JOURNAL OF FUNCTIONAL FOODS 4 (2 0 1 2) 9 7 9–98 7

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Phenolic acids, flavonoids and total antioxidant capacity

of selected leafy vegetables

Umma Khair Salma Khanama,*, Shinya Obab,1, Emiko Yanaseb,1, Yoshiya Murakamic,2
a
The United Graduate School of Agricultural Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
b
Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
c
Laboratory of Plant Production, Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan

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

Article history: Phenolic compounds and total antioxidant capacity of eight leafy vegetables, namely
Received 10 January 2012 Komatsuna, Mizuna, Pok choi, Mitsuba, Salad spinach, Lettuce, Red amaranth and Green
Received in revised form amaranth were determined. The phenolic compounds were characterized as hydroxyben-
25 July 2012 zoic acids, hydroxycinnamic acids and flavonoids. Salicylic acid was, by far, the most com-
Accepted 30 July 2012 mon hydroxybenzoic acid, ranging from 4.40 to 117.36 lg/g fresh frozen weight (ffw).
Available online 24 August 2012 Vanilic acid, gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid and
m-coumaric acid were commonly found in all of these vegetables. Isoquercetin and rutin,
Keywords: the most common flavonoids, ranged from 3.70 to 19.26 and 1.60 to 7.89 lg/g ffw, respec-
Leafy vegetables tively, and hyperoside was highest (38.72 lg/g ffw) in Mizuna. Total antioxidant capacity
Phenolics values varied widely between ABTS+ and DPPH assay methods, with values reported as
LC-MS-ESI
equivalents to trolox, quercetin and ascorbic acid. Among these vegetables, total antioxi-
HPLC-UV
dant capacity was found in the following order: Pok choi > Komatsuna > Mizuna > Mit-
DPPH
suba > Red amaranth > Lettuce > Green amaranth > Salad spinach.
ABTS+
 2012 Elsevier Ltd. All rights reserved.
Antioxidant capacity

1. Introduction
In recent decades, polyphenols have attracted considerable
interest in food technology research. Their antioxidant prop-
erties, availability in diets and probable roles in countering
deadly diseases such as cancer, neuro-degenerative and car-
diovascular diseases have been highlighted in various studies
(Manach, Scalbert, Morand, Rémésy, & Jiménez, 2004). Cur-
rently, natural antioxidants, particularly in fruits and vegeta-
bles, have gained the attention of both researchers and
consumers. Vitamins, phenolic substances and carotenoids
are the three major groups of natural antioxidants that are
available in fruits and vegetables and that are involved in
defences against several diseases (Thaipong, Boonprakob,
Crosby, Cisneros-Zevallos, & Byrne, 2006). Phenolic sub-
stances, which exist in plant tissues as secondary metabolites,
can be categorized into simple phenols, phenolic acids (both
hydroxybenzoic acids and hydroxycinnamic acids), flavo-
noids, lignins and tannins. These substances are responsible
for colour, bitterness, acerbic taste, flavour, odour and antiox-
idant properties (Kartika, Li, Wall, Nakamoto, & Iwaoka, 2007).
Antioxidants are compounds that can delay the oxidation of

