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Journal Pre-proofs
Impact of cooking and nixtamalization on the bioaccessibility and antioxidant
capacity of phenolic compounds from two sorghum varieties
Ivan Luzardo-Ocampo, Aurea K. Ramírez-Jiménez, Angel H. Cabrera-Ramirez,
N. Rodríguez-Castillo, Rocio Campos-Vega, Guadalupe Loarca-Piña, Marcela
Gaytán-Martínez
PII:
DOI:
Reference:
S0308-8146(19)31811-4
https://doi.org/10.1016/j.foodchem.2019.125684
FOCH 125684
To appear in:
Food Chemistry
Received Date:
Revised Date:
Accepted Date:
24 April 2019
7 October 2019
8 October 2019
Please cite this article as: Luzardo-Ocampo, I., Ramírez-Jiménez, A.K., Cabrera-Ramirez, A.H., Rodríguez-Castillo,
N., Campos-Vega, R., Loarca-Piña, G., Gaytán-Martínez, M., Impact of cooking and nixtamalization on the
bioaccessibility and antioxidant capacity of phenolic compounds from two sorghum varieties, Food Chemistry
(2019), doi: https://doi.org/10.1016/j.foodchem.2019.125684
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Impact of cooking and nixtamalization on the bioaccessibility and antioxidant
capacity of phenolic compounds from two sorghum varieties
Ivan Luzardo-Ocampoa , Aurea K. Ramírez-Jiménezb, Angel H. Cabrera-Ramirezc, N.
Rodríguez-Castillod, Rocio Campos-Vegaa, Guadalupe Loarca-Piñaa , Marcela GaytánMartíneza,*
a
Programa de Posgrado en Alimentos del Centro de la República (PROPAC), Research
and Graduate Studies in Food Science, School of Chemistry, Universidad Autónoma de
Querétaro, Centro Universitario, Cerro de las Campanas S/N. Santiago de Querétaro,
Querétaro, C.P. 76010, México.
b
Tecnológico de Monterrey, Escuela de Ingeniería y Ciencias, Centro de Biotecnología
FEMSA, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, NL, México
c
Instituto Politécnico Nacional, CICATA-IPN Unidad Querétaro, Cerro Blanco No. 141,
Col. Colinas del Cimatario, C.P.76090, Santiago de Querétaro, Querétaro, México
d
Facultad de Química, Universidad Autónoma de Querétaro. Centro Universitario, Cerro
de las Campanas S/N. Santiago de Querétaro, Querétaro, C.P. 76010, México.
*Corresponding Author: Marcela Gaytán Martínez ([email protected])
Authors’ e-mail:

I. Luzardo-Ocampo: [email protected]

A. Ramírez-Jiménez: [email protected]

A. H. Cabrera-Ramírez: [email protected]
1

N. Rodríguez- Castillo: [email protected]

R. Campos-Vega: [email protected]

G. Loarca-Piña: [email protected]
2
Abstract
Sorghum (Sorghum bicolor L. Moench) has been sparsely used as human food due to
certain anti-nutritional factors such as tannins that reduce its digestibility, although the
grain is an important source of bioactive compounds such as phenolic compounds (PCs).
This study aimed to assess the impact of cooking and alkaline cooking (nixtamalization) on
the bioaccessibility and antioxidant capacity of PCs of two sorghum varieties (white/red).
Nixtamalization was the most effective procedure for the reduction of tannins (74.3 %).
Gallic acid proved to be the most bioaccessible PC (6359 g/g). The total phenolics and
condensed tannins correlated with the antioxidant capacity (ABTS/DPPH; R2:0.30-0.43,
p<0.05). These results confirm the potential of thermal procedures to significantly modify
the bioaccessibility of sorghum compounds, enhancing their concentrations and reducing
anti-nutritional factors (tannins) while improving their antioxidant capacity.
Keywords: Sorghum (Sorghum spp.), phenolics, nixtamalization, in vitro gastrointestinal
digestion, antioxidant capacity.
3
Chemical compounds studied in this article: ABTS (PubChem CID: 9570474); Caffeic
acid (PubChemCID: 689043); (+)-Catechin (PubChemCID: 9064); Chlorogenic acid
(PubChemCID: 1794427); DPPH (PubChem CID: 2735032); Gallic acid (PubChem CID:
370); p-Coumaric acid (PubChemCID: 637542); Quercetin (PubChemCID: 5280343);
Rutin (PubChemCID: 5280805); Soda lime (PubChem CID: 66545795).
Abbreviations: ABTS: 2,2-azinobis-(3-ethylbenzothazoline-6-suphonic acid); ADMET:
Absorption, Distribution, Metabolism, Excretion and Toxicity; CE: (+)-catechin
equivalents; CTs: Condensed tannins; CRW: Cooked red sorghum; CWS: Cooked white
sorghum; DF: Digestible fraction; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ER: Efflux ratio;
FLEX: Flexibility; GAE: Gallic acid equivalents; HPLC-DAD: High performance liquid
chromatography coupled to Diode Array Detector; INSATU: Saturation; INSOLU:
Solubility; LIPO: Lipophilicity; ME: Methanolic extract; NDF: Non-digestible fraction;
NRS1: Nixtamalized red sorghum with 10 g Ca(OH)2/kg flour; NWS1: Nixtamalized
white sorghum with 10 g Ca(OH)2/kg flour; Papp: Apparent permeability coefficient; PCA:
Principal component analysis; POLAR: Polarity; PCs: Phenolic compounds; RE: Rutin
equivalents; RRS: Raw red sorghum; RWS: Raw white sorghum; TF: Total flavonoids;
TPCs: Total phenolic compounds; TPSA: Topological polar surface area; WF: Water flux;
WLOGP: Wildman & Crippen atomistic method score.
4
1. Introduction
Sorghum (Sorghum bicolor L. Moench) is the fifth most-produced cereal in the world,
being a primary food in several semi-arid regions of Africa and Asia (FAO, 2017).
Sorghum is a well-known source of phenolics (PCs) and other bioactive compounds.
However, due to the content of anti-nutritional factors, it is usually used for cattle feeding,
although some populations include this cereal in their diets. In the western countries,
sorghum has gradually become an important crop for human consumption, because of its
resistance to adverse conditions, and its nutritional and functional properties (de Morais
Cardoso, Pinheiro, Martino, & Pinheiro-Sant’Ana, 2015).
