Food Bioscience 61 (2024) 104491

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Bio-accessibility of phenols, flavonoid and antioxidant capacity of tropical

leafy vegetables through in-vitro gastro-intestinal digestion
Sachidananda Swain a, b , Lalit M. Bal c, d, * , Mridula Devi b , G. Kavita a , Rajiv Sinha a
a
ICAR-Central Island Agricultural Research Institute, Port Blair-744105, India
b
ICAR-Central Institute for Women in Agriculture, Bhubaneswar, Odisha-751003, India
c
Post Harvest Process and Food Engineering, College of Agriculture, Jawaharlal Nehru Agricultural University, Tikamgarh, Madhya Pradesh, 472001, India
d
Department of Dairy Engineering, Sanjay Gandhi Institute of Dairy Technology, Bihar Animal Sciences University, Patna, Bihar, 800014, India

A R T I C L E I N F O A B S T R A C T

Keywords: To provide a new insight to the nutritionist and processing industries, in-vitro digestion studies of 20 tropical
Leafy vegetables green leafy vegetables were conducted. The results indicated significant (P < 0.05) changes in phenolics, fla­
Flavonoids vonoids and antioxidants level based on food matrix properties where the leaf extracts exhibited higher anti­
DPPH
oxidant activity than corresponding stem extracts. The most flavonoids bio-accessibility were observed in
β-carotene bleaching
In-vitro digestion simulation
Ipomoea aquatic leaves (463.4%), Colocasia esculenta stem (335.5%), Alternanthera philoxeroides (260.7%). The
Antioxidant activity transformation of phenolic compounds during in-vitro digestion may lead to different structural forms possessing
different chemical properties and functions. Compared to fresh state, except for Trigonella foenum graecum,
Ipomoea aquatica stem, all vegetables exhibited decrease in antioxidant activity (DPPH) value (4.7%–76.9%),
nine vegetables exhibited a decrease in antioxidant activity (ABTS) value (3.01%–22.8%) and except for
Amarantus cruentas, all vegetables exhibited decrease in FRAP value (15.29%–85.24%). Only four vegetables
(Colocasia esculenta stem and leaf, Amaranthus cruentas and Murraya koenigii) had shown decrease (1.7%–13.2%)
and rest 16 vegetables followed 9.6%–314.13% increase in β-carotene bleaching. Interestingly, all vegetables
displayed sharp increase in Super Oxide Dismutage (SOD) value (94.99%–174.78%) after duodenal phase even
significant changes in phenolic content and flavonoid contents were observed. Correlation analysis showed weak
correlation between phenolic content with antioxidant activity in successive gastric and duodenal phase.

1. Introduction results in DNA damage, oxidation of cell membranes, inflammation and

cell death (Huang et al., 2005). The antioxidant capacity of fruits and
Promotion of balanced diets across populations of various income vegetables could be linked to their simple phenol (Feng et al., 2023),
brackets have been one of the goal of developing nations, among which flavonoids and phenolic acid, pigments such as chlorophylls, betacya­
green leafy vegetables have been gain special attention because of rich nins, betaxanthins, and carotenoids, such as beta-carotene, and xan­
natural sources of essential minerals, protein and vitamins, phyto­ thophylls, ascorbic acid, and vitamin A content (Sarker et al., 2022a,b).
chemicals and antioxidants (Sarker et al., 2022a,b; Gunathilake & This is possible due to their electron and proton donation ability (Chen
Ranaweera, 2016) and low cost of production than other vegetables. et al., 2015) to form stable compounds. The nutritional contribution of
These leafy vegetables have the potential in reducing the risks of chronic leafy vegetables has been widely documented and exploited which has
diseases (Sarker et al., 2022a,b)also designated as ‘natures anti-aging been being used for the nutritional education of the public as a means to
wonders’ (Sahoo et al., 2020). It is now widely accepted that poly­ improve the nutritional status of the population. However, scientific
phenolic compounds present in foods, specially flavonoids are respon­ supporting data for health benefits potential of these vegetables espe­
sible for curing different ailment, created by free radicals due to cially after consumption is still inadequate and also exaggerated. Hence
oxidative imbalance in the body (Bhatt & Patel, 2013). The excessive there is a need to further expand on the question of how much of the
production of Reactive Oxygen Species (ROS), so called free radicals total nutritional content is available at the cellular level (Pierre et al.,

* Corresponding author. Post Harvest Process and Food Engineering, College of Agriculture, Jawaharlal Nehru Agricultural University, Tikamgarh, Madhya
Pradesh, 472001, India.
E-mail address: [email protected] (L.M. Bal).

https://doi.org/10.1016/j.fbio.2024.104491
Received 10 March 2024; Received in revised form 31 May 2024; Accepted 3 June 2024
Available online 7 June 2024
2212-4292/© 2024 Published by Elsevier Ltd.
S. Swain et al. Food Bioscience 61 (2024) 104491

