B). The gradient elution program was as follows: 0–4 min, 6–8% B; 4–14 min, 8% B; 14–15 min, 8–10% B; 15–25 min, 10% B; 25–26 min, 10–13% B; 26–36 min, 13% B; 36–45 min, 13–30% B; 45–60 min, 30–100% B. The injection volume was 10 ␮L. The flow rate and column temperature was 1 mL/min and 30 ◦ C, respectively. Anthocyanins were detected by their absorbance at 520 nm. The conditions of MS analysis were as follows: electrospray ionization (ESI) interface, nitrogen utilized as drying, nebulizing gas and nebulizer pressure at 35 psi, dry gas flowing at 10 mL/min, 350 ◦ C dry gas temperature, capillary voltage at 3000 V and spectra recording in the positive ion mode with scans from m/z 50 to 1000. 2.4. Spectral shift and color density measurements Visible absorption spectra (450–600 nm) of juice[0] samples with and without copigments were recorded on a Shimadzu UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). Sample solutions were kept in the dark before and after their spectra were recorded. Color density was defined as the sum of absorbances at the ␭vis-max and at 420 nm with a UNICO UV-2100 spectrophotometer (UNICO, Shanghai, China). The color density was calculated as follows: color density = [(A420 nm − A700 nm ) + (A␭vis-max − A700 nm )] × DF. DF was the dilution factor. 2.5. Color properties measurements The color properties were measured by tristimulus values recommended by the Commission Internationale del’Éclairage (CIE) (Darias-Martín et al., 2007). The parameters lightness (L*), redness (a*) and yellowness (b*) in CIE1976L*a*b* color space were measured periodically using an Aoke ChromaTCP-2 Colorimeter (Aoke, Beijing, China). Chroma (C*) and hue angle (H*) were calculated by the formulas: C* = [(a*)2 + (b*)2 ]1/2 and H* = arctan(b*/a*). L* is the luminosity of a stimulus judged relative to the luminosity of that which appears as white. C* is the chromaticness of a stimulus judged relative to the white. H* is the attribute of appearance by which a color is identified according to its resemblance to red, green, yellow or blue (0◦ or 360◦ = red; 90◦ = yellow; 180◦ = green; 270◦ = blue). 2.6. Determination of total anthocyanins Total anthocyanin content was determined spectrophotometrically at 520 nm and 700 nm, according to the pH differential method described by Wrolstad et al. (2005). Total anthocyanins were quantified using equivalents of cyanidin-3-glucoside (mg/L) with a molar extinction coefficient of 29,600. 2.7. Kinetic analysis Kirca and Cemeroglu (2003) have shown that thermal degradation of anthocyanins follows a first-order reaction. The degradation of anthocyanins was calculated by using the standard equation for a first-order reaction given below: ln

C  t

C0

= −kt

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Y.-Z. Pan et al. / Industrial Crops and Products 58 (2014) 142–147

Fig. 1. HPLC chromatographic profiles of blueberry juice anthocyanins at 520 nm.

where Ct and C0 were the anthocyanins content at time t and t0 respectively, k was the reaction rate constant (day−1 ), and t was the storage time (days). Furthermore, the half-life of anthocyanins (t1/2 ) was also calculated using the equation given below: t1/2 = ln

2 k

(Prior et al., 2001), ten anthocyanins were identified in blueberry juice. It was found that delphinidin-3-glucoside, petunidin-3glucoside and malvidin-3-glucoside were the major anthocyanins in the blueberry juice.

3.2. Copigmentation effect

where k was the reaction rate constant (day−1 ). 2.8. Total phenolics and DPPH radical-scavenging activity Total phenolics content of juice samples was determined by Folin–Ciocalteu method (Singleton and Rossi, 1965). The results were expressed as milligram of gallic acid equivalent per milliliter of sample (mg GAE/mL). The free radical scavenging activity of juice samples was determined according to Sokmen et al. (2005) with minor modifications. The juice sample (0.1 mL) was added to the test tube containing mixture of 1.4 mL ethanol and 1 mL of 0.004% DPPH solution in ethanol (freshly prepared). After incubation for 70 min at room temperature, the absorbance was immediately measured at 517 nm using a UV–visible spectrophotometer. The results were expressed as microgram of gallic acid equivalent per milliliter of sample (␮g GAE/mL). 2.9. Statistical analysis The experiment was performed in triplicate and significant differences were assessed by one-way analysis of variance using SPSS version 10.0, and differences at P < 0.05 were considered significant. 3. Results and discussion

