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. 2022 Apr 2;31(8):1053–1062. doi: 10.1007/s10068-022-01071-6

Stability assessment of anthocyanins from black soybean, grape, and purple sweet potato under in vitro gastrointestinal digestion

Dayeon Ryu 1, Eunmi Koh 1,✉
PMCID: PMC9300780  PMID: 35873379

Abstract

Anthocyanins are glycosylated derivatives of anthocyanidins, whose hydroxyl groups are occasionally acylated with organic acids. The effect of anthocyanin composition on their stability under in vitro gastrointestinal digestion was investigated. Black soybean had all glycosylated anthocyanins with monosaccharide, grape contained glycosylated anthocyanins with disaccharide (23%) and acylated anthocyanins bound with two sugars (77%), and purple sweet potato had all acylated anthocyanins bound with three sugars. The order of total anthocyanins content was purple sweet potato, grape, and black soybean. Gastric digestion did not significantly decrease anthocyanins content in three samples, while intestinal digestion resulted in the significant decrease of total anthocyanins content in black soybean (40%), grape (45%), and purple sweet potato (25%). This indicates that the degree of glycosylation and acylation of anthocyanins affects their stability under the gastrointestinal conditions. Phenolic acids derived from anthocyanin degradation increased total phenolic content as well as ABTS radical scavenging activity.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-022-01071-6.

Keywords: Anthocyanin, Black soybean, Grape, Purple sweet potato, Stability, In vitro digestion

Introduction

Anthocyanins are water-soluble pigments with red, purple, and blue colors in plants. They exist mostly as glycosylated forms of 2-phenylbenzopyrylium cation, which comprises a six-membered heterocyclic ring and two benzoyl rings, and hydroxyl group of their sugar moiety is occasionally acylated with either aliphatic or aromatic acids (Escribano-Bailón et al., 2004). The stability of anthocyanins can be affected by multiple factors such as the chemical structure of anthocyanidin, the number and position of sugar moiety, and the degree of acylation. The methoxy group in the benzoyl ring enhanced the stability of anthocyanins, while the hydroxyl group reduced its stability (Kamonpatana et al., 2014). Anthocyanins glycosylated at C-3 position are more stable than those at C-5 or C-7 (Leon et al., 1931). Anthocyanins bound with disaccharide are more stable than those with monosaccharide (Bronnum-Hansen and Flink, 1985). In addition, anthocyanins acylated with phenolic acids had higher stability compared with non-acylated anthocyanins (McDougall et al., 2007; Zhao et al., 2017). These indicate that the composition and content of anthocyanins can influence their stability under gastrointestinal environment.

The content of anthocyanins in apple extract showed no significant reduction under simulated gastric digestion and decreased below the limit of detection after intestinal digestion (Bouayed et al., 2011). Anthocyanins with mono- or disaccharide in black rice had a loss of 10% after in vitro gastric digestion and a reduction of 80% after intestinal digestion (Sun et al., 2015). The recovery of total anthocyanins content in blueberry containing mono acylated anthocyanin with monosaccharide was not significantly decreased in a simulated gastric digestion, while it showed the reduction of 17% in intestinal digestion (Correa-Betanzo et al., 2014). The total anthocyanin content of pomegranate juice that contained mono glycosylated anthocyanins with mono or di-saccharide increased slightly after in vitro gastric digestion, and had a decrease of 15% after intestinal digestion (Pérez-Vicente et al., 2002). These suggest that anthocyanins stability during a gastrointestinal digestion varied depending on food source. A small portion of anthocyanins can be absorbed in the stomach. Anthocyanins are mostly absorbed through the intestinal epithelium into the systemic circulation. Bioaccessibility is the proportion of a compound that is released from food matrix and then is available for intestinal absorption. This suggests that the stability of anthocyanins under gastrointestinal tract can affect their bioavailability. The proportion of anthocyanins to reach the gastrointestinal tract needs to be investigated to predict their bioaccessibility obtained from the consumption of anthocyanin-rich foods.