An important issue for human consumption of sorghum is the presence of several antinutritional factors in the sorghum grain, such as tannins, that decrease protein digestibility
and feed efficiency in both animals and humans. Therefore, in some countries, such as the
United States, sorghum consumption has been restricted to no-tannin types, causing a
robust artificial selection against tannins, and rejecting other important bioactive
compounds that are naturally found in the grain (Wu et al., 2012). In this context, several
technological processes, such as boiling, cooking, and extrusion, have been tested to reduce
the anti-nutritional factors. Alkaline cooking or nixtamalization has shown potential to
solve this issue (Gaytán-Martínez et al., 2017). Nixtamalization has several benefits over
unprocessed grains, such as an improved nutritional value and the removal of aflatoxins and
condensed tannins (Escalante-Aburto, Mariscal-Moreno, Santiago-Ramos, & Ponce-García,
2019). Gaytán-Martínez et al. (2017) reported a significant reduction of tannins with an
optimized nixtamalization procedure (10 g lime/kg flour, 40 min of cooking) in two
sorghum varieties (red and white sorghum). Under these conditions, a maximal removal up
to 96 % was achieved for both varieties while the antioxidant capacity of sorghum was
5
preserved. However, phenolic acids, especially flavonoids (catechin/quercetin), were
reduced by up to 74-98 %. Therefore, technological processes that improve or at least
maintain the nutritional value and biological potential of sorghum are necessary to increase
the human consumption of this grain (Girard & Awika, 2018).
Given the considerable amount of bioactive compounds found in sorghum, several in vitro
and in vivo assays have been performed to confirm its health benefits, particularly those
attributed to PCs isolated from sorghum. Flavonoids, certain phenolic acids, and condensed
tannins are the major compounds, exhibiting a more diverse and higher content than those
observed for popular crops such as barley, wheat, maize, rice and oats. These benefits have
also served as an industrial stimulus for the development of sorghum-based food products
(Girard & Awika, 2018). The reported beneficial effects include the amelioration of
obesity, type 2 diabetes, dyslipidemia, cardiovascular disease, cancer, and hypertension (de
Morais Cardoso et al., 2015).
The fate of bioactive compounds along the gastrointestinal tract defines their biological
activity. Phenolic compounds interact with several micro and macromolecules in the
gastrointestinal tract;
the action of enzymes, transporters, physiological pH, and
temperature alter the pharmacokinetics and absorption parameters of these compounds
(Domínguez-Avila et al., 2017). A complete in vitro gastrointestinal digestion of sorghum
grain submitted to a thermal-alkaline procedure has not been reported. There is also a lack
of information about the bioaccessibility and antioxidant capacity of phenolic compounds.
Thus, the aim of this work was to compare the impact of cooking and nixtamalization
(alkaline cooking) on the bioaccessibility and antioxidant capacity of phenolic compounds
from two Mexican sorghum varieties (red and white sorghum). Furthermore, digested raw
sorghum grains were used for comparison purposes.
6
2. Materials and methods
2.1. Biological material
Sorghum (Sorghum bicolor L. Moench) grains var. “Tortillas y Pan” (white) were donated
by the “Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias” (INIFAP)
located in Ciudad Victoria, Tamaulipas (Mexico) in 2013. “Níquel” (red) variety was
grown and harvested in Cueramaro (Guanajuato, Mexico) in 2013.
2.2. Sample preparation
Raw sorghum grains were cleaned for the removal of husk, damaged seeds, and physical
contaminants. For the preparation of the cooked sorghum flours, the grains (1:3
sorghum/water ratio) were cooked at 94 ºC for 40 min.
The reported procedure of Gaytán-Martínez et al. (2017) was followed to obtain the
nixtamalized sorghum flours. Briefly, a Ca(OH)2 solution (10 g Ca(OH)2/kg flour) was
added to 1 kg of sorghum grains (1:3, w/v ratio). The mixture was cooked at 94 ºC for 30
min and left to stand for 12 h. Afterwards, the nixtamalized grains were separated from the
cooking water, washed to remove the excess of lime and ground (Nixtamatic®).
Subsequently, both cooked and nixtamalized sorghum flours were dehydrated (45 ºC, 24 h),
using a convective food dehydrator (3900 B, Excalibur Dehydrators, Sacramento, CA,
USA), passed through a hammer mill (Pulvex S.A. de C.V., Mexico), and sieved, using a
U.S. 60 mesh (250 m of particle size). Raw sorghum grains were milled and sieved using
the described mesh, and these flours were used for comparison purposes. All flours were
stored in amber bottles protected from light and stored at 4 ºC until used.
7
The cooking time and the calcium concentration used were based on a previous study to
allow the highest condensed tannin reduction and the maximum retention of PCs and
antioxidant capacity (Gaytán-Martínez et al., 2017). The samples were coded, depending on
the treatment (R: raw, C: cooked and N: nixtamalized), variety (W: white; R: red), and lime
concentration [1: 10 g Ca(OH)2/kg flour]. Therefore, the samples used in this study were
coded as follows: RWS (Raw white sorghum) and RRS (raw red sorghum); CWS (cooked
white sorghum) and CRS (cooked red sorghum), NWS1 (nixtamalized white sorghum, 10 g
Ca(OH)2/kg flour) and NRS1 (nixtamalized red sorghum, 10 g Ca(OH)2/kg flour).
2.3. Nutraceutical characterization
2.3.1. Methanolic extraction of phenolic compounds
Phenolic compounds were extracted by following the procedure of Cardador-Martínez,
Loarca-Piña, G., & Oomah (2002). One g of each sample was placed in a 50 ml flask and
mixed with 10 ml of methanol. The flasks were protected from light, properly closed, and
stirred with a magnetic bar at 450 rpm (IKAWorks, NC, USA) for 24 h at room temperature
(25  1 ºC). Each mixture was then centrifuged at 2166 x g, 10 min/ 4 ºC (HERMLE Z323
K, Wehingen, Germany). The supernatant was collected and stored, protected from light, at
4 ºC for further analysis.
2.3.2. Total phenolics (TPCs) content determination
The total phenolic content of the methanolic extracts was determined using the colorimetric
Folin-Ciocalteu methodology (Singleton, Orthofer, & Lamuela-Raventós, 1999). Briefly,
extract dilutions were oxidized with 125 l of the Folin-Ciocalteu reagent. After 5 min of
8
reaction, the samples were neutralized with a Na2CO3 solution (625 l, 0.07 g/ml) for 2 h.
The absorbance was read at 760 nm and compared against a blank. The results were
expressed as mg of gallic acid equivalents (GAE) per gramme of sample (mg GAE/g).
2.3.3. Condensed tannins (CTs) quantification
Condensed tannins were quantified using the method described by Feregrino-Pérez et al.
