# Phenolic Compounds and Antioxidant Activity of Rice–Tartary Buckwheat Composite as Affected by In Vitro Digestion

Authors: Nuo Li; Guifang Zhang; Dongjie Zhang
Journal: Journal of Chemistry
 (2022)

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## Abstract

The present study aimed to evaluate the phenolic compounds and antioxidant activity of rice–tartary buckwheat composite (RTBC) as affected by in vitro digestion to explore the structure-activity relationship of the release of total phenolic content (TPC) and total flavonoid content (TFC) with the antioxidant activity of RTBC during in vitro oral, gastric, and intestinal digestion stages. The release of TPC and TFC from RTBC increased significantly after in vitro digestion (P<0.05), and the change of antioxidant activity was consistent with that of TPC and TFC. Compared with the initial stage of digestion, the antioxidant activity of RTBC was increased after digestion (P<0.05), and there was a strong correlation between antioxidant activity and the release of TPC and TFC (0.954 < R < 0.997; P<0.05). The phenolic compounds released in the oral, gastric, and intestinal digestion stages varied, and eight phenolic compounds were identified by UPLC-Triple-TOF/MS, namely, quercetin-3-O-robinoside-7-O-sophoroside, quercetin-3-O-neohesperidoside-7-O-glucoside, forsythobiflavone A, forsythobiflavone B, quercetin-3-O-rutinoside-7-O-glucoside, rutin, isoquercetin, and ferulic acid. These results indicated that in vitro digestion significantly increases the release of phenolic compounds and flavonoids from RTBC and there is a higher antioxidant activity after digestion than before digestion. The phenolic compounds released after digestion of RTBC are beneficial to health protection.


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## Body

## 1. Introduction

Investigations on the digestion and metabolism of nutrients have indicated that a single ingredient cannot satisfy the nutritional needs of consumers. Experts and scholars have identified a mixture of raw materials to meet people’s requirements for comprehensive and balanced nutrition. Due to the more sufficient nutritional elements of the compound mixture, it better meets the supplementary needs of consumers for vitamins and essential amino acids. Several compounds have been studied, including multigrain steamed bread [1], compound multigrain bread [2], oatmeal-bean flour cookies [3], complex black rice porridge [4], and oat-buckwheat-pea compound multigrain flour [5].Rice is one of the staple grains in China, and it is the most important food crop and strategic material in the world. Rice is rich in vitamin B, starch, protein, and other functional components, but it has less phenolic compounds than grain. Therefore, to meet the requirements of more comprehensive nutritional components, some composite foods containing rice have appeared on the market in recent years, such as rice flour suitable for infant auxiliary consumption and composite cereal drinks. Thus, the combination of coarse grains and staple grains can better meet the requirements of a more balanced nutritional composition. Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn) originated in the western mountain areas of China, and it is mainly cultivated in China, Bhutan, northern India, Nepal, and central Europe [6]. Tartary buckwheat is a coarse grain, and it is rich in phenolic compounds and flavonoids, such as rutin and quercetin, which are important nutritional components. These active ingredients of tartary buckwheat can prevent high blood pressure, and they have hypoglycemic, hypolipidemic, and anticancer effects. Some studies have shown that tartary buckwheat flavonoids have hypoglycemic and hypolipidemic effects in type 2 diabetic rats [7]. Tartary buckwheat flavonoids show obvious antioxidant and hypoglycemic properties [8], and they have an inhibitory effect on the proliferation of human gastric cancer cells [9]. Overall, tartary buckwheat is rich in phenolic compounds and flavonoids, giving it considerable medicinal and nutritional value [10]. Although tartary buckwheat is a rare food and medicine in nature, it is considered ideal for human food.Phenolic compounds are the secondary metabolites of plants, and they have a benzene ring as the parent structure and multiple hydroxyl substituents at different positions [11]. Phenolic compounds are a class of biological components with good antiinflammatory, anticancer, and antioxidant functions, and they play an important role in human health. Phenolic compounds are considered the most important natural antioxidant. Flavonoids are an important part of phenolic compounds, and they have the common structure of diphenyl propane (C6-C3-C6) and usually form oxygen-containing heterocycles [12]. Because flavonoids have significant antioxidant activity, the intake of moderate amounts of flavonoids has potential biological significance for human health. To explore the metabolism of phenolic compounds in the digestive system, the functional effects of ingested phenolic compounds on the body are investigated. In vitro digestion technology has several advantages, including speed, reliability, and reproducibility, and it is not subject to moral and ethical constraints. In vitro digestion technology not only avoids the complexity and uncontrollability of the digestion system but also, to a certain extent, imitates the human digestion system and accurately represents the change in the target compounds during digestion. In vitro digestion technology is widely utilized by scholars globally. Lucas-González et al. used in vitro digestion technology to evaluate the recovery rate and bioavailability index of flavone in persimmon fruit powder as well as investigating the antioxidant activity of persimmon fruit powder in the in vitro digestion process [13]. Lamothe et al. combined milk and cheese with phenolic compounds to investigate the changes in antioxidant activity after in vitro digestion [14, 15]. Yang et al. conducted in vitro digestion experiments on different buckwheat samples to explore the digestion mechanism of buckwheat starch and the effects on protein and fat [16]. For experimental application, in vitro digestion is a good method to evaluate the bioavailability of food polyphenolic compounds and other nutrients. For the identification and analysis of phenolic compound components, there are common spectroscopic techniques and chromatographic detection and analysis techniques as well as mass spectrometry detection and analysis techniques [17]. These techniques include mass spectrometry detection, including liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Mass spectrometry is widely used in the separation, analysis, and identification of phenolic compounds due to its high sensitivity, accuracy, traceability, and efficiency.To avoid the simplification of nutrition, coarse cereals rich in phenolic compounds are mixed with staple grains to meet the needs of more balanced nutrition. Therefore, in the present study, rice and tartary buckwheat were used as raw materials. Along with in vitro oral, gastric, and intestinal digestion methods, we used UPLC-Triple-TOF/MS and ultraviolet visible (UV) spectrophotometer detection technology to analyze the influence of phenolic compounds and antioxidant activity during digestion, clarify their structure-activity relationship, and lay a theoretical foundation for highly efficient bioavailable polyphenolic-rich substances for developing new functional foods.


## 2. Materials and Methods

### 2.1. Materials

Rice (Dao Huaxiang variety) and tartary buckwheat were purchased from a local supermarket. Gallic acid, rutin, ferulic acid, and isoquercetin were purchased from Sinopharm Chemical Reagent (Shanghai, China). Alpha-amylase (5 U/mg), pepsin (250 U/mg), pancreatin (8 USP), and bile were purchased from Sigma Aldrich (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazylhydrate (DPPH) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Folin phenol reagent was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Methanol (HPLC) and formic acid (HPLC) were obtained from ANPEL Laboratory Technologies (Shanghai) Inc. Acetonitrile (HPLC) was purchased from Shanghai Macleans Biochemical Technology Co., Ltd. (Shanghai, China). Other reagents used were of analytical grade.