The foods that contribute the most to the consumption of anthocyanins in the Korean population are black soybean (24% of the total) and grape (20%) (Ryu, 2020). Recently, the use of purple sweet potato as a colorant in food industry has been increasing since anthocyanins in purple sweet potato are relatively stable under high temperature (Hong and Koh, 2016). Therefore, the objectives of this study were firstly to characterize the chemical structure and content of anthocyanins present in black soybean, grape, and purple sweet potato, and secondly to evaluate the stability of anthocyanins during in vitro gastrointestinal digestion. These results can be used to estimate the proportion of anthocyanins that reach the gastrointestinal tract.

Materials and methods

Chemicals

ABTS, acetic acid, Folin-Ciocalteu’s reagent, formic acid, gallic acid, hydrochloric acid, potassium chloride, potassium dihydrogen phosphate, potassium persulfate, potassium phosphate dibasic, sodium acetate, and sodium carbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cyanidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside, and petunidin-3-glucoside were purchased from Extrasynthese (Lyon, France).

Sample preparation

Black soybean (Glycine max (L.) Merr. Cheongja4ho) grown in Dalseong (Daegu, Korea) in 2016 was provided by the National Institute of Crop Science. Campbell Early grape (a hybrid of Vitis labrusca and V. vinifera) grown in Wanju (Jeollabuk-do, Korea) in 2017 was obtained from the National Institute of Horticultural and Herbal Sciences. Purple sweet potato (Ipomoea batatas L. Sinjami) grown in Muan (Jeollanam-do, Korea) in 2014 were obtained from the National Institute of Crop Science. Fresh purple sweet potato and grape were washed and freeze-dried, while black soybean was used as intact (moisture content, 8.47%). The photos of these samples are presented in the supplementary material (Fig. S1). All samples were ground using the IKA A10 basic analytical mill (Staufen, Germany) and then passed through a 30-mesh stainless sieve (≤ 600 μm) and kept at –35 °C in a deep-freezer.

Extraction

Extraction conditions were optimized for maximizing the extraction yield of total anthocyanin content, total phenol content, and ABTS radical scavenging activity in three foods using a response surface methodology. Experimental designs and results of three samples were reported in the previous studies (Ryu and Koh, 2018; Ryu et al., 2020, 2021). The difference in experimental and predicted values of total anthocyanins was less than 8%, thereby indicating that the model fits well. The optimized conditions of solid–liquid extraction were a solid–liquid ratio of 1/54.2 g/mL, a hydrochloric acid concentration of 0.359%, and a temperature of 56.8 °C for black soybean; a solid–liquid ratio of 1/50 g/mL, a temperature of 80 °C, and a time of 10 min for grape. The optimized conditions of ultrasound-assisted extraction were a time of 13 min, a temperature of 75 °C, and an amplitude of 25 μm for purple sweet potato.

In vitro gastrointestinal digestion

Simulated digestion was performed according to the method of Bouayed et al. (2011) and McDougall et al. (2005) with a slight modification. Each extract (15 mL) was mixed with 5 mL of pepsin (1260 unit/mL), which was adjusted to pH 2 and purged under nitrogen gas and then incubated in a shaking water bath at 37 °C for 2 h in the darkness. Then, the mixture was neutralized with 1 N sodium bicarbonate, followed by the addition of 5 mL of pancreatin (4 mg/mL) and bile salts (25 mg/mL). After being purged with nitrogen gas, the mixture was incubated at 120 rpm for 2 h at 37 °C under dark conditions. Extract, gastric, and intestinal digested solutions were used to determine total anthocyanins, total phenolics, ABTS radical scavenging activity, and individual anthocyanins.

Total anthocyanins

Total anthocyanins were determined by the pH differential method (Lee et al., 2005). After being mixed separately with pH 1.0 or pH 4.5 buffer solution, absorbance at 520 nm (black soybean and grape) or 530 nm (purple sweet potato) and 700 nm were measured using an ultraviolet–visible spectrophotometer (Biochrom Libra S22, Santa Barbara, CA, USA). The content was expressed as mg cyanidin-3-glucoside equivalents per 100 g of sample.