Anthocyanin copigmentation reactions can be detected by a hyperchromic effect, where absorbance at the max of the absorption spectrum increases, and/or a bathochromic shift, where the spectra of copigmented solutions display a shift of the visible max toward greater wavelengths (Malien-Aubert et al., 2001). Visible spectra were shown in Fig. 2. It was found that the addition of copigments induced the increase of absorbance in the visible band (450–600 nm). It indicated that the hyperchromic effect was elicited by studied copigments. The bathochromic shift was not significant for almost all of copigments in a molar ratio of 1:1. It could be seen that the use of FCGE (1:1) produced the highest hyperchromic effect, followed by orientin and rutin. The most obvious hyperchromic effect was observed in the juice sample with FCGE (1:1), and its magnitude was dependent on the concentration of the added FCGE. This was in accordance with the findings reported by Bacakowska et al. (2003) and González-Manzano et al. (2008) that the magnitude of copigmentation effect increases with the increase of copigment content. The hyperchromic effect and bathochromic shift should be attributed to the formation of intermolecular association, which formed the electrons ␲–␲ system (chromophore) through chargetransfer complex or ␲–␲ interaction, between anthocyanins and ˜ copigments. Castaneda-Ovando et al. (2009) have elucidated that anthocyanins and copigments formed an overlapping arrangement through intermolecular association, and the association led to the hyperchromic effect and bathochromic shift.

3.1. Identification of anthocyanins The anthocyanins profile of the blueberry juice was shown in Fig. 1. According to the mass spectrum (Table 1) and previous report Table 1 LC–MS data of anthocyanins in blueberry juice. Peak no.

Retention time (min)

(m/z) total/aglycone

Anthocyanin

1 2 3 4 5 6 7 8 9 10

19.6 21.6 23.4 24.7 27.2 28.9 30.9 32.8 34.6 37.4

465/303 465/303 449/287 435/303 449/287 479/317 479/317 449/317 493/331 493/331

Delphinidin-3-galactoside Delphinidin-3-glucoside Cyanidin-3-galactoside Delphinidin-3-arabinoside Cyanidin-3-glucoside Petunidin-3-galactoside Petunidin-3-glucoside Petunidin-3-arabinoside Malvidin-3-galactoside Malvidin-3-glucoside

Fig. 2. The absorption spectra of anthocyanins in juice samples with and without copigments. C: control; VI: vitexin; OR: orientin; RU: rutin; FCGE: flavonoid C-glycoside extracts from pigeon pea leaves.

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Table 2 Color density (CD) and DPPH radical-scavenging activity of blueberry juice samples. Samplea

CD

C C + OR C + RU C + VI C + FCGE (1:0.1) C + FCGE (1:0.5) C + FCGE (1:1)

1.12 1.30 1.27 1.18 1.14 1.25 1.32

Antioxidant capacity (␮g GAE/mL) ± ± ± ± ± ± ±

0.05g 0.04b 0.05c 0.04e 0.05f 0.05d 0.06a

44.85 75.42 55.83 49.98 45.71 53.04 64.56

± ± ± ± ± ± ±

0.95f 3.11a 1.29c 2.31e 2.20f 2.50d 2.36b

a C: control; VI: vitexin; OR: orientin; RU: rutin; FCGE: flavonoid C-glycoside extracts from pigeon pea leaves. Data in the table are means ± standard deviation of their replicates. Data with different superscript letters in the same column were significantly different (P < 0.05).

As shown in Table 2, the color density of all copigmented juice samples was enhanced. Moreover, the juice samples with copigment FCGE (1:1) (1.32) and orientin (1.30) exhibited higher color density compared with the juice sample with positive control rutin (1.27). 3.3. Effect of copigmentation on color stability We chose the CIE1976L*a*b* color space systems to quantitatively express the color change of juice samples during storage. The chromaticity parameters (L*, C*, and H*) of juice samples were shown in Table 3. It was found that the juice samples with copigments possessed lower lightness (L*) value and higher chroma (C*) value compared with control at the beginning of the assay (0 day), indicating that the color of copigmented juice samples was more intense and saturated compared with control. After two days, control and juice sample with FCGE (1:0.1) showed lower value of H* compared with their value at 0 day, whereas the juice samples with vitexin and FCGE (1:0.5) held the stable H* value until the fifth day, the juice samples with FCGE (1:1), orientin and rutin held the stable H* value until the seventh day. The trend of chroma (C*) was similar to the H*. This meant that the juice samples with vitexin and FCGE (1:0.5) could hold saturated color until the fifth day, and the juice with FCGE (1:1), orientin and rutin could keep saturated color until the seventh day. In other words, the juice samples