* Corresponding author. Present address: Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan. Tel.:
+81 058 293 2851.
E-mail addresses: skhanam04@yahoo.com (U.K.S. Khanam), soba@gifu-u.ac.jp (S. Oba), e-yanase@gifu-u.ac.jp (E. Yanase),
p8102037@edu.gifu-u.ac.jp (Y. Murakami).
1
Present address.
2
Present address: Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2012.07.006
980 JOURNAL OF FUNCTIONAL FOODS
other molecules by inhibiting the initiation or propagation of
oxidizing chain reactions caused by free radicals and thereby
may reduce oxidative damage to the human body (Namiki,
1990).
Phenolic acids as well as flavonoids are a large group of
phytochemicals that are widely available in the plant king-
dom and are produced from phenylalanine and tyrosine
via the shikimic acid pathway (Herrmann, 1995). Phenolic
compounds are involved in attracting insects for seed dis-
persion and pollination, natural defences against harmful
insects, fungi, viruses, bacteria and even controlling plant
hormones. Flavonoids are important for a wide array of bio-
logical functions. Quercetin, a major representative of the
flavonoid, prevents the oxidation of low-density lipoprotein
by scavenging free radicals (Cartea, Francisco, Soengas, &
Velasco, 2011). Ellagic acid is a naturally occurring polyphe-
nolic metabolite which is produced within vacuoles as water
soluble ellagitannins. Studies of ellagic acid have reported
its antimutagenic and anticarcinogenic responses, which
are beneficial for human health (Maas, Galletta, & Stoner,
1991).
Vegetables and fruits, in particular, are very important for
human health because they occur naturally as whole foods
and are rich in a large variety of nutrients. Leafy vegetables
are not only good sources of minerals but also contain vita-
mins, antioxidants and pigments. A large number of vegeta-
bles, such as kale, spinach, brussel sprout, broccoli, onion,
eggplant and cucumber, are rich sources of antioxidants (Gaz-
zani, Papetti, Massolini, & Daglia, 1998; Velioglu, Mazza, Gao,
& Oomah, 1998; Vinson, Hao, Su, & Zubik, 1998).
In Japan, commercially available leafy vegetables include:
Komatsuna (Japanese mustard spinach, Brassica rapa var
komatsuna, family: Brassicaceae), Mizuna (Japanese mustard,
Brassica rapa, var japonica, family: Brassicaceae), Pok choi (Chi-
nese celery cabbage, Brassica rapa var. chinenesis, family: Brass-
icaceae), Mitsuba (Japanese parsley, Cryptotaenia japonica,
family: Apiaceae), Horenso (salad spinach, Spinacea oleracea,
family: Amaranthaceae), Lettuce (condiment, Lactuca sativa,
family: Asteraceae), Red amaranth (Amaranthus tricolor, fam-
ily: Amaranthaceae) and Green amaranth (Amaranthus tricolor,
family: Amaranthaceae). Mustard group vegetables are impor-
tant food crops throughout the world (Cartea et al., 2011). Re-
cent reports have suggested that cruciferous vegetables are
good sources of natural antioxidants. They contain high lev-
els of carotenoids, tocopherols and ascorbic acid, and there
is strong epidemiological evidence that these compounds
help protect the human body against damage from reactive
oxygen species (Cartea et al., 2011). However, the phenolic
compounds of these traditional vegetables have not been ade-
quately studied. The present study selected eight leafy vege-
tables (mentioned above) and investigated their phenolic
profiles and antioxidant capacities. Therefore, the objectives
of this study were (1) to characterise the phenolic compounds
by identifying and quantifying their phenolic acids and flavo-
noids, and (2) to evaluate their antioxidant capacities using
ABTS+ and DPPH assays, with values reported as equivalents
to trolox, quercetin and ascorbic acid. The findings of this
study will improve our understanding of the antioxidant
functions of these leafy vegetables for food nutritionists and
consumers.
4 ( 2 0 1 2 ) 9 7 9 –9 8 7
2. Materials and methods
2.1. Chemicals
Standard compounds of pure phenolic acids, rutin, HPLC grade
acetonitrile and acetic acid, methanol, DPPH (2,2-diphenyl-
1-picrylhydrazyl), ABTS+ (2,2-azinobis- (3-ethyl-benzothiazo-
line-6-sulphonic acid), trolox (6-hydroxy-2,5,7,8-tetrame-
thylchroman-2-carboxylic acid), ascorbic acid, potassium per
sulphate, aluminium chloride hexahydrate, sodium carbonate
and potassium acetate were purchased from Wako Pure
Chemical Industries Ltd., Osaka, Japan. Folin–Ciocalteu re-
agent was from Nacalai tesque, Inc. (Kyoto, Japan), Isoquerce-
tin was from Kanto chemical Co. Inc. (Tokyo, Japan) and
hyperoside was from Tokiwa Phytochemical Co. Ltd. (Tokyo,
Japan).
2.2. Plant materials and extractions
Eight types of vegetables, Komatsuna, Mizuna, Pok choi, Mit-
suba, Salad spinach, Lettuce, Red amaranth and Green ama-
ranth, were purchased from a supermarket at Gifu, Japan.
The vegetables were cleaned under running tap water and
rinsed with distilled water. Fresh leaves were cut into small
pieces, frozen in liquid nitrogen and stored at 80 C until
use. The rest of the fresh leaves were dried in an electric dry-
ing oven (DRN620DB, Advantec, Chiba, Japan) for 24 h at 80 C
for further chemical analysis. Samples were collected from at
least three different vegetable batches from the supermarket.
All the vegetables used were grown during June 2011 and all
analyses were completed within 1 month.
One gram of fresh-frozen leaves was homogenized with
10 ml of 80% methanol containing 1% acetic acid. The
homogenized mixture was filtered through a 0.45 lm filter
using a MILLEX-HV syringe filter (Millipore Corporation,
Bedford, MA, USA) and centrifuged at 10,000g for 15 min.
The final filtrate was used to analyse phenolic acids and
flavonoids. Compounds were extracted from oven dried leaf
samples using 90% methanol. The leaf samples were placed
in a hot water bath (Thomastant T- N22S, Thomas kagaku
Co. Ltd., Tokyo, Japan) and shaken at 80 C. After 1 h, the ex-
tract was cooled and filtered for analyses of total polyphenol
content, total flavonoid content and total antioxidant
activity.
2.3. HPLC analysis of phenolic acids and flavonoids
The amounts of phenolic acids and flavonoids in a sample
were determined using HPLC with the method described by
Khanizadeh et al. (2008). The HPLC system (Shimadzu SCL-
10Avp, Kyoto, Japan) was equipped with LC-10Avp binary
pumps, a degasser (DGU-14A) and a variable Shimadzu
SPD-10Avp UV–vis detector. A CTO-10AC (STR ODS-II,
150 · 4.6 mm I.D., Shinwa Chemical Industries, Ltd., Kyoto, Ja-
pan) column was used for all separations. The binary mobile
phase consisted of 6% (v/v) acetic acid in water (solvent A)
and acetonitrile (solvent B) was pumped at a flow rate of
1 ml/min for a total run time of 70 min. The system was run
with a gradient program: 0–15% B for 45 min, 15–30% B for
15 min, 30–50% B for 5 min and 50–100% B for 5 min. The
 JOURNAL OF FUNCTIONAL FOODS 4 ( 20 1 2) 9 7 9–98 7 981

