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COVER LETTER
Sevilla, 17-3-2016
Dear Sir:
Common vetch, V. sativa, second to V. faba in economic importance, is mainly
used for feeding livestock, as a cover crop, and as green manure in countries of the
Mediterranean Region and more recently Australia. We have determined the
polyphenol composition of V. sativa seed extracts and have also investigated
antioxidant, metal chelating, and several enzyme inhibitory activities. These properties
were compared with those of V. faba, which was also analyzed in parallel.
Results demonstrate that V. sativa seeds have a number of health-promoting
properties related with antioxidant activity and inhibition of certain enzymatic
activities that are comparable or even higher than those observed in V. faba. This
combination of properties could allow the use of V. sativa seeds as a source of health-
promoting and therapeutic components. This manuscript is submitted based on the
offer that manuscript submitted before March-31 will be published without any
publication charges.
Sincerely,
Dr. Javier Vioque
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ANTIOXIDANT, CHELATING AND ENZYME INHIBITORY ACTIVITIES OF VICIA SATIVA
(COMMON VETCH) SEED POLYPHENOLS.
CRISTINA MEGÍAS1, GOKHAN ZENGIN2, ABDURRAHMAN AKTUMSEK2, JULIO GIRÓN-
CALLE1, ISABEL CORTÉS-GIRALDO1, MANUEL ALAIZ1 and JAVIER VIOQUE1*
1Food Phytochemistry Department, Instituto de la Grasa (C.S.I.C.), Campus
Universidad Pablo de Olavide, 41013-Sevilla, Spain.
2Department of Biology, Science Faculty, Selcuk University, 42250-Konya,
Turkey
Corresponding author:
e-mail: [email protected]
TEL. 34 954611550
FAX. 34 954616790
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ABSTRACT
It has been determined the polyphenol composition of V. sativa seeds and have
also investigated antioxidant, metal chelating, and enzyme inhibitory activities. These
properties were compared with those of V. faba. The content in total polyphenols was
higher in V. sativa than in V. faba, as were reducing power, total antioxidant activity,
and metal chelating activity. Radical scavenging activity was nevertheless higher in V.
faba. Cholinesterase and tyrosinase inhibitory activities were higher in the extracts
from V. faba seeds than in V. sativa, while inhibition of -amylase was higher in V.
sativa. Inhibition of -glucosidase was similar in V. sativa and V. faba. Analysis of the
polyphenol composition of V. sativa extracts revealed variability among populations,
but all populations had in common the presence of catechin and hydroxybenzoic
aldehyde. Two populations were characterized by the presence of glycosides of
apigenin and quercetin, while a third one had more phenolic acids.
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INTRODUCTION
Vicia genus (Fabaceae) belongs to tribe Fabeae, which also includes genera
Pisum, Lathyrus, Lens, and Vavilovia. Around 200 Vicia species can be found in
temperate areas of the Northern Hemisphere (Hanelt & Mettin, 1989), including the
ancient crops V. faba and V. ervilia (Hanelt et al., 1989). V. faba is the only Vicia
currently used for food by humans. The common vetch, V. sativa, second to V. faba in
economic importance, is mainly used for feeding livestock, as a cover crop, and as
green manure in countries of the Mediterranean Region (Robertson et al., 1996) and
more recently Australia (Enneking, 1995). V. sativa has been also used for food,
although human consumption is not very extended due to the presence in their seeds
of the neurotoxins γ-glutamyl-β-cyano-L-alanine and β-cyano-L-alanine (Megías et al.,
2014). The selection of low toxins V. sativa lines has been investigated (Firincioglu et
al., 2007) and different processing and cooking procedures have been suggested to
detoxify V. sativa seed flour (Ressler et al., 1997; Pastor-Cavada et al., 2013).
We have previously reported that V. sativa seeds are rich in polyphenols
(Pastor-Cavada et al., 2009) with potential health promoting properties, namely
antioxidant and antiproliferative activities (Megías et al., 2009). In order to further
characterize potential health-promoting properties, we have now investigated
additional antioxidant and metal chelating activities, as well as enzyme inhibitory
activities. The polyphenol composition of the seeds has also been determined.