(2008). Briefly, 200 l of acidified vanillin reagent (5 g/l vanillin mixed with 47.2 g/l of
HCl in methanol) was added to 50 l of the methanolic extract previously obtained and
placed in a 96-well plate. Condensed tannins were quantified at 492 nm, using a microplate
reader (51118307 Multiskan Ascent, Thermoscientific, USA), and reported as
milligrammes of (+)-catechin equivalents (CE) per gramme of sample (mg CE/g). A blank
was prepared using a non-vanillin reagent sample treated under the same conditions.
2.3.4. Total flavonoids (TFs) quantification
Total flavonoids were measured by following the procedure reported by Oomah, CardadorMartínez, & Loarca-Piña (2005). For this, 50 l of the methanolic extract was mixed with
180 l of methanol and 20 l of a 10 g/l solution of 2-aminoethyldiphenylborate in a 96well microtitration flat-bottom-plate (Corning, New York, USA). Absorbance was read at
404 nm in a microplate reader (51118307 Multiskan Ascent, Thermoscientific, US) and the
results were compared with a rutin standard (0-50 g/ml). The total flavonoid content was
reported as microgrammes of rutin equivalents (RE) per gramme of sample (g RE/g).
9
2.3.5. Quantification of free-phenolic compounds
The free-phenolic compounds were identified and quantified according to RamírezJiménez, Reynoso-Camacho, Mendoza-Díaz, & Loarca-Piña (2014), using a highperformance liquid chromatography system coupled to a diode array detector (HPLCDAD). An Agilent 1100 Series HPLC (Agilent Technologies, Palo Alto, CA, USA) was
used with a Zorbax Eclipse XDB-C18 column (Agilent Technologies, 4.6 mm x 250 mm x
5 m). The column was thermostatically controlled (35  0.6 ºC), maintaining a flow rate
of 1 ml/min. Two solvents were used for the mobile phase: solvent A (acidified water: 10
g/l acetic acid) and solvent B (acetonitrile). The gradient was set as follows: 80-83 % A for
7 min, 83-60 % for 5 min, 60-50 % for 1 min, 50-85 % for 2 min. The detection was
performed at 280 nm (for phenolic acids) and 320 nm (for flavonoids), using an injection
volume of 20 l. Commercial standards for gallic, chlorogenic, caffeic and p-coumaric
acids and quercetin were used for the quantification of phenolic compounds.
2.3.6. Antioxidant capacity by ABTS and DPPH methods
The antioxidant capacity was calculated using the radical 1,1-diphenyl-2-pycryhydrazyl
(DPPH) assay (Fukumoto & Mazza, 2000) and the monocation radical 2,2-azinobis-(3ethylbenzothiazoline-6-sulphonic acid) (ABTS) method (Nenadis, Wang, Tsimidou, &
Zhang, 2004). A calibration curve of trolox (50 to 800 M) was used as standard. All
results were expressed as mole equivalents of trolox/g sample.
10
2.4. In vitro gastrointestinal digestion
A simulated human gastrointestinal digestion was conducted, using the procedure reported
by Campos-Vega, Vázquez-Sánchez, López-Barrera, Loarca-Piña, Mendoza-Díaz, &
Oomah (2015). One gramme of each sample was chewed by 4 healthy participants, 15
times for ∼15 s. All subjects consumed their last meal at least 90 min prior to the test and
provided written informed consent in order to participate in the study. The chewed samples
were pooled and incubated for 10 min in an oscillating water bath (80 cycles/min, 37 ºC).
For the gastric phase, a 10 ml aliquot from the oral digestion was adjusted to pH 2.0 using a
0.5 N HCl solution and pepsin (0.055 g in 0.94 ml of 20 mM HCl) ( 2500 U/mg protein,
Sigma-Aldrich, St. Louis, MO, USA), followed by a 2 h-incubation at 37 ºC. An artificial
intestinal solution was prepared: 3 mg of bovine bile (Sigma-Aldrich, St. Louis, MO, USA)
and 2.6 mg of pancreatin (8xUSP, Sigma-Aldrich, St. Louis, Missouri) were dissolved in 5
ml of anaerobic Krebs-Ringer buffer solution [118 mM NaCl, 4.7 KCl, 1.2 mM MgSO4,
1.2 mM KH2PO4, 25 mM NaHCO3, 11 mM glucose and 2.5 mM CaCl2, pH: 6.8),
previously gasified with CO2 (30 min before use). For the intestinal phase, samples from
the gastric digestion (pH: 7.2-7.4) were transferred to a test tube containing an everted gut
sac. The gut sacs were extracted from male Wistar rats (250-300 g body weight, n=6 for
each digestion procedure). The day before the procedure, the rats were fasted overnight (12
h) with water ad libitum. Prior to the surgical procedure, the animals were anesthetized with
pentobarbital sodium (60 mg/kg, injected intraperitoneally) and the intestine was exposed
by a midline abdominal incision. A 20-25 cm segment of the proximal rat jejunum was
excised and placed into the anaerobic Krebs-Ringer buffer (37 ºC) to preserve tissue
integrity and maintain physiological conditions. The sac was cut into 6 cm segments,
11
carefully everted over a glass rod and filled with the Krebs-Ringer buffer. Afterwards, the
sacs were tied and placed into the intestinal solution.
Incubations for 30, 60, 90, and 120 min were conducted in an oscillating water bath (37 ºC,
80 cycles/min). After the incubation period, the sacs were removed, and the remaining
precipitate in the test tube (mucosal side of the rat intestine) was collected and referred to as
the non-digestible fraction (NDF). The fraction retained inside the gut sac (basolateral side)
was considered as the digestible fraction (DF). The experiments were performed in
triplicate. Additionally, a blank prepared with distilled water instead of the sample (1 g)
was treated under the same in vitro gastrointestinal digestion conditions. This study was
approved by the Universidad Autónoma de Querétaro Human Research Internal Committee
and complied with the Guide for Care and Use of Laboratory Animals of the National
Institute of Health (FQ2013-086).
2.5. Calculations
2.5.1. Bioaccessibility measurement
The bioaccessibility of individual phenolics during the simulated in vitro gastrointestinal
digestion was calculated using the following equation (D’Antuono, Garbetta, Linsalata,
Minervini, & Cardinali, 2015): B=(Cf/C0)*100, considering B as the bioaccessibility (%),
C0 the initial concentration of the compound at a certain in vitro digestion stage and Cf the
final concentration of the compound at the same stage.
