Changes in phenolic composition and bioactivity of raw and pickled cowpea (Vigna unguiculata L. Walp.) green pod after in vitro simulated gastrointestinal digestion

Abstract

Here, the changes in phenolic composition and antioxidant activities (ABTS, DPPH, and FRAP) of raw and pickled cowpea green pod during in vitro simulated gastrointestinal digestion (GID) were investigated. The inhibitory activities of the undigested and digested samples towards α-amylase, α-glucosidase and lipase were determined. Results revealed that total phenolic (TP) and flavonoid (TF) contents of both raw and pickled samples were increased after gastric and intestinal digestion, and the pickled sample had lower TP and TF content after each digestion phase. A similar trend was observed for antioxidant activities, which were significantly correlated with TP and TF contents (p < 0.05). Seven phenolic compounds were identified and quantified by HPLC-DAD. Their contents were affected by digestion and pickling, and raw cowpea pod had higher contents before and after GID. Inhibitory effects towards α-amylase, α-glucosidase and lipase were increased for raw cowpea pod after GID. Pickled cowpea pod also showed an increased inhibitory activity towards lipase, while no activity towards α-amylase and α-glucosidase. Raw cowpea pod had higher inhibitory effects towards α-amylase, α-glucosidase and lipase than the pickled one at each digestion phase. Our results showed that pickling process reduced the health-promoting properties of cowpea green pod.

Introduction

Cowpea (Vigna unguiculata L. Walp.) is a widely adapted, versatile, stress tolerant crop grown in warm to hot regions of the world (Ehlers and Hall, 1997). The crop can be used for food and animal fodder. Cowpea used for food is eaten primarily as grain seeds (also named as pulses). Moreover, its fresh peas and green pods can also be consumed as vegetables.

Cowpea is popularly grown for its immature green pods in many Southeast Asian countries (Umaharan et al., 1997). The cowpea green pods are staple vegetables in summer and autumn in China. It was reported that cowpea consumed as green pods contain more water, less protein, higher soluble carbohydrates and lower starch content than that consumed as dry pulses, making them tastier than dry pulses (Bhattacharya and Malleshi, 2012). In addition, cowpea green pods are rich in antioxidants and other health-promoting compounds, such as phenolics, carotenoids and vitamin C (Bhattacharya and Malleshi, 2012). Therefore, they could provide a more balanced nutrition (Ntatsi et al., 2018).

Previous studies have demonstrated that cowpea green pods are good sources of phenolic compounds (Karapanos et al., 2017). Growing evidence has confirmed that phenolic compounds from fruits and vegetables possess many health beneficial properties which might prevent some chronic diseases (Hachibamba et al., 2013; Qin et al., 2017). However, fresh cowpea green pods are crisp and tender with high moisture content, making them difficult to store. Pickling process has often been used in cowpea green pods to prolong their storage life in China. The pickled cowpea green pods are traditional fermented vegetable products, and widely consumed by all social groups in China due to their acidic taste, typical flavor and enhancement to people's appetite. Previous studies have indicated that pickling process was a good method for preserving phenolic acids and antioxidants of potherb mustard (Fang et al., 2008).

Although cowpea green pods contained high phenolic content, not all of the phenolics can exert their biological function after digestion. The composition and levels of phenolics could be changed during human digestion, and thus potentially alter their bioactivity (Hachibamba et al., 2013). In vitro simulating digestion model was widely used to assess the effects of food plants on their polyphenol and bioactivity stability due to its simplicity and speed (Thomas-Valdés et al., 2018; Chait et al., 2020). Unfortunately, no studies to date have reported the stability of phenolics and bioactivity of these cowpea pods after digestion process. To better understand their potential benefits, this study evaluated the changes in phenolic compositions of fresh and pickled cowpea green pods before and after in vitro gastrointestinal digestion (GID) and their effects on antioxidant activities and inhibition towards metabolic syndrome-associated enzymes (α-amylase, α-glucosidase and lipase). To our knowledge, this is the first study to evaluate the effects of simulated GID on the polyphenol composition and bioactivity of cowpea green pods, which is believed to provide new insights on the reasonable consumption of pickled cowpea pods.

