1. Introduction
Lentil (Lens culinaris Medikus) is an edible pulse, cultivated in 52
countries and recognized among the main cold season legume in the world [1].
Lentils are globally commercialized on a wild scale, and its leading producers
are Canada, India, Australia, Turkey, Nepal, and the USA, accounting for over
80% of the world’s total lentil production [2]. The cultivated lentil is a
diploid, self-pollinating, and annual legume [3]. The taxonomy of L.
culinaris presents four subspecies: subsp. culinaris;
orientalis, odemensis, and tomentosus [3]. These
pulses are also known as a significant source of dietary protein in developing
nations [4]. Lentils exist as a spectrum of colors, including black, brown,
green, red, orange or yellow, depending on the composition of the cultivar, seed
coat, along with cotyledon [5]. In the past, lentil was named “poor man’s
meat”, emerging in ancient Europe. Thereby, people have considered it a cheap
and excellent substitute for animal protein for a long time, as it contains 24.63
g/100 g of protein, and is a potential overall source of nutrient for individuals
with deficiencies in micronutrients [6, 7]. Additionally, enriched phenolic
compounds are also detected in various types of lentils [5].
Polyphenols are secondary compounds extensively distributed in the plant kingdom
[8]. It is characterized by the presence of several phenolic groups, that is,
aromatic rings with hydroxyl groups. Polyphenols can be classified as several
classes, i.e., hydroxycinnamic acids, hydroxybenzoic acids, anthocyanins,
proanthocyanidins, flavonols, flavanols, flavones, flavanones, isoflavones,
lignans, and stilbenes [9]. Due to the high antioxidant power of polyphenols,
they can have different effects, including anti-inflammatory effects, in addition
to affecting blood sugar through distinct mechanisms, such as inhibiting glucose
absorption in the intestine and improving insulin resistance [10]. Furthermore,
various potential mechanisms of polyphenols are responsible for certain disease
prevention, including inhibition of bacterial replication enzymes, induction of
apoptosis in tumor cells, and stimulation of cytokine production by
monocytes/macrophages [11]. Additionally, people who follow diets rich in
polyphenols have a low risk for a number of chronic diseases [12]. Thus,
polyphenols exhibit strong health potential for the human body.
The high content of phytochemicals in pulse-based diets including polyphenols is
related to health benefits [13]. Lentil presents a diverse phenolic profile in
which phenolic acids, flavonoids, and lignans are the majority. Phenolic acids
are phenols that own one carboxylic acid functional group, which is the major
class of phenolic compounds [14, 15]. They are usually classified as the
derivatives of hydroxy-benzoic acid and hydroxycinnamic acid [14]. These
compounds show strong antioxidant activity and have been studied for their
potential against oxidative damage [16]. Flavonoids are polyphenolic secondary
metabolites commonly linked with a cetone group [17]. These polyphenols stand for
one of the major groups of phenols and they are low molecular weight compounds
with a broad-spectrum occurrence [16]. Flavonoids are known for their disease
preventive activities including antimicrobial, antioxidant, anti-inflammatory,
and as inhibitory substances for various stages of tumor development [15, 16].
Lignans are secondary metabolites that belong to the group of diphenolic
compounds and present a dibenzylbutane skeleton [17]. Lignans are non-flavonoid
compounds, that became to be broadly investigated [15]. They exhibit their
potential health benefits through antimicrobial, and anticancer activities [15, 16].
Lens culinaris have been used in traditional practices to reduce the
prevalence of ailments such as obesity, diabetes, cancers, and cardiovascular
diseases. The seed is rich in secondary metabolites and bioactive functional
groups, including phytosterols, trypsin/protease inhibitors, lectins, defensins,
dietary fibers, polyphenols, flavonoids, phytate, triterpenoids, and saponin [5].
Some phenolic compounds, such as quercetin and populins, which belong to the
flavonoid group, were characterized in different lentils and their antioxidant
potential was also reported [18]. Moreover, a greater level of flavan-3-ols,
proanthocyanidins and some flavonols were noted in the seed coats of lentils [5].
Such active ingredients have potential advantages to be used in alternative
medicines, to act as antioxidant, antibacterial, antifungal, antiviral,
cardioprotective, anti-inflammatory, reno-protective, antidiabetic, anticancer,
anti-obesity, hypolipidemic and chemo-preventive [5]. However, there have been
few studies on red, green, brown, and black lentils in terms of phenolic
compounds, most studies have compared lentils with other legumes in terms of
phenolic compounds [19, 20, 21, 22, 23].
In-depth knowledge of the phenolic profile of different types of lentils has not
been widely studied, leading to gaps that demand further research on the phenolic
profile and antioxidant activity of these pulses. Therefore, this study aimed to
carry out the characterization of the phenolic substances and the antioxidant
potential of four different types of lentils including red, green, brown, and
black lentils. Polyphenolic components were extracted from the lentil samples and
studied regarding their TPC, TFC, TTC, TPAC, and TAC overall contents. For the
antioxidant potential evaluation, DPPH, FRAP, ABTS, •OH-RSA, FICA,
RPA, and PMA were tested. Further, the polyphenolic compounds were screened and
characterized by LC-ESI-QTOF-MS. The present research will give more
credible information on lentils’ antioxidant and health-promoting characteristics
to optimize their use in the food, supplement, and pharmaceutical industries.
2. Materials and Methods
2.1 Chemical and Regents
The majority of chemicals utilized for extraction, identification, and
quantification were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA)
and were of analytical grade, which contain potassium persulfate, aluminum
chloride hexahydrate, Folin-Ciocalteu’s phenol reagent,
2,2’-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ),
2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), quercetin,
vanillin, catechin, gallic acid monohydrate, and Trolox. The chemicals anhydrous
sodium carbonate, 3-hydroxybenzoic acid, sodium acetate, sodium hydroxide, and
potassium chloride were obtained from Chem-Supply Pty Ltd. (Adelaide, SA,
Australia). Thermo Fisher Scientific Inc (Waltham, MA, USA) supplied acetic acid,
ethanol, sulfuric acid, hydrochloric acid, n-butanol, hydrogen peroxide,
trisodium phosphate and sodium carbonate. Polyvinylpolyrrolidone, Iron(II)
sulfate heptahydrate, Iron(III) chloride, Iron(II) chloride,
ethylenediaminetetraacetic acid,
3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p’-disulfonic acid monosodium salt
hydrate, tannic acid, escin, cyanidin chloride, ammonium molybdate, sodium
phosphate monobasic monohydrate, sodium phosphate dibasic heptahydrate, potassium
ferricyanide(III), and trichloroacetic acid were purchased from Sigma-Aldrich
(St. Louis, MO, USA) as well.
2.2 Sample Preparation
The four types of lentils utilized in this research (red, green, brown, and
black) were purchased at Melbourne’s local market. All the lentil samples were
crushed into fine powders using a grinder (Breville Smart Grinder Pro,
model BCG820BSSXL, Melbourne, VIC, Australia) and reserved in a dark location at
room temperature to prevent exposure to light.
2.3 Extraction of Phenolic Compounds
The extraction process of phenolic compounds followed the study from [24] with
slight alterations. Two grams of each lentil sample powder was thoroughly mixed
with 70% ethanol (1:10, w/w) and homogenized using an Ultra-Turrax T25
Homogenizer (IKA, Staufen, Germany) at 10,000 rpm for 30 s. After that, the
mixture was incubated in a ZWYR-240 incubator shaker (Labwit, Ashwood, VIC,
Australia) at 120 rpm at 10 °C for 16 hours. Then, the extracts were
centrifuged at 8000 rpm for 15 minutes at 4 °C (ROTINA380R, Hettich
Refrigerated Centrifuge, Tuttlingen, Baden-Württemberg, Germany) and the
supernatant was collected and frozen at –20 °C for subsequent analysis.