injection volume was 10 ll. The detector was set at 254, 280
and 360 nm for simultaneous monitoring of hydroxybenzoic
acids, hydroxycinnamic acids and flavonoids. Compounds
were identified by comparing their retention time and UV–
vis spectra with those of standards (Fig 1). The phenolic acids
and flavonoids were also qualitatively confirmed using mass
spectrometry, which is discussed below in Section 2.4. The
sum of concentrations of all phenolic acids and flavonoids,
quantified by HPLC, was denoted as the total phenolic index
(TPI). From the HPLC data, TPI was obtained for each of the
vegetables according to the method described by Tsao and
Yang (2003). All samples were prepared and analysed in dupli-
cate. The results were expressed as lg/g fresh frozen weight
(ffw).
2.4. Qualitative analysis by LC-MS
Mass spectrometry analyses were performed using a JEOL
AccuTOF (JMS-T100LP, JEOL Ltd., Tokyo, Japan) time-of-flight
mass spectrometer fitted with an Agilent 1100 Series HPLC
system and a UV–vis detector coupled on-line with an Electro
Spray Ionization (ESI) source. Analytical conditions were: Neg-
ative ion mass spectra of the column elute were recorded in
the range m/z 0–1000. Needle voltage was kept at 2000 V.
The HPLC separation was done using a CTO-10AC column
(STR ODS-II, 150 · 4.6 mm I.D.) at 35 C with a solvent flow
rate of 0.7 ml/min. Solvent A was 1% acetic acid and solvent
B was acetonitrile. The chromatographic conditions were
optimised to obtain chromatograms with good resolution of

(a)

(b)

(c)

Fig. 1 – HPLC-UV–vis chromatograms of phenolic acids and flavonoids from standard compounds at (a) 254, (b) 280 and
(c) 360 nm. Peaks: 1- gallic acid, 2- p-hydroxy benzoic acid, 3- vanilic acid, 4- syringic acid, 5- salicylic acid, 6- ellagic acid.
7- chlorogenic acid, 8- caffeic acid, 9- p-coumaric acid, 10- ferulic acid, 11- m-coumaric acid, 12- sinapic acid, 13- trans-
cinnamic acid, 14- hyperoside (quercetin-3-galactoside), 15- rutin (quercetin -3-rutinoside) and 16- isoquercetin (quercetin-3-
glucoside).
982 JOURNAL OF FUNCTIONAL FOODS
adjacent peaks, for which a slight modification was made in
the method reported by Khanizadeh et al. (2008). Separation
was achieved with the initial mobile phase concentration
set at 0–15% B for 45 min, 15–30% B for 20 min, 30–50% B for
15 min and 50–100% B for 10 min. Extract constituents were
identified by LC-MS-ESI analysis.
2.5. Determination of total polyphenol content (TPC)
Total polyphenol content was determined using Folin–Ciocal-
teu (FC) method of Velioglu et al. (1998). A 100 ll sample of leaf
extract were transferred into a test tube, 0.75 ml of Folin–Cio-
calteu reagent (1:10 with deionised water) was added and the
contents was mixed. The mixture was allowed to stand at
room temperature for 5 min. Then, 0.75 ml of 6% (w/v) so-
dium carbonate was added to the mixture; it was shaken
gently and allowed to stand at room temperature for
90 min. Absorbance was read at 725 nm using a UV–vis spec-
trophotometer (Hitachi, U-1800, Tokyo, Japan). Ferulic acid
was used as the standard reference and TPC was expressed
as lg ferulic acid equivalent (FAE)/g dw.
2.6. Determination of total flavonoid content (TFC)
Total flavonoid content in vegetable extract was determined
using the aluminium chloride colorimetric method described
by Chang, Yang, Wen, and Chern (2002). For this, 500 ll of leaf
extract were transferred to a test tube followed by 1.5 ml of
methanol, 0.1 ml of 10% aluminium chloride, 0.1 ml of 1 M
potassium acetate and 2.8 ml of distilled water. After 30 min
at room temperature, the absorbance of the reaction mixture
was measured at 415 nm using a UV–vis spectrophotometer.
Rutin was used as the standard compound and TFC was ex-
pressed as lg rutin equivalent (RE)/g dw.
2.7. Determination of total antioxidant capacity (TAC)
For the ABTS+ assay the method described by Thaipong et al.
(2006) was followed. The stock solutions included 7.4 mM
ABTS+ solution and 2.6 mM potassium persulfate solution.
The working solution was prepared by mixing the two stock
solutions in equal quantities and allowing them to react for
12 h at room temperature in the dark. A 150 ll sample of leaf
extract was allowed to react with 2850 ll of ABTS+ solution
(1 ml ABTS+ solution mixed with 60 ml methanol) for 2 h in
the dark. The absorbance was taken against methanol at
734 nm using a UV–vis spectrophotometer.
For the DPPH assay, the stable DPPH radical was used to
measure the free radical scavenging activity of sample ex-
tracts as described by Alvarez-Jubete, Wijngaard, Arendt,
and Gallagher (2010).
The reaction mixture consisted of 500 ll of leaf extract and
500 ll of freshly prepared DPPH solution (0.05 mg/ml
methanol). The mixture was allowed to incubate in the dark
for 30 min at room temperature. The absorbance was
measured against methanol at 515 nm using a UV–vis
spectrophotometer.
The percentage of inhibition of ABTS+ and DPPH by leaf
extracts was calculated according to the following equation:
4 ( 2 0 1 2 ) 9 7 9 –9 8 7