2006). Many recommendations on nutritional intake are based on mere 2.3. In-vitro digestion procedure
content data, not taking into account changes happening during
gastro-intestinal (GI) digestion. In-vitro gastro-intestinal digestion method was employed to simulate
After oral intake, the bio-accessibility of bioactive compounds from physiological conditions of the stomach and small intestine i.e. pH,
food largely depends on their stability in human digestive system (Rein temperature, and enzyme conditions according to the procedures of
et al., 2013). Bio-accessibility specifically refers to the compounds, Ryan et al. (2008). Samples were transferred to clean amber bottles and
which are potentially presented to the intestinal brush border for ab­ mixed with saline to create a final volume of 20 mL. The samples were
sorption. It is therefore not sufficient to report dietary values compared acidified to pH 2.0 with 1 mL of a porcine pepsin preparation (0.04 g
against Recommended Daily Intake (RDI) values, but also a determi­ pepsin in 1 mL 0.1 mol/L HCl) and incubated at 37 ◦ C in a shaking water
nation of the bio-accessibility of vitamins and minerals in vegetables is bath at 95 rpm for 1 h. After gastric digestion, the pH was increased to
critical before valid conclusions can be drawn on the nutritional status of 5.3 with 0.9 mol/L sodium bicarbonate followed by the addition of 200
each food package, or the associated risks thereof. Most of substances μL of bile salts glycodeoxycholate (0.04 g in 1 mL saline), taurodeox­
are changed due to enzymes and the acid-base in human ycholate (0.025 g in 1 mL saline), taurocholate (0.04 g in 1 mL saline)
gastro-intestinal digestion. The absorption of the dietary compounds and 100 μL of pancreatin (0.04 g in 500 μL saline). The pH of each
liberated from the food by chewing will depend on its physicochemical sample was increased to 7.4 with 1 mol/L NaOH. Samples were incu­
properties such as molecular size, configuration, lipophilicity, solubility, bated in a shaking water bath (95 rpm) at 37 ◦ C for 2 h to complete the
and pKa. Before becoming bioavailable, they must be released from the intestinal phase of the in-vitro digestion process. After the intestinal
food matrix and modified in the gastro-intestinal (GI) tract. Therefore, it phase, 2 mL of each sample was extracted and stored at − 20 ◦ C, and all
is important before concluding on any potential health effect, to analyze the samples were analyzed within 2 weeks.
whether the digestion process affects bioactive compounds and their
stability, as this, in turn, will affect their bioavailability and their 2.4. Total phenolics content
possible beneficial effects. Numerous studies were conducted to reveal
the nutrition absorption in GI tract and their potential uptake and finally The Folin-Ciocalteu colorimetric method was used to measure the
the bioavailability in tissues (Wootton-Beard et al., 2011; Fawole & total phenolic content (Singleton et al.,1999) with minor modifications.
Opara, 2016; Chitindingu et al., 2015; Chen et al., 2014; 2015; Pavan Briefly, 200 μL of the extractions were oxidized with 1 mL of 0.5 N
et al., 2014; Bhatt & Patel, 2013; Gunathilake et al., 2018). However, the Folin-Ciocalteu reagent and then the reaction was neutralized with 1 mL
studies on leafy vegetables were rare. Although in-vivo models provide of the saturated sodium carbonate (75 g/L). The absorbance of the
more accurate results, their use has been limited due to economic and resulting blue color was measured at 760 nm with a UV-2600 spectro­
ethical restrictions (Guerra et al., 2012). Therefore, the current research photometer (Simadzu Corp., Kyoto, Japan) after incubation for 2 h at
was aimed to determine bioactive compounds and antioxidant capacity room temperature (25 ◦ C). Quantification was done on the basis of the
of 20 leafy vegetables subjected to in-vitro GI digestion. standard curve of gallic acid. Results were expressed as milligram of
gallic acid equivalent (mg GAE) per 100 g of flour weight.
2. Materials and methods
2.5. Flavonoid content
2.1. Reagents and chemicals
Total flavonoid content was determined by a colorimetric method.
Analytical grade chemicals of Porcin pepsin, Glycodeoxycholate, (Bao et al., 2005). Extract of 0.5 mL were added to 15 mL polypropylene
Taurodeoxycholate, Taurocholate, Pancreatin, Folin-Ciocalteu reagent, conical tubes containing 2 mL ddH2O and mixed with 0.15 mL 5%
2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethyl­ NaNO2. After reacting for 5 min, 0.15 mL 10% AlCl3⋅6H2O solution was
benzothiazoline-6-sulphonic acid (ABTS), 2,3,5-triphenyl-1,3,4-triaza- added. After another 5 min, 1 mL 1 M NaOH was added. The reaction
2-azoniacyclopenta-1,4 diene chloride (TPTZ), β–carotene, Chloro­ solution was well mixed, kept for 15 min and the absorbance was
form, Nitro Blue Tetrazolium (NBT), Phenazine Methosulphate (PMS), determined at 415 nm. Qualification was done using the Rutin as stan­
Phosphate Buffer, Acetate buffer, Linoleic acid, Nicotinamide Adenine dard and the results was expressed as milligrams of rutin equivalent (mg
Dinucleotide (NADH), Polyoxyethylene Sorbitan Monopalitate (Tween- RE) per 100 g of flour weight.
20), Butylated Hydroxy Toulene (BHT), 6-hydroxy-2,5,7,8-tetramethyl­
chroman-2-carboxylic acid (Trolox), Super Oxide Dismutage (SOD) and 2.6. ANTIOXIDANT ACTIVITY (DPPH) (2,2-diphenyl-1-picrylhydrazyl)
FeCl3⋅6H2O were purchased from Sigma Aldrich (Biocorporal, Chennai, activity
India) and Sodium Nitrate (NaNO2), Aluminum Chloride Hexahydrate
(AlCl3⋅6H2O), Sodium Hydroxide (NaOH), Sodium Bicarbonate Total antioxidant activity was obtained by 2,2-diphenyl-2-picrylhy­
(NaHCO3) were purchased from Hi-Media (Boon Chemicals, Port Blair, drazyl (DPPH)) method (Rattanachitthawat et al., 2010) with some
India). modification. The working solution of (DPPH) was freshly prepared by
diluting 3.9 mg of DPPH with 95% ethanol to get with an absorbance of
2.2. Samples and extract preparation 0.856 ± 0.05 at 517 nm. The different concentration of extract was
mixed with 1.5 mL of working (DPPH) and the absorbance of the
Twenty leafy vegetables were purchased from Port Blair market, A & mixture immediately measured spectrophotometrically after 10 min.
N Islands, India. All the leaves of 20 samples including both leaves and Total antioxidant activity of the leafy vegetables was expressed as mg
stems of five leafy vegetables (as their both parts are consumed) were BHA/g sample equivalent, obtained from the calibration curve.
washed well in running water and the water particles adhered to the
% inhibition of Antioxidant Activity (DPPH) radical = (Acontrol- A sample/
samples were removed using blotting paper followed by drying in cab­
A control) × 100
inet dryer at 50 ◦ C. After drying, the samples were subjected to fine
powder and passed through a 20-mesh sieve. One gram (1.0 g) of power Where Acontrol is the absorbance of the control (without extract) and
was taken ina test tube and 50 mL of double distilled water were added Asample is the absorbance in the presence of the extract/standard.
and placed in a water bath at 100 ◦ C for 20 min. The mixture was then
cooled to room temperature (25 ◦ C) and centrifuged at 13,480 g force for
10 min. The supernatant was recovered and kept at − 20 ◦ C for further
analysis.

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S. Swain et al. Food Bioscience 61 (2024) 104491

2.7. Antioxidant activity (ABTS) (2,2′-azino-bis (3-ethylbenzothiazoline- antioxidant = {[ln (a/b)]/t}, a = absorbance at time = 0, b = absor­
6-sulphonic acid) activity bance at defined time.

The total antioxidant capacity was determined by a colorimetric 2.11. Statistical analysis
method (Re et al., 1999) with a little modification. ABTS radical ion was
prepared by mixing 7 mM of ABTS stock solution with 2.45 mM of po­ Results were expressed as the mean ± standard deviation (SD), and
tassium persulphate (K2S2O8) and leaving the mixture for 4–16 h until the data were analyzed by SAS (Software Version 9.3. SAS Institute Inc.,
the reaction was complete and the solution was stable. Then ABTS.+ was Carry, NC). One-way analysis of variance (ANOVA) with Tukey’s mul­
adjusted with pH to about 0.784 ± 0.01 with 80% ethanol at 734 nm. tiple range test was used to compare the differences among various
Then, 3.9 mL (ABTS)⋅þcation solution was added to 1 mLof extracts and groups. A P-value <0.05 was considered to be statistically significant.
mixed thoroughly. The mixture incubated for 6 min at room tempera­
ture (25 ◦ C) and tested the absorbance at 734 nm. Results were 3. Results and discussion
expressed in terms of Trolox equivalent antioxidant capacity (TEAC, mM
Trolox equivalents per 100 g dry weight). 3.1. Change in phenolics