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Material and method
Material
Folin-Ciocalteu’s reagent and methanol were purchased from Merck
(Darmstadt, Germany). Gallic acid, rutin, trolox, 2,2’-azino-bis(3-ethylbenzothiazoline)-
6-sulphonic acid (ABTS), neocuproine, 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ), 3-(2-
pyridyl)-5-6-diphenly-1,2,4-triazine-4’,4”-disulfonic acid sodium salt (ferrozine), EDTA,
5.5-dithio-bis(2-nitrobenzoic) acid (DTNB), Electrophorus electricus
acetylcholinesterase Type-VI-S EC 3.1.1.7 (AchE), horse serum butyrylcholinesterase EC
3.1.1.8 (BchE), acetylthiocholine iodide (ACI), butyrylthiocholine chloride (BCI),
galanthamine, porcine pancreas α-amylase EC 3.2.1.1, starch, acarbose, glutathione,
Saccharomyces cerevisiae α-glucosidase EC 3.2.1.20, 4-N-trophenyl--D-
glucopyranoside (PNPG), L-dopa, mushroom tyrosinase EC 1.14.18.1, and kojic acid
were purchased from Sigma Chemical Co. (Sternheim, Germany). All other chemicals
were of analytical grade.
V. faba seeds were purchased in a local market. V. sativa seeds were collected
from three different populations located in southwestern Spain. Seeds at full maturity
were collected from several plants in each population. Three different populations of
V. sativa were collected during May and June 2013 in the Sierra de Aracena y Picos de
Aroche Natural Park (Huelva province, Andalucía, Spain). GPS locations of these
populations were: 1) N 37.887238, W 6.581027; 2) N 37.896836, W 6.557928; 3) N
37.846776, W 6.474205. Seeds at full maturity were collected from several plants in
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each population and were allowed to completely dry at room temperature before
storage at -20o C.
Preparation of seed polyphenols extracts
Seeds were ground to a fine powder using a laboratory mill. Five grams of seed
flour were mixed with 250 mL methanol and extracted in a Soxhlet apparatus for 6-8 h.
The extracts were concentrated under vacuum at 40 °C using a rotary evaporator and
stored at 4°C.
Determination of total polyphenols
Total polyphenols were determined according to Slinkard & Singleton (1977)
with slight modifications. Seed extracts (250 L) were mixed with 1 mL diluted Folin-
Ciocalteu reagent in water (1/9, v/v) and shaken vigorously for three minutes.
Absorbance was measured at 765 nm after addition of a Na2CO3 solution (750 L, 1%,
w/v) and incubation at room temperature for two hours. Gallic acid was used as
reference standard and total phenolic content was expressed as milligrams of gallic
acid equivalents (mg GAEs/g extract).
Determination of total flavonoids
Total flavonoids were determined using the Dowd method as modified by
Arvouet-Grand et al., (1994). Briefly, 1 mL seed extract was mixed with 1 mL AlCl3 (2 %,
w/v) in methanol. A reagent blank was also prepared by adding 1 mL seed extract to 1
mL methanol without AlCl3. Absorbance was read at 415 nm after 10 min incubation at
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room temperature. Rutin was used as a reference standard and the total flavonoid
content was expressed as milligrams of rutin equivalents (mg RE/g extract).
Cupric ion reducing activity (CUPRAC assay)
Cupric ion reducing activity was determined according to Apak et al., (2006).
Seed extracts (500 L) were added to 3 mL reagent solution containing 1 mL 10 mM
CuCl2, 1 mL 7.5 mM neocuproine and 1 mL 1 M pH 7 NH4 acetate buffer. A reagent
blank was also prepared by adding 500 L seed extract to 3 mL reaction mixture
without CuCl2. Absorbance was read at 450 nm after incubation at room temperature
for 30 min. CUPRAC activity was expressed as milligrams of trolox equivalents (mg TE/g
extract).