Total phenolics

Total phenolics was determined by the Folin-Ciocalteu method described by Singleton and Rossi (1965). Five-fold diluted sample extract was mixed with ten-fold diluted Folin-Ciocalteu solution. After 2 min, 2 mL of distilled water and 0.25 mL of saturated sodium carbonate solution were added and then placed in the darkness at room temperature for 1 h. Absorbance was measured at 725 nm, and the result was expressed as mg gallic acid equivalents per 100 g of sample.

ABTS radical scavenging activity

ABTS·+ scavenging activity was measured using the method of Shi et al. (2009) with a few modifications. An ABTS·+ solution was prepared by mixing 7 mM of the ABTS·+ solution and 2.45 mM of potassium persulfate at a ratio of 1:2 (v/v) and then allowed to react at room temperature for 12 h in the darkness. The solution was diluted with 0.1 mM phosphate buffer (pH 7.0) until the absorbance at 734 nm was approximately 0.70. After the extract reacted with the buffer solution for 5 min at room temperature, absorbance at 734 nm was measured. The ABTS·+ scavenging activity was expressed as mg gallic acid equivalents per 100 g of sample. A standard curve showed a linearity in the range of 0–10 mM gallic acid.

Anthocyanin identification and quantification

Individual anthocyanins were identified using an ultra-performance liquid chromatograph (UPLC) system (Waters Corporation, Milford, MA, USA) equipped with a quadruple-time-of-flight-tandem mass spectrometry (Q-TOF–MS/MS) system (SYNAPT™ G2, Waters Corporation, Manchester, UK). The extract was filtered with a 0.45 μm polytetrafluoroethylene syringe filter (Puradisc 13, Whatman, England) prior to injection. A reverse-phase column (CORTECS™ UPLC® C18 1.6 μm, 100 × 2.1 mm) at 40 °C was used for anthocyanins separation. The mobile phase consisted of solvent A (water/formic acid, 99.5:0.5, v/v) and solvent B (methanol/formic acid, 99.5:0.5, v/v) in the gradient program of 95% A to 0% A for 20 min at 0.3 mL/min. Electrospray ionization was operated in the positive mode with a capillary voltage of 3.1 kV and cone gas of 100 L/h. Individual anthocyanins were confirmed using retention time and ultraviolet–visible spectrum of an authentic standard as well as MS/MS spectra reported in the earlier investigations (De la Cruz et al., 2012; Kim et al., 2012; Lee et al., 2009).

Individual anthocyanins were quantitated using a high-performance liquid chromatograph (HPLC) system coupled with a diode array detector (DAD, Agilent Technologies, Waldbronn, Germany) connected with a Kintex C18 reverse-phase column (5 μm, 150 × 4.6 mm, Phenomenex, Torrance, CA, USA). The injection volume of the sample extracts and their digested solutions was 10 µL and the flow rate was 0.6 mL/min. The mobile phase consisted of solvent A (water/formic acid, 99:1, v/v) and solvent B (methanol/formic acid, 99:1, v/v). The mobile program was as follows: 0–20 min, 80–70% A; 20–50 min, 70–65% A; 50–60 min, 65–55% A. Further, the absorbance of each peak at 520 nm for black soybean and grape and 530 nm for purple sweet potato was monitored. The ranges of the calibration curve were 0.2–50 μg/mL for cyanidin-3-glucoside, 2–100 μg/mL for delphinidin-3-glucoside, 2–50 μg/mL for pelargonidin-3-glucoside, 2–50 μg/mL for peonidin-3-glucoside, and 0.2–50 μg/mL for petunidin-3-glucoside. Anthocyanins that authentic standards were not commercially available, which included acylated or diglycosidic forms, were quantified using the corresponding monoglucosides of same anthocyanidin skeleton.

Statistical analysis

Using SPSS IBM version 21.0 (Statistical Package for Social Science, IBM, Armonk, NY, USA), a one-way analysis of variance (ANOVA) was performed to determine a significant difference of anthocyanins, total phenolics, and antioxidant activity among three food samples. Pearson’s correlation analysis was conducted to evaluate the significance of the correlations between total anthocyanin content and individual anthocyanin content, total phenolic content, or ABTS activity in each sample.