2.5.2. Apparent permeability coefficients (Papp), efflux ratio (ER) and predictive
permeability screening
12
The apparent permeability coefficients (Papp) were calculated from the apical to basolateral
(Papp
A to B)
and basolateral to apical (Papp
B to A)
sides using the equation of Hubatsch,
Ragnarsson, & Artursson (2007): Papp=(dQ/dt)(1/AC0), where Papp (cm/s) is the apparent
permeability coefficient, dQ/dt (mg/s) is the relationship between the concentration of
metabolites transported across the membrane and the time unit, A is the cross intestinal area
(cm2) that represents the surface available for permeation and C0 (mg/ml) expresses the
initial concentration of the metabolites that are permeating the gut sacs. The mean and the
standard values were expressed in 10-5 cm/s units. The net apparent permeability coefficient
(Papp Net) was computed using the absolute difference of Papp A to B and Papp B to A. The efflux
ratio was considered as the ratio between Papp A to B and Papp B to A.
For the predictive permeability screening, the probabilities of selected phenolic compounds
(gallic, caffeic and chlorogenic acids; quercetin) crossing the epithelial barrier in the Caco2 cells permeability model, the predicted bioavailability radar, and the reported human
intestinal
absorption
were
calculated
using
their
SMILES
files
(http://www.swissadme.ch/index.php) generated through their official chemical structures
(https://pubchem.ncbi.nlm.nih.gov/)
via
(http://lmmd.ecust.edu.cn/admetsar2).
For
admetSAR
the
intuitive
2.0
online
absorption
software
prediction
of
compounds, the “Boiled Egg diagram” plotting WLOGP (atomistic interpretation of the
fragmental system of Wildman & Crippen: lipophilicity) and TPSA (Topological polar
surface area: apparent polarity) were generated through the SwissADME online software
(http://www.swissadme.ch/index.php#4) (Daina, Michielin, & Zoete, 2017).
2.5.3. Water flux (WF)
13
To assess the small intestine tissue viability, the water flux (WF) from both water
absorption and efflux was calculated according to the equation: WF= (W3/W2)/W1 where
WF (g water/g fresh intestine) is the water flux, W1 is the initial small intestine weight
(without Krebs-Ringer buffer filling), W2 is the buffer-filled small intestine weight before
incubation, and W3 is the small intestine segment weight after the incubation period
(Luzardo-Ocampo et al., 2017).
2.6. Statistical analysis
Data were expressed as the means  standard deviation (SD). Unless otherwise indicated,
all measurements were carried out as independent experiments in triplicate. The statistical
analysis was performed using the JMP v. 8.0 software, conducting a post-hoc TukeyKramer’s test for the analysis of variance (p<0.05). Spearman’s correlations were
calculated between the free-phenolic compounds and the antioxidant capacity
(ABTS/DPPH). A Principal Components Analysis (PCA) was conducted with the JMP v.
8.0 software, using a correlation matrix between the most significant variables: variety
(white or red), processing (raw, cooked or nixtamalized), digestion phase, time, antioxidant
capacity (ABTS/ DPPH), and the content of bioactive compounds (total phenolics,
flavonoids, condensed tannins).
3. Results and discussion
3.1.
Bioaccessibility of phenolic compounds and changes in the antioxidant capacity
during in vitro digestion
3.1.1. Total phenolic compounds (TPCs)
14
A 4-way ANOVA showed that the variety (p<0.001), treatment (p=0.0092), and digestion
phase (p=0.0007) had significant effects on PCs bioaccessibility. The raw white sorghum
(RWS) exhibited a higher amount of total phenolics than did the raw red sorghum (RRS)
(Fig. 1A). In comparison with raw samples, both cooking and nixtamalization decreased
the content of TPCs. About 60 g/kg of the total PCs are present as free forms in the
coloured sorghum grains, which are easily lost when boiling is used (Shen et al., 2018). In
the nixtamalization process, the washing step causes pericarp losses and reduction of
phenolics, which explain the lower PCs values in the processed samples. Nonetheless,
nixtamalization retained more TPCs in the white sorghum, suggesting that this process is a
suitable treatment for the release of total phenolics from non-coloured sorghum.
At the oral phase, TPCs bioaccessibility significantly dropped to negligible amounts in both
red and white sorghum (Figure 1A, B). The low bioaccessibility can be explained via
starch-PCs interactions that may interfere with their quantification. For instance, the TPCs
ability to inhibit the -amylase activity has been used as an explanation for their poor
release in the oral phase (Domínguez-Avila et al., 2017).
Compared with the oral phase, the gastric digestion increased the TPCs bioaccessibility for
both sorghum varieties, showing a maximum release at 30-60 min. Cooking was the best
process to make TPCs bioaccessible in the red variety, that reached recoveries of up to 228
mg GAE/g. The low pH in the simulated gastric fluid favoured the release of polyphenolics
from the food matrix, mostly bound polyphenols that are initially strongly associated with
macromolecules (Domínguez-Avila et al., 2017). The additional effect of thermal
treatments was enough to enhance the extractability of phenolic compounds, a trend that
15
has been reported for corn flours (Ramírez-Jiménez, Rangel-Hernández, Morales-Sánchez,
Loarca-Piña, & Gaytán-Martínez, 2019)
During the intestinal phase, PCs recovery was low in the DF of the cooked and
nixtamalized white sorghum, whereas the NDF had a higher amount of TPCs and,
especially, the NWS had the highest PCs bioaccessibility. The red variety showed an
opposite trend, a higher PCs recovery in the DF after cooking and nixtamalization, and low
amounts in the NDF. Cooking produced the highest TPCs release in red sorghum at the end
of the intestinal phase. Nixtamalization induced similar PCs release at 90 min for NRS1
(123 mg/g) and 30 and 120 min for NWS1 (103 mg/g) (data not shown). Although the
amounts of PCs quantified in the nixtamalized samples were lower than is the cooked
samples (NRS1 vs. CRS), the bioaccessibility values calculated as indicated in section
2.5.1. were higher and constant over time.
3.1.2. Total flavonoids (TFs) and condensed tannins (CTs)
White sorghum had a natural abundance of flavonoids, as observed in Figure 1C for the
non-digested samples. This abundance was noted after the thermal treatment was applied,
especially when nixtamalization was used, due to the pericarp removal during the washing
steps. Despite the initial drop during the oral stage, flavonoid bioaccessibility was
significantly enhanced by the thermal treatment during the gastric phase, especially by
nixtamalization (Figure 1C, 1D). Later, a remarkable proportion of flavonoids from all the
white sorghum samples was absorbed through the small intestine (DF, 120 min), which
indicates that flavonoids were readily transported across the small intestine. Despite this, a
significant amount of flavonoids remained in the NDF, as previously reported by ApeaBah, Minnaar, Bester, & Duodu (2016). Flavonoids play a biological role in the gut,
16
serving as a fermentation substrate for the colonic microbiota and producing several
metabolites linked to anti-inflammatory and antioxidant activities (Duodu & Awika, 2019).