Ferric reducing antioxidant power (FRAP assay)
FRAP assay was carried out as described by Benzie & Strain (1996) with slight
modifications. Seed extracts (100 L) were added to 2 mL FRAP reagent containing 0.3
M pH 3.6 acetate buffer, 10 mM TPTZ in 40 mM HCl, and 20 mM ferric chloride in a
10:1:1 ratio (v/v/v). Absorbance was read at 593 nm after incubation at room
temperature for 30 min. FRAP activity was expressed as milligrams of trolox
equivalents (mg TE/g extract).
Total antioxidant activity
Total antioxidant activity was evaluated using the phosphomolybdenum
method according to Berk et al., (2011) with slight modifications. Seed extracts (300
L) were combined with 3 mL of reagent solution containing 0.6 M sulfuric acid, 28
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mM sodium phosphate, and 4 mM ammonium molybdate. Absorbance was read at
695 nm after incubation at 95 °C for 90 min. Total antioxidant capacity was expressed
as milligrams of trolox equivalents (mg TE/g extract).
ABTS radical scavenging activity
Scavenging of the ABTS radical cation was measured according to the method
of Re et al., (1999) with slight modifications. Briefly, ABTS.+ was produced directly by
reaction between a 7 mM ABTS solution and 2.45 mM potassium persulphate. The
reaction was allowed to proceed at room temperature in the dark for 14 hours. The
resulting ABTS solution was diluted with methanol to yield an absorbance of 0.700 ±
0.02 at 734 nm. The samples (1 mL) were added to the ABTS solution (2 mL) and
thoroughly mixed. Absorbance was read at 734 nm after incubation at room
temperature for 30 min. The ABTS radical cation scavenging activity was expressed as
milligrams of trolox equivalents (mg TE/g extract).
Ferrous ion chelating activity.
Ferrous ion chelating activity was determined according to Dinis et al., (1994).
Briefly, 2 mL seed extract was added to 50 L 2 mM FeCl2 solution, and the reaction
was initiated by addition of 200 L 5 mM ferrozine. A reagent blank was also prepared
by adding 2 mL seed extract to 50 L 2 mM FeCl2 solution and 200 L water without
ferrozine. Absorbance was read at 562 nm after incubation at room temperature for
10 min. Metal chelating activity was expressed as milligrams of EDTA equivalents (mg
EDTAE/g extract).
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Enzyme inhibitory activities
Cholinesterase inhibition assay
Cholinesterase (ChE) inhibitory activity was measured using Ellman’s method
(Ellman et al., 1961). Seed extract (50 µL) were mixed with 125 µL DTNB and 25 µL of a
solution of AChE or BChE in pH 8.0 Tris-HCl buffer in 96-well microplates and incubated
at 25 C for 15 min. The enzymatic reaction was initiated by addition of 25 µL of ACI or
BCI. An enzyme blank was also prepared by mixing sample solution with all reaction
reagents except enzyme solution. Absorbance was read at 405 nm after incubation at
25 C for 10 min. Cholinesterase inhibitory activity was expressed as milligrams
galanthamine equivalents (mg GALAE/g extract).
α-amylase inhibition assay
Inhibitory of α-amylase was determined using the Caraway-Somogyi
iodine/potassium iodide method as described (Yang et al., 2012). Seed extracts (25 µL)
were mixed with 50 µL α-amylase solution in pH 6.9 phosphate buffer containing 6 mM
sodium chloride in 96-well microplates, and incubated at 37 °C for 10 min. The reaction
was initiated by addition of 50 µL starch solution (0.05%, w/v). A reagent blank was
prepared by adding seed extracts to all reagents except for the α-amylase solution. The
reaction mixture was incubated at 37 °C for 10 min and stopped by addition of HCl (25
µL, 1 M). This was followed by addition of iodine/potassium iodide solution (100 µL),
and absorbance was read at 630 nm. The α-amylase inhibitory activity was expressed
as millimoles acarbose equivalents (mmol ACE/g extract).