2.8. Ferric Reducing Antioxidant Power (FRAP) assay The highest phenolic content in fresh leafy vegetables was found in
Mentha arvensis (33.64 ± 0.84 mg GAE/g) followed by Enhydra fluctuens
It is based on the reduction of the complex of ferric iron and 2,3,5-tri­ (21.7 ± 0.85) and lowest phenolic content was found in Colocasia
phenyl-1,3,4-triaza-2-azoniacyclopenta-1,4 diene chloride (TPTZ) to the esculenta stem (2.95 ± 0.51) as shown in Table 1. After gastric phase of
ferrous form at low pH Benzie and Strain, 1999. FRAP reagent is pre­ in-vitro digestion, there was significant (P < 0.001) change in total
pared by mixing 300 mM of acetate buffer (pH 3.6) with 10 mM of TPTZ phenolic contents among tested vegetables. Among 20 vegetables, 15
solution in 40 mM of HCl and 20 mM of FeCl3⋅6H2O in the ratio of vegetables had shown increase in phenolic content where Hibiscus can­
10:1:1. Briefly, 0.9 mL of prepared FRAP reagent is mixed with 0.1 mL of nabinus had undergone increase of 198.13% followed by Ipomoea aquatic
diluted sample and the absorbance at 595 nm is recorded after a 15 min (155.42%), Colocasia esculenta (105.3%) and so on while Murraya koe­
incubation at 37 ◦ C and the results were expressed in mM of Fe2+ nigii showed highest decrease (50.62%) and Eryngium foetidum had least
equivalents per g dry weight. decrease (9.63%) (11.09 ± 0.02 to 10.02 ± 0.63 mg GAE/g) in phenolic
content. Thus, a proportion of the polyphenol compounds was trans­
2.9. Super Oxide Dismutage (SOD) activity formed into different structural forms with different chemical properties
with different bio-accessibility, bioavailability and biological activity
The influence of extracts from leafy vegetables on the generation of Bermúdez-Soto et al., 2007 This is in agreement with previous research
superoxide anion was measured according to the method described in by Wootton-Beard et al. (2011), Tagliazucchi et al. (2010), Bouayed
Yen and Chen (1995). Superoxide anion was generated in a non-enzymic et al. (2011). Chen et al. (2015) in his study among 23 edible flower
system and determined by a spectrophotometric measurement for reported 42.9% and decreased in 4.64% for Matricaria recutita and
reduction of nitro blue tetrazolium. The reaction mixture, which con­ Chamomilia flower, respectively. Bhatt and Patel (2013) reported 164%–
tained 1 mL of extract in distilled water, 1 mL of Phenazine Methosul­ 200.1% increase in phenolic content in garlic. Pavan et al. (2014) re­
phate (PMS) (60 μM) in phosphate buffer (0.1 M, pH7.4), 1 mL of ported 45.5% increase in phenolic content in jackfruit (Artocarpus het­
Nicotinamide Adenine Dinucleotide (NADH) (468 μm) in phosphate erophillus L. Cv). This increase in phenolic content could be related with
buffer 1 mL of Nitro Blue Tetrazolium (NBT) (150 μm) in phosphate the softening of the rigid cell walls and other components (vacuoles and
buffer was incubated at ambient temperature for 5 min and the color apoplasts) of the vegetable cells. At acidic pH, breaking of phenol-sugar
was read at 560 nm against blank samples. All analyses were run in glycosidic links leads to aglycons contributing increase in phenol con­
triplicate and mean values were calculated. centration (Bouayed et al., 2011; Singleton et al., 1999) or increase in
the flavylium cation in the (Pérez-Vicente et al., 2002; Tagliazucchi
2.10. β-carotene-linoleate model system et al., 2010). The significant fluctuation of phenolic content may be
linked to interaction of phenolics with other dietary constituents
The antioxidant activity of the leafy extracts was evaluated using released during digestion (iron, other minerals, dietary fibre or proteins)
β-carotene-linoleate model system as described by Jayaprakasha et al. (Manach et al., 2004). Low recovery of some polyphenolic compounds
(2001) with some modification. β-Carotene (0.1 mg) in 0.2 mL of could be due to the formation of link between some polyphenol struc­
chloroform, 10 mg of linoleic acid and 100 mg of Tween-20 (polyoxy­ tures and soluble fibers (pectin) forming gel structure (Gunathilake
ethylene sorbitan monopalitate) were mixed. Chloroform was removed et al., 2018) with intragastric gelation properties (Padayachee et al.,
at 40 ◦ C under vacuum and the resulting mixture was diluted with 10 mL 2012).
of water and was mixed well. To this emulsion, 2.0 mL of oxygenated Polyphenols are highly sensitive to alkaline conditions. In duodenal
water was added. Four milliliter aliquots of the emulsion were pipetted phase (pH = 7.3), there was again increase in phenolic content in eight
into different test tubes containing 0.2 mL of extracts and BHT (50, 100, vegetables, being highest in Bacopa monnieri stem (67.43%) (12.84 ±
150, 200 and 250 μg) in methanol. BHT was used for comparative 0.73 to 21.5 ± 0.84 mg/g) and lowest in Moringa oleifera (4.71%) (19.77
purposes. A control containing 0.2 mL of methanol and 4 mL of the ± 0.83 to 20.7 ± 0.56 mg/g) compared to gastric phase. Rest 12 vege­
above emulsion was prepared. The tubes were placed at 50 ◦ C in a water tables showed decrease in phenolic content ranged between 56.56% (9.0
bath and the absorbance at 470 nm was taken at zero time (t = 0). ± 0.44 to 3.90 ± 0.97 mg/g) in Basella alba and 9.96% (6.07 ± 0.60 to
Measurement of absorbance was continued till the color of β-carotene 5.61 ± 0.62 mg/g) in Colocasia esculenta stem. Compared to fresh state,
disappeared in the control tubes (t = 180 min) at an interval of 60 min. there were significant higher (P < 0.001) phenolic content in both
Antioxidant activity is calculated as percentage of inhibition (%) (gastric and duodenal) phase wherein 1.1 to 2.55 (gastric phase) and
relative to the control using the following equation: 1.19 to 3.53 (duodenal) fold increase in bio-accessibility was observed.
Leaves that had the highest bio-accessibility were Ipomoea aquatica
DRc − DRS
% inhibition = (253.71%) followed by Hibiscus cannabinus (227.01 %) and Colocasia
DRc
esculenta (190.0%) whereas lowest bio-accessibility was detected in
DRC – degradation rate of β-carotene in the control sample = {[ln (a/ Basella alba (48.04%) compared to fresh state (Fig. 1A). Only, seven
b)]/t}, DRS—degradation rate of β-carotene in the sample with vegetables exhibited their bio-accessibility between 48% and 90% in

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S. Swain et al. Food Bioscience 61 (2024) 104491

Table 1
Phytochemical constituents of 20 leafy vegetables at fresh, gastric and duodenal phase.
Vegetables Edible Code Total polyhenol content Flavonoid content
portion
Fresh Gastric Duodenal LSD Fresh Gastric Duodenal LSD
phase phase phase phase

Colocasia esculenta (L.) Schott Stem CES 2.95 ± 0.51 6.07 ± 0.60 05.61 ± 0.62 1.18 2.48 ± 0.34 3.40 ± 0.39 10.82 ± 0.55 0.85
Amaranthus cruentas L Leaf AC 11.20 ± 15.57 ± 16.32 ± 0.82 1.56 14.73 ± 52.32 ± 42.65 ± 0.69 0.84
0.90 0.62 0.39 0.68
Portulaca oleracea L. Leaf PO 12.75 ± 16.14 ± 7.30 ± 0.96 1.03 17.40 ± 34.15 ± 13.57 ± 0.85 1.25
0.57 0.38 0.24 0.74
Enhydra fluctuens Lour. Leaf EF 21.70 ± 14.77 ± 18.82 ± 0.81 1.23 38.98 ± 37.65 ± 34.15 ± 0.76 1.42
0.85 0.89 0.73 0.92
Colocasia esculenta (L.) Scott Leaf CEL 11.43 ± 22.61 ± 18.86 ± 0.48 1.06 13.15 ± 54.32 ± 51.98 ± 0.50 0.33
0.37 0.73 0.68 0.84
Basella alba L. Leaf BA 08.14 ± 9.00 ± 0.44 3.91 ± 0.97 1.01 19.90 ± 34.82 ± 07.73 ± 0.54 0.48
0.78 0.70 0.64
Ipomoea aquatica Forsk Leaf IA 15.20 ± 12.50 ± 19.20 ± 0.97 0.63 09.23 ± 46.57 ± 29.73 ± 0.27 0.70
0.37 0.82 0.48 0.32
Bacopa monnieri (L.) Pennell Leaf BM 12.75 ± 18.07 ± 19.11 ± 0.96 1.45 14.40 ± 55.32 ± 36.07 ± 0.36 1.28
0.61 0.92 0.64 0.76
Hibiscus cannabinus L. Leaf HC 7.30 ± 0.86 21.75 ± 16.57 ± 0.51 1.82 06.15 ± 22.90 ± 34.65 ± 0.35 0.63
0.94 0.30 0.29
Alternanthera philoxeroides Leaf AP 14.45 ± 21.70 ± 17.25 ± 0.93 0.51 12.23 ± 71.32 ± 50.90 ± 0.48 0.96
Griseb 0.76 0.59 0.36 0.62
Moringa oleifera Lam. Leaf MO 12.84 ± 19.77 ± 20.70 ± 0.56 0.81 24.07 ± 21.07 ± 27.82 ± 0.36 0.40
0.95 0.83 0.31 0.40
Murraya koenigii Spreng Leaf MK 18.27 ± 09.02 ± 22.95 ± 0.84 0.41 12.40 ± 11.32 ± 44.73 ± 0.36 0.26
0.77 0.86 0.50 0.20
Coriandrum sativum L. Leaf CS 10.73 ± 16.34 ± 07.68 ± 0.76 0.70 13.90 ± 65.98 ± 24.73 ± 0.82 0.86
0.90 0.51 0.40 0.44
Trigonella foenum graecum L. Leaf TF 05.93 ± 07.11 ± 05.34 ± 0.77 0.68 16.98 ± 22.73 ± 06.82 ± 0.61 0.74
0.56 0.54 0.83 0.54
Eryngium foetidum L. Leaf EF 11.09 ± 10.02 ± 06.20 ± 0.57 0.39 14.32 ± 26.23 ± 18.65 ± 0.54 1.11
0.96 0.63 0.79 0.39
Bacopa monnieri L. Stem BMS 09.11 ± 12.84 ± 21.50 ± 0.84 1.19 08.23 ± 32.07 ± 28.82 ± 0.91 0.45
0.66 0.73 0.53 0.59
Ipomoea aquatica Forsk Stem IAS 03.98 ± 10.16 ± 14.07 ± 0.51 0.59 06.90 ± 25.40 ± 08.90 ± 0.27 0.55
0.64 0.73 0.34 0.80
Mentha arvensis L. Leaf MA 33.64 ± 23.07 ± 33.75 ± 0.75 0.56 37.57 ± 54.65 ± 67.32 ± 0.69 1.05
0.84 0.43 0.75 0.94
Portulaca oleracea L. Stem POS 05.95 ± 06.91 ± 03.59 ± 0.21 0.50 07.23 ± 07.98 ± 11.98 ± 0.61 1.00
0.33 0.39 0.63 0.72
Amaranthus Cruentas L. Stem ACS 06.77 ± 08.07 ± 07.14 ± 0.12 0.31 16.90 ± 48.40 ± 17.65 ± 0.66 0.24
0.27 0.19 0.44 0.40

