Phenolic Compounds and Antioxidant Capacities

of 10 Common Edible Flowers from China

Lina Xiong, Jiajia Yang, Yirong Jiang, Baiyi Lu, Yinzhou Hu, Fei Zhou, Shuqin Mao, and Canxi Shen

C: Food Chemistry
Abstract: The free and bound phenolic compounds in 10 common Chinese edible flowers were investigated using
reversed phase high-performance liquid chromatography. Their antioxidant capacities were evaluated using 2,2-diphenyl-
1-picrylhydrazyl (DPPH) radical-scavenging activity, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
radical-scavenging activity, oxygen radical absorption capacity (ORAC), ferric reducing antioxidant power (FRAP), and
cellular antioxidant activity (CAA). Free factions were more prominent in phenolic content and antioxidant capacity
than bound fractions. Paeonia suffruticosa and Flos lonicerae showed the highest total phenolic content (TPC) 235.5 mg
chlorogenic acid equivalents/g of dry weight and total flavonoid content 89.38 mg rutin equivalents/g of dry weight.
The major phenolic compounds identified were gallic acid, chlorogenic acid, and rutin. P. suffruticosa had the highest
antioxidant capacity in the DPPH, ABTS, and ORAC assays, which were 1028, 2065, 990 μmol Trolox equivalents/g
of dry weight, respectively, whereas Rosa chinensis had the highest FRAP value (2645 μmol Fe2+ equivalents /g of dry
weight). The P. suffruticosa soluble phenolics had the highest CAA, with the median effective dose (EC50 ) 26.7 and
153 μmol quercetin equivalents/100 g of dry weight in the phosphate buffered saline (PBS) and no PBS wash protocol,
respectively. TPC was strongly correlated with antioxidant capacity (R = 0.8443 to 0.9978, P < 0.01), which indicated
that phenolics were the major contributors to the antioxidant activity of the selected edible flowers.

Keywords: cellular antioxidant activity, edible flowers, flavonoids, phenolic compounds, radical-scavenging activity

Practical Application: This study indicates that the 10 edible flowers in China are rich sources of phytochemicals, with
high levels of phenolic compounds and antioxidant capacity. This study not only provides useful information about the
health benefit of edible flowers for consumers, but also encourages researchers to utilize edible flowers as sources of
phytochemicals. In addition, the flower extracts are potential to be used as addictive in food to prevent the chronic
disease, help the health promotion, and prevent the food oxidization.

Introduction
Phenolics are a large and diverse group of molecules that in-
cludes many different families of aromatic secondary plant metabo-
lites. Phenolics occur in free and conjugated forms (soluble and
insoluble) with sugars, acids, and other biomolecules (Rispail and
others 2005). Accurate estimation of the total phenolics in plants
requires either a base, an acid, or enzymatic hydrolysis (Stalikas
2007). Free phenolics, which are usually stored in cell vacuoles, are
extracted with different aqueous alcohol–solvent mixtures. Bound
phenolics are extracted after hydrolysis with either a base, an acid,
or enzymes prior to extraction and analysis (Memon and others
2013). Phenolics have strong in vitro and in vivo antioxidant activi-
ties, which are associated with their ability to scavenge free radicals,
break radical chain reactions, and chelate metals (Niki 2010; Yang
and others 2010; Chiang and others 2013). Increased consump-
tion of phenolics has been correlated with anti-inflammatory ac-
tivity and a reduced risk of cardiovascular disease and certain can-
cers (Hamaguchi and others 2009; Kazłowska and others 2010;
Oueslati and others 2012; Rodrigues and others 2012).
MS 20131603 Submitted 11/4/2013, Accepted 1/20/2014. Authors are with
Zhejiang Univ., College of Biosystems Engineering and Food Science, Fuli Inst. of Food
Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang Engineering
Center for Food Technology and Equipment, Key Laboratory for Agro-Food Risk
Assessment of Ministry of Agriculture, Hangzhou, China. Direct inquiries to author
Lu (E-mail: byluzju@gmail.com).