### 2.2. Sample Preparation

The rice and tartary buckwheat were ground into powder using a universal crusher and screened by a 250μm mesh, and the powders were mixed according to a mass ratio of 1 : 1 to obtain rice–tartary buckwheat composite (RTBC).


### 2.3. In Vitro Digestion

Simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared according to the INFOGEST 2.0 digestion method [18]. The in vitro digestion method was performed according to the INFOGEST 2.0 digestion method and Ma et al. [19].In brief, 1.0 g of RTBC was weighed and dissolved in 10 mL of distilled water. Next, 10 mL of SSF withα-amylase (75 U/mL) was added followed by shaking in a water bath at 37°C for 10 min at 70 rpm. The pH of the solution was adjusted to 2.0 with 1 mol/L HCl, and 10 mL of SGF with 2000 U/mL pepsin was then added followed by shaking in a water bath at 37°C at 100 rpm for 2 h. The pH of the solution was then adjusted to 7.0 with 1 mol/L NaHCO3, and 20 mL of SIF with 100 U/mL trypsin, 10 mM bile, and 2000 U/mL pancreatic lipase were then added followed by shaking in a water bath at 37°C for 2 h at 100 rpm. For the control digestion group, no digestive enzyme was added. After the digestion was completed, the digested samples were collected at each stage and then centrifuged at 4°C and 10000 rpm for 10 min, and the supernatant was removed and stored at −80°C.


### 2.4. Total Phenolic Content (TPC)

TPC was determined by the Folin phenol reagent colorimetric method [20]. The sample solution (1.0 mL) was added to a test tube followed by the addition of 0.5 mL of folin phenol solution and 4.0 mL of 150 g/L NaCO3 aqueous solution. Distilled water was added to reach a final volume of 10 mL, and the sample was mixed well. After allowing the mixture to stand in the dark at 25°C for 1 h, the absorbance at 760 nm was measured using a T6 Series UV-visible spectrophotometer (Beijing, China). The TPC of the sample was expressed as milligrams of gallic acid equivalents per 100 grams of dry weight (mg GAE/100 g) of the sample.


### 2.5. Total Flavonoid Content (TFC)

The TFC was determined by the aluminum nitrate method [21]. Briefly, 5.0 mL of distilled water and 1.0 mL of 50 g/L sodium nitrite aqueous solution were added to 1.0 mL of the sample to be tested. After mixing the sample, 1.0 mL of 100 g/L aluminum nitrate aqueous solution was added. After 6 min of reaction, 10.0 mL of 100 g/L sodium hydroxide aqueous solution was added. After 15 min, the absorbance at 510 nm was measured using a T6 Series UV-visible spectrophotometer (Beijing, China). The TFC was expressed as milligrams of rutin equivalents per 100 grams of dry weight (mg RE/100 g) of the sample.


### 2.6. ABTS Radical-Scavenging Ability

ABTS radical-scavenging ability was measured according to the method described by Rodríguez et al. with some modifications [22]. Briefly, 7 mmol/L ABTS solution was mixed with 2.45 mmol/L potassium persulfate solution according to the volume ratio of 1 : 1, and the mixture was placed in the dark at 25°C for 16 h. The mixture was then diluted with methanol solution to obtain an absorbance of 0.70 ± 0.02 at 734 nm, yielding the ABTS reagent. The test sample (0.2 mL) was mixed with 3.8 mL of the ABTS reagent, and the absorbance was measured at 734 nm (Am). Methanol solution was used to replace the sample for the blank control (Ak). Methanol was used instead of the ABTS reagent for the sample control (An). The ABTS radical-scavenging rate of the sample was calculated as follows:(1)ABTS radical−scavenging rate=1−Am−AnAk×100%.


### 2.7. OH Radical-Scavenging Ability

To evaluate the OH free radical-scavenging ability, we used the method previously described by Zhang et al. with some modifications [23]. Briefly, 2 mL of 6 mmol/L ferrous sulfate and 2 mL of 6 mmol/L hydrogen peroxide were added to 2 mL of test sample. After mixing, the sample was placed at 25°C for 10 min, and 2 mL of 6 mmol/L salicylic acid–ethanol solution was then added. After mixing, the sample was allowed to react for 30 min at 25°C, and the absorbance at 510 nm was then measured (A1). Distilled water was used instead of hydrogen peroxide for the sample control (A2). Distilled water was used to replace the sample for the blank control (A0). The OH radical-scavenging rate was calculated as follows:(2)OH radical−scavenging rate=1−A1−A2A0×100%.


### 2.8. Superoxide Anion Radical-Scavenging Ability

To assay superoxide anion radical-scavenging ability, the pyrogallol autoxidation method was performed as previously reported [24]. Briefly, the reaction mixture contained 4.5 mL of 50 mmol/L Tris-HCl buffer solution (pH 8.2), 1 mL of the test sample, and 4.0 mL of 25 mmol/L pyrogallol solution. The mixture was placed in a water bath at 25°C for 5 min. After removing the mixture from the water bath, 1 mL of 8 mol/L hydrochloric acid solution was immediately added, and the absorbance at 325 nm was measured (Af). The blank control used distilled water instead of sample solution to determine the absorbance (A0). Distilled water was used to replace the pyrogallol solution for the sample control (An). The superoxide anion radical-scavenging rate was calculated as follows:(3)Superoxide anion radical−scavenging rate=1−Af−AnA0×100%.


### 2.9. DPPH Radical-Scavenging Ability

To determine the DPPH radical-scavenging ability, the method described by Chen et al. was utilized [25]. In brief, 1.0 mL of the test sample was mixed with 0.1 mmol/L DPPH-ethanol solution (Ai), and the blank control used absolute ethanol to replace the sample (A0). Absolute ethanol was used to replace the DPPH–ethanol solution for the sample control (Aj). Each mixture was placed in the dark at 25°C for 30 min, and the absorbance at 510 nm was then measured. The DPPH radical-scavenging rate was calculated as follows:(4)DPPH radical−scavenging rate=1−Ai−AjA0×100%.