In this sense, the presence of these compounds in the NDF may promote further production
of health-related compounds, such as phenolic metabolites and short-chain fatty acids
(Luzardo-Ocampo et al., 2018).
RRS was not bioaccessible during the entire digestion process. Only the cooking process
increased the bioaccessibility in the gastric phase and showed higher absorption in the
intestine. The low bioaccessibility in red varieties has been partially explained as a result of
the non-covalent conjugation of PCs with resistant dietary-fibre (Jakobek & Matić, 2019).
Condensed tannins (CTs) are known anti-nutritional factors that diminish the nutritive
value by binding proteins such as kafirins (Duodu & Awika, 2019). Several treatments have
been applied to remove these compounds and, so far, a previous study indicates that
nixtamalization is effective in reducing CTs when the proper lime concentrations are used
(Gaytán-Martínez et al., 2017).
In our study, undigested RRS and RWS had similar
amounts of CTs (Figures 1E, 1F). After digestion, tannins increased their bioaccessibility,
but only the nixtamalization process significantly reduced CTs in the white variety. For
RWS, no significant differences were detected between the processing methods during the
digestion stages, except for a remarkably increased absorption of CTs from RWS (DF).
Other reports confirm our results, for example, the digestibility and bioavailability were
improved in milled tannin and non-tannin sorghum grains treated with NaOH solutions (10,
20 and 40 g NaOH/kg) at 50 ºC (Adetunji, Duodu, & Taylor, 2015).
Although nixtamalization seems to be an adequate method for removing CTs in red
sorghum, the effect on white sorghum was negligible. This feature can be exploited since
CTs reduce feed efficiency through inhibition of digestive enzymes and intestinal brush17
border transporters, a property that might be used for the reduction of the caloric impact of
food. In this context, in vivo studies report a reduction in body weight gain, feed conversion
ratio, and an increased food consumption when high-tannin-sorghums are included in the
diet of rabbits (Duodu & Awika, 2019). As observed in Figure 1C for CTs, most of them
are concentrated in the NDF, and this fraction can be either fermented by gut microbiota or
excreted in feces. The maximum CT values reached during digestion are within the
reported safe limits of tannin intake for human consumption from plant sources, i.e. about
3-35 mg CE/g per day (Gaytán-Martínez et al., 2017).
3.2.
Bioaccessibility of free-phenolic compounds
Figure 2 shows the bioaccessibility of the main phenolic compounds quantified in all
sorghum samples during each digestion stage. An overall analysis of the compounds
showed that the two hydroxycinnamic acids (gallic and chlorogenic acid) had the highest
bioaccessibilities, while caffeic acid and quercetin exhibited the lowest. Gallic acid showed
a significant increase in bioaccessibility and absorption during the intestinal phase (Figure
2A), especially in the NRS1 that had the highest values in both ND and NDF. Since
carbohydrates are the major macromolecules in sorghum (760-763 g/kg and 704-713 g/kg
for red and white sorghum, respectively) (Gaytán-Martínez et al., 2017), interactions
between carbohydrates and phenolic acids are predominant in the digestion dynamics of
sorghum grains through non-covalent bonds with cellulose (Domínguez-Avila et al., 2017).
During the intestinal phase, the breakdown of carbohydrates releases an important amount
of gallic acid in its absorbable and non-digestible form. A similar trend was observed for
snacks made from nixtamalized corn and cooked common beans submitted to the same in
18
vitro gastrointestinal procedure, which showed the highest release of gallic acid at the same
stage (Luzardo-Ocampo et al., 2017).
Figure 2B and Figure 2C show a similar bioaccessibility trend for chlorogenic and caffeic
acids; however, overall values of caffeic acid were significantly lower (p<0.05). Compared
to the initial methanolic extract, the bioaccessibility of caffeic acid significantly increased
at the stomach stage. Our results are in accordance with the reduction observed in caffeic
acid during the gastric and intestinal digestion of sorghum-cowpea porridge; that was
intensified as a result of disruption of cell membranes and cell walls exerted by different
thermal treatments (Adelakun & Duodu, 2017). It is important to note that the recovery of
polyphenolics is influenced by the analytical procedure, where the extraction yields can
vary from 700 g/kg to 960 g/kg (Cendrowski, Ścibisz, Kieliszek, Kolniak-Ostek, & Mitek,
2017).
Quercetin was the main flavonoid quantified in the non-digested raw samples and CWS
(Figure 2D). At the oral stage, this compound was found only in the nixtamalized flours
(NWS1 and NRS1) and exhibited low bioaccessibility and absorption at the intestinal stage.
Regarding the intestinal phase, some publications report that quercetin is not absorbed
across the intestinal epithelial cells; its high molecular weight and fat micellization affect
the digestion process and the epithelial transport (Domínguez-Avila et al., 2017).
3.3.
Changes in the antioxidant capacity
Sorghum phytochemicals, particularly phenolic compounds, have been linked to high
antioxidant capacity and its related health benefits (Girard & Awika, 2018; StefoskaNeedham, Beck, Johnson, & Tapsell, 2015). Environmental factors, such as drought and
19
low temperature, alter the PCs profile since most bioactives are produced as a response to
plant stress (Cendrowski, Scibisz, Mitek, & Kieliszek, 2018).
As shown in Fig. 3A and B., both red and white raw sorghum flours had the highest
antioxidant capacity (ABTS and DPPH), followed by the nixtamalized and cooked samples,
which showed no significant differences between each other. At the oral phase, the
decreased antioxidant capacity of all samples was associated with the low PCs
bioaccessibility shown at this stage.
During the gastric phase, the antioxidant activity significantly increased for NWS0 and
NRS1 while the rest of the treatments did not suffer changes during the gastrointestinal
digestion. As observed in Figure 3C, the antioxidant capacity was highly correlated with
the total PCs and CTs contents. In sorghum, high molecular weight oligomers or polymers
of condensed tannins (composed of flavan-3-ol nuclei) exhibit strong radical-scavenging
activity (15-30-fold higher than simple phenolics in animal studies), chelation of transition
metals, as well as the inhibition of pro-oxidative enzymes (Stefoska-Needham et al., 2015).
The antioxidant capacity showed a remarkable increase during the intestinal phase,
especially for the cooked (CWS, CRS) and NRS1 samples, indicating that the thermal
process significantly affects this property. Dlamini, Taylor, & Rooney (2007) evaluated six
varieties of non-tannin- (NK283 and Macia) and tannin- (Red Swazi, NS5511 and Framida)
sorghum processed by different methods (decortication and milling, fermentation, and
extrusion cooking). The non-tannin varieties exhibited the lowest antioxidant capacities
(ABTS, DPPH) despite the processing type, while the Red Swazi (with the highest tannins
concentration) had the highest antioxidant capacity. The authors also reported that
fermentation and extrusion cooking, decreased the antioxidant capacity by up to 88.8 %,
20
due to the water-induced depolymerization of tannins and phenolic compounds into lowmolecular oligomers that caused a decrease in the biological radical-scavenging properties.