Fig. 3. Percent changes of total anthocyanins content in juice samples with and without copigments during storage. C: control; VI: vitexin; OR: orientin; RU: rutin; FCGE: flavonoid C-glycoside extracts from pigeon pea leaves.

copigmented with vitexin, FCGE (1:0.5), rutin, orientin, FCGE (1:1) could retain color stability until fifth to seventh day. Moreover, among all samples, the juice sample copigmented with FCGE (1:1) exhibited the most intense and saturated color. These observations indicated that these copigments, especially FCGE, could significantly improve the color stability of juice. 3.4. Effect of copigmentation on anthocyanins stability The content of anthocyanins is the key to assess the juice quality. The change in anthocyanin content of all juice samples was present in Fig. 3. It was clearly shown that the content of anthocyanins in all samples decreased over time. But the juice samples with copigment FCGE (1:1), FCGE (1:0.5), vitexin, orientin or rutin showed gentle downward trend, while control showed a faster downward trend during storage. Moreover, the first-order kinetic model was specifically used for measuring the degradation of anthocyanins. It was observed in Table 4 that the degradation of anthocyanins well fitted the first-order kinetic model (R2 > 0.97). It was also found that copigment FCGE, vitexin, orientin and rutin significantly extend half-life of anthocyanins (P < 0.05). The anthocyanins in the juice samples copigmented with FCGE (1:1), orientin, rutin,

Table 3 Change of chromatic parameters lightness (L*), chroma (C*) and hue angle (H*) of blueberry juice samples. Chromatic parameter

Samplea

Storage time (days) 0

a

2

L*

C C + OR C + RU C + VI C + FCGE (1:0.1) C + FCGE (1:0.5) C + FCGE (1:1)

7.89 5.91 5.76 7.21 7.47 6.20 5.69

± ± ± ± ± ± ±

0.25 0.19 0.20 0.25 0.26 0.25 0.24

C*

C C + OR C + RU C + VI C + FCGE (1:0.1) C + FCGE (1:0.5) C + FCGE (1:1)

304.29 307.84 308.34 306.10 304.75 308.10 307.84

± ± ± ± ± ± ±

H*

C C + OR C + RU C + VI C + FCGE (1:0.1) C + FCGE (1:0.5) C + FCGE (1:1)