Inhibition% ¼ 1  Atest sample =Ablank  100
Where Ablank is the absorbance of the methanolic blank
and Atest sample is the absorbance of the leaf extracts. Trolox,
quercetin, and ascorbic acid were used as reference standards
and the results were expressed as lg trolox/quercetin/ascor-
bic acid equivalent/g dw.
2.8. Statistical analyses
All data were reported as means ± standard deviation (SD)
using at least three independent measurements. Analysis of
variance and Tukey’s tests with a confidence interval of 95%
were performed using Excel Statistics Version 6.0 (Esumi,
Inc., Tokyo, Japan).
3. Results and discussion
3.1. Quantification of phenolic acids and flavonoids
The composition and concentrations of major phenolic sub-
stances that were determined by HPLC analysis are shown in
Table 1. A total of sixteen phenolic compounds were grouped
as hydroxybenzoic acids, hydroxycinnamic acids and flavo-
noids, with considerable variations. The hydroxybenzoic acids
possessed one functional carboxylic acid and were the most
abundant compounds in these leafy vegetables. Salicylic acid
was by far the most prominent individual hydroxybenzoic
acid, with concentrations as high as 117.36 lg/g ffw in Koma-
tsuna, followed by Pok choi (34.68 lg/g ffw) and Red amaranth
(21.17 lg/g ffw). No salicylic acid was detected in Mitsuba and
Salad spinach. The second most abundant hydroxybenzoic
acid was vanilic acid, which reached concentrations of
9.53 lg/g ffw in Red amaranth and 9.29 lg/g ffw in Green ama-
ranth. Syringic acid and gallic acid were also common in all of
the selected leafy vegetables and reached concentrations of
7.70 and 7.36 lg/g ffw in Komatsuna and Green amaranth,
respectively. Nearly all of the plants contained gallic acid, p-
hydroxybenzoic acid and vanilic acid (Robbins, 2003; Shahidi
& Naczk, 1995). Among these vegetables, ellagic acid was de-
tected only in Salad spinach (6.24 lg/g ffw).
The hydroxycinnamic acids have their biosynthetic origin
from phenylalanine; they are the most widely distributed phe-
nolic acids in plant tissues (Robbins, 2003). The most common
hydroxycinnamic acids in these selected leafy vegetables were
p-coumaric acid, ferulic acid and m-coumaric acide. Mizuna
contained high amount of p-coumaric (12.07 lg/g ffw) and m-
coumaric acid (6.23 lg/g ffw), and Pok choi contained the high-
est amount of ferulic acid (7.15 lg/g ffw). Ferulic acid has been
reported to inhibit the photo-peroxidation of linoleic acid at
high concentrations (Wang, 2003). However, caffeic acid and
chlorogenic acid were also commonly detected in the selected
leafy vegetables where Green amaranth contained the highest
amount of both acids with concentrations of 1.63 lg/g ffw and
15.34 lg/g ffw, respectively. Caffeic acid and some of its esters,
as was suggested in previous studies, possess anti-tumour
activity against colon carcinogenesis (Olthof, Hollman, & Ka-
tan, 2001; Rao et al., 1993). No cinnamic acid was detected
from Lettuce. Trans-cinnamic acid was only found in Salad
spinach (4.64 lg/g ffw) and Red amaranth (2.31 lg/g ffw). High
Table 1 – Phenolic compositions and concentrations (lg/g fresh-frozen weight) of selected leafy vegetables.

