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. 2020 Jun 19;5(25):15702–15708. doi: 10.1021/acsomega.0c02099

Timing and Pattern of Anthocyanin Accumulation during Grain Filling in Purple Waxy Corn (Zea mays L.) Suggest Optimal Harvest Dates

Jung-Tae Kim †,*, Gibum Yi †, Ill-Min Chung ‡, Beom-Young Son †, Hwan-Hee Bae †, Young Sam Go †, Jun Young Ha †, Seong-Bum Baek †, Sun-Lim Kim †
PMCID: PMC7331206  PMID: 32637845

Abstract

graphic file with name ao0c02099_0005.jpg

Purple-corn kernels contain anthocyanins, a group of antioxidants proposed to be beneficial to human health. This study investigated the concentrations of anthocyanins and amino acids and the composition of fatty acids in the kernels of purple waxy corn (Zea mays L.) “Heukjinjuchal” during grain filling to determine when the grain nutritional value is at its highest. During grain filling, anthocyanin contents increased as the kernel color darkened. Among the anthocyanins measured, cyanidin-3-β-O-glucoside reached the highest contents, 57.0–409.1 mg kg–1 fresh weight in raw kernels and 1027.6 mg kg–1 in dry seeds. Pelargonidin-3-β-O-glucoside and malvidin-3-β-O-glucoside became detectable at 21 days after silking; they occurred in the second- and third-highest amounts, respectively, among anthocyanins in the purple-corn cultivars tested. The anthocyanin accumulation pattern was strongly associated with physicochemical properties and partly associated with amino acid content. Anthocyanin contents increased in a stepwise rather than linear fashion. This study showed that kernels undergo dramatic changes that affect the nutritional value of fresh corn.

1. Introduction

Waxy corn (Zea mays L.) is increasingly consumed as a fresh vegetable in Asian countries1,2 and purple waxy corn is preferred by consumers due to the health-promoting properties of the anthocyanin pigments in the aleurone or pericarp. In purple corn, anthocyanins are reported to have antimutagenic and anticancer properties as well as antidiabetes and antiobesity activities and can scavenge free radicals.3−6

Anthocyanins, a large and diverse group of water-soluble flavonoid pigments, are glycosylated polyhydroxy or polymethoxy derivatives of 2-phenylbenzopyrilium, which contains 15 carbons in a C6–C3–C6 arrangement, with two aromatic rings connected by a three-carbon bridge. Hundreds of anthocyanins have been reported,7−9 and naturally occurring anthocyanins have various colors depending on the pH, temperature, and light intensity.10,11 Strack and Wray12 classified naturally occurring anthocyanins according to five functional groups: common basic structures, common methylated structures, 3-deoxy structures, rarely hydroxylated structures, and rarely methylated structures. Anthocyanins are biosynthesized from malonyl CoA and p-coumaroyl CoA by chalcone synthase. Malonyl CoA is derived from the fatty acid biosynthesis pathway and p-coumaroly CoA from phenylalanine. The analysis of anthocyanins is complex due to their capacity to undergo structural transformation and multiplex reactions.13

Many reports have profiled the anthocyanins of various corn cultivars and landraces. For example, Abdel-Aal et al.14 and Aoki et al.15 reported that purple corn contains the anthocyanins cyanidin-3-O-β-d-glucoside, pelargonidin-3-O-β-d-glucoside, peonidin-3-O-β-d-glucoside, cyanidin-3-O-β-d-(g-malonyl-glucoside), pelargonidin-3-O-β-d-(g-malonyl-glucoside), and peonidin-3-O-β-d-(g-malonyl-glucoside). However, the daily changes in anthocyanin profiles during seed maturation have not been previously reported. Furthermore, the relationships among anthocyanin accumulation, their precursor amino acids, and fatty acid have not yet been reported in purple waxy corn. Here, we evaluated the concentrations and profiles of anthocyanins in the kernel of the purple waxy corn “Heukjinjuchal”, one of Korea’s leading cultivars, during grain filling. Our findings suggest that anthocyanin and amino acid contents should be considered during the production and breeding of waxy purple corn to help maximize nutritional value.