### 2.10. UPLC-Triple-TOF/MS Conditions

The test sample was passed through a 0.22μm filter membrane for component analysis. The UPLC-triple-TOF/MS system was used for qualitative analysis of components (Acquity™ ultra-high-performance liquid chromatography; Waters, USA), and Triple TOF 5600 + time-of-flight mass spectrometry equipped with electrospray ion source (AB Sciex Company, USA) was used. The liquid-phase conditions were as follows: a Waters Acquity UPLC HSS T3 column (150 mm × 3.0 mm, 1.8 μm i.d.), 280 nm UV detection wavelength, 50°C column temperature, 0.3 mL/min flow rate, and 3 μL injection volume. Mobile phase A was comprised of a 0.1% formic acid aqueous solution, and mobile phase B was comprised of 0.1% formic acid acetonitrile. A binary gradient elution was employed, and the consecutive program was as follows: 95%–74% A from 0 to 25 min, 74%–5% A from 25 to 33 min, and 5%–90% A from 33 to 34 min. The column was then returned to its starting conditions for 30 min for column balance.The parameters of the MS/MS detector were as follows: triple TOF 5600 + time-of-flight mass spectrometer equipped with positive and negative-ion scanning modes; 5500 V(+) and −4500 V(−) ion sources; scan range ofm/z 100 to m/z 1500; GS1 nebulizing gas at 50 psi; GS2 nebulizing gas at 50 psi; declustering potential at 1000 V; collision energy at 10 V. The TOF MS–production and information-dependent acquisition modes were simultaneously used to collect mass spectrum data, and an automated calibration delivery system was used for automatic calibration.


### 2.11. Statistical Analysis

Each experiment was performed three times, and the data are expressed as mean ± standard deviation (SD). Excel 2010 (Microsoft Corp., Redmond, WA, USA) and SPSS software (version 20.0; IBM Corp., Armonk, NY) were used for statistical analysis of the data. Analysis of variance (ANOVA) was used to test the significant differences. Duncan’s multiple range tests were used for intragroup significance analysis.P<0.05 indicated a significant difference. The column charts were generated using Origin Pro (version 2018, OriginLab Corporation, Northampton, MA, USA).


## 2.1. Materials

Rice (Dao Huaxiang variety) and tartary buckwheat were purchased from a local supermarket. Gallic acid, rutin, ferulic acid, and isoquercetin were purchased from Sinopharm Chemical Reagent (Shanghai, China). Alpha-amylase (5 U/mg), pepsin (250 U/mg), pancreatin (8 USP), and bile were purchased from Sigma Aldrich (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazylhydrate (DPPH) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Folin phenol reagent was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Methanol (HPLC) and formic acid (HPLC) were obtained from ANPEL Laboratory Technologies (Shanghai) Inc. Acetonitrile (HPLC) was purchased from Shanghai Macleans Biochemical Technology Co., Ltd. (Shanghai, China). Other reagents used were of analytical grade.


## 2.2. Sample Preparation

The rice and tartary buckwheat were ground into powder using a universal crusher and screened by a 250μm mesh, and the powders were mixed according to a mass ratio of 1 : 1 to obtain rice–tartary buckwheat composite (RTBC).


## 2.3. In Vitro Digestion

Simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared according to the INFOGEST 2.0 digestion method [18]. The in vitro digestion method was performed according to the INFOGEST 2.0 digestion method and Ma et al. [19].In brief, 1.0 g of RTBC was weighed and dissolved in 10 mL of distilled water. Next, 10 mL of SSF withα-amylase (75 U/mL) was added followed by shaking in a water bath at 37°C for 10 min at 70 rpm. The pH of the solution was adjusted to 2.0 with 1 mol/L HCl, and 10 mL of SGF with 2000 U/mL pepsin was then added followed by shaking in a water bath at 37°C at 100 rpm for 2 h. The pH of the solution was then adjusted to 7.0 with 1 mol/L NaHCO3, and 20 mL of SIF with 100 U/mL trypsin, 10 mM bile, and 2000 U/mL pancreatic lipase were then added followed by shaking in a water bath at 37°C for 2 h at 100 rpm. For the control digestion group, no digestive enzyme was added. After the digestion was completed, the digested samples were collected at each stage and then centrifuged at 4°C and 10000 rpm for 10 min, and the supernatant was removed and stored at −80°C.


## 2.4. Total Phenolic Content (TPC)

TPC was determined by the Folin phenol reagent colorimetric method [20]. The sample solution (1.0 mL) was added to a test tube followed by the addition of 0.5 mL of folin phenol solution and 4.0 mL of 150 g/L NaCO3 aqueous solution. Distilled water was added to reach a final volume of 10 mL, and the sample was mixed well. After allowing the mixture to stand in the dark at 25°C for 1 h, the absorbance at 760 nm was measured using a T6 Series UV-visible spectrophotometer (Beijing, China). The TPC of the sample was expressed as milligrams of gallic acid equivalents per 100 grams of dry weight (mg GAE/100 g) of the sample.


## 2.5. Total Flavonoid Content (TFC)

The TFC was determined by the aluminum nitrate method [21]. Briefly, 5.0 mL of distilled water and 1.0 mL of 50 g/L sodium nitrite aqueous solution were added to 1.0 mL of the sample to be tested. After mixing the sample, 1.0 mL of 100 g/L aluminum nitrate aqueous solution was added. After 6 min of reaction, 10.0 mL of 100 g/L sodium hydroxide aqueous solution was added. After 15 min, the absorbance at 510 nm was measured using a T6 Series UV-visible spectrophotometer (Beijing, China). The TFC was expressed as milligrams of rutin equivalents per 100 grams of dry weight (mg RE/100 g) of the sample.


## 2.6. ABTS Radical-Scavenging Ability

ABTS radical-scavenging ability was measured according to the method described by Rodríguez et al. with some modifications [22]. Briefly, 7 mmol/L ABTS solution was mixed with 2.45 mmol/L potassium persulfate solution according to the volume ratio of 1 : 1, and the mixture was placed in the dark at 25°C for 16 h. The mixture was then diluted with methanol solution to obtain an absorbance of 0.70 ± 0.02 at 734 nm, yielding the ABTS reagent. The test sample (0.2 mL) was mixed with 3.8 mL of the ABTS reagent, and the absorbance was measured at 734 nm (Am). Methanol solution was used to replace the sample for the blank control (Ak). Methanol was used instead of the ABTS reagent for the sample control (An). The ABTS radical-scavenging rate of the sample was calculated as follows:(1)ABTS radical−scavenging rate=1−Am−AnAk×100%.


## 2.7. OH Radical-Scavenging Ability

To evaluate the OH free radical-scavenging ability, we used the method previously described by Zhang et al. with some modifications [23]. Briefly, 2 mL of 6 mmol/L ferrous sulfate and 2 mL of 6 mmol/L hydrogen peroxide were added to 2 mL of test sample. After mixing, the sample was placed at 25°C for 10 min, and 2 mL of 6 mmol/L salicylic acid–ethanol solution was then added. After mixing, the sample was allowed to react for 30 min at 25°C, and the absorbance at 510 nm was then measured (A1). Distilled water was used instead of hydrogen peroxide for the sample control (A2). Distilled water was used to replace the sample for the blank control (A0). The OH radical-scavenging rate was calculated as follows:(2)OH radical−scavenging rate=1−A1−A2A0×100%.