Phenolic compound Komatsuna Mizuna Pok choi Mitsuba Salad spinach Lettuce Red amaranth Green amaranth

JOURNAL OF FUNCTIONAL FOODS

Hydroxybenzoic acid
Gallic acid 2.63 ± 0.16c 2.09 ± 0.08c 3.16 ± 0.38b nd nd 2.23 ± 0.23c 2.84 ± 0.38b 7.36 ± 0.37a
Vanilic acid 8.43 ± 0.49a 8.28 ± 0.64a 8.40 ± 0.89a 7.16 ± 0.51a 6.60 ± 0.39b 6.78 ± 0.23b 9.53 ± 0.58a 9.29 ± 1.11a
Syringic acid 7.70 ± 0.77a 1.69 ± 0.11d 4.06 ± 0.16b nd nd nd 1.65 ± 0.22d 2.45 ± 0.50c
p-hydroxybenzoic acid 2.37 ± 0.01c 2.32 ± 0.02c 1.99 ± 0.04e 3.60 ± 0.01a nd 1.81 ± 0.03f 2.50 ± 0.01b 2.13 ± 0.06d
Salicylic acid 117.36 ± 2.61a 13.59 ± 0.43d 34.68 ± 0.39b nd nd 4.40 ± 0.32e 21.71 ± 1.12c 11.93 ± 0.73d
Ellagic acid nd nd nd nd 6.24 ± 0.25a nd nd nd

Hydroxycinnamic acid
Caffeic acid nd nd 1.52 ± 0.01a 1.13 ± 0.2b 1.55 ± 0.13a nd 0.67 ± 0.07c 1.63 ± 0.07a
Chlorogenic acid 1.2 ± 0.12d 1.14 ± 0.13d 2.03 ± 0.21c 2.82 ± 0.04c 1.59 ± 0.63d nd 9.06 ± 0.28b 15.34 ± 0.20a
p-Coumaric acid 2.01 ± 0.03c 12.07 ± 0.15a 5.18 ± 0.16b 1.04 ± 0.01e 1.57 ± 0.02d nd 1.17 ± 0.17e nd
Ferulic acid 2.29 ± 0.03c 2.94 ± 0.03b 7.15 ± 0.13a 2.17 ± 0.01c nd nd 2.19 ± 0.14c nd
m-Coumaric acid 2.21 ± 0.01d 6.23 ± 0.28a 3.67 ± 0.09b nd 3.16 ± 0.04c nd 1.40 ± 0.03f 1.62 ± 0.03e

4 ( 20 1 2) 9 7 9–98 7
Sinapic acid 8.45 ± 0.89c 39.22 ± 0.67a 17.11 ± 1.76b nd nd nd 1.23 ± 0.24 1.10 ± 0.05
Trans-cinnamic acid nd nd nd nd 4.64 ± 0.06a nd 2.31 ± 0.03b nd

Flavonoid
Isoquercetin (quercetin-3-glucoside) 18.17 ± 1.76a 16.99 ± 0.28a 18.25 ± 0.04a 3.65 ± 0.12c 3.70 ± 0.13c nd 10.14 ± 0.15b 19.26 ± 0.33a
Hyperoside (quercetin-3-galactoside) 13.26 ± 1.18c 38.72 ± 0.53a 19.54 ± 0.12b nd nd nd 2.70 ± 0.19d 1.70 ± 0.12e
Rutin (quercetin-3-rutinoside) 2.98 ± 0.25c 7.89 ± 0.20a 4.13 ± 0.04b 1.60 ± 0.52c 1.87 ± 0.11c nd 3.51 ± 0.13b 3.24 ± 0.23b
Total phenolic index (TPI) 189.06a 153.17b 130.87c 23.17g 30.92f 15.22g 72.61e 77.05d

Values means ± SD of triplicate assays, nd: not detected. Different letters in same rows are significantly different at p < 0.05.

983
 984
Table 2 – Molecular masses of relative ions (LC-MS-ESI) of phenolic compounds detected in selected vegetables.