2. Results and Discussion

2.1. Profiling Anthocyanins in Corn Seeds

Of 11 anthocyanin standards, cyanidin-3-β-O-glucoside (C3G), pelargonidin-3-β-O-glucoside (P3G), and malvidin-3-β-O-glucoside (M3G), were well-separated and occurred at a higher content than others in the purple-corn cultivars, whereas no anthocyanin was detected in the two yellow-corn cultivars or in B73 (Figure 1). The three specific anthocyanins were further confirmed by mass spectrometry (Supporting Information Figure S1). The purple color of the three cultivars accumulated in the aleurone but not in the pericarp, when the pericarp was filled out from a kernel. The C3G content of Heukjinjuchal, “Suwon90”, and “Miheugchal” were 1027.6 ± 26.7, 338.0 ± 13.7, and 314.2 ± 29.8 mg kg–1, respectively, in the fully matured and dried kernels (Figure 1C). Heukjinjuchal had the highest anthocyanin content and was therefore selected for further anthocyanin profiling during grain filling.

Figure 1.

Figure 1

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Anthocyanin amounts in five waxy-corn cultivars and B73. The kernels were fully matured and dried. (A) Photographs of six corn cultivars used in high-performance liquid chromatography (HPLC) analysis. Bar = 1 cm. (B) HPLC chromatograms of the six cultivars. Three major anthocyanins, cyanidin-3-β-O-glucoside (C3G), pelargonidin-3-β-O-glucoside (P3G), and malvidin-3-β-O-glucoside (M3G), are shown. The six cultivars are labeled with different colors. The retention times for three anthocyanins are indicated in the parenthesis. Standard anthocyanin chromatogram is shown in yellow. (C) Amounts of three anthocyanins in three purple-colored cultivars.

2.2. Color Changes and Anthocyanin Content during Grain Filling

Colored kernels began to appear at 12 days after silking (DAS) at the middle of the cob. Pigmentation continuously increased up to 30 DAS, spreading to the proximal and distal ends of the cob (Figure 2A). As grain filling took place, the number and peak absorbance intensity also increased (Figure 2B). C3G was first detected in kernels at 16 DAS and was the most abundant anthocyanin across the developmental stages analyzed. P3G and M3G began to be detected at 21 DAS (Figure 2C). Anthocyanin content rose continuously during the grain-filling stages, and sharp increases were detected at 24 and 29 DAS. The C3G content ranged from 57.1 to 409.7 mg kg–1 fresh weight during grain filling. The P3G and M3G contents ranged from 48.4 to 135.9 and from 21.1 to 120.3 mg kg–1 of fresh weight, respectively. The graph shows that anthocyanin mass increases took place in a stepwise manner with linear accumulation (R2 > 0.89; Figure 2C). The results show that the anthocyanin accumulation in purple corn largely occurs during grain filling.

Figure 2.

Figure 2

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Anthocyanin accumulation in kernels of the purple-corn cultivar Heukjinjuchal during grain filling. (A) Representative corn cob pictures during grain filling. (B) Ultraperformance liquid chromatography chromatogram of the three major anthocyanins in kernels of purple-corn Heukjinjuchal. Cyanidin-3-β-O-glucoside (C3G), pelargonidin-3-β-O-glucoside (P3G), and malvidin-3-β-O-glucoside (M3G) are shown. (C) Changes in anthocyanin content during grain filling. Error bars represent standard deviations of three biological replicates.

Aoki et al.15 showed that cyanidin, pelargonidin, and their malonylated derivatives are present in purple-corn seeds. Another study found that C3G and P3G were the major anthocyanins, in red and pink colored corn, respectively.14 Salinas Moreno et al.16 found that cyanidin in colored corn contained important anthocyanins and that C3G is the major anthocyanin component of the purple-corn seeds. In addition, the present study showed that C3G is a major anthocyanin component during grain filling.

Lopez-Martinez et al.17 found that the total anthocyanin content of Mexican corn strains ranges from 15.4 to 8509.0 mg cyanidin-glucoside equivalents per kg whole-grain flour. Urias-Lugo et al.18 measured 646.0–1052.0 mg kg–1 C3G elite blue corn hybrid seeds. In Heukjinjuchal, C3G totaled 1027.6 mg kg–1 corn seed, which is high enough to have nutraceutical benefits.19 The C3G amount of dry seed was over twice that measured in fresh corn at 31 DAS, likely due to the lower moisture content of the dry seed. Anthocyanins likely continue to accumulate after 31 DAS, a time frame not investigated in this study.

