Optimization of microwave-assisted extraction of anthocyanins from purple corn (Zea mays L.) cob and identification with HPLC–MS

Innovative Food Science and Emerging Technologies 11 (2010) 470–476

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Innovative Food Science and Emerging Technologies j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i f s e t

Optimization of microwave-assisted extraction of anthocyanins from purple corn (Zea mays L.) cob and identification with HPLC–MS Zhendong Yang, Weiwei Zhai ⁎ Food engineering department of Jiangsu food science college, Huaian, Jiangsu 223003, China

a r t i c l e

i n f o

Article history: Received 13 August 2009 Accepted 9 March 2010 Editor Proof Receive Date 15 April 2010 Keywords: Purple corn cob Anthocyanins Optimization Extraction Microwave HPLC–MS

a b s t r a c t A Box–Behnken design was used to obtain the optimal conditions of microwave-assisted extraction (MAE). The effects of operating conditions, such as extraction time, solid–liquid ratio, and microwave irradiation power, on the extraction yield of anthocyanins were studied through a response surface methodology (RSM). The highest total anthocyainin content (TAC) from purple corn cob (185.1 mg/100 g) was obtained at an extraction time of 19 min, a solid–liquid ratio of 1:20, and a microwave irradiation power of 555 W. Six major kinds of anthocyanins were detected and identified as cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside, and their respective malonated counterparts. In comparison with the conventional solvent extraction, MAE was highly efficient and rapid in extracting anthocyanins from Chinese purple corn cob. Industrial relevance: In the last decades, the interest in anthocyanin pigments has increased because of their possible utilization as natural food colorants and especially as antioxidant and anti-inflammatory agents. Purple corn cob was the byproduct during the corn processing. Purple corn cob is dark purple to almost black color due to its high content of anthocyanins, which makes this byproduct a good source of anthocyanins. © 2010 Published by Elsevier Ltd

1. Introduction Anthocyanins, widely found in the roots, caudexes, leaves, as well as flowers and fruits of tall plants, are used as substitutes for synthetic pigments because of their attractive color and physiological functionality (Mazza & Miniati, 1993). Anthocyanins also possess known pharmacological properties and are used by humans for therapeutic purposes (Francis, 1989). An increasing number of studies have demonstrated that anthocyanins have the ability to protect against a myriad of human diseases, such as liver dysfunction, hypertension, vision disorders, microbial infections, and diarrhea (Mazza & Kay, 2008; Smith, Marley, Seigler, Singletary, & Meline, 2000). Purple corn is a pigmented variety of Zea mays L., originally cultivated in the Andes region of South America. It was introduced to China a long time ago. This corn variety is now mainly grown in China, especially in the provinces of Shanxi and Anhui. Purple corn is an important source of anthocyanins, which have potential applications as natural food colorants and antioxidants. Such applications are widely used in Asia, South America, and Europe (Aoki, Kuze, & Kato, 2002; Jing & Giusti, 2005). The anthocyanins present in Andean purple corn, flowers, leaves, cobs, and seeds have previously been characterized, and the major anthocyanins found were cyanidin-3-dimalonylglucoside, cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside, and their respective malonated counterparts (Aoki et al., 2002; Fossen, Slimestad, &

⁎ Corresponding author. Tel./fax: +86 517 87088869. E-mail address: [email protected] (W. Zhai). 1466-8564/$ – see front matter © 2010 Published by Elsevier Ltd doi:10.1016/j.ifset.2010.03.003