2.4 Estimation of Phenolic Compounds and Antioxidant Assays
2.4.1 Total Phenolic Content (TPC)
A modified version of the Folin-Ciocalteu technique was used to access the TPC
of lentil samples [25]. Twenty-five microliters of lentil samples were added to
25 L of Folin-Ciocalteu reagent solution (1:3 dilution with water)
in a 96-well plate (Costar, Corning, NY, USA) containing 200 L of
Milli-Q water. The mixture was incubated for 5 minutes at 25 °C and
then, 25 L of sodium carbonate (10%, w/v) was added to
the reaction mixture. It was held for 60 minutes at 25 °C in the dark.
The absorbance at 765 nm was evaluated using a spectrophotometer (Thermo Fisher
Scientific, Waltham, MA, USA). A gallic acid standard curve containing 0–200
g/mL gallic acid in methanol was prepared. The TPC results were
displayed as mg of equivalent gallic acid (GAE) gram of sample.
2.4.2 Total Flavonoid Content (TFC)
The TFC of different lentils was evaluated using the aluminum chloride method
modified [26]. Eighty microliters of lentil samples were transferred to 80
L of aluminum chloride solution (2%, w/v), followed by
mixing 120 L sodium acetate (50 g/L) in the 96-well plate,
accompanied by a 150-minute incubation at 37 °C. At 440 nm, the
absorbance was determined. The quercetin standard curve was constructed utilizing
a 0–50 g/mL quercetin methanolic solution. The TFC values were displayed
as mg of quercetin equivalent (QE) per gram of sample.
2.4.3 Total Tannin Content (TTC)
The determination of TTC was measured using a polyvinylpolypyrrolidone (PVPP)
method [27]. Total tannin constituents were measured in Eppendorf tubes by
transferring 120 L of the lentil extract, 180 L of
distilled water, 150 L of Folin–Ciocalteu reagent (50%
v/v), and 675 L of sodium carbonate (20% w/v).
The mixture was vortexed and stored at room temperature for 40 minutes in the
dark. Centrifuge was then conducted and 200 L of the supernatant
was added to a 96-well plate. After that, the absorbance was read at 725 nm.
Then, a second analysis was carried out to determine the phenolic compounds left
after tannins were precipitated with PVPP. Fifty milligrams of PVPP were added,
accompanied by the addition of 0.5 mL of water along with 375 L of
sample extract. The mixture was centrifuged at 8000 g for 10 minutes at 4
°C after being vortexed and maintained at 4 °C for 15 minutes.
Using the same methods outlined above for total phenolic compounds, the
supernatant was kept for Folin-Ciocalteu analysis.
To calculate the tannin content, the values of the first and second experiments
were subtracted. A standard curve was prepared using a 0–250 g/mL
Tannic acid solution. The TTC results were displayed as mg of Tannic acid
equivalents (TAE) per gram of sample.
2.4.4 Total Condensed Tannins (TCT)
The TCT was evaluated utilizing a vanillin-sulfuric acid method [28].
Twenty-five microliters of lentil samples were transferred to 150 L
of vanillin solution (4%, w/v), followed by the addition of twenty-five
microliters of 32% sulfuric acid in a 96-well plate along with a 15-minute
incubation under 25 °C. At 500 nm, the absorbance was determined. The
standard curve was constructed using a 0–1000 g/mL catechin. The
TCT results were shown as mg of equivalent catechin (CE) per gram of sample.
2.4.5 Total Proanthocyanidin Content (TPAC)
The TPAC was measured using a modified method of [29], which relies on the
acid-catalyzed oxidative cleavage of proanthocyanidins’ C–C interflavanic bond
in butanol-HCl. Reagent A was prepared by dissolving 35 milligrams of
FeSO7HO in 2.5 mL of concentrated HCl, then making a
solution up to 50 mL with butanol. Briefly, 30 L of each extract
and 800 L of reagent A were added in Eppendorf tubes and incubated
at 95 °C for 50 minutes. After cooling down to room temperature, two
hundred microliters of the mixture was transferred into a 96-well plate and read
the absorbance at 550 nm. The standard curve was prepared using a 0–500
g/mL cyanidin chloride solution. The TPAC results were shown as mg
of cyanidin chloride equivalents (CCE) per gram of sample.
2.4.6 Total Anthocyanin Content (TAC)
The determination of TAC was performed according to the pH differential method
developed by [30]. Four hundred microliters of the sample extract were placed
into a cuvette, followed by the addition of 2.8 mL of pH 1.0 buffer (potassium
chloride, 0.025 M). Another 400 L of sample extract and 2.8 mL of
pH 4.5 buffer (sodium acetate, 0.4 M) were added into a cuvette. Absorbance was
read at 510 and 700 nm, separately. To calculate the absorbance the following
equation was used: Abs = (A – A) pH – (A
– A) pH and the molar extinction coefficient for cyanidin
3-glucoside was considered as 26,900. Results were displayed as mg of cyanidin
3-glucoside equivalents per 100 grams of sample.
2.4.7 2.2-diphenyl-1-picrylhydrazyl Assay (DPPH)
The DPPH activity was evaluated according to a modified version of [31]. In a
96-well plate, forty microliters L of lentil samples were
transferred to 260 L of DPPH methanolic solution (0.1 mM) and
incubated for 30 minutes at 25 °C. At 517 nm, the absorbance was
determined. The standard curve was constructed using a 0–200 g/mL
Trolox. The DPPH radical scavenging activity was shown as mg of Trolox
equivalents (TE) per gram of sample.
2.4.8 Ferric Reducing Antioxidant Power Assay (FRAP)
The FRAP assay was computed using a modified version of [32]. The FRAP technique
assesses a material’s capacity to convert Fe-TPTZ
(ferric-2,4,6-tripyridyl-s-triazine) to Fe-TPTZ. The FRAP reagent was
made by combining FeCl solution (20 mM), TPTZ solution (10 mM), and sodium
acetate solution (300 mM) in a volume ratio of 1:1:10. Then, twenty microliters
of lentil samples were transferred to 280 L of prepared FRAP
solution in a 96-well plate, which was incubated at 37 °C for 10
minutes. At 593 nm, the absorbance was determined. The standard curve was
prepared using a 0–200 g/mL Trolox aqueous solution. The FRAP
results were displayed as mg of Trolox equivalents (TE) per gram of sample.
2.4.9 2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)
Radical Scavenging Assay (ABTS)
The ABTS assay was evaluated using a modified form of the ABTS radical
cation decolorization test [33]. Before usage, ABTS cations were produced by
combining 5 mL of ABTS solution (7 mmol/L) with 88 L of potassium
persulfate solution (140 mM) in a dark environment for sixteen hours. To reach an
initial absorbance of 0.70 at 734 nm, the ABTS solution was further diluted
using analytical-grade ethanol. Then, 10 L of lentil samples were
combined with 290 L of prepared ABTS solution in a 96-well
plate and placed in the dark condition for six minutes at 25 °C. Under
734 nm, the absorbance was read. Utilizing the calibration curve developed with a
0–500 g/mL Trolox aqueous solution, the antioxidant capacity of
lentil samples was evaluated. The ABTS results were displayed as mg of Trolox
equivalents (TE) per gram of sample.
2.4.10 Hydroxyl Radical Scavenging Activity
(•OH-RSA)
Modifications were made to the Fenton-type reaction approach [34] to determine
•OH-RSA. In a 96-well plate, 50 L of lentil samples
were mixed with 50 L FeSO7HO (6 mM) and 50
L HO (30%, 6 mM) and incubated for ten minutes at 25
°C. Following incubation, fifty microliters of 3-hydroxybenzoic acid (6
mM) was transferred. At 510 nm, the absorbance was read. Utilizing a calibration
curve produced with a 0–300 g/mL Trolox solution, the antioxidant
capacity of lentil samples was determined. The results of the •OH-RSA
were displayed as mg of Trolox equivalents (TE) per gram of sample.