## 2.8. Superoxide Anion Radical-Scavenging Ability

To assay superoxide anion radical-scavenging ability, the pyrogallol autoxidation method was performed as previously reported [24]. Briefly, the reaction mixture contained 4.5 mL of 50 mmol/L Tris-HCl buffer solution (pH 8.2), 1 mL of the test sample, and 4.0 mL of 25 mmol/L pyrogallol solution. The mixture was placed in a water bath at 25°C for 5 min. After removing the mixture from the water bath, 1 mL of 8 mol/L hydrochloric acid solution was immediately added, and the absorbance at 325 nm was measured (Af). The blank control used distilled water instead of sample solution to determine the absorbance (A0). Distilled water was used to replace the pyrogallol solution for the sample control (An). The superoxide anion radical-scavenging rate was calculated as follows:(3)Superoxide anion radical−scavenging rate=1−Af−AnA0×100%.


## 2.9. DPPH Radical-Scavenging Ability

To determine the DPPH radical-scavenging ability, the method described by Chen et al. was utilized [25]. In brief, 1.0 mL of the test sample was mixed with 0.1 mmol/L DPPH-ethanol solution (Ai), and the blank control used absolute ethanol to replace the sample (A0). Absolute ethanol was used to replace the DPPH–ethanol solution for the sample control (Aj). Each mixture was placed in the dark at 25°C for 30 min, and the absorbance at 510 nm was then measured. The DPPH radical-scavenging rate was calculated as follows:(4)DPPH radical−scavenging rate=1−Ai−AjA0×100%.


## 2.10. UPLC-Triple-TOF/MS Conditions

The test sample was passed through a 0.22μm filter membrane for component analysis. The UPLC-triple-TOF/MS system was used for qualitative analysis of components (Acquity™ ultra-high-performance liquid chromatography; Waters, USA), and Triple TOF 5600 + time-of-flight mass spectrometry equipped with electrospray ion source (AB Sciex Company, USA) was used. The liquid-phase conditions were as follows: a Waters Acquity UPLC HSS T3 column (150 mm × 3.0 mm, 1.8 μm i.d.), 280 nm UV detection wavelength, 50°C column temperature, 0.3 mL/min flow rate, and 3 μL injection volume. Mobile phase A was comprised of a 0.1% formic acid aqueous solution, and mobile phase B was comprised of 0.1% formic acid acetonitrile. A binary gradient elution was employed, and the consecutive program was as follows: 95%–74% A from 0 to 25 min, 74%–5% A from 25 to 33 min, and 5%–90% A from 33 to 34 min. The column was then returned to its starting conditions for 30 min for column balance.The parameters of the MS/MS detector were as follows: triple TOF 5600 + time-of-flight mass spectrometer equipped with positive and negative-ion scanning modes; 5500 V(+) and −4500 V(−) ion sources; scan range ofm/z 100 to m/z 1500; GS1 nebulizing gas at 50 psi; GS2 nebulizing gas at 50 psi; declustering potential at 1000 V; collision energy at 10 V. The TOF MS–production and information-dependent acquisition modes were simultaneously used to collect mass spectrum data, and an automated calibration delivery system was used for automatic calibration.


## 2.11. Statistical Analysis

Each experiment was performed three times, and the data are expressed as mean ± standard deviation (SD). Excel 2010 (Microsoft Corp., Redmond, WA, USA) and SPSS software (version 20.0; IBM Corp., Armonk, NY) were used for statistical analysis of the data. Analysis of variance (ANOVA) was used to test the significant differences. Duncan’s multiple range tests were used for intragroup significance analysis.P<0.05 indicated a significant difference. The column charts were generated using Origin Pro (version 2018, OriginLab Corporation, Northampton, MA, USA).


## 3. Results and Discussion

### 3.1. Effect of In Vitro Digestion on the Release of Phenolic Compounds from RTBC

Using in vitro digestion technology to evaluate the release of total phenolic compounds and flavonoids during digestion can indirectly reflect the health effect of RTBC. In the present study, the dissolution of phenolic compounds in the supernatant of the in vitro digestion system was regarded as the release of total phenolic compounds and total flavonoids.Figure1 shows the release of total phenolic compounds and total flavonoids from the RTBC before and after in vitro digestion. Overall, the release of total phenolic compounds and total flavonoids from the RTBC increased significantly after in vitro digestion, and the changing trend was intestinal digestion > oral digestion > gastric digestion.Figure 1
Changes of phenolic compounds and flavonoids from the RTBC during in vitro digestion. Data are expressed as the mean ± standard deviation (n = 3). (a–d) Different lowercase letters represent significant different among digestion groups. (A–D) Different uppercase letters represent the significant different among the control groups (P<0.05).
(a)(b)Regarding the release of total phenolic compounds from the RTBC, the TPC in each digestion group was significantly higher than that in the initial digestion stage (the moment when oral digestion began). In the initial stage of digestion, the TPC of the RTBC was 1.17 g/100 g (Figure1(a)). After oral digestion, the release of total phenolic compounds increased, and the final release of total phenolic compounds was 1.52 g/100 g, which was consistent with the lower increase in the release of brown rice in oral digestion [26]. After gastric digestion, the total phenolic compounds release from the RTBC slightly decreased, and the final release of total phenolic compounds was 1.36 g/100 g. However, the release of total phenolic compounds significantly increased after simulated intestinal digestion, and the final release of total phenolic compounds was 1.92 g/100 g, which was 1.64 and 1.42 times higher than that in the initial stage digestion and gastric digestion, respectively. Similarly, the release of total phenolic compounds from red and yellow quinoa shows a decrease and then an increase during gastric and intestinal digestion [27]. In vitro digestion experiments for red, black, and white quinoa have shown that the release of phenolic compounds from white quinoa is significantly lower after gastric digestion, and there is no significant change in the phenolic compound release of red and black quinoa [28]. Tenore et al. found that some phenolic compounds may be lost in gastric fluid and may be released during intestinal digestion [29]. Therefore, these findings indicated that the release of total phenolic compounds decreases in the process of gastric digestion and increases in the process of intestinal digestion.Regarding the release of TFC from the RTBC during in vitro simulated digestion, the release of flavonoids in the digestion group was greater than that of the insitial digestion stage (Figure1(b)). After oral digestion, the release of flavonoids from the RTBC increased from 2.83 to 4.62 g/100 g. Combined with the release effect of phenolic compounds during oral digestion, we speculated that α-amylase hydrolyzes the starch coating phenolic compounds and flavonoids in this digestion stage, thereby dissolving more phenolic compounds and flavonoids. After gastric digestion, the release of flavonoids from the RTBC was 3.53 g/100 g. In this digestion stage, the release of flavonoids decreased and was significantly lower than that in oral digestion. The reason for the lower release may be due to pepsin hydrolyzing grain protein; the interaction between protein and phenolic compounds hinders the degradation and digestion in the digestion environment. For example, sorghum hull flavonoids combine with pepsin during gastric digestion, which reduces their release [30]. With the progression of digestion stages, the content of flavonoids rapidly increased in the RTBC, and the maximum release in this digestion stage was 5.98 g/100 g, which was 2.11 times that in the initial digestion and 1.70 times that in gastric digestion.Overall, during the in vitro digestion, the changing trend of total phenolic compounds and total flavonoids in the RTBC was the same. The changing trend of total phenolic compounds and total flavonoids in the control digestion group was the same as that in the digestion group, and the release in the digestion group was greater than that in the control group. Thus, these results suggested that digestion fluid and enzymes promote the release of phenolic compounds and flavonoids from the RTBC. After in vitro digestion, the release of phenolic compounds and flavonoids from the RTBC increased, similar to that from brown rice and buckwheat [31, 32].We analyzed changes in the release of total phenolic compounds and flavonoids during in vitro digestion. The reason for the release of total phenolic compounds and flavonoids may be that, with the RTBC under an extreme pH environment and the presence of digestive enzymes (such asα-amylase, pepsin, trypsin, and other digestive enzymes), the components that were originally compounded with phenolic compounds and flavonoids in the sample were hydrolyzed and destroyed, resulting in polysaccharide separation, starch hydrolysis, protein hydrolysis, and ester bond breaking. Pepsin and trypsin promote breaking of carboxyl groups. The pancreatic lipase contained in pancreatin hydrolyzes ester bonds. Because amylase hydrolyzes glycosidic bonds in starch, phenolic compounds are released without restraint [33], which may have attributed to the significant increase in phenolic compounds and flavonoids in the RTBC digestive fluid.