Edible flowers, which have been used in the culinary arts in
China for centuries, are receiving renewed interest. Flowers can
be used as an essential ingredient in a recipe, provide seasoning to
a dish, or simply be used as a garnish. Most studies have focused
on the aroma and essential oils in flowers (Gilles and others 2010;
Mekni and others 2013). Total phenolics and flavonoids content
are measured of some edible flowers in China, such as Chrysanthe-
mum morifolium, Flos carthami, Flos rosae rugosae (Miao 2010), and
although some individual phenolic compounds are identified or
quantified in flowers like Rosa chinensis (Cai and others 2005), Rosa
rugosa (Liu 2013), Lavandula pedunculata (Wu and others 2007), Flos
lonicerae (Huang and others 2005), and Paeonia suffruticosa (Fan and
others 2012), most individual phenolic compounds have not been
analyzed. Comparative studies on common edible flowers have
been performed in Thailand and India (Kaisoon and others 2011;
Banerjee and others 2013); however, biological compounds in
most Chinese common edible flowers, including phenolic acids
and flavonoids, have not been precisely analyzed. With increasing
evidence that free radicals induce oxidative stress during the de-
velopment of various diseases, the function of radical-scavenging
antioxidants has received much attention. Some selected flowers
are rich in phenolic compounds and have great antioxidant activ-
ity, as well as the capacity to inhibit chronic disease (Lin and others
2012; Besbes Hlila and others 2013; Hsu and others 2013). An-
tioxidant capacity of some flowers measured from methods such
as DPPH (Xu and others 2005), ABTS (Cai and others 2004) was
estimated, however, the method used in evaluating the antioxidant

C 2014 Institute of Food Technologists

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doi: 10.1111/1750-3841.12404 Vol. 79, Nr. 4, 2014 r Journal of Food Science C517
Further reproduction without permission is prohibited
 Edible flowers phenolics and antioxidant . . .
capacity of edible flowers is often limited to a single method in
vitro; and various methods based on different principles should be
applied to evaluate the antioxidant capacity.
The objectives of this study are as follows: (1) to measure the
total phenolics and flavonoids in the free and bound fractions of 10
Chinese edible flowers; (2) to analyze 14 phenolic compounds in
different fractions in the 10 flowers; (3) to evaluate the in vitro an-
C: Food Chemistry
tioxidant capacity and cellular antioxidant activity (CAA) of these
phenolics. This is the 1st study that analyzes phenolic compounds
in some selected flowers in China, and the 1st to evaluate CAA of
the 10 edible flowers aside from the antioxidant capacity other.
Materials and Methods
Edible flowers
Ten Chinese edible flowers were studied, namely, P. suffruticosa,
Lilium brownii var. viridulum, F. lonicerae, R. chinensis, L. pedunculata,
Prunus persica, Hibiscus sabdariffa, F. carthami, C. morifolium, and F.
rosae rugosae. The flowers were collected from their major places of
origin Luoyang, Xi’an, Xinxiang, Muyang, Yili, Heze, Kunming,
Lasa, Tongxiang, and Lanzhou in China, respectively, and were
subsequently delivered to a local laboratory for drying.
Chemicals and regents
1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2 -azino-bis (3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
2,2 -azobis (2-methylpropionamidine) dihydrochloride (granular,
AAPH), fluorescein (FL), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ),
2 ,7 -dichlorofluorescin diacetate (DCFH-DA), 2,2 -azobis (2-
amidinopropane) dihydrochloride (ABAP), 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox), Folin–Ciocalteu
reagent, phenolic compounds (gallic acid [GA], protocatechuic
acid [PCCA], p-hydroxybenzoic acid [p-HO], chlorogenic acid
[ChA], vanillic acid [VA], caffeic acid [CFA], syringic acid
[SyA], p-coumaric acid [p-CA], ferulic acid [FA]), 3-hydroxy-4-
methoxybenzaldehyde thiosemicarbazone [3-H-4-MBT], rutin,
quercetin, apigenin, and kaempferol were obtained from Sigma
(Shanghai, China). HepG2 cells were obtained from the Ameri-
can Type Culture Collection. Fetal bovine serum was obtained
from Gibco (Grand Island, N.Y., U.S.A.). High-performance
liquid chromatography (HPLC)-grade methanol, acetonitrile,
and other solvents and reagents were purchased from Merck
(Shanghai, China). All other chemicals and reagents used in the
study were of analytical grade.