Phenolic compound Identity Runtime Found [M1] m/z

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(min)
Calculated Komatsuna Mizuna Pok choi Mitsuba Salad Lettuce Red Green
molecular spinach amaranth amaranth
mass of
standard

Hydroxybenzoic acid
Gallic acid 3,4-5 Trihydroxybenzoic acid 9.05 169.12 nd nd nd nd nd nd nd nd
Vanilic acid 4-Hydroxy-3-methoxybenzoic acid 30.60 167.15 167 167 167 167 167 167 167 nd
Syringic acid 4-Hydroxy-3,5-dimethoxybenzoic acid 34.79 197.17 197 nd 197 197 197 197 197 197
p-Hydroxybenzoic 4 Hydroxybenzoic acid 31.47 137.12 nd nd nd nd nd nd nd nd
acid
Salicylic acid 2-Hydroxybenzoic acid 48.20 137.12 nd nd nd nd nd nd nd nd
Ellagic acid 2,3,7,8-Tetrahydroxy-chromeno 52.52 337.22 nd nd nd nd 301 nd nd nd
[5,4,3-cde]chromene-5,10-dione

4 ( 2 0 1 2 ) 9 7 9 –9 8 7
Hydroxycinnamic acid
Caffeic acid 3,4-Dihydroxy-trans-cinnamate) 32.00 179.16 nd nd 179 179 179 nd 179 179
Chlorogenic acid 3-(3,4-Dihydroxycinnamoyl) quinic acid 31.11 353.30 353 353 353 353 353 nd 353 353
p-Coumaric acid 4-Hydroxycinnamic acid 42.00 163.16 163 163 163 nd 163 nd 163 nd
Ferulic acid 4-Hydroxy-3-methoxycinnamic acid 47.90 193.18 193 193 193 193 193 nd 193 193
m-Coumaric acid 3-Hydroxycinnamic acid, 49.60 163.16 163 163 163 nd 163 nd 163 nd
Sinapic acid 4-Hydroxy-3,5-dimethoxycinnamic acid 49.00 223.21 223 223 223 nd 223 nd 223 nd
Trans-cinnamic 3-Phenylacrylic acid 67.33 147.16 nd nd nd nd nd nd nd nd
acid
Flavonoid
Isoquercetin Quercetin-3-glucoside 54.26 463.38 463 463 463 463 463 nd 463 463
Hyperoside Quercetin-3-galactoside 53.25 463.40 463 463 463 463 463 nd 463 nd
Rutin Quercetin-3-rutinoside 53.00 609.52 609 609 609 609 609 nd 609 609
 JOURNAL OF FUNCTIONAL FOODS
Table 3 – Total phenolic content, total flavonoid content and total antioxidant capacity (ABTS+ and DPPH method) of
selected leafy vegetables. Values are means ± SD of triplicate assays. Different letters in same columns are significantly
different at p < 0.05.
Vegetables Name TPC
c a
Komatsuna 200.87 ± 8.18
Mizuna 236.72 ± 10.34b
Pok choi 317.48 ± 5.05a
Mitsuba 128.72 ± 4.76d
Salad spinach 95.78 ± 2.95f
Lettuce 80.84 ± 4.76g
Green amaranth 117.79 ± 2.1e
Red amaranth 162.36 ± 1.64d
TPC: Total polyphenol content expressed as lg FAE/g dw, TFC: Total flavonoid content expressed as lg RE/g dw, TAC: Total antioxidant capacity
(% inhibition).
amounts of sinapic acid were detected in Mizuna (39.22 lg/g
ffw), Pok choi (17.11 lg/g ffw) and Komatsuna (8.45 lg/g ffw).
The levels of sinapic acid, p-coumaric acid and chlorogenic
acid in the selected leafy vegetables were similar or slightly
lower than those reported in Artemisia species (Carvalho, Cav-
aco, & Brodelius, 2011).
In plants, flavonoids occasionally occur as aglycone,
although the most common forms are glycoside derivatives.
These compounds account for 60% of total dietary phenolic
compounds (Harborne & Williams, 2000; Shahidi & Naczk,
2004). Flavonols are the most prevalent flavonoids in the plant
kingdom and glycosides of quercetin are the most predomi-
nant naturally occurring flavonols (Harborne & Williams,
2000). Among flavonoids, isoquercetin (quercetin-3-glucoside)
and rutin (quercetin-3-rutinoside) were the most common
forms found in the leafy vegetables used in this study. Green
amaranth contained the highest isoquercetin content
(19.26 lg/g ffw), followed by Pok choi (18.25 lg/g ffw) and
Komatsuna (18.17 lg/g ffw). Hyperoside (quercetin-3-galacto-
side) was found in Komatsuna, Mizuna, Pok choi, Red ama-
ranth and Green amaranth. Hyperoside was, by far, the
most common individual flavonol in these vegetables and
reached concentrations up to 38.72 lg/g ffw in Mizuna. Miz-
una also contained the highest amount of rutin (7.89 lg/g