Results and discussion

Anthocyanin profile

Anthocyanins were identified using UPLC-MS/MS and their content was determined by HPLC–DAD. Chemical structures of anthocyanins varied depending on black soybean, grape, and purple sweet potato (Table 1). Black soybean contained four anthocyanins including delphinidin-3-glucoside, cyanidin-3-glucoside, petunidin-3-glucoside, and pelargonidin-3-glucoside (molecular ion with an m/z value of 465, 449, 479, and 433; MS2 fragment with m/z of 303, 287, 317, and 271). Total content of four anthocyanins in black soybean was 212.22 mg per 100 g fresh weight. A predominant anthocyanin was cyanidin-3-glucoside (60% of the total), which was followed by petunidin-3-glucoside (30%) and delphinidin-3-glucoside (10%). This is similar to the result of Choung et al. (2001), who reported that black soybean (Glycine max (L.) Merr.) contained cyanidin-3-glucoside (74%), delphinidin-3-glucoside (22%), and petunidin-3-glucoside (4%). In grape, total amount of four glycosylated and four acylated anthocyanins was 290.19 mg per 100 g dry weight. The order of individual anthocyanin contents was cyanidin-3-p-coumaroyl-5-diglucoside (38%) ([M]+ with m/z of 757 and MS2 with m/z of 595, 449, and 287), delphinidin-3-p-coumaroyl-5-diglucoside (19%) ([M]+ with m/z of 773 and MS2 with m/z of 611, 465, and 303), peonidin-3-p-coumaroyl-5-diglucoside (17%), and cyaniding-3-p-coumaroyl-glucoside (3%) ([M]+ with m/z of 757 and MS2 with m/z of 595, 449, and 287). Further, acylated anthocyanins accounted for 77% of the total anthocyanins. This is in agreement with the result of Oh et al. (2008) who reported that cyanidin-3-p-coumaroyl-5-diglucoside accounted for 38% of total anthocyanins in Campbell Early grape and acylated anthocyanins accounted for 63% of the total. In addition, Choi (2010) found that cyanidin-3-p-coumaroyl-5-diglucoside was a major anthocyanin of Campbell Early grape. In purple sweet potato, eleven acylated anthocyanins including four cyanidin derivatives and seven peonidin derivatives were identified. Total amount of 11 anthocyanins was 989.61 mg per 100 g dry weight. A major anthocyanin was peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside (41%) ([M]+ with m/z of 1069 and MS2 with m/z of 907, 463, and 301), which was followed by peonidin-3-caffeoyl-feruloylsophoroside-5-glucoside (17%) ([M]+ with m/z of 1125 and MS2 with m/z of 963, 463, and 301) and cyanidin-3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside (15%) ([M]+ with m/z of 1055 and MS2 with m/z of 893, 449, and 287). This is similar to the result of Kim et al. (2012), who reported that Korean purple sweet potato had peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside (40%), peonidin-3-caffeoyl-feruloylsophoroside-5-glucoside (10%), and cyanidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside (10%). Total anthocyanin content was highest in purple sweet potato, followed by grape and black soybean. These show that the compositions of anthocyanins varied among three samples tested in this study.

Table 1.

Individual anthocyanins identified in black soybean, grape, and purple sweet potato