α-glucosidase inhibition assay
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Inhibition of α-glucosidase was determined according to Liu et al., (2004). Seed
extracts (50 µL) were mixed with glutathione (50 µL), α-glucosidase solution in pH 6.8
phosphate buffer (50 µL), and PNPG (50 µL) in 96-well microplates and incubated at 37
°C for 15 min. Similarly, a blank was prepared by adding extracts to all reagents except
for α-glucosidase solution. The reaction was stopped by addition of 50 µL 0.2 M
sodium carbonate, and absorbance was read at 400 nm. The α-glucosidase inhibitory
activity was expressed as millimoles of acarbose equivalents (mmol ACE/g extract).
Tyrosinase inhibition assay
Tyrosinase inhibitory activity was measured using the modified dopachrome
method with L-DOPA as substrate, as previously reported (Masuda et al., 2005) with
slight modifications. Seed extracts (25 µL) were mixed with 40 µL tyrosinase solution
and 100 µL pH 6.8 phosphate buffer in 96-well microplates and incubated at 25 °C for
15 min. The reaction was initiated by addition of 40 µL L-DOPA. Reagent blanks were
prepared by mixing extracts with all reagents except for tyrosinase solution.
Absorbance was read at 492 nm after incubation at 25 °C for 10 min. Tyrosinase
inhibitory activity was expressed as kojic acid equivalent (mgKAE g/ extract).
High Performance Liquid Chromatography/Mass Spectrometry.
Analysis by HPLC/MS was carried out using a 3 µm particle size, reverse-phase
20 x 0.46 cm Mediterranea Sea 18 column (Qmx Laboratories, Essex, UK). Elution at 1
mL/min was carried out using a gradient of methanol in water that was acidified using
1% (v/v) formic acid: 0 to 40 min linear gradient from 0 to 70% methanol, 40 to 55 min
70% methanol, 55 to 60 min linear gradient from 70 to 0% methanol. After monitoring
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in the UV using a diode array detector, the eluent went on to an electrospray
ionization micrOTOF-Q II mass spectrometer (Bruker Daltonics, Bremen, Germany)
operated in negative mode. Detection was recorded in the 50-1500 m/z range.
Interface parameters were as follows: capillary voltage 4.5 kV, nebulization pressure
1.2 bar, and ionization gas flow 8 L/min at 200 oC. Data was acquired in full scan mode,
and MS2 spectra were acquired in auto mode using data dependent acquisition. Target
Analysis 1.2 and Hystar 3.2 software packages (Bruker Daltonics, Bremen, Germany)
were used for data analysis.
Statistical analysis.
Statistical analysis with Sigma Plot 13.0 program was made through one way
ANOVA analysis and using the Holm-Sidak Test.
RESULTS AND DISCUSSION
The content in total seed polyphenols was significantly higher in V. sativa than
in V. faba, although the content in flavonoids was higher in V. faba (Table 1).
Polyphenols are strong antioxidants and are considered health promoting compounds
that may decrease the risk of suffering cardiovascular diseases (Duthie et al., 2000),
neurodegenerative diseases (Ramassamy, 2006), and cancer (Ramos, 2007).
Antioxidant activity was analyzed in the polyphenol seed extracts by determination of
reducing power, radical scavenging activity, and total antioxidant activity based on the
reduction of phosphate-molybdenum (VI) (Table 2). Reducing power was assayed using
the CUPRAC and FRAP methods. Although the result of both determinations were
higher in V. sativa than V. faba, differences were not statistically significant, except for
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V. sativa population 3 that showed significantly higher reducing power than all the
other samples in both assays. V. faba showed significantly higher ABTS radical
scavenging activity than V. sativa. Resembling reducing power, V. sativa population 3
showed higher radical scavenging activity than the other two V. sativa populations.
Finally, total antioxidant activity was significantly higher in V. sativa than in V. faba
seed polyphenols (Table 2).