2.4.11 Ferrous Ion Chelating Activity (FICA)
FICA was determined by modifying the method of [35]. In a
96-well plate, 15 L of lentil samples were added to fifty
microliters of ferrozine (5 mM, with extra 1:6 dilution in water), fifty
microliters of ferrous chloride (3 mM, with further addition of 1:15 dilution in
water), and eighty-five microliters of water, followed by a 10-minute incubation
under 25 °C. Under 562 nm, the absorbance was read. Utilizing a
calibration curve produced with 0–50 g/mL
Ethylenediaminetetraacetic acid (EDTA), the antioxidant capacity of lentil
samples was measured. The FICA results were shown as mg of EDTA per gram of
sample.
2.4.12 Reducing Power Assay (RPA)
The RPA was identified by following the technique of [36] with some
modifications. In a 96-well plate, 10 L of lentil samples were
added to 25 L sodium phosphate buffer (0.2 M, pH 6.6) and 25
L K[Fe(CN)] and incubated for 20 minutes at 25
°C. Due to the addition of twenty-five microliters of a 10% TCA
solution, eighty-five microliters of water, and 8.5 L of
FeCl, the reaction would be stopped. The incubation was sustained for
fifteen minutes at 25 °C before being discarded. Under 750 nm, the
absorbance was measured. Using the calibration curve constructed with 0–500
g/mL Trolox solution, the antioxidant capacity of lentil samples was
measured. The RPA results were shown as mg of Trolox equivalents (TE) per gram of
sample.
2.4.13 Phosphomolybdate Assay (PMA)
The total antioxidant capacity was determined using a modified PMA assay [37].
Blending HSO (0.6 M), NaPO (28 mM), and ammonium
molybdate (4 mM) produced the PMA reagent. In a 96-well plate, 40 L of
lentil samples were added to 260 L of the produced dye and incubated for
90 minutes at 90 °C. The plate was then allowed to cool to ambient
temperature for 10 minutes. The absorbance was read at 695 nm. The antioxidant
capacity of lentil samples was determined via a calibration curve produced with
0–300 g/mL ascorbic acid. The PMA values were shown as mg of ascorbic
acid equivalents (AAE) per gram of sample.
2.5 Determination of Total Saponins (TSC)
The determination of saponins was conducted based on the vanillin-sulfuric acid
method, modified by [38]. In brief, 10 L of sample extract was
transferred in 1.5 mL Eppendorf tubes and placed in the oven incubator at 60
°C until solvents were evaporated. Then, 200 L of 4%
vanillin (dissolved in ethanol) and 1000 L of 72% sulfuric acid
were added, followed by 15-min incubation under 60 °C. After cooling
down to the ambient temperature, 0.25 mL of the mixture was transferred to a
96-well plate, followed by measuring the absorbance under 560 nm. The total
saponin content of lentils was evaluated by using a calibration curve produced
with 0–25 g/mL Aescin. The TSC results were displayed as mg of
Aescin equivalents (AE) per gram of sample.
2.6 LC-ESI-QTOF-MS Characterization of Phenolic Compounds
Samples used for LC-ESI-QTOF-MS analysis were extracted utilizing two
different extraction methods. First, extracts were produced using 70% ethanol
and mixed using an Ultra-Turrax T25 Homogenizer (IKA, Staufen, Germany) at 10,000
rpm for thirty seconds. Then the prepared extracts were placed in a ZWYR-240
incubator shaker (Labwit, Ashwood, VIC, Australia) at 120 rpm at 10 °C
for 16 hours. Then, another extraction method was performed using ultrasonic for
5 minutes in an ice water bath with a cell disruptor (Branson, model Digital
Sonifier 450) at an amplitude of 40%. After both extraction methods, extracts
were centrifuged at 8000 rpm for 15 minutes at 4 °C (Hettich ROTINA
380R, Tuttlingen, Baden-Württemberg, Germany) and the supernatant was
collected and frozen at –20 °C for subsequent analysis.
Phenolic characterization was made by following the method of [39] with some
modifications and was conducted by Agilent 1200 series HPLC (Agilent
Technologies, CA, USA) connected with an Agilent 6520 Accurate Mass Q-TOF
LC-MS (Agilent Technologies, Santa Clara, CA, USA). Compound Separation was
carried out using a Synergi Hydro-RP 80 Å, LC Column (250 mm 4.6
nm, 4 m) (Phenomenex, Lane Cove, NSW, Australia) with Phenomenex
C18 ODS (4.0 2.0 mm) guard column to protect the column. Mobile phase
A was made by water/acetic acid (98:2, v/v), and mobile phase B was made
by acetonitrile/water/acetic acid (100:99:1, v/v/v). The degassing
process was performed under 25 °C for 15 min. The gradient program was
conducted by a mixture of mobile phase A and B as follow: 90% A and 10% B (0
min); 75% A and 25% B (20 min); 65% A and 35% B (30 min); 60% A and 40% B
(40 min); 45% A and 55% B (70 min); 20% A and 80% B (75 min); 100% B (77
min); 90% A and 10% B (85 min). The flow rate was set to be 0.8 mL/min and
five-microliter was the sample injection volume. The peak was identified by
positive and negative modes and nitrogen gas was used as a nebulizer and drying
gas at 45 psi, with a flow rate of 0.5 mL/min. Capillary and nozzle voltage was
placed at 3.5 kV and 500 V respectively, while the mass spectra were obtained in
the range of 50–1300 amu with collision energy (10, 15, and 30 eV) for
fragmentation. Data collection and assays were performed using Agilent
LC-ESI-QTOF-MS Mass Hunter Data Acquisition Software Version B.03.01
(Agilent Technologies, Santa Clara, CA, USA). Compounds identified by
LC-ESI-QTOF-MS/MS that had library identification scores greater than 80 were
selected for characterization and m/z verification.
2.7 Statistical Analysis
The polyphenol content and antioxidant assay data were reported as means
standard deviation (SD), and studies were conducted in triplicate (n = 3).
Minitab Statistical Software for Windows Version 18.0 was used to conduct a
one-way analysis of variance (ANOVA) accompanied by Tukey’s honestly significant
differences (HSD) multiple rank test at a significance level of p
0.05 (Minitab Inc., State College, PA, USA). The correlation between phenolic
compounds and antioxidant activities was performed via XLSTAT-2019.1.3 (Addinsoft
Inc. New York, NY, USA).
3. Results and Discussion
The antioxidant potential and the association between phenolic substances and
antioxidant activity in the lentil samples were estimated following different
assays. In addition, LC-ESI-QTOF-MS/MS was used as a tool to determine and
characterize the phenolic compounds. The phenolic content, antioxidant activity,
and saponin content results are listed in Table 1.
Table 1.Assessment of the phenolic content, antioxidant capacity, and
saponin content existing in different lentil samples.