Andersen, 2001; Harborne & Self, 1997; Pascual-Teresa, Santos-Buelga, & Rivas-Gonzalo, 2002). Although purple corn cob can obviously be used as a natural colorant, existing literature and recent reports have focused only on analyzing the stability and structure of anthocyanins found in Peruvian purple corn varieties or their derivatives. Usually, conventional solvent extraction of anthocyanins is time and solvent-consuming and has low efficiency (Sun, Liao, Wang, Hu, & Chen, 2007). Moreover, thermal extraction over a long time can cause the degradation of anthocyanins and decrease the antioxidant activity of the extracts (Camel, 2000; Lapornik, Prošek, & Wondra, 2005). Anthocyanins also may also undergo denaturalization when they are extracted from a natural source. The extraction process involves a loss of color followed by the formation of brownish degradation products and insoluble compounds (Castillo-Saánchez, Mejuto, Garrido, & García-Falcón, 2006). Microwave-assisted extraction (MAE) utilizes the energy of microwaves to cause molecular movement and rotation of liquids with a permanent dipole, leading to rapid heating of the solvent and the sample. It has advantages over conventional extraction techniques, such as improved efficiency, reduced extraction time, low solvent consumption, and high level of automation (Buldini et al., 2002; Eskilsson & Bjorklund, 2000). Aside from these advantages, a wider range of solvents can be used in MAE, as the technique is less dependent on high solvent affinity (Eskilsson & Bjorklund, 2000). Recently, many reports have been made on the application of MAE on the extraction of natural products, such as artemisinin (Hao, Han, Huang, Xue, & Deng, 2002), ginseng saponins (Kwon, Bélanger, & Paré, 2003), essential oils (Gomez & Witte, 2001), and anthocyanins (Sun et al., 2007). However, no information has yet

Z. Yang, W. Zhai / Innovative Food Science and Emerging Technologies 11 (2010) 470–476

been found on the application of MAE for the extraction of anthocyanin in purple corn cob. Response surface methodology (RSM) is an affective statistical technique for optimizing complex processes. It is widely used in optimizing the process variables. Upon reviewing the basic theoretical and fundamental aspects of RSM, color was found to be one of the most important attribute of natural colorants (Chandrika & Fereidoon, 2005; Farooq, Imran, & Khaled, 1997). The application of colorimetric systems, based on uniform color spaces (CIELUV and CIELAB) and non-uniform color spaces (CIEXYZ), is of great value in the quantification and characterization of the color properties of pigments and foods. The correlation between some color parameters and pigment content in food has been evaluated in many studies (Lee, 2001; MeléndezMartínez, Vicario, & Heredia, 2003; Montes, Vicario, Raymundo, Fett, & Heredia, 2005). In this paper, MAE parameters, such as the ratio of solvents to materials, microwave irradiation power, and extraction time, were optimized by RSM in order to obtain the optimal extraction yield of anthocyanin extracts from purple corn cob. In addition, the anthocyanins composition of the extracts was determined by HPLC–MS, and the effect of MAE on the extraction efficiency was evaluated in comparison to the conventional solvent extraction method. 2. Materials and methods 2.1. Material Purple corn (Z. mays L. cv Heizhenzhu) cob was generously supplied by Prof. Zhong Zhang of the Anhui Technical Teachers College (Fengyang City, Anhui Province, China). The cobs were dried in a heated air dryer (50 °C) (ZT-3, Jiangdu City, Jiangsu Province, China), pulverized by a disintegrator (FSD-100A, Taizhou City, Zhejiang Province, China), and sifted through a 100-mesh sieve. The purple corn cob powder (water content b12%) was sealed in a brown bottle and kept at 4 °C.

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(Table 1). The complete design consisted of 17 combinations, including five replicates of the center point (Table 2), and the response function (Y) was partitioned into linear, quadratic, and interactive components, k