2.3. Correlations between Anthocyanin Content and Physicochemical Properties

We measured the physical properties of kernels during grain-filling stages (Table 1). Dry weight continuously increased from 17 to 30 DAS. Fresh weight tended to increase during grain filling, but there were several decrements related to moisture content. An analysis of the correlation among the three anthocyanins detected in Heukjinjuchal and physicochemical characteristics (Table 2) showed that the anthocyanin content displayed a strong positive correlation coefficient with fresh weight and dry weight and a negative correlation with the moisture content. We attribute two sharp C3G increments observed at 29 DAS in part to the moisture content, which was lower at that time. Increased fresh weight due to starch accumulation and decreased moisture in purple-corn kernel during grain-filling stages has previously been shown.20

Table 1. Physical Parameters of Purple-Corn Kernels at Different Ripening Stagesd.

DASa fresh weight (g/100 kernel) moisture content (%) dry weight (g/100 kernel) dry matter accumulationb (%)
15 15.0 ± 0.2 65.7 ± 1.4 5.1 ± 0.1 33.8 ± 1.2
16 16.5 ± 1.2 54.6 ± 3.6 7.4 ± 0.5 45.0 ± 4.6
17 18.5 ± 0.9 62.2 ± 0.9 6.8 ± 0.4 37.2 ± 3.7
18 17.0 ± 0.5 57.3 ± 1.4 7.2 ± 0.4 42.1 ± 1.4
19 20.1 ± 0.5 61.0 ± 0.9 8.1 ± 0.3 40.1 ± 2.7
20 20.8 ± 1.4 57.6 ± 2.5 8.8 ± 0.7 42.3 ± 2.7
21 18.7 ± 0.6 51.8 ± 1.3 8.9 ± 0.3 47.7 ± 2.2
22 19.5 ± 0.7 52.2 ± 0.6 9.2 ± 0.5 47.5 ± 3.6
23 18.6 ± 1.0 49.7 ± 0.8 9.2 ± 0.4 49.7 ± 0.5
24 19.7 ± 1.1 51.4 ± 2.7 9.5 ± 1.1 48.3 ± 2.6
25 20.9 ± 0.4 53.4 ± 0.8 9.7 ± 0.2 46.3 ± 1.4
26 19.9 ± 1.4 49.9 ± 1.4 9.8 ± 0.7 49.4 ± 5.4
27 21.4 ± 1.2 52.5 ± 1.1 9.7 ± 0.4 45.7 ± 4.3
28 22.3 ± 1.2 53.3 ± 1.2 10.1 ± 0.3 45.2 ± 2.6
29 23.1 ± 3.9 50.1 ± 3.1 11.3 ± 1.8 49.2 ± 4.1
30 25.1 ± 2.4 50.0 ± 0.8 12.3 ± 0.4 49.6 ± 5.4
31 27.9 ± 1.1 54.0 ± 0.9 12.3 ± 0.4 44.0 ± 0.5
LSDc (0.05) 2.9 3.5 1.3 6.6
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a

Days after silking (DAS).

b

Dry matter accumulation (%) = (dry weight/fresh weight) × 100.

c

Least significant difference (LSD) at 5% probability.

d

Values represent means of three independent replicates ± standard deviation.

Table 2. Correlation between Anthocyanin Contents and Fresh Weight, Dry Weight, and Dry Matter Accumulation during Grain-Filling Stagese.

  FWa MCb DWc DMAd
C3G 0.79** –0.63** 0.89** 0.52**
P3G 0.74** –0.67** 0.86** 0.54**
M3G 0.75** –0.53** 0.82** 0.43**
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a

Fresh weight (FW) (g/100 kernel).

b

Moisture content (MC) (%).

c

Dry weight (DW) (g/100 kernel).

d

Dry matter accumulation (DMA) (%) = (dry weight/fresh weight) × 100.

e

* and ** represent significance at p < 0.05 and p < 0.01, respectively.

For the analysis of seed color, the correlation analysis of lightness, redness, and yellowness with the anthocyanin content is shown in Table 3. Lightness (L*) and yellowness (b*) had negative correlations with the anthocyanin content, whereas redness (a*) had a positive correlation with the anthocyanin content, showing that the corn kernel color is derived from the accumulation of anthocyanins and is a good indicator of anthocyanin accumulation.

Table 3. Correlation among Anthocyanin Contents and Lightness, Redness, and Yellownessd.