### 3.2. Effect of In Vitro Digestion on the Antioxidant Activity of the RTBC

As natural antioxidants, phenolic compounds scavenge free radicals, inhibit the production of oxygen free radicals, activate antioxidant enzymes, or regulate the synthesis of related endogenous antioxidants [34]. To evaluate the effect of in vitro digestion on the antioxidant function of the RTBC, we measured the ABTS-, OH-, superoxide-, and DPPH radical-scavenging abilities (Figure 2).Figure 2
Effect of antioxidant activity of the RTBC in vitro digestion. Data are expressed as the mean ± standard deviation (n = 3). (a–d) Different lowercase letters represent significant different among digestion groups. (A–D) Different uppercase letters represent the significant different among the control groups (P<0.05).
(a)(b)(c)(d)Regarding the ABTS radical-scavenging ability, the scavenging rate of ABTS radicals by the RTBC digestive solution was consistent with the release of phenolic compounds during in vitro digestion. After the oral, gastric, and intestinal digestion stages, the scavenging rates of the RTBC digestive solution for ABTS radicals were 1.11, 1.01, and 1.24 times higher than that in the initial stage of digestion, respectively (Figure2(a)). Thus, the antioxidant activity of the RTBC was significantly enhanced during oral, gastric, and intestinal digestion. In addition, the ABTS radical-scavenging ability of the RTBC was the strongest after intestinal digestion with the final scavenging rate reaching 93.27%.The superoxide radical-scavenging ability of the RTBC digestive solution was consistent with that of the ABTS-scavenging ability as shown in Figure2(b). After the oral digestion stage, the rate of superoxide radical scavenging was 87.24%, which was 1.06 times higher than that in the initial stage of digestion. In the gastric digestion stage, the antioxidant activity of the RTBC digestive solution showed a weakening trend, and the scavenging rate of superoxide radicals was 83.68%, which was 1.02 times higher than that of the initial digestion. In the intestinal digestion stage, the superoxide radical-scavenging rate of the RTBC digestive solution significantly increased, and the final scavenging rate was 91.56%, which was 1.11, 1.04, and 1.09 times higher than that in the initial digestion stage, oral digestion stage, and gastric digestion stage, respectively.The OH radical-scavenging ability of the RTBC digestive solution was consistent with that of the ABTS- and superoxide radical-scavenging abilities. After oral, gastric, and intestinal in vitro digestion, the scavenging rates of OH radicals by the RTBC were 1.54, 0.92, and 2.65 times higher than the initial digestion stage, respectively, and the scavenging rate of the intestinal digestion stage was 83.81% (Figure2(c)). Compared with the first two antioxidant evaluation models, in vitro digestion had the greatest effect on the scavenging of OH radicals by the RTBC.The DPPH-radical scavenging abilities of the RTBC during the in vitro digestion stages are shown in Figure2(d). In the oral and gastric digestion stages, the DPPH radical-scavenging ability of the RTBC had a changing trend similar to those of the ABTS-, OH- and superoxide-scavenging abilities. After the oral digestion stage, the DPPH radical-scavenging ability increased to 78.07%, which was 1.06 times higher than that of the initial digestion stage. After the gastric digestion stage, the scavenging rate of DPPH radicals slightly decreased with a scavenging rate of 75.58%, which was 1.03 times higher than that of the initial digestion stage. However, compared with the oral digestion stage, the DPPH radical-scavenging rate slightly decreased, resulting in a rate 0.97 times higher than that of the oral digestion stage. The radical-scavenging rate also decreases in the gastric digestion stage of the seed of Job’s tears [35]. In contrast to the changing trends of the other three antioxidant models, the DPPH radical-scavenging ability of the RTBC was decreased after the intestinal digestion stage, and the final scavenging rate was 37.24%. These results indicated that the sample had a stronger effect on the DPPH-radical scavenging ability at the oral and gastric digestion stages but a weaker effect at the intestinal digestion stage. The rapid loss of antioxidant activity after the intestinal digestion stage occurs in other foods, such as bamboo leaf soup [20], persimmon fruit [13], pomegranate peel [36], and some Brazilian fruits [37].In summary, the antioxidant ability of the RTBC was higher in all the digestion stages compared with that in the control digestion group. Regarding the antioxidant models, the changing trends of the ABTS-, OH-, and superoxide radical-scavenging rates of the RTBC during in vitro digestion were the same, showing that the radical-scavenging ability was significantly enhanced after in vitro digestion, which was consistent with the antioxidant level of the in vitro digestive solution of most grain crops. In contrast, the scavenging rate of DPPH radicals decreased after the intestinal digestion stage. Chen et al. showed that 84% of the antioxidant activity of blackberry is lost after in vitro digestion [38], which may be related to the interaction of phenolic compounds produced by digestion with polysaccharides and active peptides, thereby weakening the activity of phenolic compounds [39].