Many polyphenols chelate transition metals such as iron (Yoshino & Murakami,
1998) and copper (Brown et al., 1998), an activity that may result in protection against
DNA damage (Perron et al., 2008) and inhibition of free radical formation (Guo et al.,
2007). Thus, flavonoids such as quercetin and rutin protect cells from toxicity due to
exposure to iron and copper (Aherne & O’Brien, 2000). Interestingly, the use of
biomolecules with antioxidant and chelating activities has been suggested, for the
treatment of neurodegenerative diseases such as Parkinson´s and Alzheimer´s disease
(Mandel et al., 2006). As shown in Table 3, iron chelating activity was significatively
higher in V. sativa than in V. faba polyphenols.
Enzyme inhibitors are used for the treatment of many pathological conditions.
These include inhibitors of acetylcholinesterase such as galathamine and tacrin for the
treatment of Alzheimer’s disease (Stepankova & Komers, 2008), the glucosidase
inhibitors acarbose and viglibose for the treatment of diabetes mellitus, and the
tyrosinase inhibitor kojic acid for the treatment of skin diseases. Due to side-effects
such as diarrhea, abdominal discomfort, and cell toxicities (Baek et al., 2012; Dong et
al, 2012; Schulz, 2003), there is an increasing interest in the search for new and safer
enzyme inhibitors from natural sources. Although several species belonging to the
Fabaceae family have been screened in the search for enzyme inhibitors (Boaduo et
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al., 2014; Orhan et al., 2004), this is the first report on the presence of enzyme
inhibitory activities in V. sativa seed polyphenol extracts.
As shown in Table 4, polyphenol extracts inhibited to some extent all the
enzymatic activities that were assayed. Data is given as equivalents of well-known
inhibitors, facilitating an evaluation of the intensity of the inhibitory activities. Using
either ACI or BCI as substrate, cholinesterase inhibition was higher in V. faba than in V.
sativa. This was true for the average activity in the V. sativa extracts, but the inhibitory
activity in population 2 was actually significative higher than in V. faba (P<0.05).
The enzymes α-amylase and α-glucosidase are directly linked to the
concentration of glucose in blood. Managing blood glucose levels is the goal of any
treatment for diabetes, and the use of natural α-amylase and α-glucosidase inhibitors
would be a powerful tool to manage diabetes disease (Gray, 1995). Inhibition of -
amylase and -glucosidase by polyphenol extracts from soybean, pea, Vigna sp., and
Phaseolus sp. has been reported (Saito et al., 2007), and a strong correlation between
content in polyphenols and -amylase inhibitory activities has been reported as well
(Premakumara et al., 2013).
Our data shows that the α-amylase inhibitory activity was quite variable in V.
sativa populations, ranging from 0.4 to 1.1 mmol ACEs/g extract, and even lower in V.
faba at 0.3 mmol ACEs/g extract (Table 4). On the other hand, the -glucosidase
inhibitory activity was very similar in all V. sativa populations and in V. faba, namely
0.3 mmol ACEs/g extract.
Tyrosinase is a copper-containing, key enzyme in melanin biosynthesis. It has
been reported that tyrosinase inhibition plays a crucial role in the prevention of skin
disorders (Kim & Uyama, 2005) and hence, that inhibition of tyrosinase activity may be
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useful for the treatment of disorders associated with melanin hyperpigmentation
(Masamoto et al., 2003). Polyphenols extracts from persimmon (Xue et al., 2011), and
the flavonoids kaempferol and quercetin (Kim et al., 2005; Orhan et al., 2004) have
been shown to inhibit tyrosinase. Certain soybean products such as cheonggukjang
have also been shown to have tyrosinase inhibitory activity (Choi et al., 2008). Our
data demonstrates that both V. faba and V. sativa polyphenol seed extracts have
strong tyrosinase inhibitory activity, exciding several fold the activity of kojic acid
(Table 4).