Materials and Methods

Chemicals and standards    Potassium dihydrogen phosphate (KH₂PO₄), aluminium nitrate, potassium sodium tartrate, ferric chloride, 3,5-dinitrosalicylic acid (DNS), ethanol, sodium hydroxide, and dipotassium phosphate were analytical grade and purchased from Xilong Scientific Co., Ltd. (Shantou, China). Trition X-100, HCl, methanol, formic acid, n-hexane, sodium chloride, glacial acetic acid, magnesium chloride, and sodium bicarbonate were obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Folin-ciocalteau reagent, soluble starch, α-amylase, α-glucosidase, lipase, and the phenolic standards (HPLC grage, purity ≥ 98%): protocatechic, gallic, chlorogenic, vanillic, caffeic, and p-hydroxybenzoic acid, rutin, catechin, and epicatechin were obtained from Hefei Bomei Biotechnology Co., Ltd. (Hefei, China). 2,4,6-Tri(2-pyridyl)-1,3,5-triazine (TPTZ) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Pepsin and p-nitrophenyl palmitate (pNPP) were from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Pancreatin was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Acarbose, orlistat, bile salts p-nitrophenyl-α-D-glucopyranoside, 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Other chemicals were obtained from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China).

Preparation of raw (RCP) and pickled cowpea pods (PCP)    Fresh cowpea (Vigna unguiculata L.Walp.) green pods were obtained from a local farm in Xiangtan, Hunan Province, in July 2018 and transported to our laboratory within 2 h. The cowpea pods were washed and air-dried until no visible water droplet. Subsequently, the cowpea pods were divided into two portions. One portion was retained raw; the other was macerated in an earthenware pot with NaCl brine (50 g/L) for 15 d. The pot were sealed and kept in the shadows at room temperature. The raw and pickled cowpea pods were dried in oven at 60 °C for 12 h. The samples were then ground into powder, sieved by a 80 mesh sieve and stored in refrigerator at 4 °C for the future analysis.

Extraction of total phenolics for undigested samples    The total phenolics were the mixture of free phenolics and bound phenolics. The free and bound phenolics for undigested samples were obtained according to the methods described by Gutiérrez-Uribe et al. (2011). Content of total phenolics was the sum of free and bound phenolics content. The obtained values were taken as 100% for calculations.

In vitro simulated GID    In vitro simulated GID of samples was performed according to the method suggested by Minekus et al. (2014), consisting of three successive phases of oral, gastric and intestinal digestion. The corresponding electrolyte stock solutions of simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared freshly as described in the literature (Minekus et al., 2014), respectively. During oral phase, 5 g sample (flour from raw and pickled cowpea pod) was mixed well with 8.4 mL SSF electrolyte stock solution, and the pH was adjusted to 7 with NaOH solution (1 mol/L). Then 1.2 mL α-amylase solution (1 500 U/mL, prepared in SSF electrolyte stock solution), 60 μL CaCl₂ solution (0.3 mol/L) and 2 340 μL of distilled water were added in turn and fully mixed again. The final volume was made up with distilled water to 24 mL in order to make the enzyme and substrate fully contact. The obtained mixture was placed in an oscillating water bath at 37 °C and 100 rpm for 5 min. After adding 18 mL SGF electrolyte stock solution, 12 μL CaCl₂ solution (0.3 mol/L), and 1 668 µL distilled water to the mixture and adjusting the pH to 3.0 with HCl (1 mol/L), the gastric digestion began. Next, 3.84 mL porcine pepsin solution (1 500 U/mL, prepared in SSF electrolyte stock solution) was added, and kept continuous shaking (100 rpm) for 2 h at 37 °C. Subsequently, intestinal digestion was followed by adding 26.4 mL SIF electrolyte stock solution, 96 µL CaCl₂ solution (0.3 mol/L) and 6 mL bile salts solution (160 mmol/L) to the mixture. After the pH was adjusted to 7 with NaOH solution (1 mol/L), 12 mL pancreatin solution (800 U/mL, prepared in SSF electrolyte stock solution) and 3 144 µL distilled water were added, and stirred at 100 rpm and 37 °C for 2 h. The individual digestions were carried out for each digestion phase.The digestion mixtures were collected at the end of each phase and kept in a water bath at 85 °C for 10 min to inactivate the digestive enzymes. The samples were cooled to room temperature, and centrifuged for 10 min at 6 000 rpm. Then, the supernatant was collected and stored at −80 °C for further analysis. All samples were analysed within 2 weeks.