Figure 4 shows the principal component analysis (PCA). Five components explained more
than 80 % of the total variation (Figure 4A). The variables with most influence were the
antioxidant capacity (ABTS/DPPH) in the first component, CTs in the second, TFs and the
gastric phase in the third, process in the fourth and total PCs in the fifth (Figure 4B).
Figure 4C shows the loading and score plots of PC1 and PC4 for the analyzed variables.
Data were separated by process and variety. The process was the variable that influenced
TFs the most, whereas phase and PCs explained the variation of the antioxidant capacity.
According to the analysis, the thermal procedure (cooking/nixtamalization) were the most
significant variables that influenced the PCs content and the antioxidant capacity during the
in vitro gastrointestinal digestion. Nixtamalization had a greater effect on white sorghum.
3.4.
Apparent permeability coefficients (Papp)
The apparent permeability coefficients are factors that express the absorption from the
apical (intestinal lumen) to the basolateral (blood flux) side of the intestine. This
measurement is meant to predict the bioavailability of phenolic compounds (Campos-Vega
et al., 2015; Luzardo-Ocampo et al., 2017). Total phenolic compounds and TFs (Figures AD) showed a similar trend in the net Papp (Papp), exhibiting low values at the end of the
intestinal incubation.
For the TPCs (Figure 5A-B), a stabilization of the flux was shown between 90 and 120
min. The efflux ratio confirmed that both apical and basolateral sides reached an
equilibrium. Flavonoids and condensed tannins (Figure 5C-E) presented their maximum
transport rates between 30 and 60 min for all samples.
21
For the selected phenolic compounds, Supplementary Table 1 indicates the apparent
permeability values and efflux ratios of gallic, caffeic, chlorogenic acids, and quercetin
during the intestinal incubation for each sorghum flour. Permeation to the basolateral side
was reached between 60 and 90 min for gallic acid, indicating a saturation of the system in
this time interval. Caffeic acid showed a peak at 90 min for RWS and NRS, chlorogenic
acid at 60 min (RRS) and quercetin between 60 and 90 min. There are no reports of the
apparent permeability coefficients and efflux ratio for phenolic compounds of sorghum
grains, yet gallic and caffeic acids values are similar to those reported by Luzardo-Ocampo
et al. (2017) for a nixtamalized corn-cooked common bean chip subjected to the same in
vitro gastrointestinal digestion procedure.
The apparent permeability coefficients can be associated with permeation speeds that also
correlate with the similarity of in vitro systems to in vivo systems. For instance, Hubatsch et
al. (2007) defined the range for low (Papp < 1 x 10-7 cm/s) and high (Papp > 1 x 10-6)
permeation using the Caco-2 cell monolayer model. On this basis, it can be inferred that the
transport of phenolic compounds is at high permeation during the intestinal phase.
Nevertheless, further studies for evaluating the bioavailability of these compounds, using in
vivo systems, should be performed to confirm these results.
It has been reported that efflux ratios (ER) lower than 0.5 and higher than 2.0 are indicative
of active or passive transportation, respectively (Hubatsch et al., 2007). Figure 5 depicts
ER trends of TPCs, TF, and CTs for all sorghum treatments. Overall, nixtamalized samples
showed a decrease in the ER values of TF and CTs, but not in TPCs, which showed an
increase at the end of the intestinal incubation (Figure 5A). Except for NRS1, TPCs
exhibited an active transportation mechanism. ER values of TFs and CTs (Figures 5C-F)
did not show significant differences (p>0.05) in the cooked samples, indicating that this
22
thermal procedure can maintain both the speed and absorption of phenolic compounds
through the small intestine.
Low molecular weight phenolics are mainly transported by passive diffusion; however, the
food matrix influences have an important effect on these mechanisms (Domínguez-Avila et
al., 2017). These effects can be seen in the ER values of gallic acid (Supplementary Table
1), that showed values between 0.5 and 2.0 for most of the treatments, except for the CWS
which presented a value higher than 2.0 at 60 min of incubation. These results are in
agreement with the reported structural affinity of gallic acid with transporters through the
enterocyte and paracellular diffusion (Konishi, Kobayashi, & Shimizu, 2003). Despite the
fact that ER of caffeic acid could not have been calculated, it is well known that
monocarboxylic acid transporters are involved in its transportation for the recognition of
monoanionic carboxylic groups or non-polar chains in the polyphenol structure (Bohn,
2016).
Figure 6 shows the intuitive prediction of permeability for the selected phenolic acids. The
Papp Net values of gallic, chlorogenic and caffeic acids, and quercetin were similar to those
reported in the Caco-2 cell predictive model for processed and raw white and red sorghum
(Figure 6A). This result validates the experimental in vitro gastrointestinal model used in
this research. The bioavailability radar of each compound (Figure 6B), is a scheme that
contains the descriptors for lipophilicity (LP), size, polarity (PO), solubility (IN), saturation
(IS) and flexibility (FL). The pink colour depicts the oral limits for the five properties,
which are within the limits of log P -0.7-5, size (molecular weight) of 150-500, polarity 20130, water solubility score 1-3 (1 is the highest and 5 is the lowest), and the number of
rotatable bonds (flexibility) 0-9. Except for the IS, all the evaluated phenolic compounds
23
fitted within the limits of all parameters. Chlorogenic acid showed a different polarity,
explaining the different trend for this molecule in the “Boiled Egg” diagram. A higher
saturation is associated with enhanced absorption, which partially agrees with our results
for gallic and caffeic acids that exhibited high concentrations in the digestible fraction
(Figures 2A, 2C).
The “Boiled Egg” diagram (Figure 6C) showed that caffeic and gallic acids, and quercetin
were located in the white region, while chlorogenic acid was plotted outside this region.
The white region correlates with a high probability of passive gastrointestinal absorption
(Daina et al., 2017), which was shown in Supplementary Table 1B for the efflux ratio of
gallic acid during the in vitro intestinal absorption. The location of chlorogenic acid outside
this area is an indicator of another type of transport mechanism; however, the model does
not consider potential interactions with the food matrix.
Additionally, some ADMET (Adsorption, distribution, metabolism, excretion, and toxicity)
parameters were calculated for the target phenolics. Caffeic acid had a high probability of
being absorbed and bioavailable (Figure 6D). This absorption was also significant for
quercetin, as confirmed by the higher content of this compound in the digestible fraction
(Figure 2D).