Values represented with mean ± standard deviation of the means of four independent experiment (n = 4).

duodenal phase compared to fresh state. The reduction of polyphenols in by Chen et al. (2015), Podsędek et al. (2014) and Tagliazucchi et al.
the digesta after duodenal digestion could be due to high instability in (2010). Variations in concentration within plant tissues, cell wall
the mild alkaline condition in small Intestine (Gunathilake et al., 2018). structure and site of glycosides in cells could justify the distinctive
The low bio-accessibility may also be as a result of phenolic compounds susceptibility of polyphenols from different morphological parts of
in plants being conjugated to other molecules like carbohydrates, cel­ selected vegetables.
lulose, lignin etc that are resistant to digestion (Manach et al., 2004) or Overall, there were continuous increase in phenolic content in
binding of polyphenols with components of the pancreatin/bile salts Bacopa monnieri (41.71% and 5.78%), Amaranthus cruentas (38.9% and
mixture forming insoluble complexes (Jakobek, 2015). Bouayed et al. 4.81%), Moinga oleifera (53.98% and 4.71%), Ipomoea aquatica
(2011) stated that the additional time of extraction was also a crucial (155.42% and 38.47%) compared to gastric and duodenal phase of
factor facilitating release phenolics bound to the matrix. Our results are digestion. From this, it may be inferred that gastric enzymes were more
in consistent with the studies by Chitindingu et al. (2015) (66%–77% in effective in accessibility of polyphenols in the GI tract. Interestingly, bio-
seven cereals), Fawole and Opara (2016) (72%–84% in pomegranate accessibility of Mentha arvensis is almost same in final duodenal stage
co-products) and Pérez-Vicente et al. (2002) (20% in pomegranate), compared to fresh state though there had 31.4% decrease in gastric
Fazzari et al. (2008) (27%–29% in sweet cherries) and Bouayed et al. phase. It could be concluded that Mentha arvensis had highest phenolic
(2011) (44.6%–62.7% in apples), Gunathilake et al. (2018) (14.4%– content no matter they were before or after the gastric and duodenal
77.4% in edible green leaves) compared to fresh undigested state. phases of digestion. This discrepancy might be related to difference the
The reason for high bio-accessibility in 13 vegetables may be pre­ food matrix characteristics (Pérez-Vicente et al., 2002) and the presence
sumed that bound phenolics or the ratio of bound and free phenolics of more bioavailable polyphenols in the leaves of Mentha arvensis
(Pinelo et al., 2006) in food matrix were not digested with the selected (Gunathilake et al., 2018) for Centella asiatica leaves. Again, it was seen
in-vitro procedure leading to higher phenolic content after duodenal that though phenolic content in fresh and its accessibility after gastric
phase. Friedman and Jürgens (2000) reported that the reduction in total phase were least in Colocasia esculenta, its bio-accessibility in duodenal
phenolic content could be linked to the instability of phenolic com­ phase were third (1.9 fold) after Bacopa monnieri (3.53 fold) and Ipomoea
pounds in high pH, as they speculated, larger molecules may be more aquatica (2.35 fold) among 20 vegetables.
stable, but when hydrolyzed to smaller molecules, such as gallic acid, The stability of phenolic content depends upon chemical structure,
were not stable at high pH. Our results are in agreement with the report molecular size, solubility, glycosylation and esterification with other

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S. Swain et al. Food Bioscience 61 (2024) 104491

Fig. 1. Change (%) in phytochemicals in gastric and duodenal phase with respect to fresh state. A. Total phenolic content; B. Flavonoid content; C. DPPH activity, D.
ABTS activity; E. FRAP activity; F. SOD activity; G. β-carotene bleaching.

compounds and its release from food matrix (Karaś et al., 2017). In our 3.2. Change in flavonoid contents
study, the variations in concentration within plant tissues, cell wall
structure and site of glycosides in cells, degree of precipitation with Total flavonoid content was varied between 2.48 ± 0.34 (Colocasia
other constituents, (Jakobek, 2015) and porous structure (void space) esculenta) and 38.98 ± 0.73 mg GAE/g (Enhydra fluctuens) before
(Gunathilake et al., 2018) could justify the distinctive susceptibility of digestion. After gastric phase, there was a significant (P < 0.001) change
polyphenols from same/different morphological parts of selected leafy in flavonoid content. 17 vegetables displayed increase in flavonoid
vegetables. content ranged between 10.36% (Portulaca oleracea stem) and 404.33%

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S. Swain et al. Food Bioscience 61 (2024) 104491