[M]+ (m/z) MS/MS (m/z) Compound Contenta (mg/100 g)
Black soybean
465.103 303.049 Delphinidin-3-glucoside 21.64 ± 0.29
449.108 287.055 Cyanidin-3-glucoside 127.04 ± 2.10
479.118 317.067 Petunidin-3-glucoside 63.54 ± 4.56
433.112 271.060 Pelargonidin-3-glucoside –b
Total 212.22
Grape
627.156 465.103/303.050 Delphinidin-3,5-diglucoside 14.96 ± 0.09
625.176 463.122/301.070 Peonidin-3,5-diglucoside 37.95 ± 0.12
655.186 493.136/331.081 Malvidin-3,5-diglucoside 5.19 ± 0.14
611.140 287.054 Cyanidin-3-sophoroside 9.95 ± 0.37
773.193 611.139/465.101/303.051 Delphinidin-3-p-coumaroyl-5-diglucoside 54.42 ± 0.90
757.198 595.146/449.200/287.057 Cyanidin-3-p-coumaroyl-5-diglucoside 110.20 ± 1.01
771.211 609.160/463.120/301.071 Peonidin-3-p-coumaroyl-5-diglucoside 48.30 ± 0.23
595.144 287.056 Cyanidin-3-p-coumaroyl-glucoside 9.22 ± 0.03
Total 290.19
Purple sweet potato
893.236 731.182/449.108/287.055 Cyanidin-3-p-hydroxybenzoylsophoroside-5-glucoside 26.20 ± 4.65
907.252 745.200/463.123/301.071 Peonidin-3-p-hydroxybenzoylsophoroside-5-glucoside 7.55 ± 0.59
949.261 787.207/463.123/301.070 Peonidin-3-caffeoylsophoroside-5-glucoside 52.63 ± 10.50
949.263 787.208/449.106/287.055 Cyanidin-3-feruloylsophoroside-5-glucoside 11.03 ± 1.26
963.277 801.224/463.123/301.070 Peonidin-3-feruloylsophoroside-5-glucoside 17.32 ± 2.71
935.254 773.192/449.108/287.054 Cyanidin-3-caffeoylsophoroside-5-glucoside 34.60 ± 6.49
1055.267 893.214/449.107/287.055 Cyanidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside 150.27 ± 27.18
1111.294 949.254/787.204/463.122/301.070 Peonidin-3-dicaffeoylsophoroside-5-glucoside 61.63 ± 9.64
1069.283 907.230/463.124/301.071 Peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside 404.81 ± 81.92
1125.309 963.256/463.123/301.071 Peonidin-3-caffeoyl-feruloylsophoroside-5-glucoside 167.10 ± 35.76
1083.289 921.244/463.124/301.070 Peonidin-3-feruloyl-p-hydroxybenzoylsophoroside-5-glucoside 26.47 ± 0.02
Total 989.61
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Individual anthocyanins were identified using UPLC-ESI/MS/MS and the content was determined using HPLC–DAD

aFresh weight for black soybean and dry weight for grape and purple sweet potato

bNot detected

Anthocyanin stability

Anthocyanin is an unstable molecule when there is a change in pH (Fossen et al., 1998). After anthocyanin-containing foods were consumed, anthocyanins pass through stomach (pH 2) and small intestine (pH 7–8). Acylated anthocyanins from red cabbage were notably more stable than non-acylated anthocyanins (McDougall et al., 2007). In addition, monoacylated petanin showed a comparable stability to other anthocyanins with aromatic mono- or di-acylation (Goto et al., 1983). Take into consideration that anthocyanins varied in the chemical structure such as the extent of glycosylation and acylation, the stability of anthocyanins existing in three foods needs to be assessed under simulated gastrointestinal conditions. The contents of individual anthocyanin before and after in vitro digestion are presented in Fig. 1 and Table 2.

Fig. 1.

Fig. 1

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Changes in individual anthocyanin content of black soybean (A), grape (B), purple sweet potato (C) before and after in vitro gastrointestinal digestion. Values with different letters on the bar indicate significantly different among digestion processes (p < 0.05). Cy, cyanidin; dp, delphinidin; mv, malvidin; pn, peonidin; p-cou, p-coumaroyl; caf, caffeoyl; fer, feruloyl, p-HB, p-hydroxybenzoyl; glu, glucoside; sop, sophoroside

Table 2.

Total anthocyanin content, total phenol content, and antioxidant activity in three food extracts before and after in vitro digestion (mg/100 g)