Total phenolic content (TPC) and total flavonoid content (TFC)    The TPC of different samples was determined by using the method of Majheniè et al. (2007). Gallic acid was used to make a calibration curve. Results were expressed as mg of gallic acid equivalents (mg GAE)/g sample (dry weight, DW). The TFC was determined using the method reported by Qin et al. (2018), with slight modification (Chen et al., 2015). Briefly, 1 mL of sample was mixed with 4 mL 600 mL/L ethanol solution and 1 mL 50 g/L NaNO₂ solution, and left at room temperature for 6 min. Next, 1 mL 100 g/L Al(NO₃)₃ solution was added, and the mixture was held for another 6 min at room temperature. A total of 2 mL 1 mol/L NaOH was then added. The solution was mixed well and kept for 30 min at room temperature. The absorbance was recorded at 510 nm using 600 mL/L ethanol solution as a blank. TFC were determined as rutin equivalents according to the standard calibration curve and expressed as mg rutin equivalents/g sample DW (mg RE/g DW).

DPPH radical scavenging activity    DPPH radical scavenging activity was determined by using the method of Binsan et al. (2008), with minor modification. Sample (3.0 mL) was mixed with 3.0 mL of 0.2 mmol/L DPPH in methanol and kept out of light at room temperature for 30 min. The absorbance was measured at 517 nm. The blank was prepared by replacing the sample with distilled water. Trolox was used to prepare the standard curve. The result was expressed as µmol Trolox equivalents (TE)/g sample.

Ferric reducing antioxidant power (FRAP)    FRAP assay was performed as described by Benzie and Strain (1996), with slight modification. Firstly, the FRAP reagent was prepared as reported in the literature. Then 4.5 mL FRAP solution was mixed with 1 mL sample and the mixture was held at room temperature for 30 min in dark. The absorbance was recorded at 593 nm. Trolox was used to prepare a standard curve. The result was expressed as µmol TE/g sample in DW.

ABTS radical scavenging activity    This assay was performed as reported by Binsan et al. (2008), with slight modification. Firstly, the ABTS•⁺ working solution was prepared as reported in the literature. Subsequently, the solution was diluted with methanol until it had an absorbance of 0.700 ± 0.02 at 734 nm. Next, 5.7 mL diluted ABTS solution was taken out and mixed with 0.3 mL sample, and the mixture was kept out of light at room temperature for 2 h. The absorbance was then measured at 734 nm. Distilled water was used to replace the sample and prepare the control by the same method. Trolox was used to make a standard curve. Result was expressed as µmol TE/g sample in DW.

Determination of phenolic profile by HPLC-DAD analysis    Phenolic content of undigested and digested samples was determined by using a Shimadzu LC-20AT HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a LC-20AT pump, a SPD-M20A UV photodiode array detector (PDA) and a LabSolution software. Samples were filtered through 0.22 µm nylon filters, and separated on a JADE-PAK^® ODS-AQ column (250 mm × 4.6 mm, 5 µm particle size; Guangzhou Techway Biological Technology Co., Ltd., Guangzhou, China) using two solvents system: (A) 5 mL/L formic acid in ultrapure water and (B) 100% methanol. Phenolic compounds were analyzed at 40 °C with 20 µL of sample injection and a gradient elution at 1 mL/min according to the following gradient program: 0–15 min (15–25% B), 15–25 min (25% B), 25–40 min (25–40% B), 40–45 min (40–15% B) and 45–50 min (15% B). The chromatograms were recorded at 280 nm. The available standards were analyzed under the same conditions. Phenolic compounds in samples were then identified by comparing their retention times with those of standards. Their quantification was obtained by the calibration curves of standards. Content of the identified phenolic compound was expressed in µg per gram of dry weight of cowpea pod (raw or pickled) flour.

α-Amylase inhibition assay    The measurement was carried out according to the method reported by Tan et al. (2017). Results were expressed as the sample amount that could inhibit 50% enzyme activity (IC₅₀, mg sample/mL). Acarbose was included as a positive control.

α-Glucosidase inhibition assay    The assay was carried out as described by Tan et al. (2017). Results were expressed as IC₅₀ values (mg sample/mL). Acarbose was included as a positive control.

Lipase inhibition assay    The measurement was performed according to the method reported by Burgos-Edwards et al. (2017). Results were expressed as IC₅₀ values (mg sample/mL). Orlistat was included as a positive control.