Extraction of phenolic compounds
The phenolics in the flowers were extracted using modified
method described by Sun and others (2002). Briefly, each sample
was ground to 40 mesh size on a grinding mill; and then 2 g was
weighed and extracted thrice with 50 mL of 80% acetone using
a Polytron homogenizer for 5 min each time. The homogenates
were filtered using nr. 2 Whatman paper on a Buchner funnel
under a vacuum. Phenolics in the filtrate were designated as soluble
free phenolic (SF). The SF extracts contained free aglycones and
soluble conjugates (glycosylated forms). The residue from soluble
fractions described above were drained off and hydrolyzed directly
with 20 mL of 4 M sodium hydroxide, at room temperature for 1 h
with shaking under nitrogen gas. The solution was then acidized to
pH 2, with an appropriate amount of 4 M hydrochloric acid, and
extracted with 20 mL hexane to remove lipids. The final solution Determination of antioxidant activity
was extracted 5 times with 20 mL ethyl acetate. Phenolics extracted Radical-scavenging activity (DPPH, ABTS, and oxy-
by ethyl acetate were designated insoluble bound-E (ISBE). The gen radical absorption capacity [ORAC]). The hydrogen-
remaining water-soluble portion was neutralized to pH 7 and or electron-donation ability of the corresponding extracts was 1st
C518 Journal of Food Science r Vol. 79, Nr. 4, 2014
was applied to a column with D101 macroporous resin. One-
hundred-milliliter 90% ethanol was used as mobile phase to wash
phytochemicals out of the column. Phenolics in the washout were
designated as insoluble bound-W (ISBW). The SF, ISBE, and
ISBW portions were evaporated at 45 °C under a vacuum to
dryness, and recovered with water to a final volume of 10 mL, and
then stored at –40 °C until use. All extractions were performed in
triplicate.
Determination of total phenolic content (TPC) and total
flavonoid content (TFC)
TPC was determined by Folin–Ciocalteu method described
previously (Pinelo and others 2005; Dudonne and others 2009)
and modified by our laboratory. Briefly, 500 μL flower extract
(previously diluted a suitable fold with distilled water) was mixed
with 100 μL Folin–Ciocalteu reagent, and the mixture was al-
lowed to stand at room temperature for 5 min. One-milliliter 7%
sodium carbonate solution was added and the fully mixed mixture
was recovered with water to a final volume of 6 mL. After 90 min
at room temperature, absorbance was read at 765 nm using a spec-
trophotometer. Results were expressed as milligram of chlorogenic
acid equivalents per gram of dry weight (mg CAE/g DW).
TFC was determined using colorimetric method described by
Tel and others (2012). Briefly, 2.5 mL of the extract (diluted with
distilled water to a suitable concentration) was mixed with 0.15 mL
of 5% NaNO2 solution. After 6 min, 0.15 mL of 10% Al(NO3 )3
solution was added and allowed to stand for another 5 min, before
1.0 mL of 1 M NaOH was added. The mixture was mixed well
on a vortex and was then recovered with water to a final volume
of 6 mL. The absorbance was measured immediately at 510 nm
using a spectrophotometer. The results are expressed as milligrams
of rutin equivalents per gram of dry weight (mg RE/g DW).