(Ipomoea aquatica leaf) and only three has shown non-significant environment (pH 7.3) as phenolic compounds, in particular flavonoids,
decrease in content by 3.4% (Enhydra fluctuens), 8.73% (Murraya koe­ are highly sensitive to alkaline conditions (Bouayed et al., 2011;
nigii) and 12.46% (Moringa oleifera). In the duodenal phase, 18 vegeta­ Pérez-Vicente et al., 2002). Apart from this, there was dissociation of
bles had shown decrease in flavonoid content, being highest in Trigonella anthocyanin content (component of flavonoid group) where the flavy­
foenum graecum (70.01%) and lowest in Colocasia esculenta stem (4.29%) lium cation changed to colourless chalcone at the alkaline pH of the
compared to gastric phase. However, compared to fresh state, only four medium. Similar studies were also reported by Gunathilake et al. (2018).
vegetables exhibited decrease in content (5.9%–22.03%) and rest fol­ Second reason may be presence of higher flavonoid glycoside in the
lowed 4.4% (Colocasia esculenta stem) to 463.41% (Ipomopea aquatica) selected vegetables than its aglycone which cannot be hydrolyzed by the
increase in flavonoid content (Fig. 1B). It was interesting to note that enzymes usually employed in simulated digestion model leading to
Cococasia esculenta leaf and stem showed differential properties in both decrease in flavonoid content (Kamiloglu et al., 2014). The pattern of
phases. This may be due to matrix effect of different constituents of plant releasing flavonoids at the end of GI digestion was similar to total
parts (Podsędek et al., 2014). As the composition of different plant parts polyphenols for eight leafy vegetables only where rest vegetable
are different (matrix effect), the compound formations between plant exhibited different trend.
parts with enzymes at two different pH are different, leading to wider
flavonoid content. Molecular size, their basic structure, degree of poly­ 3.3. Change in antioxidant activities
merization, esterification, glycosylation, solubility, and conjugation
with other phenolics can be considered critical factors (Bouayed et al., 3.3.1. Antioxidant activity (DPPH) activity
2011) for the bio-accessibility of polyphenolic content. As expected, the antioxidant activities were found to vary in the same
Overall flavonoid content continuously decreased in four vegetables manner as the phenolic concentration before in-vitro digestion. The
(Colocasia esculenta, Hibiscus cannabinus, Mentha arvensis and Portulaca DPPH values of 20 leafy vegetables are shown in Table 2. In fresh state,
oleracea) in both gastric and duodenal phase. This indicated the effec­ DPPH values varied from 93.3 ± 0.92 (Trigonella foenum graecum) to
tiveness gastric enzymes and alkaline phase for bioavailability these four 696.66 ± 1.27 (Mentha arvensis) mg BHT/100 g (Table 2). It is a fact that
vegetables like five vegetables for phenolic content. The observed the antioxidant activities are correlated to the flavonoid content. In our
decline in total flavonoid concentration could be linked to the decrease study, the discrepancy may be due to more hydrophilic phenolic content
in total phenolic concentration at the duodenal phase of in-vitro diges­ in these vegetables having antioxidant activities (Wu et al., 2004). In the
tion. This may be attributed to its degradation in the weak alkaline gastric phase, Eryngium foetidum exhibited highest (DPPH) value (658.3

Table 2
Antioxidant activity of leafy vegetables (Antioxidant DPPH and Antioxidant ABTS).
Vegetables Edible Code DPPH ABTS
portion
Fresh Gastric phase Duodenal LSD Fresh Gastric Duodenal LSD
phase phase phase

Colocasia esculenta (L.) Schott Stem CES 260.00 ± 330.00 ± 60.00 ± 1.33 0.86 4.40 ± 0.24 11.22 ± 05.95 ± 0.24 0.5
1.02 1.65 0.35
Amaranthus Cruentas L Leaf AC 563.33 ± 421.60 ± 326.66 ± 2.11 1.17 42.85 ± 43.56 ± 41.65 ± 0.66 0.92
1.16 1.44 0.48 0.37
Portulaca oleracea L. Leaf PO 626.66 ± 550.00 ± 330.00 ± 1.75 0.66 46.34 ± 41.94 ± 45.91 ± 0.42 0.68
1.66 1.32 0.47 0.30
Enhydra fluctuens Lour. Leaf EF 665.01 ± 598.30 ± 265.00 ± 1.49 1.66 46.88 ± 41.96 ± 46.92 ± 0.31 0.11
1.61 1.70 0.35 0.38
Colocasia esculenta (L.) Scott Leaf CEL 610.00 ± 623.33 ± 295.00 ± 1.31 1.22 46.43 ± 40.68 ± 37.61 ± 0.46 0.85
1.65 2.00 0.46 0.37
Basella alba L. Leaf BA 446.66 ± 211.66 ± 203.30 ± 1.20 1.42 46.16 ± 16.43 ± 35.64 ± 0.39 0.31
1.67 1.64 0.51 0.38
Ipomoea aquatica Forsk Leaf IA 628.33 ± 583.33 ± 338.30 ± 1.58 1.58 44.45 ± 43.02 ± 33.79 ± 0.512 0.43
1.46 2.52 0.41 0.42
Bacopa monnieri (L.) Pennell Leaf BM 678.33 ± 541.66 ± 513.30 ± 2.62 3.01 43.82 ± 45.08 ± 51.30 ± 0.42 0.60
1.41 2.17 0.47 0.49
Hibiscus cannabinus L. Leaf HC 378.36 ± 603.33 ± 271.6 0 ± 1.40 1.83 40.95 ± 45.35 ± 36.15 ± 0.51 0.25
1.59 1.59 0.55 0.38
Alternanthera philoxeroides Leaf AP 538.30 ± 581.66 ± 408.30 ± 1.87 1.24 47.33 ± 42.03 ± 45.91 ± 0.47 0.71
Griseb 1.02 1.70 0.73 0.78
Moringa oleifera Lam. Leaf MO 640.00 ± 538.33 ± 481.60 ± 2.02 2.85 47.60 ± 43.38 ± 51.08 ± 0.60 0.27
1.91 2.41 0.74 0.61
Murraya koenigii Spreng Leaf MK 636.30 ± 515.00 ± 573.30 ± 2.20 1.34 41.40 ± 41.04 ± 52.87 ± 0.38 1.21
1.46 1.68 0.72 0.61
Coriandrum sativum L. Leaf CS 660.01 ± 451.60 ± 535.00 ± 2.29 1.52 43.02 ± 40.50 ± 47.48 ± 0.58 0.51
1.68 2.36 0.45 0.62
Trigonella foenum graecum L. Leaf TF 93.330 ± 401.60 ± 341.66 ± 0.91 1.83 29.18 ± 28.38 ± 40.86 ± 0.60 0.55
0.92 1.12 0.19 0.47
Eryngium foetidum L. Leaf EF 666.60 ± 658.30 ± 498.30 ± 1.28 0.51 46.70 ± 42.21 ± 52.87 ± 0.55 0.77
0.93 1.30 0.68 0.43
Bacopa monnieri L. Stem BMS 651.60 ± 570.00 ± 396.60 ± 1.16 1.42 42.48 ± 44.27 ± 39.40 ± 0.68 0.27
0.59 0.83 0.57 0.52
Ipomoea aquatica Forsk Stem IAS 315.00 ± 500.10 ± 363.30 ± 1.34 1.64 24.16 ± 36.01 ± 46.03 ± 0.42 0.66
1.01 1.28 0.55 0.70
Mentha arvensis L. Leaf MA 696.60 ± 536.60 ± 663.60 ± 1.13 0.59 45.53 ± 37.36 ± 56.92 ± 0.74 0.79
1.27 1.08 0.40 0.90
Portulaca oleracea L. Stem POS 413.30 ± 545.00 ± 303.30 ± 1.40 1.34 22.99 ± 45.53 ± 50.85 ± 0.63 0.68
1.12 0.82 0.40 0.43
Amaranthus Cruentas L. Stem ACS 340.00 ± 416.60 ± 86.660 ± 1.14 0.72 24.96 ± 18.86 ± 33.34 ± 0.79 0.98
1.61 1.40 0.69 0.91