The V. sativa seed extracts were analyzed by HPLC/DAD/MS in order to
determine the composition in polyphenols. As shown in Table 5, this analysis revealed
variability among populations, but all populations had in common the presence of high
levels of catechin and hydroxybenzoic aldehyde. Populations 2 and 3 were
characterized by the presence of glycosides of apigenin and quercetin, while
population 1 had more phenolic acids. Kaempferol rhamnoside was only found in
population 2. Chelation of iron by kaempferol, quercetin, apigenin, and catechin has
been reported (Mira et al., 2002), which might explain the high iron chelating activity
of the V. sativa polyphenol extracts. There are very few previous reports on the
polyphenol composition of V. sativa plants, which are in general consistent with our
results. Webb & Harborne (1991) carried out a taxonomical study of flavonoid
aglycones in the leaves of genus Vicia, reporting the presence of quercetin and
kaempferol in V. sativa ssp. sativa, ssp. macrocarpa, ssp. nigra and ssp. cordata. Torck
& Pinkas (1992) reported the presence of glycosil derivatives of quercetin and
kaempferol, as well as minor amounts of apigenin and luteolin heterosides, in V.
sativa.
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In conclusion, the polyphenol extracts from V. sativa seeds have high
antioxidant and metal chelating activity, comparable or even higher than the activity of
V. faba seed extracts. They also significantly inhibit the activity of the enzymes
acetylcholinesterase, α-amylase, α-glucosidase, and tyrosine. This combination of
properties could allow the use of V. sativa seeds as a source of health-promoting and
therapeutic components.
ACKNOWLEDGEMENTS
This work was carried out with the financial support of Junta de Andalucía to
the Laboratory of Bioactive and Functional Components of Plant Products (Ayudas
interanuales to AGR 257, Instituto de la Grasa, C.S.I.C.). Cristina Megías is recipient of a
JAE-Doc contract (C.S.I.C. and European Social Fund). Isabel Cortés-Giraldo was
recipient of a JAE-Pre fellowship (C.S.I.C. and European Social Fund). We are thankful
to J. J. Rios and A. Sánchez assistance with HPLC/DAD/MS analyses.
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Table 1. Polyphenol and flavonoid content in V. sativa and V. faba seeds. Results are expressed as the mean standard deviation of three determinations. Same superscripts means significative differences (P<0.05).
aGAEs: gallic acid equivalents. bREs: rutin equivalents.
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SamplesPolyphenols
(mg GAEs/g extract)aFlavonoids
(mg REs/g extract)b
V. sativa 1 11.32 ± 0.34de 2.07 ± 0.07ae
V. sativa 2 10.51 ± 0.32cf 2.62 ± 0.21de
V. sativa 3 13.32 ± 0.31aceg 2.35 ± 0.11c
Average V. sativa 11.72 ± 1.18bg 2.35 ± 0.23b
V. faba 8.89 ± 0.19abdf 3.81 ± 0.21abcd
1
2
3
45
6
7
8
9
10
11
1213
14
15
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Table 2. Reducing power, radical scavenging activity and total antioxidant activity of V. sativa and V. faba seed polyphenols. Results are expressed as the mean standard deviation of three experiments. Same superscripts means significative differences (P<0.05).
Samples
Reducing power Radical scavenging activity
Total antioxidant activity
CUPRAC assay (mg TEs/g extract)a
FRAP assay (mg TEs/g extract) (mg TEs/g extract) (mg TEs/g extract)
V. sativa 1 18.63 ± 0.38a 8.62 ± 0.06ad 15.67 ± 0.35af 203.86 ± 0.64ac
V. sativa 2 19.73 ± 0.77b 9.94 ± 0.19b 15.50 ± 0.32be 135.98 ± 7.06cef
V. sativa 3 29.89 ± 0.92abcd 15.38 ± 0.43abc 18.60 ± 0.33cef 189.78 ± 6.10be
Average V. sativa 22.75 ± 5.07d 12.66 ± 2.72d 16.59 ± 1.42d 176.54 ± 29.3df
V. faba 20.78 ± 0.32c 10.01 ± 0.29c 23.36 ± 1.44abcd 118.38 ± 0.76abd
aTEs: trolox equivalents.