ffw). Similar amounts of quercetin were found in collard
greens, mustard greens, kale, okra, sweet potato greens, pur-
ple hull peas and purslane (Huang, Wang, Eaves, Shikany, &
Pace, 2007). Our results suggested that flavonols, like isoqu-
ercetin and rutin, together with hyroxybenzoic acids (salicylic
acid, gallic acid, vanillic acid and p-hydroxybenzoic acid) and
hydroxycinnamic acids (p-coumaric acid, ferulic acid, m-cou-
maric acid, caffeic acid and chlorogenic acid) are frequently
detectable in the selected leafy vegetables. The existence of
such flavonoids, hydroxybenzoic and hydroxycinnamic acids
in daily diets may enhance cellular antioxidant defences and
prolong healthy life (Carvalho et al., 2011).
3.2. Mass spectrometry confirmation
The parent ion masses of standard phenolic acids and flavo-
noids were compared with values for the same components
that were separated by LC from the selected leafy vegetables
by observing the particular peaks of the relative components.
4 ( 20 1 2) 9 7 9–98 7 985
TFC TAC (% inhibition)
ABTS+ DPPH
a
149.89 ± 4.40 88.85 ± 13.97 57.75 ± 17.84a
151.77 ± 2.46a 47.28 ± 3.16b 67.71 ± 5.06a
147.92 ± 3.55a 96.97 ± 0.88a 72.16 ± 8.35a
102.77 ± 3.97c 35.6 ± 10.71b 56.35 ± 2.55ab
44.85 ± 1.56e 29.56 ± 1.09b 33.07 ± 6.52b
31.67 ± 0.86f 33.5 ± 6.36b 40.28 ± 5.3b
77.54 ± 4.89d 31.37 ± 2.01b 36.53 ± 8.94b
125.97 ± 3.04b 38.18 ± 0.63b 37.23 ± 3.91b
The relative intensity of each component’s parent ion mass
could be easily determined during its elution. In this way, each
component in the sample was carefully monitored and con-
firmed, with the exception of gallic acid, p-hydroxybenzoic acid
and salicylic acid. These results are summarized in Table 2.
3.3. TPC, TFC and TPI
The amount of TPC, estimated using FC method, varied
widely among the selected leafy vegetables (Table 3). Total
phenol content ranged from 80.84 to 317.48 lg FAE/g dw.
The highest level of TPC was observed in Pok choi followed
by Mizuna (236.72 lg FAE/g dw) and Komatsuna (200.87 lg
FAE/g dw), while the lowest concentration was observed in
Lettuce. High levels of phenolics were also found in Red ama-
ranth (162.39 lg FAE/g dw) and Mitsuba (128.27 lg FAE/g dw).
Among all of the samples, Mizuna contained the highest
amount of TFC (151.77 lg RE/g dw), followed by Komatsuna
(149.89 lg RE/g dw), Pok choi (147.92 lg RE/g dw) and Red ama-
ranth (125.97 lg RE/g dw) (Table 3). In this study TFC values
were lower than TPC values.
The TPI values, determined by HPLC, also varied widely
and were lower than the TPC values (Table 3). These results
appeared to contradict a previous report by Khanizadeh
et al. (2008). Although the FC method does not provide a de-
tailed view of the quantity and quality of the phenolic com-
pounds in an extract, this method is widely used and
provides a rapid and useful overall estimate of phenolic con-
tents. It is likely that any of the phenolic acids and flavonoids
that were not detected by HPLC would have contributed to the
lower number of phenolic profiles in these leafy vegetables.
In this study, eight types of leafy vegetable species from
four different families were examined. Vegetables from Brass-
icaceae family (Komatsuna, Mizuna, and Pok choi) had signif-
icantly higher levels of TPC, TFC and TPI than those of
Amaranthaceae and Apiaceae families. Lettuce, a condiment
from Asteraceae family, had significantly lower TPC and TPI
contents among the selected leafy vegetables.
3.4. Total antioxidant capacity (TAC)
Total antioxidant capacity (TAC) of a leafy vegetable reflects
its ability to inhibit the production of free radicals. We
986 JOURNAL OF FUNCTIONAL FOODS 4 ( 2 0 1 2 ) 9 7 9 –9 8 7