3.5.
Water flux dynamics
As a way to assess the small intestine viability during the intestinal digestion, the water flux
dynamics can provide information about the intestinal water permeability and the behaviour
of the associated water transporters (Khemiss, Saidane, & Moshtaghie, 2005).
Supplementary Figure S1 shows the water flux during the intestinal digestion. All
samples exhibited similar behaviour, that confirmed the intestinal viability of the tissues
24
during the ex vivo procedure. At the beginning of the incubation, the blank (salivary
solution subjected to in vitro gastrointestinal digestion) displayed the lowest value while
the raw samples showed the highest. The differences are more prominent at 60 min, where
the nixtamalized samples exhibited the highest water flux values, a trend that was
maintained during the remaining incubation time. Despite the lack of reports of water flux
for ex vivo measurements from cereals and grains, the behaviour of the water flux for
sorghum samples is similar to the reported values of Luzardo-Ocampo et al. (2017) for
nixtamalized corn-cooked common bean chips during similar incubation times for the same
in vitro gastrointestinal digestion.
Rising values of water flux are associated with a higher intestinal wettability and absorption
capacity; thus, it can be inferred that the peak of absorption for the nixtamalized samples
occurred during the 60 min of intestinal incubation. This is in agreement with the majority
of the reported Papp
Net
from the selected phenolic compounds and condensed tannins.
Nevertheless, the absorption dynamics of the compounds tend toward an equilibrium at the
end of the incubation. Furthermore, the variability of the water flux is also a function of the
variable distribution of aquaporin and protein channels along the intestinal epithelium that
contribute to the regulation of the osmotic pressure gradient (Zhu, Chen, & Jiang, 2016).
4. Conclusion
In this study, we showed that the bioaccessibility of phenolic compounds from sorghum
dynamically changed during the gastrointestinal digestion. The changes in bioaccessibility
were mainly correlated to variety, thermal treatment, and the digestion phase. Cooking and
nixtamalization increased the bioaccessibility of total phenolics and flavonoids.
25
The variety determined the extent of change. For instance, cooking was better to increase
PCs in the red variety whereas, for flavonoids, it was the best treatment for the white
grains. Both thermal treatments increased the gastric bioaccessibility and intestinal
absorption of PCs and flavonoids. Only nixtamalization significantly decreased the
condensed tannin contents to safe intake values for humans.
The thermal treatment was the most important variable influencing the antioxidant capacity,
a variable that must be taken into account for industrial processes. During the intestinal
transport dynamics, permeability coefficients displayed similarities between this
gastrointestinal digestion procedure and some reported in vitro models, indicating different
cellular transport mechanisms. The information provided in this work gives insight about
the impact of thermal treatment on the bioaccessibility of phenolic compounds from
sorghum, which allow us to better understand the derived health benefits of sorghum
phenolics and their potential as a functional ingredient. Control and optimization of the
thermal treatments is essential for keeping safe values of anti-nutritional compounds
without affecting the health-derived properties of the food matrix.
Acknowledgements
Ivan Luzardo-Ocampo (grant number: 384201) and Angel H. Cabrera-Ramirez (grant
number: 734975) were supported by a scholarship from the Consejo Nacional de Ciencia y
Tecnología (CONACYT-Mexico) [The authors would like to thank B.S. Jairo Cruz for the
preparation and processing of samples. English was edited by Subhiska Chandrasekaran.
Conflict of interest statement
The authors declare that there is no conflict of interest.
26
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32
WHITE SORGHUM
RED SORGHUM
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10
5
0
d d
e e e
UND
Mouth Stomach DF
Raw
RRS
CTs (mg CE/ g sample)
E
CTs (mg CE/ g sample)
b
c
0
C
a
80
60
40
20
d c
0
NDF
Cooked
CRS
a
b
UND
e e e
b b
b
c
Mouth Stomach
b b
c
DF
c
NDF
Nixtamalized
NRS1
Figure 1. Bioaccessibility of total phenolic compounds (A, B), total flavonoids (C, D) and
condensed tannins (E, F) from raw, cooked and nixtamalized sorghum [10 g Ca(OH)2/kg]
during the different digestion phases.
33
Data are expressed as the means  SD of two independent experiments. Different letters indicate significant
differences (p<0.05) by Tukey-Kramer’s test. GAE: gallic acid equivalents; CE: (+)-catechin equivalents;
CTs: Condensed tannins; DF: Digestible Fraction; NDF: Non-digestible fraction; RE: rutin equivalents; TF:
Total flavonoids; TPCs: Total phenolic compounds; UND: Undigested; RWS: raw white sorghum; RRS:
raw red sorghum; CWS: cooked white sorghum; CRS: cooked red sorghum; NWS1: nixtamalized white
sorghum with 10 g Ca(OH)2/kg flour; NRS1: nixtamalized red sorghum with 10 g Ca(OH)2/kg flour. The
stomach,
DF,
and
NDF
samples
were
taken
34
at
120
min
of
incubation
(37
ºC).
A)
B)
7000
400
a
Chlorogenic acid (µg/g sample)
Gallic acid (µg/g sample)
6000
b
5000
c
c
4000
c
c
d
3000
e
f
g
h
2000
i
1000
a
350
i
jjj j
k
0
ME
kj k
k
k
k k
l
j
i
j
Mouth
Estomach
DF
In vitro gastrointestinal digestion stages
300
250
200
a
b
c
RWS
dd
RRS
e
NWS0
NRS0
150
100
50
j
0
NWS1
f
h h
i i
NRS1
g
jj
g
h
ME
Mouth Estomach
DF
NDF
In vitro gastrointestinal digestion stages
NDF
35
C)
D)
100
700
a
a
90
600
Quercetin (µg/g sample)
Caffeic acid (µg/g sample)
80
70
60
50
b
40
30
b b
500
RWS
400
RRS
NWS0
300
NRS0
NWS1
200
c
20
d
d
e
10
h g
0
ME
h
g
f
Mouth
Estomach
DF
In vitro gastrointestinal digestion stages
NRS1
c
100
f
g fg
0
ef
c
d
f
e
f
ME
Mouth Estomach
DF
NDF
In vitro gastrointestinal digestion stages
NDF
Figure 2. Bioaccessibility of free-phenolic compounds from raw, cooked and nixtamalized white and red sorghum (Sorghum spp.): A)
Gallic acid; B) Chlorogenic acid; C) Caffeic acid; D) Quercetin
36
Data are the means  SD of two independent experiments. Different letters express significant differences (p<0.05) for each sample between the in vitro
gastrointestinal digestion stages. The concentration of each free-phenolic compound is expressed as microgramme equivalents of each free-phenolic/g sample.