Total anthocyanins Total phenolics Antioxidant activity*
Black soybean
Extract 133.68 ± 1.93a (100%) 1343.51 ± 192.64b (100%) 165.52 ± 32.90b (100%)
Gastric 119.69 ± 3.19b (90%) 1499.29 ± 152.91b (112%) 270.06 ± 21.19a (163%)
Intestinal 79.71 ± 8.52c (60%) 1954.52 ± 177.26a (145%) 177.60 ± 26.92b (107%)
Grape
Extract 181.35 ± 4.96b (100%) 1150.36 ± 20.29c (100%) 244.24 ± 11.04a (100%)
Gastric 199.06 ± 3.49a (110%) 1285.76 ± 23.42b (112%) 251.94 ± 21.43a (103%)
Intestinal 100.24 ± 10.46c (55%) 1575.10 ± 86.35a (137%) 110.13 ± 17.26b (45%)
Purple sweet potato
Extract 556.26 ± 14.48b (100%) 1752.33 ± 59.78c (100%) 403.14 ± 27.72a (100%)
Gastric 579.10 ± 19.82a (104%) 1,952.71 ± 50.89b (111%) 423.17 ± 29.79a (105%)
Intestinal 417.98 ± 8.46c (75%) 2,372.09 ± 234.77a (135%) 343.17 ± 68.50b (85%)
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*Different letters indicate statistical differences (p < 0.01) in the digestion processes

In black soybean, total anthocyanins content after gastric digestion was 119.69 mg fresh weight, which corresponded to 90% of anthocyanins present in black soybean (Table 2). The losses of petunidin-3-glucoside and cyanidin-3-glucoside were 67% and 9%, respectively (Fig. 1). In the intestinal digestion, anthocyanins content was 79.71 mg/100 g fresh weight, which amounted for 60% of the extract. These results revealed that the anthocyanins in black soybean were stable after the acidic gastric digestion and relatively less stable after intestinal digestion. Similarly, Sun et al. (2015) reported that the total anthocyanin content had no significant change in vitro gastric digestion of black rice (Oryza sativa L.), whereas 76% of anthocyanins remained after an intestinal digestion. This can be explained by stating that anthocyanins were stable under the gastric environment (pH 2) and became degraded under the intestinal digestion (pH 7). Anthocyanins exist as a flavylium cation in the acidic condition, whereas they are degraded by the opening of flavylium ring in the alkaline condition to form colorless chalcone (Valdez and Bolling, 2019). The stability of anthocyanins was enhanced with the increase in the number of methoxyl groups (McDougall et al., 2005). In this study, delphinidin-3-glucoside and cyanidin-3-glucoside resulted in a reduction of 29% and 26%, whereas petunidin-3-glucoside was not significantly changed under simulated intestinal digestion compared with the gastric digestion (Fig. 1). This implies that petunidin-3-glucoside with one methoxyl group was more stable than delphinidin and cyanidin that have no methoxyl group. Correa-Betanzo et al. (2014) demonstrated that the reduction of delphinidin-3-glucoside and cyanidin-3-glucoside in wild blueberry were 70% and 89%, respectively, by the intestinal digestion. Pearson’s correlation analysis was conducted to elucidate the effect of the individual anthocyanins on the change in the total anthocyanins content during simulated gastrointestinal digestion. Delphinidin-3-glucoside and cyanidin-3-glucoside contents were correlated with total anthocyanin content, but petunidin-3-glucoside was not correlated during simulated gastrointestinal digestion (Table 3).

Table 3.

Pearson’s correlation coefficients between individual anthocyanins and total anthocyanin content in black soybean, grape, and purple sweet potato during simulated gastrointestinal digestion

Anthocyanin Correlation coefficient
Black soybean
 Delphinidin-3-glucoside 0.905**
 Cyanidin-3-glucoside 0.968**
 Petunidin-3-glucoside 0.630
Grape
 Delphinidin-3,5-diglucoside  − 0.697*
 Peonidin-3,5-diglucoside  − 0.326
 Malvidin-3,5-diglucoside 0.884**
 Cyanidin-3-sophoroside 0.994**
 Delphinidin-3-p-coumaroyl-5-diglucoside 0.995**
 Cyanidin-3-p-coumaroyl-5-diglucoside 0.990**
 Peonidin-3-p-coumaroyl-5-diglucoside 0.992**
 Cyanidin-3-p-coumaroyl-glucoside 0.993**
Purple sweet potato
 Cyanidin-3-p-hydroxybenzoylsophoroside-5-glucoside 0.453
 Cyanidin-3-feruloylsophoroside-5-glucoside  − 0.229
 Cyanidin-3-caffeoylsophoroside-5-glucoside  − 0.171
 Cyanidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside 0.780*
 Peonidin-3-caffeoylsophoroside-5-glucoside 0.989**
 Peonidin-3-p-hydroxybenzoylsophoroside-5-glucoside  − 0.629
 Peonidin-3-feruloylsophoroside-5-glucoside 0.523
 Peonidin-3-dicaffeoylsophoroside-5-glucoside 0.761*
 Peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside 0.810**
 Peonidin-3-caffeoyl-feruloylsophoroside-5-glucoside 0.881**
 Peonidin-3-feruloyl-p-hydroxybenzoylsophoroside-5-glucoside 0.982**
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Levels of significance *p < 0.05, ** p < 0.01