Identification and quantification of individual phenolic
compounds
The individual phenolic compounds were subjected to reversed
phase HPLC analysis using Waters 2695 pumps (Waters, Mass.,
U.S.A.), with a W2996 diode array detector. Chromatographic
separations were performed on a BDS C18 column (4.6 mm × 250
mm, 5 μm i.d.). The solvent compositions and gradient elution
conditions used were as previously described (Proestos and others
2005; Kaisoon and others 2011; Engida and others 2013) with
some modifications. The mobile phase consisted of purified water
with acetic acid (pH 2.74) (solvent A) and acetonitrile (solvent
B) at a flow rate of 0.8 mL/min. Gradient elution was performed
as follows: from 0 min to 2 min, linear gradient from 5% to 8%
solvent B; from 2 min to 25 min, linear gradient from 8% to 15%
solvent B; from 25 min to 30 min, linear gradient from 15% to 60%
solvent B; from 30 min to 40 min, linear gradient from 60% to 90%
solvent B; from 40 min to 45 min, 90% solvent B; from 45 min to
46 min, from 90% to 5% solvent B, and a re-equilibration period
of 5 min with 5% solvent B were used between individual runs.
The operating conditions were as follows: column temperature of
38 °C, injection volume of 20 μL, and UV-diode array detection
at 280 nm. Spectra were recorded from 210 to 400 nm. Phenolic
compounds in the samples were identified by comparing their
retention times and UV spectra with those of standard compounds
and were quantified using an external standard method.
Edible flowers phenolics and antioxidant . . .
measured from the bleaching of a purple methanolic DPPH solu-
tion. The antioxidant activity of the extracts was determined based
on the scavenging activity of the stable DPPH radical according
to the method described by a previous study with some modifi-
cations (Tai and others 2011). Exactly 100 μL of flower extract
(previously diluted a suitable fold with distilled water) was added to
3.9 mL of a 40 mg/L DPPH solution in methanol. Absorbance at
517 nm was read after the mixture was allowed to react for 60 min
at room temperature in the dark. The standard curve was linear
between 25 and 800 μM Trolox. Results are expressed in μmol
of Trolox equivalents per gram of dry weight (μmol TE/g DW).
For the ABTS assay, the procedure followed the method of
previous assays (Thaipong and others 2006; Tai and others 2011)
with some modifications. The stock solutions included 7.4 mM
ABTS·+ solution and 2.6 mM potassium persulfate solution. The
working solution was then prepared by mixing the 2 stock so-
lutions in equal quantities, and allowing them to react for 12 h
at room temperature in the dark. The solution was then diluted
by mixing 1 mL of ABTS·+ solution with 27 mL of methanol
to obtain an absorbance of 0.7 ± 0.03 units at 734 nm using a
spectrophotometer. Fresh ABTS·+ solution was prepared for each
assay. Then, 100 μL flower extract (previously diluted a suitable
fold with distilled water) was added to 3.0 mL of ABTS·+ solution.
Absorbance at 734 nm was read after reacting for 60 min at room
temperature in the dark. The standard curve was linear from 25 to
800 μM Trolox. Results are expressed in μmol TE/g DW.
The ORAC procedure was performed using a Fluoroskan As-
cent FL plate reader (Thermo scientific, Franklin, Mass., U.S.A.)
with 96-well plates (Carbonneau and others 2014). Analyses were
conducted in phosphate buffer (pH 7.4) at room temperature.
Peroxyl radical was generated using AAPH, which was prepared
fresh for each run. Fluorescein was used as the substrate. Then,
20 μL of flower extract (diluted with distilled water to a suitable
concentration) and 120 μL of fluorescein (70 nM final concen-
tration) were added to 96-well plates and allowed to mix for 5
min at room temperature in the dark, and 60 μL of AAPH (12
mM final concentration) was then added before recording. The
fluorescence assay conditions were as follows: excitation at 485 nm
and emission at 520 nm, recording for 180 min every 2 min. The
standard curve was linear from 0 μM to 80 μM Trolox. Results
are expressed as μmol TE/g DW.