6
S. Swain et al. Food Bioscience 61 (2024) 104491

exhibited lowest ABTS value in all three stages (Fresh, gastric and
± 1.30) and Basella alba exhibited the lowest one (211.66 ± 1.64 mg
duodenal). Compared to its leaf fraction, 10.5, 3.62 and 6.32 fold in­
BHT/g). Although Trigonella foenum graecum had least DPPH value
crease in activity was observed in fresh, gastric and duodenal phase.
before digestion, its value increased to 4.3 fold (highest) after gastric
Similar observations were made by other extracts except for Portulaca
digestion. Here, eight vegetables had shown increase in DPPH value
oleracea where 8.5% and 10.1% decrease in accessibility was found in
(1.02–4.3 fold) and among rest 12 vegetables, Basella alba and Eryngium
leaf over stem after gastric and duodenal phase.
foetidum had highest (52.61%) and lowest decrease (1.24%) in DPPH
value. When exposed to mild-alkaline pH, polyphenols suffer structural
3.3.3. FRAP activity
transformations that result in metabolites with different chemical
The initial FRAP values of the tested vegetables varied between
structures/properties and, in general, lower bioactivities (Bouayed
9042.5 ± 2.53 (Mentha arvensis) and 183.55 ± 1.73 mM Fe+2/g (Colo­
et al., 2012; Chen et al., 2015; Tagliazucchi et al., 2010). As a result of
casia esculenta) as shown in Table 3. After gastric phase, FRAP values
digestion process, the antioxidants could not react effectively or their
remained significantly (P < 0.05) lower, with between 2.8 and 14.8 fold
reducing capacities were impaired (Podsędek et al., 2014). In duodenal
decrease in FRAP value. Though FRAP value of Mentha arvensis was
phase, there was decrease in DPPH value of 13 vegetables, being highest
significantly decreased (P < 0.05), it exhibited the highest antioxidant
in Colocasia esculenta stem (81.81%) and lowest in Basella alba (3.94%)
capacity (771.26 ± 1.75) among tested vegetables, followed by Enhydra
compared to gastric phase. However, only three vegetables have shown
fluctuens (486.52 ± 0.60), Moringa oleifera (344.68 ± 0.43) and Colo­
increase (Murraya koenigii - 11.32%; Coriandrum sativum - 18.16% and
casia esculenta (339.52 ± 0.52). Chen et al. (2015) reported decrease in
Mentha arvensis - 23.66%) in DPPH value. Compared to fresh state,
FRAP value in 15 out of 23 flowers after gastric phase. In duodenal
23.07%–95.26% decrease was exhibited by all samples except for Ipo­
phase, 13 vegetables exhibited increase in FRAP activity, being highest
moea aquatica stem and Trigonella foenum graecum (Fig. 1C). Hence, it
in Mentha arvensis (629%) followed by Bacopa monnieri (576.55%),
could be concluded that Trigonella foenum graecum exhibited strongest
Murraya koenigii (536%) while lowest increase was found in Portulaca
antioxidant activity (4.3 and 3.63 fold) no matter it was after gastric and
oleracea (46.49%) compared to gastric phase. The reason may be the pH
duodenal digestion. The most notable of which was once again observed
dependent radical scavenging activity of polyphenols which increased
for Mentha arvensis, which despite remaining high, decreased from
with increase in pH (Tagliazucchi et al., 2010). It is known that pH of a
696.60 ± 1.27 (fresh state) to 663 ± 1.13 mg BHT/100 g (after the
substance is known to affect racemization of molecules, possibly
duodenal phase). The loss in DPPH value may be attributed by corre­
creating two chiral enantiomers with different reactivity. As a result,
sponding degradation of polyphenols (complex bonding with carbohy­
some antioxidants could be rendered more reactive at acidic pH and less
drates or gel formation) possessing efficient radical scavenging ability in
reactive in mild alkaline phase which was a trend observed in this cur­
alkaline intestinal conditions (Bermudez-Soto et al., 2007). Other
rent study. Due to structural changes of polyphenolic compounds due to
possible reason may be the change in pH from 2.0 to 7.3 in gastric and
change in pH leading to change in their chemical properties, the
duodenal phase which affects racemization of molecules, possibly
reducing power was also changed in most of the tested vegetables.
creating two chiral enantiomers with different reactivity in the respec­
Wootton-Beard et al. (2011) suggested that metabolites formed as a
tive reagent. This could alter their biological reactivity and may render
result of structural changes in the alkaline condition could have reacted
the antioxidants more reactive early in the digestive process and less
differently in the FRAP assay. Amongst rest seven vegetables, Baselaalba
reactive at pH 7.3 in the duodenal phase (Wootton-Beard et al., 2011).
and Bacopa monnieri stem had shown highest and lowest decrease
As a result, the antioxidants could not react effectively or their
(92.57%, 14.47%) in FRAP value indicating incomplete release/de­
de-protonation capacity was impaired at the duodenal phase (Chen
gradation of phytochemicals. Compared to fresh state, all vegetables
et al., 2014).
exhibited decrease in FRAP value in duodenal phase, ranged between
15.29% and 99.29% except for Amaranthus cruentas leaf and Hibiscus
3.3.2. Antioxidant activity (ABTS) activity
cannabinus which showed 36.13% and 249.60% increase in FRAP value
Moringa oleifera had highest (47.6 ± 0.74 mM/g) ABTS value fol­
(Fig. 1E) indicating their low redox potential for converting ferric to
lowed by Alternanthera philoxeroides (47.33 ± 0.73), Enhydra fluctuens
ferrous ion (stable form). Our study was agreed with the report by
(46.88 ± 0.35) in fresh state. The lowest value was observed in Colocasia
Bouayed et al. (2011) for four apple variety. The peculiar trend by
esculenta (4.4 ± 0.24 mM/g). Interestingly there was a trend for ABTS
Amaranthus cruentas leaf and Hibiscus cannabinus may be due to food
activity that were slightly increased following the gastric phase and
matrix properties and formation of complex compounds with minerals,
slightly decreased following the duodenal phase, although there were
dietary fibers and protein etc making lesser bio-accessibility of their
some except for ions to this trend. In gastric phase, there was significant
polyphenols as evident from their phenolic content. Another reason may
(P < 0.05) increase in ABTS value of seven vegetables, with between
be the presence of higher content flavonoid glycosides than aglycone
1.02 (Bacopa monnieri leaf) and 2.55 fold (Colocasia esculenta stem). Rest
having lower scavenging activity (Kamiloglu et al., 2014). However, the
13 vegetables had followed decrease in value ranged between 64.4%
present results also demonstrate that Mentha arvensis exhibited the
(Basella alba) and Trigonella foenum graecum (2.74%) but there was non-
strongest FRAP activity no matter they were before or after the gastric
significant difference (P > 0.05) was observed in Murraya koenigii.
and duodenal phases of digestion.
Similarly in duodenal phase, 13 vegetables had shown increase in ABTS
value, being highest in Basella alba (116.92%) and lowest in Alter­
3.3.4. SOD activity
nanthera philoxeroides (9.23%) compared to gastric phase. Among
As shown in Table 3 and in fresh state, the SOD values were ranged
decrease in ABTS value, Ipomoea aquatica had highest decrease (21.45%)
from 29.95 ± 1.13 QE mg/g (Colocasia esculenta stem) to 59.95 ± 2.10
and Amaranthus cruentas had shown lowest decrease (4.38%). Compared
(Mentha arvensis). Following the gastric phase, there was1.4–55.09%
to fresh state, 12 vegetables exhibited increase in ABTS value between
increase in SOD values in 18 vegetables except for Ipomoea aquatica and
7.31% (Moringa oleifera) to 121.18% (Fig. 1D). It could be concluded
Mentha arvensis which exhibited 1.2% and 8.8% decrease in SOD value.
that Ipomoea aquatica leaf and Portulaca oleracea exhibited the strongest
After the duodenal phase of digestion, all vegetables again exhibited
antioxidant capacities no matter they were before or after the gastric and
increase in SOD value (94.99%–174.78%), being highest in Portulaca
duodenal phases of digestion. It was interesting to note that leaf and
oleracea stem (142.84 ± 3.29) and lowest in Colocasia esculenta stem
stem extract all five tested vegetables such as Bacopa monnieri, Ipomoea
(98.4 ± 2.32). Thus, the bio-accessibility of Colocasia esculenta stem is
aquatica, Portulaca oleracea, Colocasia esculenta, Amaratnthus cruentas
least amongst tested vegetables. Compared to fresh state, 121.77%–
exhibited different trend in their bio-accessibility of ABTS value in
306.06% increase in SOD value was observed by all leafy vegetables
gastric and duodenal phase. In addition to this, Colocasia esculenta stem
(Fig. 1F). Despite low SOD value in fresh state, Portulaca oleracea