k

i=1

i=1

2

k

Y = β0 + ∑ Bi Xi + ∑ Bii Xi + ∑ Bij Xi Xj

ð1Þ

i N j

where Y stands for total anthocyanin yield; β0 denotes the model intercept; Bi, Bii, and Bij represent the coefficients of the linear, quadratic, and interactive effects, respectively; Xi, and Xj are the coded independent variables; and k equals the number of tested factors (k = 3). Tables of the analysis of variance (ANOVA) were generated, and the effect and regression coefficients of individual linear, quadratic, and interactive terms were determined. The significances of all terms in the polynomial were evaluated statistically by computing the F-value at the probability (p) of 0.001, 0.01, or 0.05. The regression coefficients were then used to make statistical calculations to generate contour maps from the regression models. 2.4. Determination of the total anthocyanin content (TAC) The TAC was determined according to the pH-differential methods (Lee, Durst, & Wrosltad, 2005). An aliquot of the sample (1 mg) was placed into a 25 mL volumetric flask and made up to the final volume with pH 1.0 buffer (0.025 M potassium chloride). Another 1 mg of the sample was placed into a 25 mL volumetric flask and made up to a final volume with pH 4.5 buffer (0.4 M sodium acetate). Absorbance was measured by a spectrophotometer (UV-2802, UNICO) at 510 and 700 nm. Absorbance was calculated as Abs=[(A510 −A700) at pH 1.0]−[(A510 −A700) at pH 4.5] with a molar extinction coefficient of 26,900 for cyanindin-3glucoside. The TAC was calculated using Eq. (2) and expressed as milligrams of cyanindin-3-glucoside equivalents per 100 g dry cods. All TCA shown was normalized with the amount of cyanindin-3-glucoside. TAC ðmg=100gÞ ¼

AB V × MW × D × × 100 eL G

ð2Þ

2.2. Extraction of purple corn cob anthocyanin The MAE procedure used in the experiment was a modified version of the method developed by Sun et al. (2007). Briefly, the powders of purple corn cob were put into a 50 mL double-necked flask with a cooling system. After the flask was filled with a known volume of solvent, which was selected according to the designed solvent-tosample ratio [1.5 M HCl–95% ethanol (15:85)], it was exposed in a microwave extractor (Model NJL07-3, Jiequan microwave equipment Co., Ltd., Nanjing, China) for a certain period of time under a given microwave power. The flask was taken out. The solution was cooled with water under room temperature, filtered through Whatman No. 1 paper under vacuum, and then collected in a volumetric flask. The residue was taken back and extracted again under the same conditions. The anthocyanin extracts from the two extraction steps were mixed and used for the determination of the total anthocyanin content (TAC). About 5 g of the samples was used for each treatment. 2.3. Experimental design RSM was used to determine the optimum conditions for extraction of anthocyanin in purple corn cob. The experimental design and statistical analysis were done using Stat-Ease software (Design-Expert 6.0.10 Trial, Delaware, USA Echip, 1993). A three-level three-factor Box– Behnken design was chosen to evaluate the combined effect of three independent variables, extraction time, solid–liquid ratio, and microwave irradiation power. There were termed as X1, X2 and X3, respectively. The minimum and maximum values for extraction time were set at 10 and 30 min. The solid–liquid ratio was set at 1:10 and 1:30. The microwave irradiation power was set between 400 and 600 W

where Abs is absorbance, e is the cyanindin-3-glucoside molar absorbance (26,900), L is the cell path length (1 cm), MW is the molecular weight of anthocyanin (449.2 Da), D is the dilution factor, V is the final volume (mL), and M is the dry cob weight (mg). 2.5. Color coordinates Tristimulus parameters (L*, C *, and h°) were calculated using CRS4w software (CR400, Konica Minolta, Japan), based on the CIELAB color space. A standard illuminator D65 was used at a Δλ of 5 nm (Fan, Han, Gu, & Gu, 2008). 2.6. Conventional extraction of anthocyanins from purple corn cob The conventional extraction procedure used in this study was a modified version of the process described by Fuleki and Francis (1968). A total of 1 g of samples was mixed with 30 mL of 1.5 N HCl–95% ethanol (15:85) in a 50 mL round bottom flask fitted with a cooling system. The extraction temperature was set at 55 ± 1 °C, equivalent to the mean temperature under optimized conditions determined through the MAE Table 1 Independents variables and their coded and actual values used for optimization. Independents variables