### 3.3. Correlation Analysis of the Release of TPC and TFC with Antioxidant Activity

Table1 shows the correlation analysis of the release of total phenolic compounds and total flavonoids with antioxidant activity of the RTBC after in vitro digestion. In the ABTS-, OH-, superoxide, and DPPH radical antioxidant models, there was a certain correlation between the antioxidant activity and the release of phenolic compounds with greater releases of phenolic compounds indicating stronger antioxidant activity. Among the evaluation methods for ABTS-, OH-, and superoxide radicals, the release of both total phenolic compounds and total flavonoids had the strongest correlation with the superoxide radical-scavenging ability (R2 = 0.995, 0.992; P<0.05). In the DPPH radical-scavenging model, the oral and gastric digestion solution of the RTBC had a strong correlation with the release of total phenolic compounds and total flavonoids, but the intestinal digestion solution did not correlate with the release of these components. The four antioxidant evaluation models demonstrated that the oral, gastric, and intestinal digestion stages helped to release the phenolic compounds from the RTBC and enhanced the antioxidant activity. These results were similar to those obtained by Lu et al. [40] and Li et al. [28].Table 1
Correlation analysis between the release of total phenolic compounds or total flavonoids and antioxidant activities of the RTBC.
ItemABTS radicalOH radicalSuperoxide radicalDPPH radical (oral;, gastric)Release of phenolic compounds0.965∗0.962∗0.995∗∗0.997∗Release of flavonoids0.985∗0.954∗0.992∗0.997∗∗Significantly correlated at the 0.05 level (two-sided). ∗∗Significantly correlated at the 0.01 level (two-sided).


### 3.4. Analysis of Phenolic Compound Components

There were many types of phenolic compounds. Ambigaipalan et al. [41] showed that the changes in the TPC of plant extracts and antioxidant activity may be due to changes in a single phenolic compound component. To explore the changes of phenolic compound components in the RTBC during in vitro digestion, the RTBC digestive solution was analyzed by UPLC-Triple-TOF/MS. The spectrum of the identified phenolic compound components, as shown in Figure 3 and Table 2 shows the specific components.Figure 3
Chromatogram of phenolic compounds in the RTBC during in vitro digestion.Table 2
Phenolic compounds of the RTBC during in vitro digestion.
PeakRt (min)Tentative identificationMolecular formulam/zArea% (oral)Area% (gastric)Area% (intestinal)18.17Quercetin-3-O-robinoside-7-O-sophorosideC39H50O26933.25+++28.77Quercetin-3-O-neohesperidoside-7-O-glucosideC33H40O21771.19++−311.41Forsythobiflavone AC54H58O321217.28+−+412.98Forsythobiflavone BC54H58O321217.28+−+513.38Quercetin-3-O-rutinoside-7-O-glucosideC33H40O21771.19+−−615.04RutinC27H30O16609.28+++715.59IsoquercetinC21H20O12463.08+++817.44Ferulic acidC10H10O4195.06+++Based on SciFinder and Reaxys database searches, eight types of phenolic compounds (quercetin-3-O-robinoside-7-O-sophoroside, quercetin-3-O-neohesperidoside-7-O-glucoside, forsythobiflavone A, forsythobiflavone B, quercetin-3-O-rutinoside 7-O-glucoside, rutin, isoquercetin, and ferulic acid) were identified in the RTBC after in vitro digestion, and the release conditions were slightly different in each digestion stage as shown in Figure 3 and Table 2. At the oral digestion stage, these eight types of phenolic compounds were released. At the gastric digestion stage, forsythobiflavone A, forsythobiflavone B, and quercetin-3-O-rutinoside-7-O-glucoside were not detected. At the intestinal digestion stage, quercetin-3-O-neohesperidoside-7-O-glucoside and quercetin-3-O-rutinoside-7-O-glucoside were not detected. Regarding the entire digestion process, the relative content of detectable phenolic compounds was the largest in the intestinal digestion stage, which was consistent with the strongest antioxidant activity of the RTBC in the intestinal digestion stage. During the entire digestion process, phenolic monomers were released at different digestion stages. After in vitro digestion, the RTBC mainly released quercetin-3-O-rutinoside-7-O-glucoside, rutin, isoquercetin, and ferulic acid, which was similar to the in vitro digestion results for quinoa [27] and wheat seed [42]. In most studies, the proportion of bound phenolic compounds in cereals accounts for more than 90% of the TPC [43]. Thus, we speculated that a high proportion of bound phenolic compounds in the RTBC is released by the destruction of ester bonds and ether bonds through enzymatic hydrolysis and acid hydrolysis during in vitro digestion, resulting in good antioxidant activity.


## 3.1. Effect of In Vitro Digestion on the Release of Phenolic Compounds from RTBC

Using in vitro digestion technology to evaluate the release of total phenolic compounds and flavonoids during digestion can indirectly reflect the health effect of RTBC. In the present study, the dissolution of phenolic compounds in the supernatant of the in vitro digestion system was regarded as the release of total phenolic compounds and total flavonoids.Figure1 shows the release of total phenolic compounds and total flavonoids from the RTBC before and after in vitro digestion. Overall, the release of total phenolic compounds and total flavonoids from the RTBC increased significantly after in vitro digestion, and the changing trend was intestinal digestion > oral digestion > gastric digestion.Figure 1
Changes of phenolic compounds and flavonoids from the RTBC during in vitro digestion. Data are expressed as the mean ± standard deviation (n = 3). (a–d) Different lowercase letters represent significant different among digestion groups. (A–D) Different uppercase letters represent the significant different among the control groups (P<0.05).
(a)(b)Regarding the release of total phenolic compounds from the RTBC, the TPC in each digestion group was significantly higher than that in the initial digestion stage (the moment when oral digestion began). In the initial stage of digestion, the TPC of the RTBC was 1.17 g/100 g (Figure1(a)). After oral digestion, the release of total phenolic compounds increased, and the final release of total phenolic compounds was 1.52 g/100 g, which was consistent with the lower increase in the release of brown rice in oral digestion [26]. After gastric digestion, the total phenolic compounds release from the RTBC slightly decreased, and the final release of total phenolic compounds was 1.36 g/100 g. However, the release of total phenolic compounds significantly increased after simulated intestinal digestion, and the final release of total phenolic compounds was 1.92 g/100 g, which was 1.64 and 1.42 times higher than that in the initial stage digestion and gastric digestion, respectively. Similarly, the release of total phenolic compounds from red and yellow quinoa shows a decrease and then an increase during gastric and intestinal digestion [27]. In vitro digestion experiments for red, black, and white quinoa have shown that the release of phenolic compounds from white quinoa is significantly lower after gastric digestion, and there is no significant change in the phenolic compound release of red and black quinoa [28]. Tenore et al. found that some phenolic compounds may be lost in gastric fluid and may be released during intestinal digestion [29]. Therefore, these findings indicated that the release of total phenolic compounds decreases in the process of gastric digestion and increases in the process of intestinal digestion.Regarding the release of TFC from the RTBC during in vitro simulated digestion, the release of flavonoids in the digestion group was greater than that of the insitial digestion stage (Figure1(b)). After oral digestion, the release of flavonoids from the RTBC increased from 2.83 to 4.62 g/100 g. Combined with the release effect of phenolic compounds during oral digestion, we speculated that α-amylase hydrolyzes the starch coating phenolic compounds and flavonoids in this digestion stage, thereby dissolving more phenolic compounds and flavonoids. After gastric digestion, the release of flavonoids from the RTBC was 3.53 g/100 g. In this digestion stage, the release of flavonoids decreased and was significantly lower than that in oral digestion. The reason for the lower release may be due to pepsin hydrolyzing grain protein; the interaction between protein and phenolic compounds hinders the degradation and digestion in the digestion environment. For example, sorghum hull flavonoids combine with pepsin during gastric digestion, which reduces their release [30]. With the progression of digestion stages, the content of flavonoids rapidly increased in the RTBC, and the maximum release in this digestion stage was 5.98 g/100 g, which was 2.11 times that in the initial digestion and 1.70 times that in gastric digestion.Overall, during the in vitro digestion, the changing trend of total phenolic compounds and total flavonoids in the RTBC was the same. The changing trend of total phenolic compounds and total flavonoids in the control digestion group was the same as that in the digestion group, and the release in the digestion group was greater than that in the control group. Thus, these results suggested that digestion fluid and enzymes promote the release of phenolic compounds and flavonoids from the RTBC. After in vitro digestion, the release of phenolic compounds and flavonoids from the RTBC increased, similar to that from brown rice and buckwheat [31, 32].We analyzed changes in the release of total phenolic compounds and flavonoids during in vitro digestion. The reason for the release of total phenolic compounds and flavonoids may be that, with the RTBC under an extreme pH environment and the presence of digestive enzymes (such asα-amylase, pepsin, trypsin, and other digestive enzymes), the components that were originally compounded with phenolic compounds and flavonoids in the sample were hydrolyzed and destroyed, resulting in polysaccharide separation, starch hydrolysis, protein hydrolysis, and ester bond breaking. Pepsin and trypsin promote breaking of carboxyl groups. The pancreatic lipase contained in pancreatin hydrolyzes ester bonds. Because amylase hydrolyzes glycosidic bonds in starch, phenolic compounds are released without restraint [33], which may have attributed to the significant increase in phenolic compounds and flavonoids in the RTBC digestive fluid.