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S. Swain et al. Food Bioscience 61 (2024) 104491

Table 3
Antioxidant activity of leafy vegetables (FRAP and SOD).
Vegetables Edible Code Ferric Reducing Antioxidant Power (FRAP) value Super Oxide Dismutage (SOD) value
portion
Fresh Gastric phase Duodenal LSD Fresh Gastric Duodenal LSD
phase phase phase

Colocasia esculenta (L.) Schott Stem CES 183.95 ± 1.71 67.38 ± 1.96 26.60 ± 1.72 1.15 29.95 ± 43.95 ± 98.4 ± 2.32 2.45
1.13 1.47
Amaranthus Cruentas L Leaf AC 607.27 ± 0.76 319.15 ± 826.68 ± 0.86 1.87 39.45 ± 55.22 ± 126.36 ± 2.02 2.29
0.83 2.32 2.02
Portulaca oleracea L. Leaf PO 884.31 ± 0.68 207.80 ± 164.01 ± 0.96 0.87 42.36 ± 55.50 ± 108.4 ± 3.79 4.81
0.84 2.79 2.14
Enhydra fluctuens Lour. Leaf EF 2617.46 ± 486.52 ± 1631.21 ± 0.86 51.45 ± 55.18 ± 118.63 ± 2.79 1.18
0.78 0.60 0.87 1.69 1.62
Colocasia esculenta (L.) Scott Leaf CEL 1057.18 ± 339.36 ± 622.78 ± 0.67 0.96 46.09 ± 57.59 ± 135.22 ± 2.14 1.95
0.67 0.52 2.02 1.52
Basella alba L. Leaf BA 627.22 ± 0.66 135.82 ± 4.43 ± 0.95 1.27 44.13 ± 55.13 ± 107.5 ± 2.69 4.82
0.95 3.02 3.31
Ipomoea aquatica Forsk Leaf IA 1668.88 ± 126.60 ± 210.55 ± 0.62 0.74 57.68 ± 56.95 ± 124.88 ± 1.69 1.68
0.85 0.23 2.60 2.26
Bacopa monnieri (L.) Pennell Leaf BM 1615.69 ± 176.24 ± 1192.38 ± 1.27 46.31 ± 54.18 ± 148.88 ± 1.62 2.36
0.88 0.52 0.64 3.29 3.50
Hibiscus cannabinus L. Leaf HC 281.47 ± 0.49 210.28 ± 984.04 ± 0.91 0.74 41.54 ± 58.63 ± 138.4 ± 2.73 3.9
0.85 3.23 2.46
Alternanthera philoxeroides Leaf AP 2526.60 ± 293.26 ± 1008.42 ± 1.09 53.50 ± 57.90 ± 133.75 ± 2.02 0.75
Griseb 0.49 0.43 0.53 2.21 2.10
Moringa oleifera Lam. Leaf MO 1597.96 ± 344.68 ± 1194.59 ± 0.75 42.59 ± 52.45 ± 126.47 ± 1.52 2.9
0.45 0.43 0.41 2.91 2.52
Murraya koenigii Spreng Leaf MK 1999.11 ± 121.28 ± 775.71 ± 0.46 1.16 39.13 ± 39.68 ± 124.54 ± 4.96 1.36
0.67 0.43 2.45 2.14
Coriandrum sativum L. Leaf CS 1294.33 ± 227.30 ± 425.53 ± 0.51 0.91 45.36 ± 54.36 ± 114.43 ± 3.02 2.15
0.51 0.57 2.56 2.35
Trigonella foenum graecum L. Leaf TF 454.34 ± 0.52 179.79 ± 15.51 ± 0.61 0.43 37.95 ± 52.77 ± 119.09 ± 3.31 1.20
0.65 2.69 2.81
Eryngium foetidum L. Leaf EF 999.56 ± 0.78 281.21 ± 846.63 ± 0.50 0.94 37.36 ± 51.86 ± 130.22 ± 2.72 5.05
0.49 2.92 2.87
Bacopa monnieri L. Stem BMS 693.71 ± 0.37 235.82 ± 201.68 ± 1.26 0.37 43.59 ± 55.68 ± 139.88 ± 2.0 2.03
0.42 2.29 2.35
Ipomoea aquatica Forsk Stem IAS 281.47 ± 0.39 100.35 ± 19.95 ± 0.45 0.29 36.81 ± 57.09 ± 129.31 ± 2.26 3.62
0.26 1.22 1.80
Mentha arvensis L. Leaf MA 9042.55 ± 771.26 ± 5622.78 ± 0.53 59.95 ± 54.63 ± 132.95 ± 3.56 1.44
2.53 1.75 1.26 2.10 2.61
Portulaca oleracea L. Stem POS 525.27 ± 1.25 278.37 ± 407.80 ± 1.85 0.92 35.18 ± 53.90 ± 142.84 ± 3.29 1.20
1.53 1.32 1.29
Amaranthus Cruentas L. Stem ACS 410.02 ± 1.53 149.29 ± 11.08 ± 0.99 0.49 32.27 ± 52.31 ± 117.84 ± 3.5 2.89
1.85 1.13 1.47

displayed highest SOD value after duodenal phase. SOD, being a key (47.52%) compared with gastric phase. 15 vegetables had shown higher
cellular antioxidant, is highly responsible for the elimination of O−2 . This inhibition capacity compared to fresh stage, ranging from 9.2 (Mentha
indicated higher presence of dismutage enzymes in these leafy vegeta­ arvensis) to 234.22% (Ipomoea aquatica stem). Interestingly, though
bles for converting superoxide ion to molecular oxygen and hydrogen Mentha arvensis had higher phenolic content with higher DPPH and
peroxide. SOD is enzyme linked antioxidant assay and pH plays a ABTS value, its bio-accessibility in inhibiting linoleate radical was
prominent role in enzymatic activity. However, due to change in pH, the lowest after duodenal phase compared to fresh stage. Dawidowicz and
structural and functional properties could be altered leading to change Olszowy (2010) reported hydrophobicity properties that influence the
in SOD activity like DPPH and ABTS. dispersion (or solubility) of the emulsion or of plants components and
presence of metal ions may influence the dispersion (or solubility) of the
3.3.5. β–carotene bleaching activity emulsion. Valgimigli et al. (2009) reported that the presence of small
The highest β–carotene bleaching activity was observed in Enhydra amounts of acid leads to increase in the rate reactions between phenols
fluctuens (88.22%) followed by Murrayakoenigii (86.9%), Mentha arvensis and chain-carrying peroxyl radicals. As a consequence, the reaction
(86.57%) and the lowest value was exhibited by Bacopa monnieri stem velocity of extracts changed leading to decrease/increase in inhibition
(18.9%). Significant variation (P < 0.05) was observed in some vege­ activity. The observed changes are difficult to explain in an unequivocal
tables (Table 4). In the gastric phase, Colocasia esculenta and Ipomoea way. Here, as the plants were of different genotypes grown in varied soil
aquatica stem had highest and lowest bleaching activity of 59.89% and conditions, antioxidant compounds (flavonoids, carotenoids and
14.93%, respectively. Here, all vegetables exhibited decrease in β–carotene etc) were significantly different having different trend in
bleaching activity ranged between 2.24% (Hibiscus cannabinus) and inhibition activities. The change in inhibition of β–carotene in tested
84.91% (Trigonella foenum graecum) except for Bacopa monnieri stem and vegetables at gastric and duodenal phase may by accrued to varying
Colocasia esculenta stem compared to fresh state. However, in the hydrophobicity properties at two different pH affecting solubility of
duodenal phase, Colocasia esculenta leaf and stem exhibited highest phenolic compounds as well as the presence of metal ions. Thus, the
(95.69%) and lowest (34.34%) inhibition activity (Fig. 1G). It was resulting metabolites may react differently across different assays
interesting to observe that except for Colocasia esculenta there was sig­ (Wootton-Beard et al., 2011).
nificant increase in bleaching activity in all vegetables, being highest in
Trigonella foenum graecum (664.16%) followed by Ipomoea aquatica stem 3.3.5.1. Correlation analysis. Table 5 shows the correlation analysis
(522.92%) and lowest increase was observed in Enhydra fluctuens