Ferric reducing antioxidant power (FRAP). The FRAP
assay was done according to Thaipong and others (2006) with some
modifications. The stock solutions included 300 mM acetate buffer
(3.1 g C2 H3 NaO2 3H2 O and 16 mL C2 H4 O2 ; pH 3.6), 10 mM
TPTZ solution in 40 mM HCl, and 20 mM FeCl3 6H2 O solution.
The fresh working solution was prepared by mixing acetate buffer,
TPTZ solution, and FeCl3 6H2 O solution at a ratio of 10:1:1
(v/v/v). The solution was warmed to room temperature before
use. Then, 100 μL of flower extract (diluted with distilled water to
a suitable concentration) was added to 3.0 mL of FRAP solution.
Absorbance at 593 nm was read after allowing the mixture to react
for 60 min at room temperature in the dark. The standard curve
was linear from 0 to 1500 μM FeSO4 ·7H2 O. Results are expressed
in μmol Fe2+ /g DW.
Cellar antioxidant activity (CAA). The CAA assay was
done according to Wolfe and Liu (2007). Briefly, human
hepatocellular carcinoma HepG2 cells were seeded at a density
of 6 × 104 /well on a 96-well microplate in 100 μL of growth
medium/well. The growth medium was removed at 24 h after
seeding and the wells were washed with 100 μL of phosphate
buffered saline (PBS). Triplicate wells were treated for 1 h with
100 μL of quercetin solution or 100 μL of the flower extracts
with 25 μM DCFH-DA dissolved in treatment medium. Then,
600 μM ABAP was added to the cells in 100 μL of Hanks’ bal-
anced salt solution (HBSS), and the 96-well microplate was placed
into a Fluoroskan Ascent FL plate reader at room temperature.
Emission at 538 nm was measured with excitation at 485 nm ev-
ery 5 min for 1 h. Each plate included triplicate control and blank
wells: the control wells contained cells treated with DCFH-DA
C: Food Chemistry
and oxidant; the blank wells contained cells treated with dye and
HBSS. Results are expressed in μmol of quercetin equivalents per
100 gram of dry weight (μmol QE/100 g DW).
Statistical analysis
Each value was demonstrated as mean ± SD. Data were ana-
lyzed using an analysis of variance (ANOVA), and different letters
indicated that the values were statistically different from each other
(P < 0.05). Correlations between total phenolic contents, TFCs
and antioxidant capacity were assessed using Pearson’s correlation
analysis, and statistical significance was expressed as ∗ P < 0.05 and
∗∗
P < 0.01. In the CAA assay, the median effective dose (EC50 )
was determined for the flowers from the median effect plot of log
(fa /fu ) compared with log (dose), where fa is the fraction affected
(CAA unit) and fu is the fraction unaffected (1 – CAA unit) by the
treatment. Antioxidant index = antioxidant capacity/average an-
tioxidant capacity (Formula 1), average antioxidant index (AAI) =
(DPPH index + ABTS index + ORAC index + FRAP index)/4
(Formula 2).
Results and Discussion
TPC and TFC
Chlorogenic acid was used as the external standard for quan-
tification of TPC for it widely existed in 10 edible flowers. The
TPC values, expressed as mg CAE/g DW, are shown in Figure 1A.
Significant differences (P < 0.01) were noted between soluble free
and insoluble bound phenolics tested. The TPC of different frac-
tions of the 10 edible flowers ranged from 0.31 mg CAE/g DW
to 235.5 mg CAE/g DW. P. suffruticosa and R. chinensis had the
highest TPC, at 235.5 mg CAE/g DW and 205.5 mg CAE/g DW,
respectively, followed by F. rosae rugosae (116.6 mg CAE/g DW)
and F. lonicerae (76.1 mg CAE/g DW). C. morifolium had the lowest
TPC at 16.29 mg CAE/g DW. Previous study (Li and others 2009)
reported that P. suffruticosa had 38.5 to 114.5 milligrams of gallic
acid equivalents per gram of dry weight (mg GAE/g DW), which
was much lower than we have found. Despite of the different stan-
dards, we used 80% acetone as extraction solvents while they used
70% methanol. As reported by Liu and others (2009), the acetone
extract of lychee flowers had a higher antioxidant compounds in-
cluding polyphenols, flavonoids, and condensed tannins than the
methanolic extract and the water extract.