Statistical analysis    All determinations were performed in triplicate and the results were expressed as mean values ± standard deviation (SD). The statistical analyses were carried out with SPSS 14.0 software (IBM, Armonk, NY). Statistical differences between the undigested and digested samples for RCP and PCP were analyzed by one-way analysis of variance (ANOVA) and Duncan's multiple comparison tests. The significance level was p < 0.05. The relationships between TPC or TFC and the antioxidant activity was determined by Pearson's correlation coefficient with a significance level of p < 0.05.

Results and Discussion

Effect of simulated GID on TPC and TFC    Table 1 showed TPC and TFC in RCP and PCP obtained before and after in vitro GID. The TPC of RCP was determined to be 10.24 mg GAE/g DW, significantly higher than that of PCP (8.84 mg GAE/g DW) (p < 0.05). The results indicated that TPC of cowpea green pod was reduced upon pickling, with a loss of about 14% total phenolics. Similar results were obtained by Kiai and Hafidi (2014), who reported a loss of TPC in olive flesh during spontaneous fermentation due to diffusion of phenolic compounds into brine. Fang et al. (2008) also reported that total phenolics in potherb mustard decreased during pickling process. The authors think this may be due to phenolics degradation by polyphenol oxidase during pickling (Fang et al., 2008).

Table 1. Changes in total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities of raw and pickled cowpea pods before and after in vitro gastrointestinal digestion

Sample	TPC (mg GAE/g DW)	TFC (mg RE/g DW)	ABTS (µmol TE/g DW)	DPPH (µmol TE/g DW)	FRAP (µmol TE/g DW)
Raw cowpea pod (RCP)
Undigest	10.24 ± 0.77d*	4.68 ± 0.18f	140.84 ± 3.85c	36.87 ± 0.11e	21.84 ± 0.68d
Oral	9.79 ± 0.28de	8.21 ± 0.15d	67.29 ± 6.54d	30.70 ± 1.10fg	15.02 ± 0.34e
Gastric	18.40 ± 0.36c	14.22 ± 0.54b	126.14 ± 2.77c	58.67 ± 1.47c	33.10 ± 3.55b
Intestinal	30.26 ± 1.69a	17.29 ± 1.65a	451.80 ± 1.28b	66.99 ± 1.66b	43.75 ± 0.81a
Pickled cowpea pod (PCP)
Undigest	8.84 ± 0.47e	6.03 ± 0.43e	53.05 ± 3.66d	34.63 ± 0.07ef	18.71 ± 0.32de
Oral	4.98 ± 0.40f	4.44 ± 0.16f	47.93 ± 11.34d	27.06 ± 2.00g	15.64 ± 2.31e
Gastric	10.10 ± 0.06de	7.57 ± 0.35d	119.98 ± 27.96c	51.28 ± 3.99d	20.52 ± 1.55d
Intestinal	27.63 ± 0.64b	9.87 ± 0.40c	531.58 ± 14.71a	86.22 ± 4.41a	27.41 ± 4.43c

*  Values are means ± standard deviations (n = 3) and different letters within the same column indicate significant differences at p < 0.05 from Duncan's test.

After oral digestion, the TPC of RCP showed no significant change, while 43.7% decrease occurred in PCP. The amounts of released phenolics increased significantly for RCP and PCP during gastric and intestinal digestion (p < 0.05). After intestinal digestion, the TPC significantly increased (p < 0.05) to 2.96- and 3.13-fold for RCP and PCP, respectively. Similar results were obtained by Pellegrini et al. (2017) who reported that TPC and TFC of quinoa seeds were increased after gastric and intestinal digestion. Zhang et al. (2017) found that the amount of released phenolics for cooked green lentil increased gradually during gastric to intestinal phase. Results also showed that the TPC in PCP was lower than that of RCP after each digestion phase. The underlying mechanism for the decreased TPC in PCP may be due to diffusion of phenolic compounds into brine during pickling (Kiai and Hafidi, 2014).

As regard TFC, PCP had a higher value than RCP. The TFC changes of RCP and PCP were significant after in vitro GID (p < 0.05). The amount of total flavonoid significantly increased throughout the digestion for RCP (p < 0.05), whereas the change pattern of TFC in PCP was similar to its total phenolics. After oral digestion, PCP had a lower TFC than its undigested sample, whereas an opposite result was obtained for RCP. After intestinal phase, the TFC significantly increased to 3.69- and 1.64-fold for RCP and PCP (p < 0.05), respectively. The increase of TPC and TFC after GID could be caused by the pH and digestive enzymes, which could break the bond of phenolic compounds with proteins, fibre or sugar residues, thus making the bound phenolics released into the digestive juice (Celep et al., 2015). The results also showed that the TFC of PCP was lower than that of RCP after each digestion phase.