ME: Methanolic extract; DF: Digestible Fraction (120 min); NDF: Non-digestible fraction (120 min). RWS: Raw white sorghum; RRS: Raw red sorghum;
CWS: Cooked white sorghum; CRS: Cooked red sorghum; NWS1: Nixtamalized white sorghum with 10 g Ca(OH)2/kg flour; NRS1: Nixtamalized red sorghum
with 10 g Ca(OH)2/kg flour. The stomach, DF, and NDF samples were taken at 120 min of incubation (37 ºC).
37
A)
B)
35
35
a
30
25
20
15
10
a
b
a
b
c
5
b
0
ME
b
a
Mouth
Stomach
c
d
c
DF
NDF
ABTS (µmol eq. Trolox/g sample)
DPPH (µmol eq. Trolox/g sample)
40
a
30
25
RWS
RRS
20
a
15
10
a
c
a
0
ME
CRS
Mouth
b
c
Stomach
b
c
b
c
d
e
DF
NDF
C)
Free-phenolic
compounds
Total Phenolic
compounds
Total Flavonoids Total Phenolics ABTS DPPH
Quercetin
0.36*
0.29* 0.28*
p-Coumaric acid
0.21
-0.51* 0.08
Gallic acid
0.08
0.55* 0.19*
Chlorogenic acid
0.31*
0.32* 0.31*
Caffeic acid
0.35*
0.27* 0.29*
Total Phenolics
0.43* 0.42*
Total Flavonoids
0.07
0.10
Condensed Tannins
0.30*
0.07
38
CWS
NWS1
b
5
a
NRS1
Antioxidant
Capacity
ABTS
DPPH
1.00
0.63*
1.00
Figure 3. Antioxidant capacity of all sorghum samples by A) DPPH and B) ABTS methods during the in vitro gastrointestinal
digestion; C) Correlation coefficients between the antioxidant capacity (ABTS/DPPH) and the chemical composition of the samples
through the in vitro gastrointestinal digestion.
Data are the means  SD of two independent experiments. ME: Methanolic extract; DF: Digestible Fraction (120 min); NDF: Non-digestible fraction (120 min).
RWS: Raw white sorghum; RRS: Raw red sorghum; CWS: Cooked white sorghum; CRS: Cooked red sorghum; NWS1: Nixtamalized white sorghum with 10 g
Ca(OH)2/kg flour; NRS1: Nixtamalized red sorghum with 10 g Ca(OH)2/kg flour. The stomach sample was taken at 120 min of incubation (37 ºC). Different
letters express significant differences (p<0.05) for each in vitro gastrointestinal digestion stage among all samples. For the correlation coefficients, the asterisks
indicate
significant
differences
(p<0.05)
39
using
Spearman’s
correlation.
A)
Principal
Component
1
2
3
4
5
6
7
8
9
Percent
27.457
20.777
13.463
10.531
8.626
7.930
5.145
4.074
2.176
Cumulative
Percent
27.457
48.233
61.696
72.227
80.853
88.784
93.929
98.003
100.179
B)
Variable
PC1
Gallic acid
-0.213
Chlorogenic acid
0.340
Quercetin
0.497
Total Phenolics
0.421
Total Flavonoids
0.044
Condensed Tannins 0.042
DPPH
0.463
ABTS
0.443
PC2
0.326
-0.323
-0.074
-0.018
0.460
0.706
0.214
0.168
C)
40
PC3
0.365
0.441
-0.047
0.465
0.494
-0.132
-0.178
-0.402
PC4
PC5
0.693 0.054
0.325 0.361
-0.182 0.015
-0.075 -0.694
-0.534 0.473
0.044 -0.278
0.266 0.094
0.130 0.273
Figure 4. Principal component analysis (PCA) of white and red sorghum (Sorghum bicolor
L. Moench) clustered by treatments. A) Eigenvalues and their participative percentage
among the total variation of the analyzed variables; B) Participation of each variable on
each component from the PCA analysis; C) Loading and scatter plots of the first and fourth
component for all sorghum samples and the assessed variables.
PC: Principal component; RWS: Raw white sorghum; RRS: Raw red sorghum; CWS0: Cooked white
sorghum with 10 g Ca(OH)2/kg flour; CRS0: Cooked red sorghum; NWS1: Nixtamalized white sorghum
with 10 g Ca(OH)2/kg flour; NRS1: Nixtamalized red sorghum. The procedures: 1, 2, 3 corresponded,
respectively, to raw (RWS, RRS), cooked (CWS, CRS) and nixtamalized (NWS1, NRS1) samples.
41
Figure 5. Net apparent permeability coefficients (Papp Net) and efflux ratio (ER) of total
phenolics of total phenolics (A, B), total flavonoids (C, D) and condensed tannins (E, F)
from raw, cooked and nixtamalized white and red sorghum.
42
RWS: Raw white sorghum; CWS: Cooked white sorghum; NWS1: Nixtamalized white sorghum with 10 g
Ca(OH)2/kg flour; RRS: Raw red sorghum; CRS: Cooked red sorghum; NRS1: Nixtamalized red sorghum
with 10 g Ca(OH)2/kg flour; Papp Net: Net apparent permeability; ER: Efflux ratio. The results were expressed
as means  SD. Different letters express significant differences by Tukey-Kramer’s test (p<0.05) for the Papp
Net. The
asterisks indicate significant differences (pz0.05) by Tukey-Kramer’s Test for the ER charts.
43
Figure 6. Intuitive prediction of absorption and permeability of selected phenolic
compounds. A) Comparison between the obtained Papp Net values and the predicted ADMET
(Absorption, Distribution, Metabolism, Excretion and Toxicity) Caco-2 permeability; B)
Bioavailability radar of selected phenolic compounds; C) Prediction of passive human
44
gastrointestinal digestion via BOILED-Egg model; D) Prediction of the absorption’s
probability from phenolic compounds through several in vitro gastrointestinal models.
FL: Flexibility; LP: Lipophilicity; IN: Solubility; IS: Saturation; PO: Polarity; SZ: Size; TPSA:
Topological Polar Surface Area; WLOG: Wildman & Crippen atomistic method score. The bold values
indicate either a high probability of absorption or acting as a substrate/inhibitor (Figure 2D).
Highlights

Nixtamalization significantly reduced condensed tannins from white/red sorghum.

Phenolics from sorghum are significantly absorbed in the small intestine.

The non-digestible fraction of sorghum retains considerably amounts of phenolics.

The antioxidant capacity is governed by condensed tannins/total phenolics.

Permeability coefficients from phenolics are linked to transportation mechanisms.
45