The ISBE and ISBW phenolics were fewer than soluble free
form. F. rosae rugosae (26.64 mg CAE/g DW) and R. chinensis
(19.68 mg CAE/g DW) had the highest ISBE TPC, followed by L.
pedunculata (17.22 mg CAE/g DW). C. morifolium exhibited high
ISBE TPC (14.20 mg CAE/g DW) compared to its low soluble
free TPC (16.29 mg CAE/g DW), whereas F. lonicerae (0.31 mg
CAE/g DW) had the lowest ISBE form TPC despite its high
soluble free TPC. F. rosae rugosae had the highest ISBW TPC at
32.69 mg CAE/g DW, whereas F. lonicerae had the lowest. Kaisoon
and others (2011) studied common edible flowers in Thailand and
found that the some flowers extracts had higher bound phenolic
contents than their soluble free forms, ranging from 37 to 89 mg
GAE/g dry extraction and from 56.04 to 148.73 mg GAE/g dry
extraction.
Vol. 79, Nr. 4, 2014 r Journal of Food Science C519
 Edible flowers phenolics and antioxidant . . .
The edible flowers exhibited significant differences in TFC. The
TFCs, expressed as mg RE/g DW, are shown in Figure 1B. The
soluble free TFCs varied from 7.67 to 89.38 mg RE/g DW, with
the highest in F. lonicerae, which was consistent with the findings of
Liu and others (2008), which indicated that F. lonicerae had a TFC
of 74.93 mg RE/g DW, followed by R. chinensis (39.54 mg RE/g
DW), and the lowest in F. carthami (7.67 mg RE/g DW). The
C: Food Chemistry
ISBW and ISBE phenolics ranged from 0.42 to 2.32 mg RE/g
DW and from 0.42 to 2.39 mg RE/g DW, respectively. Results
implied that most phenolics in the 10 edible flowers existed in
nonflavonoid form.
Identification and quantification of phenolic compounds
The distribution of phenolic compounds in 3 forms is presented
in Table 1. A representative HPLC profile of P. persica polyphenol
extract and UV spectrogram of phenolic compounds detected are
presented in Figure 2. The highest concentrations of total phe-
nolic acids and flavonoids were in the soluble free fraction, and
lower in their insoluble bound fractions. However, despite of a
considerable TPC, few phenolic acids or flavonoids we have de-
tected were in the ISBW form (data not shown), and it may be
caused by some nonphenolic compounds that are soluble in water,
such as ascorbic acid, organic acids, which can also be reduced by
the Folin–Ciocalteu phenol reagent (Ndhlala and others 2010). F.
lonicerae and P. suffruticosa contained the most abundant SF acids
(16.63 and 12.18 mg/g DW, respectively), followed by R. chinensis
(11.92 mg/g DW), H. sabdariffa (8.44 mg/g DW), and P. persica
C520 Journal of Food Science r Vol. 79, Nr. 4, 2014
(7.36 mg/g DW). The most kinds of phenolic compounds were
detected in P. persica, which contained 10 of the 14 phenolic com-
pounds. Among phenolic acids, the most widely occurring was
chlorogenic acid, which was found in 9 of the 10 edible flowers
and was particularly concentrated in F. lonicerae(16.04 mg/g DW)
and P. persica (6.54 mg/g DW). Gallic acid was also widely present
at high concentrations in these flowers: it was detected in 7 of
the 10 flowers and reached 7.99 mg/g DW in P. suffruticosa. The
main SF acids also included caffeic acid and protocatechuic acid.