Assays |
BKL |
GWL |
RWL |
BWL |
TPC (mg GAE/g) |
0.84 0.03 |
0.96 0.07 |
0.79 0.02 |
0.81 0.12 |
TFC (mg QE/g) |
0.05 0.01 |
0.02 0.02 |
0.06 0.01 |
0.04 0.01 |
TTC (mg TAE/g) |
1.56 0.04 |
1.11 0.03 |
1.03 0.03 |
2.05 0.04 |
TCT (mg CE/g) |
0.03 0.12 |
0.01 0.24 |
- |
0.02 0.07 |
TPAC (mg CCE/g) |
0.09 0.01 |
- |
- |
0.01 0.01 |
TAC (mg/100 g) |
3.32 0.01 |
2.78 0.01 |
1.72 0.01 |
3.04 0.01 |
DPPH (mg TE/g) |
4.32 0.08 |
5.24 0.02 |
4.65 0.02 |
3.21 0.08 |
FRAP (mg TE/g) |
2.13 0.12 |
3.38 0.05 |
2.05 0.09 |
2.09 0.01 |
ABTS (mg TE/g) |
6.47 0.81 |
9.82 0.71 |
8.06 0.47 |
7.94 0.12 |
•OH-RSA (mg TE/g) |
3.14 0.09 |
3.87 0.13 |
2.49 0.22 |
3.74 0.02 |
FICA (mg EDTA/g) |
0.19 0.07 |
0.08 0.01 |
0.11 0.01 |
0.09 0.01 |
RPA (mg TE/g) |
2.10 0.17 |
1.66 0.05 |
1.96 0.04 |
1.59 0.05 |
PMA (mg AAE/g) |
3.18 0.21 |
3.39 0.17 |
4.01 0.31 |
2.31 0.14 |
TSC (mg AE/g) |
0.01 0.01 |
0.02 0.01 |
0.02 0.01 |
0.03 0.01 |
The results are displayed in mg equivalents per gram in terms of fresh weight
and expressed as mean standard deviation (SD) (n = 3); the lettering
() indicated the significant difference (p 0.05)
utilizing one-way analysis of variance (ANOVA) and Tukey’s HSD test. TPC, total
phenolic content; TFC, total flavonoid content; TTC, total tannin content; TCT,
total condensed tannins; TPAC, total proanthocyanidin content; TAC, total
anthocyanin content; DPPH, 2,2′-diphenyl-1-picrylhydrazyl assay; FRAP, ferric
reducing antioxidant power assay; ABTS,
2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid assay;
•OH-RSA, hydroxyl-radical scavenging activity; FICA, ferrous ion
chelating activity; RPA, reducing power assay; PMA, phosphomolybdate assay; TSC,
total saponin content. GAE, gallic acid equivalents; QE, quercetin equivalents;
TAE, tannic acid equivalents; CE, catechin equivalents; CCE, cyanidin chloride
equivalents; TE, Trolox equivalents; EDTA, ethylenediaminetetraacetic acid; AAE,
ascorbic acid equivalents; AE, aescin equivalents. RWL, red whole lentil; GWL,
green whole lentil; BWL, brown whole lentil; BKL, black whole lentil.
3.1 Phenolic Compounds and Saponin Estimation (TPC, TFC, TTC, TCT,
TPAC, TAC, and TSC)
Phenolics are essential secondary metabolites widely found in nature. Most
studies correlate phenolic compounds with their potential health benefits,
including antioxidant capacity and other health-promoting properties [40].
According to a recent review of polyphenols in lentils [13], the most frequently
found polyphenols in lentils comprise phenolic acids, flavonols, flavan-3-ol,
proanthocyanidins, anthocyanidins, or condensed tannins, and anthocyanins.
Therefore, this study intended to evaluate the TPC, TFC, TTC, TCT, TPAC, and TAC,
besides the saponin estimation. The Folin-Ciocalteu method was utilized to
quantify the total phenolic content of lentils (Table 1). There was no
statistically significant difference in the results of red and brown whole
lentils, which showed a value of 0.79 0.02 mg GAE/g and 0.81 0.12
mg GAE/g, separately. However, the TPC value of green whole lentils was
significantly different (p 0.01) from the other three kinds
of lentils and displayed the greatest total phenolic content (0.96 0.07
mg GAE/g), accompanied by black whole lentils (0.84 0.03 mg GAE/g). This
outcome is in accordance with the results found by [41], that observed the
highest TPC values in green lentils (737.32 mg/100 g dry weight (d.w.) in aqueous-organic
extract) when compared to red lentils, chickpeas, and peas.
Flavonoids are considered the most significant polyphenol in human diets, and it
is among the main phenolic compounds present in lentils [42, 43]. As shown in
Table 1, flavonoids in lentils ranged from 0.02 0.02 mg QE/g to 0.06
0.01 mg QE/g. This research revealed that red whole lentils had the
greatest total flavonoid content (0.06 0.01 mg QE/g), accompanied by
black whole lentils (0.05 0.01 mg QE/g), and brown whole lentils (0.04
0.01 mg QE/g). Moreover, green whole lentils (0.02 0.02 mg QE/g)
displayed the lowest flavonoid content among samples. There was no significant
statistical difference between black and brown whole lentils in terms of
flavonoid concentration. However, the above results are challenged by [44], who
examined 33 samples of cool-season legumes and found that lentils’ TPC values
varied from 4.86 to 9.6 mg GAE/g and that green lentil had a greater quantity of
flavonoids. Variations in total phenolics and flavonoid concentration may be
influenced by the solvent used in the extraction, origin, harvesting year, and
storage conditions of lentils.
Tannins are complex phenolic compounds that are typically separated into two
groups, hydrolysable and condensed tannins [42]. Total tannin content (TTC) was
measured utilizing the polyvinylpolypyrrolidone (PVPP) method. This method is
based on tannin complexation/precipitation, instead of proteins [27]. Assuming
that the phenolics that bind to proteins are identical to those that bind to
PVPP, this method separates tannins from non-tannins by using this solid matrix.
According to Table 1, brown lentils showed the highest total tannins (2.05
0.04 mg TAE/g), accompanied by black (1.56 0.04 mg TAE/g), green
(1.11 0.03 mg TAE/g), and red (1.03 0.03 mg TAE/g) lentils.
Similarly, Menga et al. [45] discovered that the brown lentils (4.45 mg
CE/g) showed higher values of TTC than the green lentils (2.92 mg CE/g). Irakli
et al. [46] reported that the total tannin content ranged from 2.86 to
3.20 mg GAE/g for five different kinds of lentils. The PVPP method applied in our
study cannot determine the presence or absence of certain types of tannins in a
mixture, such as condensed or hydrolysable tannins, but could be seen as the
measurement of total tannins [47].
Condensed tannins are among the main phenolic compounds present in legume seeds
and are usually discovered in lentils among other pulses [43]. Total condensed
tannin content (TCT) was shown as mg/g equivalents of catechin (CE) per gram of
material. As depicted in Table 1, three out of four lentils had total condensed
tannins, namely black whole lentils (0.03 0.12 mg CE/g), green whole
lentils (0.01 0.24 mg CE/g) and brown whole lentils (0.02 0.07 mg
CE/g). Nonetheless, no tannins were detected in whole red lentils. In contrast,
[48] observed a total condensed tannin concentration of 0.012 to 0.014 mg/g fresh
weight in red lentils extracted using an ethanolic solution. The variation in the
condensed tannin content of red lentils might be attributed to the concentration
of the extraction solvents used, as our study used 70% ethanolic extraction
while [48] applied 80% ethanolic extraction. In addition, lentil origin type
may have a role in the final results, since [48] applied red lentils from India
while the Australian variety was used for the red lentil in our research.
Flavanols occur either in a monomeric form (catechins) or in a polymeric form
(proanthocyanidins) [49]. The oligomers of catechin and epicatechin molecules
known as proanthocyanidins are mainly found in lentils with colored seed coats
[3]. The determination of TPAC was conducted according to the HCl-butanol method.
This method can also be used to measure the condensed tannin contents in
different samples [47]. This is because condensed tannins when heated in an acid
alcohol solution can be degraded into anthocyanidins through an acid-catalyzed
oxidation process, therefore condensed tannins are also known as
proanthocyanidins [42]. Based on the results, black whole lentils had the
greatest proanthocyanidins’ level, demonstrating a value of 0.09 0.01 mg
CCE/g accompanied by brown lentils (0.01 0.01 mg CCE/g). However, no
proanthocyanidins were detected for green and red whole lentils. Compared with
the TCT values, a similar tendency has appeared. [46] reported a mean TPAC value
for five lentils with different genotypes of 7.93 mg procyanidin B
equivalents/g. Using different standard compounds to display the results leads to
results that cannot be directly compared. In our study, the calibration curve was
based on measurements of cyanidin chloride. In the prior study, the results were
shown as catechin or procyanidin B equivalents [45, 46].