  L*a a*b b*c
C3G –0.79** 0.72** –0.90**
P3G –0.78** 0.72** –0.88**
M3G –0.79** 0.72** –0.86**
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a

L*: Lightness.

b

a*: Redness.

c

b*: Yellowness.

d

* and ** represent significance at p < 0.05 and p < 0.01, respectively.

2.4. Amino Acid Profiles of the Kernels

Quantities of 17 amino acids were measured during grain-filling stages. The amino acid content fluctuated rather than accumulating steadily. Glutamate was detected in the highest amount, while cysteine was measured in the lowest amount (Figure 3A). The amounts of most amino acids started to increase at 24 DAS and peaked at 26 DAS (Figure 3A). Leucine and isoleucine showed higher standard deviations from three biological replicates; this is thought to be the characteristics of these amino acids. Phenylalanine and tyrosine are converted to cinnamic acid and p-coumaric acid, respectively, by ammonia-lyase. Cinnamic acid is further converted to p-coumaric acid and p-coumaric acid to p-coumaroyl-CoA by 4-coumarate-CoA ligase, the first-step substrate of the flavonoid biosynthesis pathway. Phenylalanine and tyrosine, the two precursors of anthocyanins, were the second and the third lowest in mass and also showed similar fluctuations in mass during grain filling. During 20–25 DAS, these amino acids showed a clear oscillation pattern. This pattern could be related to anthocyanin accumulation during grain filling, as cyclic decrements of these amino acids could limit the quantities available for anthocyanin biosynthesis.

Figure 3.

Figure 3

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Amino acid profiles in kernels of purple-corn Heukjinjuchal during grain filling. (A) Amount of each amino acid during grain filling. Error bars represent standard deviations of three biological replicates. (B) Correlations among amino acids. Cluster I, II, and III were designated with cluster analysis. (C) Correlations among amino acid and cyanidin-3-β-O-glucoside (C3G). Color scales for B and C represent Pearson’s R.

Correlation analysis among amino acid showed three clear clusters during grain filling. Phenylalanine and tyrosine showed one of the strongest correlations to each other and belonged to cluster I along with methionine, leucine, and isoleucine. Cluster I correlated weakly with other clusters, whereas clusters II and III had strong positive correlations with each other (Figure 3B). We also analyzed the correlations between anthocyanins and amino acids, since phenylalanine and tyrosine are precursors of anthocyanins (Figure 3C). Cysteine showed a significant positive correlation, while lysine, glycine, and aspartic acid showed significant negative correlations (p < 0.05). Phenylalanine and tyrosine showed a positive but nonsignificant correlation. Intermediate products should be further analyzed to elucidate the relationships among amino acids and anthocyanins.

Kernels of colored corn, including red, blue, and purple varieties, have a higher antioxidant capacity and ability to scavenge free radicals compared with light-colored corns,17,21−23 suggesting that those cultivars can be used as nutraceuticals. Although anthocyanins accumulated throughout grain filling in our study, the best harvest time could be 29 DAS when kernel softness and amino acid content are also taken into account. If an earlier harvest is required, timing it to occur during 24–28 DAS would not reduce the quality with regard to anthocyanin and amino acid contents. Since anthocyanin accumulation can be affected by temperature and light intensity, harvest time could be changed according to environmental conditions. More research on this topic is needed.

2.5. Changes of Fatty Acids during Grain Filling

The other precursor of the anthocyanin biosynthesis pathway is malonyl CoA, which is also a precursor of fatty acid biosynthesis. The exact contents of the fatty acids were not determined in this study; however, fluctuations among the three fatty acids measured (palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1)) showed dynamic changes in grain filling. In purple waxy-corn kernels, the fatty acid composition was stable from 15 to 22 DAS but fluctuated between stearic acid (C18:0) and oleic acid (C18:1) during 24–29 DAS (Figure 4). Oleic acid made up about half of the observed total of three fatty acids and was the highest at 24 DAS. The stearic acid composition was the highest, making up 36.2% of the total contents of the three fatty acids at 27 DAS. The palmitic acid (C16:0) showed a relatively stable composition throughout grain filling, ranging from 16.6 to 19.9% of the three fatty acids (Figure 4). Other fatty acids such as C14:0, C18:2, C18:3, C20:0, and C22:0 were not detected in any of the samples analyzed. Palmitic acid is elongated to stearic acid, which is then transformed to C18:1 unsaturated fatty acid by stearoyl-CoA desaturase.24 The fatty acid composition after 30 DAS could be explained by the transfer of palmitic acid to stearic acid, but stearic acid is rarely transferred to oleic acid as the corn kernel reaches the late stages of grain filling.