## 3.2. Effect of In Vitro Digestion on the Antioxidant Activity of the RTBC

As natural antioxidants, phenolic compounds scavenge free radicals, inhibit the production of oxygen free radicals, activate antioxidant enzymes, or regulate the synthesis of related endogenous antioxidants [34]. To evaluate the effect of in vitro digestion on the antioxidant function of the RTBC, we measured the ABTS-, OH-, superoxide-, and DPPH radical-scavenging abilities (Figure 2).Figure 2
Effect of antioxidant activity of the RTBC in vitro digestion. Data are expressed as the mean ± standard deviation (n = 3). (a–d) Different lowercase letters represent significant different among digestion groups. (A–D) Different uppercase letters represent the significant different among the control groups (P<0.05).
(a)(b)(c)(d)Regarding the ABTS radical-scavenging ability, the scavenging rate of ABTS radicals by the RTBC digestive solution was consistent with the release of phenolic compounds during in vitro digestion. After the oral, gastric, and intestinal digestion stages, the scavenging rates of the RTBC digestive solution for ABTS radicals were 1.11, 1.01, and 1.24 times higher than that in the initial stage of digestion, respectively (Figure2(a)). Thus, the antioxidant activity of the RTBC was significantly enhanced during oral, gastric, and intestinal digestion. In addition, the ABTS radical-scavenging ability of the RTBC was the strongest after intestinal digestion with the final scavenging rate reaching 93.27%.The superoxide radical-scavenging ability of the RTBC digestive solution was consistent with that of the ABTS-scavenging ability as shown in Figure2(b). After the oral digestion stage, the rate of superoxide radical scavenging was 87.24%, which was 1.06 times higher than that in the initial stage of digestion. In the gastric digestion stage, the antioxidant activity of the RTBC digestive solution showed a weakening trend, and the scavenging rate of superoxide radicals was 83.68%, which was 1.02 times higher than that of the initial digestion. In the intestinal digestion stage, the superoxide radical-scavenging rate of the RTBC digestive solution significantly increased, and the final scavenging rate was 91.56%, which was 1.11, 1.04, and 1.09 times higher than that in the initial digestion stage, oral digestion stage, and gastric digestion stage, respectively.The OH radical-scavenging ability of the RTBC digestive solution was consistent with that of the ABTS- and superoxide radical-scavenging abilities. After oral, gastric, and intestinal in vitro digestion, the scavenging rates of OH radicals by the RTBC were 1.54, 0.92, and 2.65 times higher than the initial digestion stage, respectively, and the scavenging rate of the intestinal digestion stage was 83.81% (Figure2(c)). Compared with the first two antioxidant evaluation models, in vitro digestion had the greatest effect on the scavenging of OH radicals by the RTBC.The DPPH-radical scavenging abilities of the RTBC during the in vitro digestion stages are shown in Figure2(d). In the oral and gastric digestion stages, the DPPH radical-scavenging ability of the RTBC had a changing trend similar to those of the ABTS-, OH- and superoxide-scavenging abilities. After the oral digestion stage, the DPPH radical-scavenging ability increased to 78.07%, which was 1.06 times higher than that of the initial digestion stage. After the gastric digestion stage, the scavenging rate of DPPH radicals slightly decreased with a scavenging rate of 75.58%, which was 1.03 times higher than that of the initial digestion stage. However, compared with the oral digestion stage, the DPPH radical-scavenging rate slightly decreased, resulting in a rate 0.97 times higher than that of the oral digestion stage. The radical-scavenging rate also decreases in the gastric digestion stage of the seed of Job’s tears [35]. In contrast to the changing trends of the other three antioxidant models, the DPPH radical-scavenging ability of the RTBC was decreased after the intestinal digestion stage, and the final scavenging rate was 37.24%. These results indicated that the sample had a stronger effect on the DPPH-radical scavenging ability at the oral and gastric digestion stages but a weaker effect at the intestinal digestion stage. The rapid loss of antioxidant activity after the intestinal digestion stage occurs in other foods, such as bamboo leaf soup [20], persimmon fruit [13], pomegranate peel [36], and some Brazilian fruits [37].In summary, the antioxidant ability of the RTBC was higher in all the digestion stages compared with that in the control digestion group. Regarding the antioxidant models, the changing trends of the ABTS-, OH-, and superoxide radical-scavenging rates of the RTBC during in vitro digestion were the same, showing that the radical-scavenging ability was significantly enhanced after in vitro digestion, which was consistent with the antioxidant level of the in vitro digestive solution of most grain crops. In contrast, the scavenging rate of DPPH radicals decreased after the intestinal digestion stage. Chen et al. showed that 84% of the antioxidant activity of blackberry is lost after in vitro digestion [38], which may be related to the interaction of phenolic compounds produced by digestion with polysaccharides and active peptides, thereby weakening the activity of phenolic compounds [39].