Anthocyanins are a broadly studied flavonoid subgroup that is present in
different foods as pigments for the pink, red, purple, or cyan color of such
foods [42]. The determination of TAC in four kinds of lentils was performed and
displayed as mg of cyanidin 3-glucoside equivalents per 100 g of lentil samples.
TAC values in lentils varied from 3.32 0.01 to 1.72 0.01 mg/100 g
based on our findings. Black lentils had the greatest total anthocyanin content
(3.32 0.01 mg/100 g), accompanied by brown lentils (3.04 0.01
mg/100 g), green lentils (2.78 0.01 mg/100 g), and red lentils (1.72
0.01 mg/100 g). A range of 13.67–15.99 mg/100 g of anthocyanins in mixed
lentils was reported, according to the study by [50]. Anthocyanins, a class of
water-soluble flavonoids, broadly exist in fruits as well as vegetables. Several
factors may have an impact on the stability of anthocyanins, containing light, pH
values, temperature, ascorbic acid, oxygen, and enzymes, leading to different
results [51].
Saponins are amphiphilic glycosidic secondary metabolites produced by several
plants that possess emulsifying and foaming properties [52]. Lentils are one of
the main sources of saponins in the human diet [13]. According to our findings,
the highest saponin level was displayed in brown lentils (0.03 0.01 mg
AE/g). No significant differences were shown between green and red whole lentils
(p 0.05), while black lentils possessed the lowest saponin content
among all the lentil samples. The amount of saponin in lentils varies depending
on the cultivar, location, soil type, weather, and season of harvest, extraction
methods, as well as processing methods, such as soaking, cooking, and blanching
[13, 53]. The total amount of saponins in 44 different genotypes of lentils was
estimated by [54], who observed a concentration from 1.70 to 3.50 mg/g. Pure
ethanol used in ultrasonic dried lentil extracts had the highest total saponin
content (1.063 mg/g), followed by ethanol: water (0.328 mg/g) and water extract
(0.019 mg/g), according to the study of [55].
3.2 Antioxidant Activities (DPPH, FRAP, ABTS, RPA,
•OH-RSA, FICA, and PMA)
Antioxidants play an essential part in the preservation of human health,
preventing oxidative processes related to the deterioration of food quality, and
in the treatment of diseases, due to their ability to delay, control and reduce
oxidative stress [56]. Among the antioxidant assays, the DPPH, ABTS,
•OH-RSA, and FICA have been applied to measure the electron transfer
capacity of bioactive compounds related to the presence of polyphenols, while
FRAP and RPA have been used to evaluate the ability of samples to donate
electrons to reduce a Fe [57]. Therefore, the antioxidant studies of
lentil samples were carried out by testing DPPH, FRAP, ABTS, RPA,
•OH-RSA, FICA, and PMA assays, which illustrated the potential to
scavenge the free radical results from various kinds of lentils (Table 1). The radical scavenging activity of DPPH focuses on the hydrogen
ability donation to eliminate free radicals [58]. As dispalyed in Table 1, the
radical scavenging activity varied from 3.21 to 5.24 mg TE/g, with green whole
lentils exhibiting the highest value (5.24 0.02 mg TE/g) and brown
lentils the lowest (3.21 0.08 mg TE/g). No significant differences
(p 0.05) were shown between black and red whole lentils, noting
values of 4.32 0.08 and 4.65 0.02 mg TE/g, respectively. This
result was consistent with a previous study in which there were significant
differences in DPPH between lentils, black soybeans, and soybean legumes
(p 0.05). Lentils exhibited the highest level of DPPH, with French
green lentils (19.87 mol TE/g) presenting the highest concentration among
the lentil subgroups [44].
The reducing power of FRAP is on the basis of the reduction of
ferric-tripyridyltriazine [FeIII(TPTZ)], to produce an intense
blue-colored ferrous complex [FeII(TPTZ)] [59]. The ferric reducing
antioxidant power in lentils was between 2.05 to 3.38 mg TE/g. The greatest
antioxidant capacity was discovered in green lentils with 3.38 0.05 mg
TE/g. While red whole lentils had the lowest FRAP value, of 2.05 0.09
TE/g. This conclusion is comparable to the findings by [41], who observed the
greatest FRAP in green lentils (140.32 mol/g d.w. in aqueous-organic
extract) when compared to red lentils, beans, soybeans, and chickpeas. FRAP
values for black and brown lentils showed no significant difference (p 0.05). Similarly, there were no significant differences between Brewer’s
lentils (12.02 0.33 mmol Fe equivalents/100 g) and Red Chief
lentils (11.37 0.66 mmol Fe equivalents/100 g) in terms of the
study of [44].
The radical scavenging activity of ABTS is based on the measurement of the
electron transfer capacity of the antioxidant evaluated [60]. In the ABTS
analysis, a variation from 6.47 0.81 to 9.82 0.71 mg TE/g was
observed among all the evaluated lentils. Green whole lentils had the highest
ABTS value (9.82 0.71 mg TE/g) among the lentils, followed by the red
lentils (8.06 0.47 mg TE/g), whereas the black lentils showed the lowest
radical scavenging activity of 6.47 0.81 mg TE/g. The ABTS content of
Pardina lentils (green lentils, 14.8 mol TEAC g) was greater than
that of Crimson lentils (red lentils, 14.0 mol TEAC g), according
to prior research [18], which is consistent with our results. Moreover, there
were statistically significant variations in ABTS levels between red and brown
lentils (p 0.05).
In the •OH-RSA analyses, hydroxyl radicals (•OH) are
formed due to the existence of Fe ion and hydrogen peroxide via the Fenton
reaction [61]. While FICA assay measure the antioxidant potential by the
chelating ability of ferrous ion [59]. Whereas RPA causes the reduction of the
Fe/ferricyanide complex to the ferrous form in the existence of reducers
(i.e., antioxidants) [36].
In •OH-RSA, FICA, and RPA assay, there were no significant
differences between green and brown whole lentils. Meanwhile, in
•OH-RSA assay, green whole lentils had the strongest antioxidant
potential (3.87 0.13 mg TE/g), accompanied by the brown whole lentils
(3.74 0.02 mg TE/g), black whole lentils (3.14 0.09 mg TE/g), and
red whole lentils (2.49 0.22 mg TE/g), respectively. However, red whole
lentils showed highly significant antioxidant activity compared to the green and
brown lentils in the FICA and RPA assays. Besides, the FICA and RPA values
followed the same trend, with black lentils performing the best, followed by red,
green, and brown lentils. To our knowledge, this is the first time that the
antioxidant potential of different lentils has been analyzed by
•OH-RSA, FICA, and RPA, and limited data are available for
comparison.
PMA is often used to measure the TAC of liquid food extracts based on an
electron transfer mechanism. This test uses phenolic chemicals to convert
molybdenum (VI) to molybdenum (V). The total antioxidant capacity of lentils
ranged from 2.31 0.14 to 4.01 0.31 mg AAE/g, with red whole
lentils having the highest PMA value at 4.01 0.31 mg AAE/g, accompanied
by green whole lentils with 3.39 0.17 mg AAE/g, black whole lentils with
3.18 0.21 mg AAE/g, and brown whole lentils with 2.31 0.14 mg
AAE/g. Further, significant differences were observed among all different types
of lentils (p 0.05). This result parallels the findings from [62],
in which beans with red or black pigmentation exhibited greater PMA
concentrations. The amount of flavonol glycosides, anthocyanins, and condensed
tannins (proanthocyanidins) determines the color of lentil seed coats [62].
3.3 Correlations of Phenolic Contents and Antioxidant Activities
It is generally recognized that the antioxidant activity of plants is related to
their phenolic composition [44]. Some recent studies suggest this correlation,
such as the study by [63] and [64].