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2
3
4
5
67
8
910
11
12
13
14
12
Table 3. Iron chelating activity of V. sativa and V. faba seed polyphenol extracts. Results are expressed as the mean standard deviation of three experiments. Same superscripts means significative differences (P<0.05).
SamplesChelating activity
(mg EDTAEs / g extract)a
V. sativa 1 5.90 ± 0.04c
V. sativa 2 5.88 ± 0.02d
V. sativa 3 6.25 ± 0.64a
Average V. sativa 6.01 ± 0.17b
V. faba 3.99 ± 0.13abcd
aEDTAEs: EDTA equivalents.
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1
2
3
4
5
6
78
910111213
12
Table 4. Enzyme inhibitory activity of V. sativa and V. faba seed polyphenols. Results are expressed as the mean standard deviation of three experiments. Same superscripts means significative differences (P<0.05).
Cholinesterase inhibition
SamplesAChE inhibition(mg GALAEs/g
extract)a
BChE inhibition(mg GALAEs/g
extract)a
α-amylase inhibition (mmol ACEs/g extract)b
α-glucosidase inhibition (mmol ACEs/g extract)b
Tyrosinase inhibition ( mg
KAEs/g extract)
V. sativa 1 0.645 ± 0.001a 0.645 ± 0.079ac 1.114 ± 0.066abcd 0.369 ± 0.118 4.24 ± 0.03adh
V. sativa 2 0.674 ± 0.007b 0.876 ± 0.004cd 0.553 ± 0.036c 0.337 ± 0.126 5.67 ± 0.03bf
V. sativa 3 0.650 ± 0.013c 0.645 ± 0.007bd 0.399 ± 0.025b 0.337 ± 0.112 7.87 ± 0.10defg
Average V. sativa 0.656 ± 0.013d 0.722 ± 0.109e 0.689 ± 0.307de 0.348 ± 0.015 5.93 ± 1.49cgh
V. faba 1.130 ± 0.033abcd 0.895 ± 0.033abe 0.307 ± 0.008ae 0.357 ± 0.068 10.82 ± 0.23abce
aGALAEs: galanthamine equivalents. bACEs: acarbose equivalents. cKAEs: kojic acid equivalents.
26
123
4
5
6
789
10111213
12
Peak area(Abs units)
Compound RT (min)1 [M-H]- (m/z) Formula Fragments MS/MS λmax (nm) V. sativa 1 V. sativa 2 V. sativa 3
Arbutin 8.9 272 C12H16O7 71, 108, 109 260, 278 24020
Vanillic acid hexoside 10.6 330 C14H18O9 167, 123 47752
Gallic acid 11.7 170 C7H6O5 2067
Oleoside dimethyl ester 12.4 418 C18H26O11 403, 255, 389 12512
Dihydroxybenzoic acid 14 154 C7H6O4 136, 137 1479 511
type procyanidin dimer 17.1 578 C30H26O12 279 4878
Catechin 3-O-glucoside 17.5 452 C21H24O11 276 11436
p-Hydroxybenzoic acid 19.0 138 C7H6O3 182226 7458
Hydroxybenzoic acid hexoside 19.1 C13H16O8 137 149872 6054
(+)-Catechin 19.8 290 C15H14O6 180, 109 280 25634 19061 20243
Apigenin di-C-hexoside 25.7 594 C27H30O15 473, 353, 293 216, 340 14860 31010
Apigenin C-hexoside-pentoside 27.7 564 C26H28O14 443 2162 9537
Kaempferol rhamnoside-hexose-hexose 28.5 756 C33H40O20 284, 285 ,609 23797
Hydroxybenzoic aldehide 29.9 122 C7H6O2 90, 91, 92, 93 59434 867802 434415
Quercetin rutinoside 32.7 610 C27H30O10300, 301, 463, 179,
180 7325 45652
Table 5. V. sativa polyphenols composition.
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1Retention time. 2Retention time of standards.
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