In grape, eight anthocyanins in the extract and gastric digested solution and four anthocyanins in the intestinal digested mixture were found (Fig. 1). Anthocyanins in the grape were diglycosylated with sugars or acylated with p-coumaric acid (Table 1). There was an increase of 10% in the content of total anthocyanins after gastric digestion (Table 2). This is consistent with the result of Pérez-Vicente et al. (2002), who demonstrated that the anthocyanins content in pomegranate juice had an increase of 10% after in vitro gastric digestion. This can be attributed to the presence of flavylium cation under the gastric environment of pH 2. On the other hand, the content of total anthocyanins by intestinal digestion was reduced to 45%. Cyanidin-3-p-coumaroyl-5-diglucoside and delphinidin-3-p-coumaroyl-5-diglucoside showed the highest reduction and delphinidin-3,5-diglucoside and peonidin-3,5-diglucoside were more stable than the other anthocyanins (Fig. 1). Delphinidin-3,5-diglucoside had a negative relationship with total anthocyanins content, while another anthocyanins except peonidin-3,5-diglucoside showed positive correlations (Table 3).

In purple sweet potato, the content of total anthocyanins slightly increased after gastric digestion and 75% of total anthocyanins was retained after intestinal digestion (Table 2). In comparison with the reduction of 40% in black soybean, purple sweet potato was relatively more stable under the intestinal digestion. All anthocyanins were not significantly changed by gastric digestion, while four major anthocyanins including cyanidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside, peonidin-3-dicaffeoyl-sophoroside-5-glucoside, peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside, and peonidin-3-caffeoyl-feruloylsophoroside-5-glucoside showed significant changes after intestinal digestion (Fig. 1). Major anthocyanins with a substantial reduction after intestinal digestion were peonidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside (44%) and peonidin-3-caffeoyl-feruloylsophoroside-5-glucoside (42%) (Fig. 1). Peonidin-3-caffeoylsophoroside-5-glucoside and peonidin-3-feruloyl-p-hydroxybenzoylsophoroside-5-glucoside were not found after the intestinal digestion. Other anthocyanins such as peonidin-3-p-hydroxybenzoylsophoroside-5-glucoside and peonidin-3-feruloylsophoroside-5-glucoside had no significant change. This is consistent with the result of Yang et al. (2019), who reported that peonidin-3-p-hydroxybenzoylsophoroside-5-glucoside and peonidin-3-feruloylsophoroside-5-glucoside were more stable than other anthocyanins. Among eleven anthocyanins found in purple sweet potato, six anthocyanins showed significant correlations with total anthocyanin content during simulated gastrointestinal digestion (Table 3).

Total phenolic content

The content of total phenolics in black soybean did not show a significant change during simulated gastric digestion and an increase of 45% after intestinal digestion (Table 2). Polyphenols including flavanone and flavonol are known to be stable during gastric digestion (Bermúdez-Soto and Tomás-Barberán, 2004; Boyer et al., 2005; Gil-Izquierdo et al., 2001). A negative correlation between total anthocyanins and total phenolics was found in black soybean (r = –0.881) (Table 4). Anthocyanins could be changed into phenolic compounds such as catechol, caffeic acid, ρ-coumaric acid, and protocatechuic acid by the digestion process steps (Braga et al., 2018; Woodward et al., 2011). Total phenol content in grape also significantly increased after gastrointestinal digestion (Table 2), thereby resulting in a negative correlation between total anthocyanins and total phenolics (r = –0.837). The total phenolic contents of purple sweet potato after gastric and intestinal digestion resulted in an increase of 11% and 35%, respectively (Table 2). McDougall et al. (2007) found that there was no reduction of total phenolics in red cabbage extract after intestinal digestion. They demonstrated that anthocyanins were degraded and their breakdown products were detected as total phenolics that have reducing properties. McDougall et al. (2005) reported that pelargonidin-3-glucosylrutinoside, pelargonidin-3-rutinoside, and pelargonidin-3-sophoroside in raspberry was hydrolyzed into pelargonidin-3-glucoside and sugar moiety. The negative relationship between total anthocyanins and total phenolics in purple sweet potato (r = –0.786) indicates that the degradation of anthocyanins may contribute to the increase of total phenolic content after gastrointestinal digestion.