Pearson’s correlation test established a relationship between phenolic levels
and antioxidant tests (Table 2). FRAP showed the strongest association with TPC
in the Person Correlation study (r = 0.975; p 0.01). Meanwhile,
Pearson’s correlation coefficient r = –0.897 (p 0.05) demonstrated
a strong negative relation between total phenolic content and total flavonoid
content. The significant association between FRAP and TPC implies that the
phenolic content of lentil extract is primarily responsible for its antioxidant
properties. This outcome is comparable to the study of [44]. It was discovered
that the connection between ABTS and FRAP was strongly correlated (p
0.05) (r = 0.825). By scavenging ABTS radicals, ABTS determines the
hydrogen-donating and chain-breaking ability of antioxidants. Correlations showed
that the ability to scavenge free radicals was determined by the polyphenol
content of the samples, while antioxidants with strong hydrogen-donating ability
to scavenge radicals were also effective in improving antioxidant and anti-free
radical capacity and contributed significantly to the total antioxidant capacity
of lentils. Saharan et al. [65] examined the link between the phenolic
content of several types of beans and their corresponding antioxidant activities,
reporting that a significant correlation
(p 0.01) was obtained among total
phenolic, flavonoid contents with radical scavenging activity (maximum in pigeon
pea; i.e., r = 0.955 and
r = 0.976, separately).
Table 2.Pearson’s correlation coefficients (r) between antioxidant
assays.
Variables |
TPC |
TFC |
TCT |
DPPH |
FRAP |
ABTS |
•OH-RSA |
FICA |
RPA |
TFC |
–0.897 |
|
|
|
|
|
|
|
|
TCT |
–0.274 |
0.255 |
|
|
|
|
|
|
|
DPPH |
0.646 |
–0.278 |
–0.496 |
|
|
|
|
|
|
FRAP |
0.975 |
–0.889 |
–0.478 |
0.683 |
|
|
|
|
|
ABTS |
0.684 |
–0.729 |
–0.847 |
0.499 |
0.825 |
|
|
|
|
•OH-RSA |
0.660 |
–0.904 |
0.050 |
–0.144 |
0.611 |
0.463 |
|
|
|
FICA |
–0.289 |
0.518 |
0.820 |
–0.05 |
–0.467 |
–0.867 |
–0.412 |
|
|
RPA |
–0.354 |
0.703 |
0.442 |
0.247 |
–0.448 |
–0.702 |
–0.763 |
0.870 |
|
PMA |
0.050 |
0.45 |
–0.494 |
0.793 |
0.134 |
0.163 |
–0.703 |
0.093 |
0.550 |
Significant correlation with p 0.05 and
Significant correlation with p 0.01.
Furthermore, substantial negative associations were found between
•OH-RSA and TFC (r = –0.904; p 0.01), ABTS and FICA (r
= –0.867; p 0.05), as well as between FRAP and TFC (r = –0.889,
p 0.05). Moreover, a strong positive association existed between
FICA and RPA (r = 0.870, p 0.05). Few studies have previously used
•OH-RSA, FICA, and RPA to establish the antioxidant potential of
lentils. To our knowledge, this is the first time that •OH-RSA, FICA,
and RPA studies have been done on various lentil samples, and comparative data
are scarce. Several investigations using RPA and •OH-RSA methods
concluded that antioxidant activity is positively correlated with phenolic
content, which is not in agreement with our study [66, 67, 68].
3.4 LC-ESI-QTOF-MS Characterization of Phenolic Compounds from
Different Lentil Samples
LC-ESI-QTOF-MS analysis has been widely utilized to identify phenolic
compounds from several plant-based samples [67, 69, 70]. In this research,
LC-ESI-QTOF-MS was used to evaluate the phenolic components in ethanolic
and ultrasonic extracts of lentils. On the basis of retention time (RT), mass to charge
(m/z) values, and MS spectra in negative and positive ionization
modes ([M – H]/[M + H]), the phenolic compounds in four lentil
samples were identified and characterized utilizing Agilent LC-MS/MS MassHunter
Qualitative Software and Personal Compound Database and Library (PCDL) (Table 3).
By combining negative and positive modes, more compounds present in the lentils
with broader chemical diversity would be identified and characterized. Compounds
having a PCDL score over 80 and a mass error less than 5 ppm were chosen
for further MS identification and m/z characterization and
verification purposes. More information about the total ion chromatograms of the
lentil samples can be found at supplementary materials (Supplementary Fig. 1).
Table 3.Characterization of phenolic compounds in various kinds of
lentil samples by LC-ESI-QTOF-MS.
No. |
Proposed compounds |
Molecular formula |
RT (min) |
Ionization (ESI/ESI) |
Molecular weight |
Theoretical (m/z) |
Observed (m/z) |
Error (ppm) |
MS production |
Sample |
Phenolic acid |
Hydroxybenzoic acids |
1 |
Ellagic acid |
CHO |
8.012 |
[M–H] |
302.0036 |
300.9963 |
300.9962 |
–0.3 |
284, 229, 201 |
BWL |
Hydroxycinnamic acids |
2 |
Ferulic acid |
CHO |
4.099 |
[M–H] |
194.0575 |
193.0502 |
193.0500 |
–1.0 |
178, 149, 134 |
*BWL |
3 |
Caffeic acid |
CHO |
4.790 |
[M–H] |
180.0405 |
179.0332 |
179.0331 |
–0.6 |
143, 133 |
BWL |
4 |
Ferulic acid 4-O-glucoside |
CHO |
5.029 |
[M–H] |
356.1117 |
355.1044 |
355.1041 |
–0.8 |
193 |
BWL |
5 |
Caffeoyl glucose |
CHO |
47.840 |
[M–H] |
342.0949 |
341.0876 |
341.0878 |
0.6 |
179, 161 |
*BKL, GWL, RWL |
Hydroxyphenylpropanoic acids |
6 |
Dihydrocaffeic acid 3-O-glucuronide |
CHO |
4.499 |
[M–H] |
358.0897 |
357.0824 |
357.0818 |
–1.7 |
181 |
RSL |
Flavonoids |
Flavanols |
7 |
Procyanidin dimer B7 |
CHO |
4.288 |
[M–H] |
578.1379 |
577.1306 |
577.1302 |
–0.7 |
451 |
RWL, BWL, GWL |
8 |
Procyanidin trimer C1 |
CHO |
4.310 |
[M–H] |
866.2056 |
865.1983 |
865.1985 |
0.2 |
739, 713, 695 |
*GWL, BWL |
9 |
4”-O-Methylepigallocatechin 3-O-gallate |
CHO |
4.916 |
[M–H] |
472.0989 |
471.0916 |
471.0917 |
0.2 |
169, 319 |
BWL |
Flavanones |
10 |
Neohesperidin |
CHO |
4.485 |
[M+H] |
610.1895 |
611.1968 |
611.1969 |
0.2 |
593, 465, 449, 303 |
GWL |
11 |
Sakuranetin |
CHO |
5.750 |
[M+H] |
286.0841 |
287.0914 |
287.0920 |
2.1 |
269, 203, 175 |
BWL |
Flavonols |
12 |
Quercetin 3-O-rutinoside |
CHO |
4.069 |
[M–H] |
610.1556 |
609.1483 |
609.1495 |
2.0 |
301 |
BWL |
13 |
Myricetin 3-O-rhamnoside |
CHO |
4.234 |
[M–H] |
464.0983 |
463.0910 |
463.0909 |
–0.2 |
317 |
RWL |
14 |
Quercetin 3-O-glucosyl-xyloside |
CHO |
4.293 |
**[M–H] |
596.1412 |
595.1339 |
595.1343 |
0.7 |
265, 138, 116 |
*GWL, BWL |
15 |
Quercetin 3-O-rhamnoside |
CHO |
4.350 |
[M–H] |
448.1047 |
447.0974 |
447.0972 |
–0.4 |
447, 287 |
*GWL, BKL, RWL |
16 |
Quercetin 3-O-xyloside |
CHO |
4.406 |
[M–H] |
434.0859 |
433.0786 |
433.0788 |
0.5 |
301 |
*GWL, BKL, BWL |
Dihydroflavonols |
17 |
Dihydroquercetin |
CHO |
5.448 |
[M–H] |
304.0609 |
303.0536 |
303.0539 |
1.0 |
285, 275, 151 |
RWL |
Anthocyanins |
18 |
Cyanidin 3-O-(6′-p-coumaroyl-glucoside) |
CHO |
4.714 |
**[M–H] |
595.1446 |
594.1373 |
594.1394 |
3.5 |
287 |
*BWL |
Isoflavonoids |
19 |
Dalbergin |
CHO |
5.087 |
[M–H] |
268.0734 |
267.0661 |
267.0665 |
1.5 |
252, 224, 180 |
GWL |
Other polyphenols |
Alkylmethoxyphenols |
20 |
4-Vinylsyringol |
CHO |
5.066 |
[M+H] |
242.0946 |
243.1019 |
243.1026 |
2.9 |
225, 211, 197 |
RWL |
Lignans |
21 |
Secoisolariciresinol-sesquilignan |
CHO |
45.717 |
[M–H] |
558.2503 |
557.2430 |
557.2422 |
–1.4 |
539, 521, 509, 361 |
BWL |
22 |
Deoxyschisandrin |
CHO |
55.151 |
[M–H] |
416.2218 |
415.2145 |
415.2159 |
3.4 |
402, 347, 361, 301 |
*RWL, BWL |
*Compound was scanned in more than one lentil samples, Compound was scanned in
more than one lentil samples, data presented in this table are from lentil
samples. **Compounds were detected in both negative [M-H] and
positive [M+H] mode of ionization whereas only single mode data was
presented. As displayed in the table, sample lentils are mentioned in abbreviated
form. RWL, red whole lentil; GWL, green whole lentil; BWL, brown whole lentil;
BKL, black whole lentil.