Figure 4.

Figure 4

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Fatty acid composition changes during grain filling. Error bars represent standard deviations of three biological replicates.

3. Conclusions

Commercial fresh corn is harvested at 25–30 DAS, depending on growth conditions. Nutritional value is not typically considered when determining the harvest time. This study showed that, during development, corn kernels undergo dramatic changes that can affect their nutritional value. This study showed that kernels at 26 and 29 DAS have more amino acids and anthocyanin, respectively. Taking these changes into account may help ensure high-quality fresh corn production.

4. Materials and Methods

4.1. Preparing Kernels

Five waxy-corn cultivars, three purple and two yellow, plus B73 were used to investigate anthocyanin profiles in fully matured and dried seeds (Figure 1A). The five waxy-corn cultivars, “Ilmichal”, “Mibaek2”, Heukjinjuchal, Suwon90, and Miheugchal, are single-cross hybrids dominating the fresh waxy-corn seed market in South Korea and were developed by the National Institute of Crop Science or by Gangwon-do Agricultural Research and Extension Services.25−28 The corn was grown in the field of National Institute of Crop Science, Suwon, Korea (37°15′47″N, 126°59′16″E). Preplant broadcast manure at a dose of 15 000 kg ha–1 and basal fertilizer containing 145, 30, and 60 kg ha–1 for N, P2O5, and K2O, respectively, were applied for field preparation. Corn seeds were sown on May 2, 2013, at 30 and 65 cm spacing, within and between rows, respectively. During the growth period, the average of the daily lower and the higher temperature was 18.1–27.2 °C and 70% relative humidity on average. Kernels were harvested at 15 to 31 days after silking (DAS). Each day, over 20 kernels from three different cobs were weighed and immediately placed in liquid nitrogen (N2) and stored at −72 °C. To minimize variation, kernels were sampled in the middle of the cob.

4.2. Determining Color

Kernel color values L* (0 = black, 100 = white), a* (negative = green, positive = red), and b* (negative = blue, positive = yellow) were measured using a color difference meter (Minolta Co. Ltd., Japan). Calibration was carried out on a standard white plate. Fresh corn kernels were placed in a round cuvette and measured nine times for each time point (3 technical repeats × 3 biological replications).

4.3. Chemicals and Reagents

Eleven anthocyanin standards were purchased from Extrasynthese Co. (Genay, France): 3,4′,5,7-tetrahydroxy-3′,5′-dimethoxyflavylium chloride (malvidin chloride), 3,3′,4,5,7-pentahydroxyflavylium chloride (cyanidin chloride), 3,3′,4′,5,5′,7-hexahydroxyflavylium chloride (delphindin chloride), 3,4′,5,7-tetrahydroxy-3′-methoxyflavylium chloride (peonidin chloride), 3,3′,4′,5,7-pentahydroxy-5′-methoxyflavylium chloride (petunidin chloride), 3,5-bis(glucosyloxy)-4′,7-dihydroxyflavylium chloride (pelargonidin chloride), cyanidin-3-O-glucoside chloride (kuromanin chloride), peonidin-3-O-glucoside chloride, delphinidin-3-β-O-glucoside chloride (myrtillin chloride), malvidin-3-β-O-glucoside chloride (oenin chloride), and pelargonidin-3-O-glucoside chloride (callistephin chloride). All chemicals and regents were of analytical grade.

4.4. Extracting Samples

Anthocyanins were extracted from ground kernels according to the method described by Aoki et al.15 Approximately 2 g of powder from each type was added to a flask containing 20 mL of 0.1% hydrochloric acid (HCl) aqueous solution. This flask was shaken on a platform shaker at 150 rpm at 37 °C for 24 h. Extracts of each sample were filtered through Whatman No. 42 filter paper. The extracted solution was stored at 4 °C in darkness until analyzed.