Effect of simulated GID on antioxidant activity    In this study, the antioxidant activities of undigested and digested samples were evaluated by using DPPH, ABTS and FRAP assays. Table 1 shows antioxidant activity values obtained after each digestion phase for RCP and PCP. No significant difference was observed in antioxidant activities between the undigested samples of RCP and PCP, except ABTS values. The overall trend of antioxidant activity followed TPC and TFC. There are significant differences in the tested antioxidant activities among the digested samples of RCP and PCP (p < 0.05), and the activities increased gradually throughout the digestion. After intestinal phase, the activities were much higher than those of the corresponding undigested one, indicating an increase in phenolics after the digestive process (Celep et al., 2017).

For ABTS assay, RCP had significantly higher values than PCP before GID (p < 0.05). The ABTS•⁺ scavenging capacity of PCP did not significantly change after oral phase, while the ABTS value of RCP decreased, showing 0.52-fold change. However, there was no significant difference in ABTS value between RCP and PCP. After gastric phase, the ABTS•⁺ scavenging capacity of RCP did not significantly change, while the ABTS values of PCP significantly increased (p < 0.05), with 1.26-fold change. Again the ABTS values showed no significant difference between RCP and PCP. At the end of intestinal phase, ABTS•⁺ scavenging capacity significantly increased in RCP and PCP (p < 0.05), with 2.21- and 9.02-fold changes, respectively. PCP had significantly higher ABTS values than RCP (p < 0.05). Similar results were obtained by Wootton-Beard et al. (2011), who found that gastric and intestinal digestion increased ABTS values of 23 commercially available vegetable juices. Chandrasekara and Shahidi (2012) also reported that ABTS values of millet grains increased after gastric and intestinal digestion.

As for DPPH values, there was no significant difference between RCP and PCP before digestion. After oral digestion, DPPH values of RCP and PCP significantly decreased with respect to their undigested samples (p < 0.05). After gastric and intestinal digestion, DPPH value increased 59.13% and 81.69% for RCP, respectively, and increased 48.08% and 148.97% for PCP. In addition, RCP had a higher value than PCP after gastric phase, but lower after intestinal phase. Chen et al. (2014) also reported that gastric and intestinal digestion increased DPPH inhibition activity of 33 fruits.

Regarding FRAP, again there was no significant difference between RCP and PCP prior to digestion. After oral digestion, FRAP value significantly decreased (p < 0.05) for RCP, but did not change for PCP. No significant difference was observed between RCP and PCP. FRAP value showed a similar trend after gastric digestion, except RCP having higher values than PCP. After intestinal phase, the FRAP value significantly increased 100.30% and 46.50% for RCP and PCP (p < 0.05), respectively. Again, RCP has a higher FRAP value than PCP.

The above results demonstrated that the antioxidant activities of both samples increased during gastric and intestinal digestions, probably due to the release of unextractable antioxidants from the matrix and/or their conversion into other compounds with greater antioxidant activity (Gullon et al., 2015). Pellegrini et al. (2017) obtained similar results which showed that the antioxidant activities increased during in vitro GID.

Many studies have indicated that antioxidant activities of natural plants are related to their TPC and TFC. In order to explain the relationship between TPC or TFC and antioxidant values of RCP and PCP, correlation coefficients (r) were calculated and the results were shown in Table 2. For RCP, there was a strong and positive correlation between TPC or TFC and the antioxidant activities, and the strongest correlation was found between TPC and FRAP (p < 0.01). TPC significantly correlated with TFC (p < 0.01), indicating that flavonoids contribute significantly to the antioxidant activity of RCP. Many studies (Chandrasekara and Shahidi, 2012; Gullón et al., 2015; Lucas-Gonzalez et al., 2016) also showed that there was a high correlation between TPC and antioxidant activity.

Table 2 Pearson's correlation coefficient (r) analysis between total phenolic content (TPC) or total flavonoid content (TFC) and antioxidant activities (ABTS, DPPH, FRAP) from raw and pickled cowpea pods during in vitro gastrointestinal digestion.