Extraction time Solid–liquid ratio Microwave irradiation power

Units

min 1: X W

Symbol

X1 X2 X3

Code levels −1

0

1

10 10 400

20 20 500

30 30 600

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Table 2 Experimental data and the observed response value with different combinations of extraction time (A), solid–solvent ration (B) and microwave irradiation power (C) used in the Box–Behnken design. No

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Independent variables

Dependent variables

X1 (min)

X2 (1:X)

X3 (W)

TAC (mg/100 g)

L*

C*

h°

10 30 10 30 10 30 10 30 20 20 20 20 20 20 20 20 20

10 10 30 30 20 20 20 20 10 30 10 30 20 20 20 20 20

500 (0) 500 (0) 500 (0) 500 (0) 400 (− 1) 400 (− 1) 600 (1) 600 (1) 400 (− 1) 400 (−1) 600 (1) 600 (1) 500 (0) 500 (0) 500 (0) 500 (0) 500 (0)

129.9 ± 5.5 129.8 ± 3.5 133.1 ± 6.4 110.8 ± 7.7 143.4 ± 3.1 125.1 ± 4.4 148.4 ± 5.8 155.5 ± 7.7 132.6 ± 2.7 125.6 ± 8.9 153.2 ± 5.4 149.6 ± 3.1 182.3 ± 5.6 183.3 ± 4.8 180.8 ± 2.7 183.5 ± 5.4 184.6 ± 3.7

32.11 ± 0.08 32.47 ± 0.10 32.82 ± 0.09 19.94 ± 0.07 31.28 ± 0.14 33.10 ± 0.04 30.36 ± 0.11 30.54 ± 0.08 30.47 ± 0.06 32.31 ± 0.04 30.11 ± 0.07 30.86 ± 0.09 29.32 ± 0.07 29.49 ± 0.06 29.31 ± 0.15 29.35 ± 0.07 29.31 ± 0.01

30.24 ± 0.05 30.34 ± 0.07 30.02 ± 0.07 4.62 ± 0.03 31.14 ± 0.11 30.73 ± 0.09 31.77 ± 0.07 31.17 ± 0.05 32.54 ± 0.16 32.35 ± 0.18 32.37 ± 0.07 31.37 ± 0.01 32.85 ± 0.09 32.98 ± 0.15 32.58 ± 0.03 32.99 ± 0.04 32.85 ± 0.02

10.93 ± 0.12 12.98 ± 0.07 9.53 ± 0.13 12.02 ± 0.03 10.60 ± 0.09 10.27 ± 0.01 8.87 ± 0.18 12.72 ± 0.07 12.03 ± 0.06 12.92 ± 0.05 12.09 ± 0.09 12.49 ± 0.14 13.92 ± 0.07 13.85 ± 0.08 13.24 ± 0.11 14.03 ± 0.08 13.30 ± 0.02

(− 1) (1) (− 1) (1) (− 1) (1) (−1) (1) (0) (0) (0) (0) (0) (0) (0) (0) (0)

(− 1) (− 1) (1) (1) (0) (0) (0) (0) (− 1) (1) (− 1) (1) (0) (0) (0) (0) (0)

process. Extractions were carried out for 30, 60, 90, and 120 min. The anthocyanin extracts were cooled to room temperature, filtered through Whatman No. 1 paper in a vacuum, and collected in a volumetric flask. The residue was gathered and extracted again under the same conditions. The anthocyanin extracts of the two extractions were combined and used to determine the TAC.

with solvent A at 95–80% solvent A from 0 to 20 min and, at 80–60% from 20 to 50 min. The flow rate was 0.8 mL/min. The identity of anthocyanins was checked confirmed by using electrospray mass spectrometry (MS) with a Waters Platform ZMD 4000 mass spectrometer equipped with an ion spray interface (ISV = 4400) operated in the positive-ion mode. Spectra were recorded in positive ion mode between m/z 200 and 1200.