Table 4 – Total antioxidant capacity equivalent with trolox, quercetin and ascorbic acid measured by ABTS+ and DPPH
method.

Vegetable name Total antioxidant capacity (lg/g dw)

Trolox Quercetin Ascorbic acid
ABTS+ DPPH ABTS+ DPPH ABTS+ DPPH
a a a a a
Komatsuna 184.33 53.73 150.99 51.84 108.21 39.07a
Mizuna 83.69b 65.53a 70.13b 63.99a 50.43b 54.76a
Pok choi 203.95a 70.81a 166.78a 69.56a 119.51a 61.77a
Mitsuba 55.39b 52.06a 47.39b 49.75a 34.32b 36.87a
Salad spinach 40.09b 24.49ab 35.64b 20.58ab 25.75b 3.61abc
Lettuce 50.31b 27.94ab 43.3b 29.91ab 31.23b 11.56ab
Green amaranth 45.16b 28.58ab 39.17b 24.91ab 28.28b 9.83ab
Red amaranth 61.63b 29.42ab 52.4b 25.79ab 37.74b 6.88ab
Values are means. Different letters in same columns are significantly different at p < 0.05.

measured TAC using DPPH and ABTS+ radical cation assays to
evaluate free radical-scavenging properties (Table 3).The se-
lected leafy vegetables were evaluated by quantifying percent
inhibition, and they exhibited considerable variation. TAC, as
measured by the ABTS+ method, ranged from 29.56 to 96.97%;
Pok choi had the highest value followed by Komatsuna
(88.85%), Mizuna (47.28%), Red amaranth (38.18%) and the
lowest value was found in Salad spinach. DPPH is a relatively
stable organic radical that has been widely used to determine
the antioxidant activity of single compounds, as well as dif-
ferent plant extracts (Katalinic, Milos, & Jukic, 2006). TAC, as
measured by the DPPH method, did not show the same trend
as the ABTS+ results. With the DPPH method, TAC ranged
from 33.07 to 72.17% and Pok choi had the highest value, as
was observed with the ABTS+ method. Mizuna exhibited a
higher antioxidant capacity with the DPPH method (67.71%),
as compared to the ABTS+ method (47.28%), and Mitsuba also
showed a similar trend.
The antioxidant capacities of the leafy vegetables, which
were expressed as equivalents to trolox, quercetin and ascor-
bic acid, three different antioxidants that are widely used as
standard references, varied widely between the analysis meth-
ods (Table 4). The antioxidant capacities of highly pigmented
foods, such as cherry, spinach, red cabbage, plums (Floegel,
Kim, Chung, Koo, & Chun, 2011) and several varieties of toma-
toes (Martı́nez-Valverde, Periago, Provan, & Chesson, 2002) var-
ied widely between analyses that used DPPH and ABTS+
methods. Most of the leafy vegetables used in this study had
lower antioxidant capacities, as equivalents to trolox, querce-
tin and ascorbic acid, when using the DPPH assay, as compared
to the ABTS+ assay. The ABTS+ assay is based on the genera-
tion of blue/green ABTS+, which is applicable to both hydro-
philic and lipophilic antioxidant systems. In contrast, the
DPPH assay uses a radical that is dissolved in an organic media
and is, therefore, applicable to hydrophobic systems (Kim, Lee,
Lee, & Lee, 2002). Some compounds that have ABTS+ scaveng-
ing activity may not show DPPH scavenging, and some prod-
ucts that show ABTS+ scavenging reactions may have a
higher antioxidant capacity and can continually react with
ABTS+ (Arts, Haenen, Voss, & Bast, 2004; Wang et al., 1998).
In this study, trolox equivalent TAC values were higher
than quercetin and ascorbic acid equivalent values with both
the ABTS+ and DPPH methods (Table 4). In all of the cases, Pok
choi had significantly higher values, as compared to the other
vegetables. Lettuce and Salad spinach contained significantly
lower amounts of TPC, TFC and TPI, and also had very low
TAC values that were equivalent with trolox, quercetin and
ascorbic acid. These results are consistent with the data re-
ported by Cai, Luo, Sun, and Corke (2004). Antioxidant capac-
ities of plants are commonly evaluated using more than one
method to measure various oxidation products. Since the re-
sults differ depending on the methods used, it is better to use
at least two methods (Schlesier, Harwat, Böhm, & Bitsch,
2002). However, the present study provides a new approach
by examining relative antioxidant capacity equivalent with
trolox, quercetin and ascorbic acid in the selected eight leafy
vegetables using two different TAC methods that rely on dif-
ferent biological mechanisms.
4. Conclusions
Phenolic compounds and antioxidant capacity varied widely
among the eight selected leafy vegetables. Salicylic acid, van-
ilic acid, gallic acid and p-hydroxybenzoic acid were the most
common hydroxybenzoic acids. Caffeic acid, chlorogenic
acid, p-coumaric acid, ferulic acid and m-coumaric acid were
the prevalent hydroxycinnamic acids in these leafy vegeta-
bles. Among the flavonoids, isoquercetin and rutin frequently
occurred in all of the selected vegetables. Therefore, the se-
lected eight leafy vegetables are important sources of pheno-
lic antioxidants. This study also provided the detailed view of
the antioxidant capacities of these vegetables. The total anti-
oxidant capacities of Pok choi (Brassica rapa, var chinensis),
Komatsuna (Brassica rapa, var komatsuna) and Mizuna (Brassica
rapa, var japonica) were significantly higher than in the other
vegetables, which implies that these leafy vegetables can be
consumed as potential sources of natural antioxidants. In
addition, the phenolic profiles and antioxidant capacities of
these vegetables indicate their considerable dietary and nutri-
tional value.
Acknowledgements
This work was supported by the Plant Production Laboratory,
Gifu University, Japan. The authors gratefully thank all of the
 JOURNAL OF FUNCTIONAL FOODS
students in this laboratory and the staffs of Analytical center,
Gifu University for their spontaneous cooperation, which
helped us complete this study.
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