4.5. Anthocyanin Quantification by Liquid Chromatography and Mass Spectrometry

Qualitative and quantitative anthocyanin analyses were performed with ultraperformance liquid chromatography (UPLC) with a Waters ACQUITY BEH C18 column (particle size 1.7 μm, 2.1 mm × 100 mm, Waters, MA) and with a photodiode array detector at 530 nm. The HPLC-grade solvents such as water and acetonitrile for UPLC analysis were purchased from J. T. Baker (New Jersey). Sample extracts were filtered through a 0.2 μm membrane syringe filter before injection. Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile. HPLC analysis for anthocyanin was performed using an LC system (Waters e2695 Separation Module) equipped with a PDA detector (Waters 2487 Dual λ Absorbance Detector) and with a C18 column (YMC-Pack ODS-AM). A gradient mobile phase of A (5:95, formic acid/water, v/v) and B (5:95, acetonitrile/water, v/v) were set to 90% A in 5 min, gradual decrement to 60% A until 35 min, and back to 90% A until 36 min at a flow rate of 0.7 mL min–1. For mass spectrometry (MS) analysis, a tetraquadrupole detector equipped with an electrospray ionization-tandem MS/MS (Waters) was used in the positive-ion mode, voltage 30 V, capillary 4 kV, and drying N2 gas at 800 L h–1.

4.6. Analyzing Amino Acids in Kernels

Amino acid quantification was performed by the method of Kim et al.29 with some modifications. Three milligrams of lyophilized fine purple-corn powder was added to 5 mL of 6N HCl. The digestion of the corn powder was incubated for 24 h at 110 °C with flushing by nitrogen gas. The resulting hydrolyzed solution was diluted with 10 mL of pure water and filtered through Millipore 0.45 μm syringe filters (Milford). Each hydrolyzed solution was loaded onto an automatic L-8800 high-speed amino-acid analyzer (Hitachi, Japan) with an ion-exchange column no. 2622SC PH. Amino acid calibration mixture solutions (Ajinomoto-Takara, Japan) were used as standards.

4.7. Analyzing Fatty Acids in Kernels

The fatty acids were analyzed as described by Garces and Mancha.30 The procedure was as follows: 0.5 g of lyophilized purple-corn powder was heated with an extraction solution containing methanol/heptane/benzene/2,2-dimethoxypropane/sulfuric acid (37:36:20:5:2, v/v). The digestion and lipid transmethylation took place at the same time at 80–85 °C and slowly cooled to room temperature. The supernatant containing the fatty acid methyl esters (FAMEs) was analyzed by capillary gas chromatography (GC) using the HP 6890 GC system (HP Co.) equipped with a FID detector and an HP-Innowax capillary column (cross-linked poly(ethylene glycol), 0.25 μm × 30 m). The initial oven temperature of 150 °C was sequentially raised to the final temperature of 280 °C at a rate of 4 °C min–1. Carrier gas nitrogen flowed at a rate of 10 mL min–1. During the determination of the composition of fatty acids of lipids from purple-corn kernel, the temperatures of the inlet and the detector were continuously maintained at 250 and 300 °C, respectively. The standard was used in the FAME mix (C14–C22) and obtained from Supelco Co. (Bellefonte).

4.8. Statistical Analysis

Statistical analysis was performed using Excel (Microsoft Office 2016) and SAS ver. 9.3 for Windows (Statistical Analysis Systems Institute Inc.). Correlation analysis of amino acid contents was performed with the web-based tool Metaboanalyst (http://metaboanalyst.ca).31

Acknowledgments

A part of the Ph.D. thesis of J.-T.K., titled “Analysis of Physicochemical Characteristics, Antioxidants, Anthocyanins and Proteome during the Ripening Stage of Purple Corn (Zea mays L.) Heukjinjuchal”, is used for this study.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02099.

  • LS–MS confirmation of major anthocyanin detected in the Heukjinjuchal (A) LC chromatogram of 15, 20, 25, and 30 DAS kernel samples; cyanidin-3-β-O-glucoside (C3G), pelargonidin-3-β-O-glucoside (P3G), and malvidin-3-β-O-glucoside (M3G) are indicated with dashed box; and (B–D) mass spectrometry of C3G, P3G, and M3G (PDF)

Author Contributions

§ J.-T.K., G.Y., and I.-M.C. contributed equally to this work.

This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development in Rural Development Administration (Project No. PJ01249702), Republic of Korea.

The authors declare no competing financial interest.

Supplementary Material

ao0c02099_si_001.pdf (214KB, pdf)

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