2.7. Purification of anthocyanin from purple corn cob extracts Anthocyanin extracts of purple corn were purified according to the procedure described by Sun et al. (2007). Amberlite CG-50 resins (50 g each time) were hydrated by placing in a beaker with water and performing repeated decantation. The fine particles were removed with distilled water. An aqueous slurry of the hydrated resin was poured into a 26 × 400 mm column, and the excess water was allowed to drain out without letting the column dry. Approximately 15 mL of the aqueous extract was poured on the top of the column until the entire resin bed became red due to the absorbed anthocyanin. Anthocyanins were absorbed onto the resin column, while sugars, acids, and other water-soluble compounds were removed by washing the column with 100 mL distilled water. This was confirmed by the refractive index of the liquid coming out of the column. The pigments were eluted by adding ethanol containing 0.01% HCl (approximately 50 mL) until the resin returned to its original color. The eluate was concentrated with a rotary evaporator at 45 °C in a vacuum until the ethanol evaporated, and the residue was dissolved in 0.5% HCl solvent. The solution was stored at −20 °C until further analysis. 2.8. Identification of anthocyanin from purple corn cob extracts by HPLC–MS Identification was performed by hA High-pPerformance lLiquid cChromatography (HPLC), Waters 2690) and with a Waters 2996 photodiode array detector (Waters 2996) were used. Data analysis was performed with Waters HPLC chem-station software. Solvents and samples were filtered through a 0.45 μm filter. Chromatographic analysis was carried outdone using a Lichrospher C-18 (5 μm 2.1 mm × 250 mm i.d.) prodigy. Simultaneous monitoring was performed at 520 nm at a flow rate of 0.8 mL/min. The running temperature was at 35 °C, and the injection volume was 10 μL. The mobile phase Phase A was a mixture of 0.05% (v/v) trifluoroacetic acid (TFA) in distilled water, whereas Mobile B consisted of 100% HPLCgrade acetonitrile. The separation of anthocyanins was carried outdone for 40 min. The elution profile was a linear gradient elution

2.9. Statistical analysis All trials were carried out in triplicate and all the data were reported as mean ± standard deviation (n = 3). The statistical significance was established using the Students's t-test at p b 0.05. 3. Results and discussion 3.1. Statistical analysis The results of each dependent variable with their coefficients of determination (R2) are summarized in Table 3. Statistical analysis indicated that the proposed model was adequate, possessing no significant lack of fit and with satisfactory values of the R2 for the TAC and h°. The R2 values for the TAC, L*, C*, and h° were 0.996, 0.958, 0.942, and 0.936, respectively. The coefficients of variances (Table 3) for the TAC, L*, C*, and h° were 1.55, 1.40, 1.20, and 4.93, respectively. In general, a high coefficient of variances indicates that variation in the mean value is high and does not satisfactorily develop an adequate response model (Ravikumar, Ramalingam, Krishnan, & Balu, 2006). The probability (p) values of all the regression models were less than 0.05. According to the model (Table 3), the linear terms of extraction time (X1, p b 0.01), solid–liquid ratio (X2, p b 0.01), microwave irradiation power (X3, p b 0.001), the quadratic terms of extraction time (X21, p b 0.01), solid–liquid ratio (X22, p b 0.01), microwave irradiation power (X23, p b 0.001) reached statistical significance. The interaction of extraction time and solid–liquid ratio (X1X2, p b 0.01), and the extraction time and microwave irradiation power (X1X3, p b 0.001) also reached statistical significance. The results suggested that the change in the above three factors had a significant effect on the TAC in the extracts. In contrast, the interaction between the solid– liquid ratio and the microwave irradiation power (X2X3) was not statistically significant. Meanwhile, the whole model, including the linear level (p b 0.01), quadratic level (p b 0.001), and cross-product level (p b 0.01), all reached statistical significance (p b 0.01), but the

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Table 3 Regression coefficients, R2, and CV values for four dependents variables for the extraction of purple corn cob. Coefficient