According to the literature, lentils are an important dietary source of
extractable polyphenols, such as phenolic acids, condensed tannins
(proanthocyanidins), anthocyanidins, flavan-3-ols, flavanols, flavones,
flavanones, and stilbenes [6]. In this study, 22 phenolic compounds, comprising 6
phenolic acids, 13 flavonoids, 2 lignans, and 1 other polyphenol, were
tentatively characterized by LC-ESI-QTOF-MS and are summarized in Table 3.
Polyphenols, containing flavonoids, phenolic acids, and stilbenes, have
considerable antioxidant activity and are indicated to help control free radicals
arising from oxidative stress. Other bioactive properties including
antimicrobial, anti-inflammatory, antineoplastic, estrogenic, hepatoprotective,
and hypolipidemic activities have also been reported [71].
3.4.1 Phenolic Acids
Phenolic acids are simple phenolics and their subclasses are hydroxybenzoic acid
derivatives and hydroxycinnamic acid derivatives [6]. In this research six
phenolic acids were identified in each of the four lentil samples. Hydroxybenzoic
acids (1), hydroxycinnamic acids (4), and hydroxyphenylpropanoic acids (1) were
tentatively characterized in various lentil samples.
3.4.1.1 Hydroxybenzoic Acids and Hydroxycinnamic Acids
One type of hydroxybenzoic acid along with four kinds of hydroxycinnamic acids
was identified in selected lentil samples. Compound 1 with [M – H]m/z at 300.9963 was only detected from brown whole lentils and
characterized as ellagic acid based on the product ion at 284 m/z, 229
m/z, and 201 m/z. Ellagic acid was also present in the spices
from Australia in terms of previous research [57]. Three hydroxycinnamic acid
derivatives (Compound 2, 3, 4) were only found in the brown whole lentils. These
three compounds were tentatively characterized in negative mode, containing
ferulic acid (Compound 2), caffeic acid (Compound 3), and ferulic acid
4-O-glucoside (Compound 4) with [M – H] at m/z 193.0502,
179.0332, and 355.1044, respectively. And ferulic acid was confirmed by the
characteristic ions at m/z 178, m/z 149, and m/z 134
due to the loss of CH (15 Da), CO (44 Da), and CH with CO
(59 Da) [72], was also identified in bitter cumin by [73]. Caffeic acid was
identified with MS spectrum by the characteristic ions of m/z 143
(loss of two molecules of water, 36 Da) and m/z 133 (loss of HCOOH, 46
Da) [74], which was also detected in black spices and pepper [75, 76]. Moreover,
ferulic acid (Compound 2) and caffeic acid (Compound 3) both were previously
found in 6 different varieties of lentils, namely CDC green land, CDC invincible,
3493-6, CDCSB-2, maxim, and black lentils, according to the study of [77]. The
loss of CHO (177 Da) and CHO (206 Da) from
the precursor ion of ferulic acid 4-O-glucoside produced the fragment
peaks (m/z 178 and m/z 149) [78], which was also found in hops
and juniper berries [24]. Caffeoyl glucose (Compound 5), identified based on [M
– H], was found in black whole lentils, red whole lentils, and green whole
lentils. The molecular ion of caffeoyl glucose (m/z 341.0878) produced
the major fragment ion at m/z 179, corresponding to the loss of
glucoside (162 Da) from the product ion [57]. The exsitence of caffeoyl glucose
in Australian grown apples was also previously reported [79].
3.4.1.2 Hydroxyphenylpropanoic Acids
Compound 6 was tentatively characterized as dihydrocaffeic acid
3-O-glucuronide, and only found in black whole lentil based on [M –
H]m/z at 357.0818 in negative ionization mode, which
identification was additionally aided by the MS spectrum. The identity of
dihydrocaffeic acid 3-O-glucuronide was verified by the product ions at
m/z 181 [M – H – 176], related to the neutral loss of hexuronyl moiety
(glucuronyl moiety), which was also found in Australian grown berries, according
to the previous research [80].
3.4.2 Flavonoids
Flavonoids are characterized by a C6–C3–C6
backbone structure. Its classification includes distinct sub-groups of
flavan-3-ols, flavones, flavanols, flavanones, anthocyanidins, and isoflavones,
and oligomers such as proanthocyanins [6]. A total of 13 flavonoids were
discovered and characterized in all four lentil samples. In this study, we
tentatively characterized 6 subclasses in different lentil samples, including
flavanols (3), flavanones (2), flavonols (5), dihydroflavonols (1), anthocyanins
(1), and isoflavonoids (1).
3.4.2.1 Flavanols
Three flavanols (Compound 7, Compound 8, and Compound 9) were tentatively
identified according to specific criteria: precursor and product ions, all of
which in negative ionization mode in this study. Procyanidin dimer B7 (Compound
7, m/z 577.1302, RT = 4.288 min) was identified in red whole lentil,
green whole lentil, and brown whole lentil samples, procyanidin trimer C1
(Compound 8, m/z 865.1985) was found in green whole lentil and brown
whole lentil. 4”-O-Methylepigallocatechin 3-O-gallate
(Compound 9, m/z 471.0917) was only characterized in brown whole
lentils. The precursor ions of procyanidin dimer B7 produced the product ions at
m/z 451; the precursor ions of procyanidin trimer C1 produced the
product ions at m/z 739, m/z 713, and m/z 695,
indicating the expected loss of heterocyclic ring fission (HRF) reaction (126
Da), retro-Diels-Alder (RDA) (152 Da) and HO [81]; the precursor ions of
4”-O-methylepigallocatechin 3-O-gallate produced the product
ions at m/z 169 and m/z 319. Previous research also mentioned
the presence of procyanidin trimer C1 (Compound 8) in six different
lentil samples [77].
3.4.2.2 Flavanones
Neohesperidin (Compound 10 with [M + H] at m/z 611.1969, RT =
4.485 min) was presented in green lentils. The MS spectrum of neohesperidin
displayed the product ions at m/z 593, m/z 465, m/z
449, and m/z 303. The presence of neohesperidin in grapefruit and lime
peel was also previously reported by [82]. Compound 11 (Sakuranetin)
displayed the [M + H]m/z at 281.092, RT = 5.75 min, and was
observed in brown whole lentil, and confirmed by the product ions at m/z
269, m/z 203, and m/z 175. Sakuranetin is one of the most
distinctive natural products of the plant, as previously reported in sweet
cherries [83].