Table 4.

Pearson’s correlation coefficients among anthocyanin monomer content, total phenol content, and ABTS radical scavenging activity in black soybean, grape, and purple sweet potato during simulated gastrointestinal digestion

Total phenolics ABTS radical scavenging activity
Black soybean Anthocyanin monomera  − 0.881** 0.203
ABTS radical scavenging activity  − 0.287
Grape Anthocyanin monomer  − 0.837** 0.953**
ABTS radical scavenging activity  − 0.833**
Purple sweet potato Anthocyanin monomer  − 0.786** 0.603**
ABTS radical scavenging activity  − 0.483**
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aAnthocyanin monomer was determined by the pH differential method

Level of significance **p < 0.01

ABTS radical scavenging activity

Structural change of anthocyanins during simulated gastrointestinal digestion could affect the antioxidant activity of anthocyanin-containing extract. Antioxidant activity was generally determined by the combination of radical scavenging capacity, reducing power, and metal chelating activity. High correlations between ABTS, reducing power, and metal chelating activity have been reported in anthocyanin-rich foods (Sabraoui et al., 2020; Zhoa et al., 2015). Accordingly, the ABTS radical scavenging activity was used as an indicator of anthocyanin stability during in vitro digestion in this study. 

ABTS radical scavenging activity showed an increasing trend in gastric digestion and then decreased in intestinal digestion (Table 2). Podsędek et al. (2014) reported that 87% of total anthocyanin content and 50% of ABTS radical scavenging activity was reduced after intestinal digestion in red cabbage containing acylated anthocyanins. The ABTS radical scavenging activity in black soybean had no correlation with total phenolic content and anthocyanin monomer (Table 4). Earlier studies reported that radical scavenging activity positively correlated with total phenolic content (Dudonné et al., 2009; Lingua et al., 2018). Šaponjac et al. (2013) demonstrated a positive relationship (r = 0.953) between ABTS radical scavenging activity and total anthocyanin content in cranberry residues. In grape, ABTS radical scavenging activity was not significantly changed after gastric digestion, but had a decrease of 55% after intestinal digestion (Table 2). This is similar to the result of Wang et al. (2017), who demonstrated that there was no change in the ABTS radical scavenging activity of grape pomace after gastric digestion and a reduction of 30% in intestinal digestion. A significant positive relationship was found between ABTS radical scavenging activity and total anthocyanin content in grape (r = 0.953) and purple sweet potato (r = 0.603) (Table 4). This suggests that anthocyanins in grape and purple sweet potato extracts highly contributed to the ABTS radical scavenging activity.

Anthocyanins in black soybean, grape, and purple sweet potato varied in glycosylation and acylation and showed a varying stability of anthocyanins under simulated gastrointestinal conditions. In particular, anthocyanins with two sugar molecules or acylated with phenolic acids were more stable than anthocyanins with one sugar during in vitro gastrointestinal digestion. These results indicate that detailed information regarding anthocyanin’s structure can be useful for estimating their bioaccessibility as well as functional potential from the consumption of anthocyanin-rich foods.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1153 KB) (1.1MB, docx)

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03028841).

Declarations

Conflict of interest

Eunmi Koh and Dayeon Ryu declared that there is no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Dayeon Ryu, Email: dryu4@naver.com.

Eunmi Koh, Email: kohem7@swu.ac.kr.

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