TAC (mg/100 g)

β0 (intercept)

182.90

Liner X1 X2 X3

− 4.20⁎⁎ − 3.30⁎⁎ 10.00⁎⁎⁎

Quadratic X11 X22 X33 Cross product X12 X13 X23 R2 CV Probability (p) Lack of fit

L*

C* 32.85

13.67

0.38⁎ 0.41⁎ − 0.68⁎⁎

−0.22 −0.11 − 0.01

1.01⁎⁎ − 0.13 0.04

−1.72⁎⁎⁎ −0.76⁎⁎ 0.07

− 2.04⁎⁎ − 0.27 − 1.02⁎⁎

−0.24 − 0.05 − 0.20 0.942 1.20 0.0015⁎⁎ 0.0209⁎

0.11 1.05⁎⁎ − 0.12 0.936 4.93 0.0021⁎⁎ 0.0833

−27.08⁎⁎⁎ −29.93⁎⁎⁎ −12.72⁎⁎⁎

1.77⁎⁎⁎ 1.31⁎⁎⁎ 0.25

− 5.55⁎⁎ 6.35⁎⁎⁎

0.03 − 0.46 −0.30 0.958 1.40 0.0005⁎⁎⁎ 0.0006⁎⁎⁎

0.85 0.996 1.55 b 0.0001⁎⁎⁎ 0.0811

h°

29.36

⁎ Significant at 0.05 level. ⁎⁎ Significant at 0.01 level. ⁎⁎⁎ Significant at 0.001 level.

lack of fit was not significant, indicating excellent agreement of the experiment values with the predicted values. 3.2. Analysis of Box–Behnken experiment When one factor was fixed as the optimal value calculated from the Box–Behnken experiment, the effects of both factors on the extraction of anthocyanins were shown by the contour optimizer plots. Contour plots for the TAC are shown in Figs. 1–3. The effects of extraction time (X1) and solid–liquid ratio (X2) on the TAC of the extracts are reflected in Fig. 1. With an increase of X1 and X2, the TAC sharply increased, achieving a saturated value when the extraction was conducted for 20 min at 1:20. It remained at this value despite further increases in X1 and X2. The relationship between the TAC and extraction time (X1) as well as the microwave irradiation power (X3) is illustrated in Fig. 2. The factor X3 is as significant as X1 in relation to the TAC. Therefore, we can conclude that the microwave irradiation power and extraction time is important for attaining a highly efficient MAE extraction of anthocyanin. This result is in agreement with a study done by Eskilsson and Bjorklund (2000) on analytical-scale MAE. Due to the acceleration of the disruption of tissues and the immigration of solutes from tissues under microwave power, extraction became more efficient. The effects of the solid– liquid ratio (X2) and microwave irradiation power (X3) on the TAC in the extracts are shown in Fig. 3. When the microwave irradiation power is about 550 W, X2 becomes the critical factor for improving the TAC. The fluctuation of X2 can lead to a large difference in the TAC. The optimal X2 and X3 for anthocyanin extraction were about 1:20 and 550 W. If the X 3 was lower than 550 W, the dissolution of anthocyanins did not reach an equilibrium and part of the anthocyanins remained in purple corn cob. If the X3 was higher than 550 W, the TAC in the extracts slightly decreased. The contour plots show the optimum conditions of the extraction process for anthocyanins. There are a number of combinations of variables that can give maximum levels of the TAC. During the anthocyanin extraction, the extraction time, solid–liquid ratio, and microwave irradiation power were the most important process variables needed to optimize the response in this study. The best combination of process variables for the best set of response properties included an extraction time of 19 min, a solid–liquid ratio

Fig. 1. Contour plots for the effects of extraction time and solid–liquid ratio at a constants microwave irradiation power of 500 W on the TAC from purple corn cob.