277.45 277.45 277.45 277.45 277.45 277.45 277.45

± ± ± ± ± ± ±

5 8.83 5.91 6.69 8.01 8.52 7.23 6.09

± ± ± ± ± ± ±

0.32 0.21 0.24 0.29 0.28 0.28 0.25

9.50 9.99 10.94 10.78 11.01 12.36 12.66

303.59 307.66 306.31 304.22 303.66 308.83 307.66

± ± ± ± ± ± ±

8.96 8.93 9.89 9.82 10.02 11.13 11.53

277.44 277.64 277.57 277.65 277.60 277.53 277.74

± ± ± ± ± ± ±

7 12.20 8.51 9.03 10.10 11.55 9.72 8.14

± ± ± ± ± ± ±

0.44 0.30 0.28 0.34 0.39 0.38 0.32

9.78 10.62 11.17 10.99 10.17 11.41 11.64

46.42 304.60 303.03 301.57 46.80 304.10 304.60

± ± ± ± ± ± ±

9.21 9.63 9.71 9.48 9.62 9.62 11.16

32.90 277.63 277.53 277.30 33.00 277.44 277.63

± ± ± ± ± ± ±

10

20

14.10 9.66 9.19 10.01 13.63 9.87 8.90

± ± ± ± ± ± ±

0.47 0.36 0.32 0.35 0.48 0.37 0.33

16.70 9.85 9.85 11.35 15.80 10.88 9.48

± ± ± ± ± ± ±

0.50 0.33 0.33 0.42 0.51 0.45 0.41

23.00 15.43 16.28 18.23 22.06 18.17 14.85

± ± ± ± ± ± ±

0.86 0.49 0.56 0.69 0.76 0.73 0.63

1.56 11.61 12.42 9.55 1.67 10.88 12.26

46.26 303.31 302.28 49.08 46.32 41.00 303.31

± ± ± ± ± ± ±

1.69 10.05 12.13 1.52 1.77 1.49 11.80

40.95 50.00 45.36 44.11 41.09 41.68 53.00

± ± ± ± ± ± ±

1.33 1.71 1.92 1.59 1.65 1.56 2.06

26.83 32.90 34.60 30.29 27.29 30.00 34.90

± ± ± ± ± ± ±

1.01 1.19 1.13 1.06 1.04 1.17 1.45

1.14 10.24 9.31 10.11 1.29 10.71 11.47

30.75 277.54 277.47 35.62 30.90 32.20 277.64

± ± ± ± ± ± ±

1.00 9.59 9.58 1.16 1.24 1.36 11.00

24.72 30.00 28.85 27.77 25.10 26.80 29.50

± ± ± ± ± ± ±

0.86 0.97 0.98 0.99 0.97 1.06 1.13

23.71 25.20 26.24 26.22 23.90 24.40 26.40

± ± ± ± ± ± ±

0.84 0.80 0.96 0.92 0.94 0.94 1.04

C: control; VI: vitexin; OR: orientin; RU: rutin; FCGE: flavonoid C-glycoside extracts from pigeon pea leaves.

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Table 4 Relative degradation kinetics, rate constant (k) and half-life time (t1/2 ) of anthocyanins in blueberry juice samples. Samplea

Equation

R2

k

t1/2 (days)

C C + OR C + RU C + VI C + FCGE (1:0.1) C + FCGE (1:0.5) C + FCGE (1:1)

y = 0.1158x − 0.0927 y = 0.0631x − 0.074 y = 0.0646x − 0.0385 y = 0.0709x − 0.0847 y = 0.1022x − 0.0103 y = 0.0703x − 0.0595 y = 0.0603x − 0.0738

0.9797 0.9856 0.9930 0.9873 0.9879 0.9925 0.9831

0.1158 0.0631 0.0646 0.0709 0.1022 0.0703 0.0603

6.8f 12.2b 11.3c 11.0d 6.9f 10.7e 12.7a

a C: control; VI: vitexin; OR: orientin; RU: rutin; FCGE: flavonoid C-glycoside extracts from pigeon pea leaves. Data in the table are means ± standard deviation of their replicates. Data with different superscript letters in the same column were significantly different (P < 0.05).

vitexin and FCGE (1:0.5) had a longer half-life (t1/2 = 12.7, 12.2, 11.3, 11.0, 10.7 days, respectively), and smaller degradation rate constant (k = 0.0603, 0.0631, 0.0646, 0.0709, 0.0703, respectively) compared with control (t1/2 = 6.8 days, k = 0.1158). Based on above results, it could be concluded that flavonoid C-glycoside orientin, vitexin and rutin (positive control) played an equally important role in enhancing the stability of anthocyanins. Furthermore, FCGE showed a strongest positive effect on anthocyanins stability. 3.5. Possible mechanism for copigmentation reaction The color and anthocyanins of juice were excellently stabilized by copigment FCGE, vitexin and orientin, so it was necessary to study their possible mechanism for the copigmentation reaction. FCGE mainly contained vitexin and orientin, thus the study on vitexin and orientin could sufficiently illustrate the mechanism for copigmentation reaction in the FCGE-copigmented juice. According to the theory proposed by Brouillard et al. (1989), the possible mechanism for the copigmentation reaction was as follows: K

AH+ + nCPAH(CP)n + where AH+ was flavylium cation, CP was copigment. The copigmentation reaction was established by the equation given below: ln

A − A  0

A0

Fig. 4. The graph of ln[CP]0 against ln[(A − A0 )/A0 ] of vitexin, orientin and rutin in copigmentation reaction model.

3.6. Total phenolics and radical scavenging activity of juice = ln(Kr1 ) + n ln [CP]0

where A and A0 were the absorbance of the maximum absorption with and without copigment, respectively; [CP]0 was the concentration of copigment; r1 represented the ratio of the molar absorption coefficient between the complex and the flavylium cation; K was the complexation constant, which represented the strength of the association between the copigment and the flavylium form of anthocyanins. This mathematical model above was used for our data analysis. The graph of ln[CP]0 against ln[(A − A0 )/A0 ] in model gave a straight line with slope n, which meant the number of copigment molecules that formed a complex with one flavylium cation. The results for vitexin, orientin and rutin were shown in Fig. 4. Good linear fits for these copigments were obtained (R2 ≥ 0.95). The slope of graph of vitexin, orientin and rutin was 0.93, 0.99 and 0.99, respectively, which meant that the complex formed between studied copigment and anthocyanins with an approximate ratio of 1:1. According to Asen et al. (1972) and Chen and Hrazdina (1981), the main mechanistic driving forces for complex formation are hydrogen bonding and hydrophobic interactions. This causes an overlapping arrangement of the two molecules, preventing nucleophilic attack of water on the anthocyanins molecule.