3.4.2.3 Flavonols
Compounds 12, 14, 15, and 16 were tentatively identified the presence in red,
green, brown, and black lentils, including quercetin 3-O-rutinoside
(Compound 12) with [M – H] at m/z 609.1495, quercetin
3-O-glucosyl-xyloside (Compound 14) with [M – H] at m/z
595.1343, quercetin 3-O-rhamnoside (Compound 15) with [M – H] at
m/z 447.0972 and quercetin 3-O-xyloside (Compound 16) with [M
– H] at m/z 433.0788, respectively. The presence of quercetin
3-O-rutinoside in jelly palm and leaves of fishtail palm were discovered
in previous studies [84, 85]. Quercetin 3-O-glucosyl-xyloside could be
detected in both ionization modes, identified based on the fragment peaks at
m/z 265, m/z 138, and m/z 116, present in
mango peel by product from Australia according to [86]. Meanwhile, myricetin
3-O-rhamnoside was dispalyed with the molecular formula
CHO and the precursor ion [M – H] at m/z
463.0909 (Compound 13). The MS spectrum of myricetin
3-O-rhamnoside showed the product ions at m/z 317, indicating
the presence of a desoxyhexose sugar [87]. Myricetin 3-O-rhamnoside was
also discovered in black spices and tobacco [76, 88]. Quercetin
3-O-rutinoside and quercetin 3-O-xyloside were detected in both
brown whole and black whole lentil samples; quercetin
3-O-glucosyl-xyloside and quercetin 3-O-xyloside were
identified in brown and green whole lentils; quercetin 3-O-rhamnoside
was characterized in both red and green whole lentils.
3.4.2.4 Dihydroflavonols and Anthocyanins
Dihydroquercetin ([M – H] ion at m/z 303.0539 was considered as
Compound 17 found in red lentil, yielding product ions at m/z 275 [M –
H – CO], m/z 285 [M – H – HO], and m/z 151 [M – H –
RDA cleavage] [89], was one of the highly specific polyphenols in pears [90]. By
contrast to cyanidin 3-O-(6′-p-coumaroyl-glucoside), dihydroquercetin
was only detected in the red whole lentils. Cyanidin
3-O-(6′-p-coumaroyl-glucoside) (Compound 18 with [M – H] at
m/z 594.1394, RT = 4.714 min) was the only anthocyanins present in brown
whole lentil. The MS spectrum of cyanidin
3-O-(6′-p-coumaroyl-glucoside) showed the product ions at m/z
287. The existence of cyanidin 3-O-(6′-p-coumaroyl-glucoside) in
blackberry fruit and kiwifruit was also previously reported [66, 91]. Both
compounds (Compound 17 & 18) have been found before in the black lentil samples
according to the study of [92].
3.4.2.5 Isoflavonoids
Compound 19, with the molecular formula CHO and possessing
the precursor ion [M – H] at m/z 267.0665 in the negative
ionization mode in green whole lentil, was characterized as dalbergin. Dalbergin
produced fragments at m/z 252, m/z 224, and m/z 180,
due to the loss of CH (15 Da), CHO (43 Da),
CHO (87 Da) from the precursor ion, separately [93]. To our
knowledge, this is the first time that isoflavonoid derivatives have been
identified and characterized in lentils.
3.4.3 Other Polyphenols
One other polyphenol was detected and characterized in the red lentil sample,
classified as alkylmethoxyphenols.
Alkylmethoxyphenols
Compound 20 was identified as 4-vinylsyringol according to the precursor ion [M
+ H]m/z 243.1026 and was only found in the red lentil sample. The
identity of 4-vinylsyringol was confirmed by the product ions at m/z
225, m/z 211, and m/z 197, respectively. 4-vinylsyringol was
also present in the giant reed (Arundo Donax L.) and edible lotus
(Nelumbo nucifera G.), in terms of the previous research [70, 94].
3.4.4 Lignans
Lignans are represented by two phenylpropane units connected by a C6-C3 bond
between the central atoms of the respective side chains [95]. A total of 2
lignans were detected and characterized in two out of four lentils.
Compound 21 with [M – H]m/z at 557.2422, RT = 45.717 min was
only discovered from brown whole lentil and characterized as
secoisolariciresinol-sesquilignan in terms of the product ion at m/z
539, m/z 521, m/z 509 and m/z 361, corresponding to
the loss of CO (44 Da) from precursor
ion. Secoisolariciresinol-sesquilignan was identified as the dominant lignans
present in flaxseeds according to the previous study [96]. The presence of
secoisolariciresinol-sesquilignan in spices from Australia and palm fruits was
also noted [57, 97]. Deoxyschisandrin (Compound 22, m/z 415.2159) was
identified in brown and red lentil samples in negative mode, previously
characterized in schisandra [98]. The molecular ions of deoxyschisandrin produced
the product ions at m/z 402, m/z 347, m/z 361, and
m/z 301, corresponding to the loss of CH (15 Da), CH
(70 Da), CH (56 Da) and CHO (152 Da) from the precursor
ion [99]. Both secoisolariciresinol-sesquilignan and deoxyschisandrin could be
found in the brown whole lentils, whereas deoxyschisandrin was only detected in
the red whole lentils.
3.5 Distribution of Phenolic Compounds—Venn Diagram
Lentils possess a large diversity of phenolic compounds, which vary across
varieties. Hence, researchers have developed a strong interest in the
distribution of phenolic chemicals in lentils. The distribution of phenolic
compounds in lentils, identified in various hues such as BWL (yellow), GWL
(green), BKL (red), and RWL (blue), is shown using Venn diagrams (Fig. 1). In
terms of the Venn diagram of total phenolic compounds, there are 46 (23.8%), 20
(10.4%), 14 (7.3%), and 12 (6.2%) distinct compounds in brown, green, black,
and red whole lentils, respectively. Nineteen (9.8%) compounds were shared by
all four lentil samples. The highest number of overlapping total phenolic
compounds in BWL and RWL was 13 (6.7%), while the lowest number of overlapping
total phenolics was found in red, green, and black lentils (2.6%). In the
majority of overlapped compounds and all unique compounds, lentil samples have
more flavonoids than phenolic acids. The greatest concentrations of distinct
phenolic acids and flavonoids were still shown in brown whole lentils, 21.6%,
and 28.3%, respectively. No common overlapping phenolic acids exist between red
and black lentils, red and green lentils, and brown and green lentils, which is a
considerable divergence, while flavonoids in this area overlapping by 3 (3%), 4
(4%), and 6 (6.1%), respectively. Additionally, three phenolic acids and seven
flavonoids were found in each of the four lentils. Unique polyphenols were
detected in brown whole lentils (17.5%), green whole lentils (3.5%), black
whole lentils (7%) and red whole lentils but not in other polyphenols (5.3%).
Four lentil samples had a total of nine (15.8%) polyphenols on average. The
highest number of overlapping compounds was six in brown along with red lentils,
while the lowest number was three in green, brown, along with black whole
lentils. Differences in phenolic composition require further studies to
investigate the effects of specific phenolics.
Fig. 1.
Venn diagram of phenolic compounds exist in four lentil samples
(red, green, brown, and black whole lentils). (A) displays the relations of
total phenolic compounds present in different lentil samples. (B) displays the
relations of phenolic acids in present in different lentil samples. (C) displays
the relations of flavonoids present in different lentil samples. (D) displays the
relations of other phenolic compounds present in different lentil samples. As
shown in the graph, sample lentils are mentioned in abbreviated form. RWL, red
whole lentil; GWL, green whole lentil; BWL, brown whole lentil; BKL, black whole
lentil.