of 1:20, and a microwave irradiation power of 555 W. The responses calculated from the final polynomial functions were 185.1 mg/100 g for the TAC, 29.05 for L*, 32.88 for C*, and 13.34 for h°, respectively. In this paper, the purple corn (Z. mays L. cv Heizhenzhu) cob contained higher amounts of anthocyanins than pigmented corns from the United States (30.7 mg/100 g), Mexico (72.1 mg/100 g), and Canada (127.7 mg/100 g) (Dep Pozo-Insfran, Brenes, Serna Saldivar, & Talcott, 2006). Thus, the variety and growth conditions of purple corn could influence their anthocyanin content. To prove this, Moreno and co-workers (Moreno, Sánchez, Hernáadez, & Lobato, 2005) found that the TAC of four Mexican blue corn varieties ranged from 54 to 115 mg/ 100 g. In the present study, the anthocyanin yield in the purple corn cob was relatively high, which made this material a good source of anthocyanin.

Fig. 2. Contour plots for the effects of extraction time and microwave irradiation power at a constants solid–liquid ratio of 1:20 on the TAC from purple corn cob.

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Z. Yang, W. Zhai / Innovative Food Science and Emerging Technologies 11 (2010) 470–476 Table 5 The comparison of the extraction efficiency by MAE method to conventional solvent extraction. MAEa

Conventional solvent extractionb 19

30

40

60

TAC (mg/100 g) 184.8 ± 4.2 125.3 ± 2.7 132.7 ± 4.4 148.8 ± 3.1 158.2 ± 3.8 a

The MAE was carried out at optimized conditions (19 min, 1:20, 555 W). The conventional solvent (1.5 N HC–95% ethanol, 15:85) extraction was carried out 55 °C, solid–solvent ration 1:30. b

Torre, Pereiro, & Torrijos, 2004; Zhou & Liu, 2006). Furthermore, the direct interaction of microwaves with solvent also results in the rupture of the plant cells and a quick release of intracellular products into the solvent (Lay-Keow & Michel, 2003) quickly. 3.5. Identification of anthocyanin from purple corn cob extracts by HPLC–MS analysis

Fig. 3. Contour plots for the effects of microwave irradiation power and solid–liquid ratio at a constants extraction time of 20 min on the TAC from purple corn cob.

3.3. Correlations between the TAC and the color parameters The correlations between the TAC and the color parameters were explored in this study (Table 4). A negative correlation (r = −0.899) was found with L*, indicating that higher L* values corresponded to lower TAC. In contrast, a positive correlation was found between the yield of anthocyanins and C* (r = 0.730). This means that high C* values were correlated to high TAC. These results were in agreement with a study done by Montes et al. (2005), who evaluated the correlations between the anthocyanin yield and C* and L* in Jaboticaba fruit. There are no correlations between the TAC and h°. 3.4. Comparison of MAE with conventional extraction of anthocyanins in purple corn cob Samples were extracted by MAE and conventional extraction, respectively, in order to evaluate the effects of MAE on the extraction efficiency. As shown in Table 5, the MAE method was more efficient than the conventional extraction. When purple corn cob samples were extracted for 10 min, the TAC in the extracts obtained by conventional method was only 67.8% that of MAE. Even when the conventional extraction was done for a longer time like 60 min, the TAC was just 85.6% that of MAE. The TAC did increase as the extraction time increased. Microwave energy is an electromagnetic source of radiation that causes molecular motion through the migration of ions and rotation of dipoles (Beejmohun et al., 2007). Conventional solvent extraction without microwave assistance is a time-consuming process that uses heat to increase the mass transfer rate of the extraction system (Proestos & Komaitis, 2008). In contrast, microwave-assisted extraction is a fast extraction process where microwave energy is delivered efficiently delivered to materials through molecular interaction with in the electromagnetic field. It offers a rapid transfer of energy to the extraction solvent and raw plant materials (Criado,