The total phenolics contents of juice samples with and without copigment were presented in Fig. 5. The results showed that the addition of copigments produced the increase in the total phenolics content. The highest and the lowest total phenolics contents

Fig. 5. The change of total phenolics content of juice samples with and without copigments during storage. C: control; VI: vitexin; OR: orientin; RU: rutin; FCGE: flavonoid C-glycoside extracts from pigeon pea leaves.

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were found in the juice sample with FCGE (1:1) and control, respectively. Furthermore, juice samples copigmented with FCGE and its main components vitexin and orientin always showed higher total phenolics content compared with control during storage. In conclusion, the addition of copigments, especially FCGE, could enhance the nutritional value of juice. The free radical scavenging activities of juice samples were assayed in the DPPH system. Table 2 illustrated the DPPH radical scavenging capacities of juice samples ranged from 44.85 to 75.42 ␮g GAE/mL. The highest and lowest antioxidant capacities were found in the juice sample with orientin and control, respectively. Obviously, the addition of copigments increased the radical scavenging activity of juice (Markovic et al., 2003a, 2003b). In summary, the addition of copigments increased the total phenolics content and the radical scavenging activity of juice, especially FCGE. FCGE proved to be superior in improving the juice quality, and had an excellent application potential. 4. Conclusions The color density of blueberry juice anthocyanins could be enhanced through intermolecular copigmentation using copigment vitexin, orientin and FCGE. The complexes formed between anthocyanins and copigments resulted in an enhancement of absorbance at maximum wavelength, and gave saturated and more stable color on blueberry juice anthocyanins. In addition, the stability of anthocyanins was significantly enhanced through copigmentation of anthocyanins with vitexin, orientin and FCGE. Among copigments studied in this study, FCGE showed a strongest positive effect on stabilizing color and anthocyanins of blueberry juice. Therefore, FCGE as a natural, available and inexpensive resource has great potential applications as copigment in food industry. Acknowledgements The authors gratefully acknowledge the financial supports by the Program for Importation of International Advanced Forestry Science and Technology, National Forestry Bureau (2012-4-06), Agricultural Science and Technology Achievements Transformation Fund Program (2012GB23600641), Special Fund of National Natural Science Foundation of China (31270618) and Project for Distinguished Teacher Abroad, Chinese Ministry of Education (MS2010DBLY031). References Asen, S., Stewart, R.N., Norris, K.H., 1972. Copigmentation of anthocyanins in plant tissues and its effect on color. Phytochemistry 11, 1139–1144. ´ J., 2003. The effects of heating UV Bacakowska, A., Kucharska, A.Z., Oszmianski, irradiation, and storage on stability of the anthocyanin–polyphenol copigment complex. Food Chem. 81, 349–355. ´ N.A., Dimitric-Markovi ´ ´ J.M., 1997a. Spectrophotometric Baranac, J.M., Petranovic, c, study of anthocyanin copigmentation reactions. 2. Malvin and the nonglycosidized flavone quercetin. J. Agric. Food Chem. 45, 1694–1697. ´ N.A., Dimitric-Markovi ´ ´ J.M., 1997b. Spectrophotometric Baranac, J.M., Petranovic, c, study of anthocyanin copigmentation reactions. 3. Malvin and the nonglycosidized flavone morin. J. Agric. Food Chem. 45, 1698–1700. ´ N.A., Dimitric-Markovi ´ ´ J.M., 1996. Spectrophotometric Baranac, J.M., Petranovic, c, study of anthocyanin copigmentation reactions. J. Agric. Food Chem. 44, 1333–1336. Barba, F.J., Esteve, M.J., Frigola, A., 2013. Physicochemical and nutritional characteristics of blueberry juice after high pressure processing. Food Res. Int. 50, 545–549. Bloor, S.J., Falshaw, R., 2000. Covalently linked anthocyanin–flavonol pigments from blue Agapanthus flowers. Phytochemistry 53, 575–579. Boulton, R., 2001. The copigmentation of anthocyanins and its role in the color of red wine: a critical review. Am. J. Enol. Viticult. 52, 67–87. Breness, C.H., Pozo-Insfran, D.D., Talcott, S.T., 2005. Stability of copigmented anthocyanins and ascorbic acid in a grape juice model system. J. Agric. Food Chem. 53, 49–56.

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