Gallic acid and chlorogenic acid have attracted considerable at-
tention for their antioxidant activity, neuroprotective effects, and
anticarcinogenic properties (Kwon and others 2010; Lu and oth-
ers 2010; Sato and others 2011). Among flavonoids, P. suffruticosa
exhibited the highest content at 25.77 mg/g DW. Although F.
lonicerae had a high TFC, it only contained 0.64 mg/g DW of
flavonoid compounds; thus, it may contain other flavonoid com-
pounds. The main flavonoid compounds detected were rutin and
quercetin, which existed in 7 and 6 of 10 edible flowers, respec-
tively, and were especially abundant in P. suffruticosa (18.12 and 7.24
mg/g DW, respectively). Kaisoon and others (2011) indicated that
quercetin was a potential active compound in the flowers. Rutin
and quercetin are effective in scavenging free radical (Pekal ˛ and
others 2011) and anti-inflammatory (Lee and others 2013). Api-
genin was only detected in F. rosae rugosae, at 0.65 mg/g DW.
Kaempferol content ranged from 0.19 to 2.78 mg/g DW in 3
edible flowers. For insoluble bound phenolic compounds, R. chi-
nensis exhibited the highest total insoluble bound phenolic acids
Figure 1–Total phenolic content (A) and total
flavonoids content (B) of soluble free phenolic
(SF), insoluble bound-ester phenolic (ISBE) and
insoluble bound-water phenolic (ISBW) of 10
edible flowers.
 Table 1–Phenolic compounds contents of 10 edible flowers.
Phenolic acids (μg/g DW) Flavonoids (μg/g DW) Total
phenolics
Phenolic forms Flowers GA PCCA ChA p-HO CFA VA SyA 3-H-4-MBT p-CA FA Rutin Quercetin Apigenin Kaempferol (μg/g DW)
Soluble free Paeonia suffruticosa 7986.0 ± ND 2226.8 ± ND ND ND 699.8 ± 9.4b ND ND 1270.9 ± 18119.5 ± 7237.6 ± ND 411.8 ± 37952.5 ±
phenolic 166.8a 26.8d 6.8a 174.03a 62.7a 10.1b 456.6a
Rosa chinensis 6873.4 ± ND 2661.1 ± 783.8 ± 96.5 ± 3.2b ND ND 1085.6 ± 423.1 ± 2.3a ND ND ND ND ND 11923.5 ±
2.2b 44.2cd 79.6b 13.4b 144.9c
Chrysanthemum ND 17.8 ± 2.1f 810.6 ± 1.0e 82.8 ± 0.2c ND ND ND ND ND ND ND ND ND ND 911.2 ± 3.3g
morifolium
d d d b b b
Prunus persica ND 116.1 ± 0.5 6539.0 ± ND 44.6 ± 0.4 181.6 ± 284.5 ± ND 1.0 ± 0.0 222.7 ± 4.8 704.4 ± 2.3 266.2 ± 0.8 ND 2775.2 ± 11135.4 ±
21.2b 13.3b 15.4c 1.9a 60.7c
c
Edible flowers phenolics and antioxidant . . .