Gradient reversed-phase HPLC with UV detection and MS analysis were used to rapidly identify anthocyanins in purple corn cob extracts. The HPLC profile is shown in Fig. 4, and the mass spectral fragmentation patterns are shown in Fig. 5. If the standard anthocyanins were unavailable, identification was performed by comparing their retention time and molecular weight with data from literature (Pascual-Teresa et al., 2002; Pedreschi & Cisneros-Zevallos, 2007). Six anthocyanins were identified from the purple corn cob extracts (Fig. 5): Peak 1 was cyanidin-3-glucoside with a molecular ion peak [M + H]+ at m/z 449 and a fragment ion peak [M + H-162]+ at m/z 287. This corresponded to the loss of a glucoside residue from cyanidin-3glucoside, yielding cyanidin. Peak 2, with [M]+ at m/z 433, was identified as pelargonidin-3-glucoside. The fragment ion peak [M-162]+ at m/z 271 corresponded to a loss of glucoside from pelargonidin-3-glucoside, yielding pelargonidin. Peak 3, with [M]+ at m/z 463, is identified as peonidin-3-glucoside. The fragment ion peak at m/z 301, indicating a loss of 162 from peonidin-3-glucoside, was identified as peonidin. Peak 4, with [M]+ at m/z 535, was identified as cyanidin-3-(6-malon)-glucoside. The fragment ion peak [M-162–86]+ at m/z 287 was referred to as cyanidin, from a loss of glucoside (162) and a malonic acid (86) from cyanidin-3(6-malon)-glucoside. Peak 5, with [M]+ at m/z 519, was identified as pelargonidin-3-(6-malon)-glucoside. The fragment ion peak at m/z 433, indicating a loss of 162 from pelargonidin-3-glucoside, was referred to as pelarginidin. Peak 6, with [M]+ at m/z 549, was identified as peonidin-3-(6-malon)-glucoside. The fragment ion peaks at m/z 463 and 301 referred to peonidin-3-glucoside and peonidin, respectively. In this study, the anthocyanins in the purple corn (Z. mays L. cv. Heizhenzhu) cob extracts showed little differences with those reported by Pascual-Teresa et al. (2002), such as cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside, and their respective

Table 4 Correlation coefficients for relations between TAC and color parameters. Index

L*

C*

h°

TAC

0.899

0.730

0.489

Fig. 4. HPLC chromatogram at 520 nm corresponding to purple corn cob extract.

Z. Yang, W. Zhai / Innovative Food Science and Emerging Technologies 11 (2010) 470–476

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malonated derivatives in the purple corn (Z. mays L. cv. Heizhenzhu) cob extract. Cyaniding-3-6-ethylmalonylglucoside, cyaniding-3-6-ethylmalonylglucoside, and cyaniding-3-6-ethylmalonylglucoside were not found. The difference may be attributed to different variants and growing conditions (Ferreyra, Vina, Mygridge, & Chaves, 2007; Fossen et al., 2001).

Fig. 5 (continued).

4. Conclusions In this study, process variables, such as extraction time, solid– liquid ratio, and microwave irradiation power, significantly affected the anthocyanin yield. When MAE was conducted at an extraction time of 19 min, a solid–liquid ratio of 1:20, and a microwave irradiation power of 555 W, the TAC in the extract reached 185.1 mg/100 g (98.85%). Six kinds of anthocyanins were identified using HPLC–MS analysis: cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside, and their respective malonated counterparts. In this study, the anthocyanins identified in the purple corn cob extract showed a small difference with those reported by PascualTeresa et al. (2002). References

Fig. 5. Positive ion mass spectra or anthocyanin constituents in purple corn cob extract: (peak 1) cyanidin-3-glucoside; (peak 2) pelargonidin-3-glucoside; (peak 3) penodin3-glucoside; (peak 4) cyanidin-3-(6-malonylglucoside); (peak 5) pelargonidin -3-(6malonylglucoside); (peak 6) penodin-3-(6-malonylglucoside). Glu = glucoside; Mal = malonic acid.

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