		TFC	ABTS	DPPH	FRAP
Raw cowpea pod (RCP)	TPC	0.898**	0.920**	0.930**	0.952**
	TFC		0.698*	0.913**	0.864**
Pickled cowpea pod (PCP)	TPC	0.921**	0.987**	0.969**	0.890**
	TFC		0.885**	0.957**	0.862**

*  Correlation was significant at the 0.05 level.

**  Correlation was significant at the 0.01 level.

For PCP, again a high and positive correlation was found between TPC or TFC and antioxidant activity, with p < 0.01 in all the cases. The strongest correlation was found between TPC and ABTS (p < 0.01). TPC significantly correlated with TFC (p < 0.01). These results suggested that the phenolics and flavonoids released from RCP and PCP during simulated GID play key roles in antioxidant activities of these foods.

Effect of simulated GID on the levels of phenolic compounds    HPLC-DAD analysis was used to determine the effect of in vitro GID on the phenolic composition of RCP and PCP. The HPLC chromatograms of the soluble digesta obtained from raw and pickled cowpea pods at each digestion are presented in Fig. 1. Table 3 shows the phenolic compounds identified and quantified in RCP and PCP before and after GID. Five phenolic acids (p-hydroxybenzoic, protocatechuic, chlorogenic, vanillic and caffeic acid) and two flavanols (catechin and epicatechin) were identified in RCP. However, vanillic acid and epicatechin were not detectable in PCP, probably because these compounds were degraded during pickling or their contents were lower than the quantitation limit (Jara-Palacios et al., 2018). The identified polyphenols have also been detected in dry pulses by different authors (Cai et al., 2011).

Fig. 1.

HPLC chromatogram of a standard phenolics mixture (A) and the soluble digesta obtained from raw cowpea pods (B) or pickled cowpea pods (C) at each digestion (280 nm). Peaks: 1, protocatechuic acid; 2, catechin; 3, p-hydroxybenzoic acid; 4, chlorogenic acid; 5, vanillic acid; 6, caffeic acid; 7, epicatechin.

Table 3 Contents (µg/g) of individual phenolic compounds in raw and pickled cowpea pods during in vitro gastrointestinal digestion

	Protocatechuic acid	Catechin	p-Hydroxybenzoic acid	Chlorogenic acid	Vanillic acid	Caffeic acid	Epicatechin
Raw cowpea pod (RCP)
Undigest	138.33 ± 5.63b*	64.13 ± 6.24d	193.89 ± 11.73a	145.73 ± 8.52d	106.04 ± 26.32b	298.44 ± 18.47a	97.02 ± 5.18c
Oral	52.72 ± 5.01c	146.35 ± 22.85c	41.55 ± 1.89c	43.80 ± 4.81f	90.43 ± 2.45b	10.85 ± 0.60c	146.99 ± 11.05c
Gastric	151.09 ± 21.23b	2110.42 ± 59.72a	73.82 ± 27.78bc	270.80 ± 51.82b	115.04 ± 4.68b	53.24 ± 4.64b	815.11 ± 61.57a
Intestinal	750.94 ± 9.45a	1266.72 ± 32.06b	163.99 ± 30.92a	437.75 ± 11.84a	401.80 ± 6.86a	38.79 ± 3.48b	530.12 ± 32.47b
Pickled cowpea pod (PCP)
Undigest	56.14 ± 4.49c	59.28 ± 7.29d	79.18 ± 3.20b	99.02 ± 0.78e	Nd	0.78 ± 0.42c	Nd
Oral	0.17 ± 0.02d	33.23 ± 0.25d	Nd	65.87 ± 6.44ef	Nd	Nd	Nd
Gastric	2.57 ± 0.55d	113.36 ± 3.23c	Nd	168.95 ± 2.83d	Nd	Nd	Nd
Intestinal	3.35 ± 0.04d	140.01 ± 8.34c	Nd	224.07 ± 13.13c	Nd	Nd	Nd

*  Values are means ± standard deviations (n = 3) and different letters within the same column indicate significant differences at p < 0.05 from Duncan's test. Nd: not detected.

Levels of the identified phenolic compounds were affected by pickling and digestion process. In general, RCP had higher contents than PCP before and after GID. This suggested that pickling could significantly decrease the levels of the individual phenolic compounds quantified in RCP (p < 0.05). There was 22% reduction in total phenolics. The reason may mainly be due to diffusion of phenolic compounds into the brine during pickling (Kiai and Hafidi, 2014).