## 3.3. Correlation Analysis of the Release of TPC and TFC with Antioxidant Activity

Table1 shows the correlation analysis of the release of total phenolic compounds and total flavonoids with antioxidant activity of the RTBC after in vitro digestion. In the ABTS-, OH-, superoxide, and DPPH radical antioxidant models, there was a certain correlation between the antioxidant activity and the release of phenolic compounds with greater releases of phenolic compounds indicating stronger antioxidant activity. Among the evaluation methods for ABTS-, OH-, and superoxide radicals, the release of both total phenolic compounds and total flavonoids had the strongest correlation with the superoxide radical-scavenging ability (R2 = 0.995, 0.992; P<0.05). In the DPPH radical-scavenging model, the oral and gastric digestion solution of the RTBC had a strong correlation with the release of total phenolic compounds and total flavonoids, but the intestinal digestion solution did not correlate with the release of these components. The four antioxidant evaluation models demonstrated that the oral, gastric, and intestinal digestion stages helped to release the phenolic compounds from the RTBC and enhanced the antioxidant activity. These results were similar to those obtained by Lu et al. [40] and Li et al. [28].Table 1
Correlation analysis between the release of total phenolic compounds or total flavonoids and antioxidant activities of the RTBC.
ItemABTS radicalOH radicalSuperoxide radicalDPPH radical (oral;, gastric)Release of phenolic compounds0.965∗0.962∗0.995∗∗0.997∗Release of flavonoids0.985∗0.954∗0.992∗0.997∗∗Significantly correlated at the 0.05 level (two-sided). ∗∗Significantly correlated at the 0.01 level (two-sided).


## 3.4. Analysis of Phenolic Compound Components

There were many types of phenolic compounds. Ambigaipalan et al. [41] showed that the changes in the TPC of plant extracts and antioxidant activity may be due to changes in a single phenolic compound component. To explore the changes of phenolic compound components in the RTBC during in vitro digestion, the RTBC digestive solution was analyzed by UPLC-Triple-TOF/MS. The spectrum of the identified phenolic compound components, as shown in Figure 3 and Table 2 shows the specific components.Figure 3
Chromatogram of phenolic compounds in the RTBC during in vitro digestion.Table 2
Phenolic compounds of the RTBC during in vitro digestion.
PeakRt (min)Tentative identificationMolecular formulam/zArea% (oral)Area% (gastric)Area% (intestinal)18.17Quercetin-3-O-robinoside-7-O-sophorosideC39H50O26933.25+++28.77Quercetin-3-O-neohesperidoside-7-O-glucosideC33H40O21771.19++−311.41Forsythobiflavone AC54H58O321217.28+−+412.98Forsythobiflavone BC54H58O321217.28+−+513.38Quercetin-3-O-rutinoside-7-O-glucosideC33H40O21771.19+−−615.04RutinC27H30O16609.28+++715.59IsoquercetinC21H20O12463.08+++817.44Ferulic acidC10H10O4195.06+++Based on SciFinder and Reaxys database searches, eight types of phenolic compounds (quercetin-3-O-robinoside-7-O-sophoroside, quercetin-3-O-neohesperidoside-7-O-glucoside, forsythobiflavone A, forsythobiflavone B, quercetin-3-O-rutinoside 7-O-glucoside, rutin, isoquercetin, and ferulic acid) were identified in the RTBC after in vitro digestion, and the release conditions were slightly different in each digestion stage as shown in Figure 3 and Table 2. At the oral digestion stage, these eight types of phenolic compounds were released. At the gastric digestion stage, forsythobiflavone A, forsythobiflavone B, and quercetin-3-O-rutinoside-7-O-glucoside were not detected. At the intestinal digestion stage, quercetin-3-O-neohesperidoside-7-O-glucoside and quercetin-3-O-rutinoside-7-O-glucoside were not detected. Regarding the entire digestion process, the relative content of detectable phenolic compounds was the largest in the intestinal digestion stage, which was consistent with the strongest antioxidant activity of the RTBC in the intestinal digestion stage. During the entire digestion process, phenolic monomers were released at different digestion stages. After in vitro digestion, the RTBC mainly released quercetin-3-O-rutinoside-7-O-glucoside, rutin, isoquercetin, and ferulic acid, which was similar to the in vitro digestion results for quinoa [27] and wheat seed [42]. In most studies, the proportion of bound phenolic compounds in cereals accounts for more than 90% of the TPC [43]. Thus, we speculated that a high proportion of bound phenolic compounds in the RTBC is released by the destruction of ester bonds and ether bonds through enzymatic hydrolysis and acid hydrolysis during in vitro digestion, resulting in good antioxidant activity.


## 4. Conclusion

In summary, the present study considered the phenolic compound content and antioxidant activity in the RTBC as the indexes, used in vitro simulated digestion technology, and performed qualitative analysis by UPLC-triple-TOF/MS detection technology to investigate the changes in phenolic compound content and structure-activity relationship of the composite of tartary buckwheat (coarse cereal) and rice (staple grain) during in vitro digestion. The in vitro digestion analysis showed that the release of total phenolic compounds and total flavonoids significantly increased and the release occurred mainly in the process of oral and intestinal digestion. Regarding the antioxidant activity, the digestive solution of the RTBC exhibited strong antioxidant activity. Overall, the release of total phenolic compounds and total flavonoids was consistent with the changing trend of antioxidant activity with a strong positive correlation. The results were consistent with the changes in quinoa phenolic compounds and antioxidant activity during in vitro digestion [44]; after in vitro digestion, the concentration of quinoa flavonoids is higher, and the antioxidant activity is enhanced. In addition, qualitative analysis of phenolic compounds in the digestion solution of the RTBC by UPLC-triple-TOF/MS found that the phenolic compounds released in each digestion stage varied. A total of eight types of phenolic compounds were identified. Among these compounds, quercetin-3-O-robinoside-7-O-sophoroside, rutin, isoquercetin, and ferulic acid were continuously and significantly released in the oral, gastric, and intestinal digestion stages. The correlation analysis of phenolic compounds and flavonoids with antioxidant activity in each digestion stage further confirmed that phenolic compounds and flavonoids influence the antioxidant function. Singh et al. [45] showed that phenolic compounds, especially flavonoids, are powerful antioxidants and effective free radical scavengers, and they suggested that they have broad prospects in antiinflammatory studies, antioxidant studies, heart protection, and other types of health protection. These results suggest that flavonoid-rich foods should be developed in the future. Tartary buckwheat, as the crop with the highest content of flavonoids in common coarse cereals, plays an important role in the development of functional food and the application of healthy food. The consumption of tartary buckwheat and rice composite has broad research potential, and this composite can make up for the lack of nutritional components of rice as well as improving the problems of coarse taste and limited processing methods of cereals. The RTBC not only satisfies the staple grain providing energy for body metabolism but also improves the bioavailability of unique antioxidant active components in coarse cereals. The present study investigated and analyzed the changes in characteristic components in the digestion process of the RTBC, and it explored the relationship between its functional components and antioxidant ability, which is of great significance to the research and development of new tartary buckwheat-rice compound food and the development of health functional food. The present study also laid a foundation for further exploring the antioxidation of the RTBC and understanding its potential mechanism of digestion and absorption.



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*Source: 2472513-2022-06-23.xml*