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S. Swain et al. Food Bioscience 61 (2024) 104491

Table 4 fourteen bioactive compounds. As expected, positive significant (P <

β-Carotene bleaching activity of leafy vegetables. 0.001) correlations (R2 = 0.38 to 0.91) could be found between all pa­
Vegetables Edible Code β-carotene bleaching activity rameters with total phenolic and flavonoid content. Differences in cor­
portion relations among parameters could be explained by different responses of
Fresh Gastric Duodenal LSD
phase phase phenolic compounds to different antioxidant activity evaluation tests as
well as to the variable nature of product generated by the reaction
Colocasia Stem CES 38.39 59.90 37.34 ± 3.2
esculenta (L.) ± 1.36 ± 1.92 1.69
system. After the gastric phase, the correlations between total phenolic
Schott content and rest bioactive compounds were lower (R2 = 0.06–0.60) and
Amaranthus Leaf AC 80.64 25.67 67.43 ± 1.80 nonsignificant negative correlation was found between β–carotene
Cruentas L ± 1.58 ± 1.72 1.73 bleaching with flavonoid. Flavonoid were significantly correlated with
Portulaca Leaf PO 45.95 21.96 70.96 2.12
±
only SOD activity, indicated the de-protonation activity of flavonoid
oleracea L. ± 1.29 ± 1.77 1.73
Enhydra Leaf EF 88.82 52.01 76.75 ± 1.17 rather electron transfer mechanism in quenching free radical activity.
fluctuens Lour. ± 1.29 ± 1.81 1.71 Lower correlation between Antioxidant Activity (DPPH), Antioxidant
Colocasia Leaf CEL 85.62 36.81 95.70 ± 1.61 Activity (ABTS), FRAP with flavonoid (0.21–0.35) indicated presence of
esculenta (L.) ± 1.09 ± 1.82 1.58 non-phenolic compounds (carotenoids and tannin) showing antioxidant
Scott
Basella alba L. Leaf BA 80.16 14.45 69.56 ± 2.24
activity. Following the duodenal phase of digestion, all of the correla­
± 1.77 ± 1.02 1.62 tions between total antioxidant capacities and total phenolic contents
Ipomoea Leaf IA 49.43 15.62 94.50 ± 0.72 were positive, although the correlations between all parametres and
aquatica Forsk ± 1.57 ± 1.10 1.20 total phenolic contents were poor than the initial values. Both ABTS and
Bacopa monnieri Leaf BM 74.87 51.77 93.69 1.62
±
FRAP values were poorly correlated with phenolic content. This may be
(L.) Pennell ± 1.83 ± 1.52 1.25
Hibiscus Leaf HC 60.87 59.50 94.81 ± 1.22 because FRAP and ABTS assays are both electron transfer based methods
cannabinus L. ± 1.18 ± 1.99 1.06 (Chen et al., 2015). Both of the assays take advantage of
Alternanthera Leaf AP 64.96 56.48 93.03 ± 2.71 electron-transfer reactions, therefore, there is not much difference be­
philoxeroides ± 1.51 ± 1.21 1.37 tween ABTS assay and the FRAP assay except for ABTS assay is carried
Griseb
Moringa oleifera Leaf MO 50.69 39.32 88.12 ± 3.62
out at neutral pH and FRAP assay under acidic (pH 3.6) condition. DPPH
Lam. ± 1.89 ± 1.71 1.57 were poorly correlated with SOD, FRAP and β–carotene bleaching in
Murraya koenigii Leaf MK 86.96 65.28 85.43 ± 2.8 gastric phase but significant correlation (P < 0.05) was observed in
Spreng ± 1.54 ± 1.48 1.24 duodenal phase. This may be due to change in pH of GI tract where mild
Coriandrum Leaf CS 80.85 18.79 95.65 2.88
±
alkaline condition could facilitate more de-protonation. The DPPH
sativum L. ± 1.54 ± 1.44 1.27
Trigonella Leaf TF 82.55 12.45 95.17 ± 3.16 scavenging activity assay is based on the normal hydrogen atom transfer
foenum ± 1.68 ± 1.79 1.19 (HAT) reaction whereas ABTS is based on singlet electron transfer (SET)
graecum L. mechanism. Interestingly, only SOD activity was significantly (P <
Eryngium Leaf EF 53.21 40.39 83.59 ± 2.09 0.001) correlated with ABTS value though free radical generation
foetidum L. ± 1.49 ± 1.81 1.12
Bacopa monnieri Stem BMS 18.90 31.85 78.28 ± 3.01
mechanism were different. The correlation between SOD and bleaching
L. ± 1.52 ± 1.39 1.44 activity was very poor even negative in gastric phase. The reason may be
Ipomoea Stem IAS 27.82 14.93 93.0 ± 3.35 due to their free radical generation system. FRAP is a SET assay whereas
aquatica Forsk ± 1.4 ± 1.93 1.73 bleaching activity is a HAT assay.
Mentha arvensis Leaf MA 86.57 27.41 94.94 ± 2.49
L. ± 1.27 ± 1.71 1.10
Portulaca Stem POS 70.46 43.51 94.28 ± 2.9 4. Conclusions
oleracea L. ± 1.41 ± 1.22 1.72
Amaranthus Stem ACS 70.75 56.65 85.29 ± 2.91 Among 20 vegetables, 15 vegetables had shown increase in phenolic
Cruentas L. ± 1.41 ± 1.43 1.49
content varied from 10.56% to 198.13% and rest five vegetables had
shown decrease in phenolic content (9.63%–50.62%) after gastric phase
of digestion. The pattern of releasing flavonoids at the end of GI diges­
tion was similar to total polyphenols for eight leafy vegetables only

Table 5
Correlation analysis of seven independent parameters.
Parameters Phenolic content Flavonoid content DPPH ABTS SOD FRAP β-carotene bleaching

Phenolic content 1 0.76***1 0.66*** 0.57*** 0.77*** 0.91*** 0.43**

0.60***2 0.49** 0.53** 51** 0.58** 0.06
0.84***3 0.57*** 0.37 0.45** 0.70*** 0.31
Flavonoid content 1 0.38* 0.47** 0.51** 0.68*** 0.51***
0.14 0.21 0.54** 0.33 − 0.19
0.51** 0.35 0.45** 0.71*** 0.31
DPPH 1 0.75*** 0.59*** 0.55* 0.11
0.81*** 0.29 0.36 0.25
0.80*** 0.41 0.57*** 0.53**
ABTS 1 0.68*** 0.24 0.39
0.36 0.31 0.01
0.28 0.46** 0.65***
SOD 1 0.70*** 0.25
0.26 − 0.43
0.67** 66***
FRAP 1 0.34
− 0.02
0.23

**, *** - Significant at 5 and 5 %, respectively. Superscript 1, 2 and 3 represent Fresh, Gastric and Duodenal phase of digestion, respectively.

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S. Swain et al. Food Bioscience 61 (2024) 104491

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(2012). Binding of polyphenols to plant cell wall analogues – Part 2: Phenolic acids.
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