The GID had different influence on the stability of the phenolics detected in RCP and PCP. After GID, protocatechuic acid, chlorogenic acid and catechin identified in RCP and PCP were detected, while p-hydroxybenzoic acid and caffeic acid only detected in RCP. This indicated that these compounds could be degraded completely under the digestion conditions, or were not released from cell wall polysaccharidesthe by the enzyme digestion (Nderitu et al., 2013), or were converted into other compounds during GID and thus not detected (Chandrasekara and Shahidi, 2012).

After oral digestion only catechin in RCP was increased, whereas its concentration didn't change in PCP. A significant decrease was found among four phenolic acids (protocatechuic, p-hydroxybenzoic, caffeic and chlorogenic acid) in RCP and PCP (p < 0.05), except chlorogenic acid detected in PCP, whose content didn't change as compared to undigested sample. Vanillic acid and epicatechin in RCP were also not changed. The decrease of these compounds after oral phase (5 min of digestion) could be due to their low solubility in salivary fluid and to the short duration of this phase (Ydjedd et al., 2017).

After gastric digestion, significant increases (p < 0.05) were observed for chlorogenic acid and catechin in RCP and PCP and for protocatechuic acid, caffeic acid and epicatechin in RCP, while no significant differences were observed for p-hydroxybenzoic and vanillic acids in RCP, and for protocatechuic acid in PCP as compared with oral digestion. Similar behavior was also observed for chlorogenic acid and catechin in RCP and PCP and for vanillic acid and epicatechin in RCP with respect to the undigested samples. The rest polyphenols detected in RCP and PCP decreased their concentrations, except protocatechuic acid in RCP, whose content showed no change as compared with undigested sample.

After intestinal digestion, significant increases (p < 0.05) were observed for chlorogenic acid in RCP and PCP and for p-hydroxybenzoic acid, protocatechuic acid, and vanillic acid in RCP, while decrease for catechin in RCP and epicatechin in PCP, and no change for protocatechuic acid and catechin in PCP and caffeic acid in RCP with respect to the gastric digestion. In comparison to the undigested samples, protocatechuic acid, chlorogenic acid, vanillic acid and catechin significantly increased (p < 0.05), while caffeic acid significantly decreased (p < 0.05) in RCP. As regard to p-hydroxybenzoic acid and epicatechin, their contents showed no change. In PCP, there were significant increases (p < 0.05) found for chlorogenic acid and catechin, whereas protocatechuic acid significantly decreased (p < 0.05).

Many studies showed that the same phenolic compound from different sources exhibited a variable behavior in its content after GID. For example, Jara-Palacios et al. (2018) reported that catechin and epicatechin have no significant change after GID for grape seed extracts, while increased significantly (p < 0.05) and gradually for grape stems and pomace. However, they were reduced for grape skin and were not detected after gastric and intestinal digestion. For another example, Chait et al. (2020) found that contents of protocatechuic, chlorogenic, vanillic and caffeic acid and catechin in carob free phenolic were significantly increased (p < 0.05) after GID, whereas their contents in conjugated and bound polyphenols were decreased and vanillic and caffeic acid were not quantifiable. These results showed that the food matrix composition could highly affect the variation tendency for contents of phenolic compounds after GID. Phenolic compounds may be released from food matrix during digestion due to breaking the bond of these compounds to proteins, fibre or sugar residues by the acidic pH and digest enzyme (Celep et al., 2015). Moreover, they could be converted from other compounds under in vitro digestion conditions (Mosele et al., 2015). At the same time, these compounds could be also degraded, transformed, oxidated and polymerized during digestion (Bermúdez-Soto et al., 2007; Sánchez-Patán et al., 2011). In this study, protocatechuic acid in RCP significantly increased (p < 0.05) after intestinal digestion. The increase could be due to a dehydroxylation of gallic acid (Mosele et al., 2015) or the degradation of flavonoids (Sánchez-Patán et al., 2011). Since no gallic acid was detected in this work, the increase in protocatechuic acid content after intestinal digestion could be attributed to the flavonoids degradation. Similar results were reported by Jara-Palacios et al. (2018) who found an increase in protocatechuic acid concentration of different white winemaking byproducts extracts during in vitro digestion. Zhao et al. (2020) reported protocatechuic acid in Chaenomeles speciosa and Crataegus pinnatifida significantly increased (p < 0.05) after intestinal digestion. A large amount of flavonoids in cowpea green pod could diffuse into the pickling water during pickling (Kiai and Hafidi, 2014), resulting in less flavonoids degradation to form protocatechuic acid. Therefore, protocatechuic acid in PCP significantly decreased (p < 0.05) after intestinal digestion.

Effect of simulated GID on the inhibition of α-amylase, α-glucosidase and lipase    Table 4 shows the effects of RCP and PCP on activities of α-amylase, α-glucosidase and lipase during simulated GID. The undigested sample of RCP inhibited α-amylase and α-glucosidase, with IC₅₀ values of 34.05 and 74.34 mg/mL, respectively. Whereas, it was inactive towards lipase. After GID, RCP showed inhibitory effects towards the three enzymes, and the inhibitory activities were significantly increased with respect to the undigested sample (p < 0.05). The inhibitory effects on α-amylase and α-glucosidase were significantly reduced from oral to intestinal phase (p < 0.05), while the inhibitory effects on lipase were significantly increased (p < 0.05). However, their inhibition activities towards α-amylase and α-glucosidase were lower than those of acarbose, and the inhibition on lipase was lower than that of orlistat after GID. He et al. (2017) reported that the inhibitory activities of twenty-two fruit juices towards α-amylase and α-glucosidase significantly increased (p < 0.05) after digestion, and found that the digested polyphenols and polysaccharides synergistically contributed to the inhibitory activity.

Table 4 Effects of raw and pickled cowpea green pods before and after in vitro gastrointestinal digestion towards α-amylase, α-glucosidase and lipase.

Samples	α-Amylase (IC50, mg/mL)	α-Glucosidase (IC50, mg/mL)	Lipase (IC50, mg/mL)
Raw cowpea pod (RCP)
Undigest	34.05 ± 0.26a*	74.34 ± 3.54a	>100
Oral	4.80 ± 0.92d	23.73 ± 0.62d	23.80 ± 0.65b
Gastric	7.09 ± 0.17c	37.27 ± 1.24c	14.85 ± 1.15d
Intestinal	10.61 ± 1.63b	49.73 ± 0.29b	7.46 ± 0.42f
Pickled cowpea pod (PCP)
Undigest	>100	>100	>100
Oral	>100	>100	54.17 ± 1.05a
Gastric	>100	>100	16.80 ± 0.38c
Intestinal	>100	>100	12.40 ± 1.01e
Positive control
Acarbose	7.59 ± 0.12c	18.63 ± 1.00e	-
Orlistat	-	-	0.05 ± 0.01g

*  Values are means ± standard deviations (n = 3) and different letters in the same column show significant differences at p < 0.05.

The undigested sample of PCP did not show inhibitory activity towards three enzymes, with IC₅₀ values greater than 100 mg/mL. PCP did not show inhibitory activity towards α-amylase and α-glucosidase after GID, while a significant inhibition (p < 0.05) on lipase was observed, and the inhibitory activity significantly increased (p < 0.05) during simulated GID. But the inhibition activities towards lipase were lower than that of orlistat. Gutiérrez-Grijalva et al. (2019) also reported that gastric and intestinal digestions increased the inhibitory activity of oregano against lipase. Our results also showed RCP had significantly higher (p < 0.05) inhibitory effects towards three enzymes than PCP at each digestion phase. These results suggested that picking could reduce the inhibitory effect of cowpea green pods against the three enzymes. PCP could increase the inhibition on lipase and decrease the inhibition on α-amylase and α-glucosidase after GID. Hence, PCP could increase the digestibilities of carbohydrates and decrease the digestibilities of lipids after GID.

Conclusion

This work reported the effects of in vitro GID on the bioactivity and phenolic profiles of RCP and PCP. GID could increase their TPC, TFC, and antioxidant activities. The antioxidant activities were significantly correlated with the TPC and TFC (p < 0.05). These parameters of cowpea green pod were reduced by pickling during GID. Contents of the phenolic compounds identified in cowpea green pod were decreased by pickling after GID. Pickling could also reduce the inhibitory activity of cowpea green pod towards α-amylase and α-glucosidase. GID increased the inhibitory activity of RCP towards α-amylase, α-glucosidase and lipase, and the activity of PCP toward lipase. These present results indicated that pickling significantly affected the bioactivity and phenolic profiles of cowpea green pod during GID and might reduce its health-promoting properties.

Acknowledgements    This work was supported by the Research Foundation of Education Bureau of Hunan Province, China (Grant No. 19A470).

Conflict of interest    There are no conflicts of interest to declare.

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