Hibiscus sabdariffa ND 1380.7 ± 3108.1 ± 3162.8 ± 52.0 ± 0.5 ND 740.5 ± ND ND ND ND ND ND ND 8444.2 ±

3.2b 4.8c 97.0a 10.1b 115.5d
Flos carthami 347.8 ± ND ND 104.7 ± 5.1c 20.1 ± 0.1e 62.1 ± 2.8c ND ND 18.1 ± 0.5c 80.1 ± 5.9c 372.5 ± 133.3 ± ND ND 1138.8 ±
10.0d 36.2b 13.0b 73.4g
Lavandula 25.4 ± ND 191.7 ± ND ND ND ND 1827.6 ± ND ND 827.7 ± 222.2 ± 3.0b ND 193.1 ± 1.7c 3287.6 ±
b
pedunculata 2.0e 44.8e 46.8a 28.5 126.7f
ef b
Lilium brownii var. 127.0 ± 22.8 ± 2.1 307.6 ± 4.6e ND 267.9 ± ND ND ND 22.7 ± 0.2 ND 335.5 ± 1.4b ND ND ND 1083.4 ±
Viridulum 3.4e 10.0a 21.6g
e a b
Flos rosae rugosae 2122.4 ± 1608.2 ± 596.1 ± 9.4 ND ND 356.4 ± 1.4 1174.0 ± ND ND ND 389.2 ± 0.5 140.0 ± 651.83 ± ND 7038.1 ±
40.5c 14.8a 55.8a 0.19b 11.56 122.6e
d c d
Flos lonicerae 25.8 ± 253.9 ± 2.4c 16042.9 ± ND 35.2 ± 1.6 147.4 ± ND 230.1 ± 0.2 4.9 ± 0.4 ND 623.0 ± 16.8 ± 1.6c ND ND 17380.0 ±
5.1e 776.5a 24.4b 63.1b 875.4b
Insoluble bound Paeonia suffruticosa 663.1 ± ND ND 13.7 ± 1.1b ND ND ND ND 7.4 ± 0.2a ND ND ND 112.04.7a 24.11.6b 820.
phenolic 16.6b 324.26a
Rosa chinensis 738.0 ± ND ND ND ND ND ND ND ND ND ND ND ND ND 738.0 ±
23.4a 23.4b
a a
Chrysanthemum ND ND ND ND 291.4 ± 9.9 ND ND ND ND 54.8 ± 3.1 96.1 ± 2.8 35.3 ± 1.8 ND ND 477.7 ±
morifolium 13.0d
d b a c b b
Prunus persica 5.7 ± 0.0 5.4 ± 0.0 13.3 ± 1.0 ND 53.5 ± 2.1 ND ND ND ND 2.6 ± 0.0 ND ND 5.6 ± 0.2 7.8 ± 0.5c 93.9 ± 3.8f
Hibiscus sabdariffa 6.5 ± 0.0d 1.1 ± 0.0c ND ND 13.3 ± 0.7d ND ND ND ND ND ND ND 2.0 ± 0.0b ND 22.8 ± 0.7g
Flos carthami 10.0 ± 0.0d ND ND 2.2 ± 0.0c 0.7 ± 0.0d ND ND ND ND 5.3 ± 1.0b ND ND ND 11.3 ± 0.9c 29.4 ± 1.0g
Lavandula 15.0 ± 0.2d 31.0 ± 1.0a 12.3 ± 2.1a 7.5 ± 0.5c 5.3 ± 0.0d 7.8 ± 0.0 ND ND 7.9 ± 0.2b 3.9 ± 1.0b ND ND ND ND 90.6 ± 4.2f
pedunculata
Lilium brownii var. ND ND ND ND 2.0 ± 0.0d ND ND ND ND ND ND ND ND ND 2.0 ± 0.0g
Viridulum
c b a b a
Flos rosae rugosae 552.3 ± 9.0 4.6 ± 0.1 ND 47.6 ± 2.7 ND ND ND ND ND ND ND ND 0.7 ± 0.0 46.1 ± 1.4 651.2 ±
13.3c
c b b
Flos lonicerae ND 2.2 ± 0.2 5.3 ± 0.1 ND 109.4 ± 1.0 ND ND ND ND ND ND ND ND ND 116.9 ± 0.4e
Values are shown as the mean ± SD (n = 3).
Different superscript letters indicate that the values are statistically different from each other at the level P < 0.05 and values marked by the same letter within a column are not statistically different.
ND, not detected; GA, gallic acid; PCCA, protocatechuic acid; p-HO, p-hydroxybenzoic acid; ChA, chlorogenic acid; VA, vanilic acid; CFA, caffeic acid; SyA, syringic acid; p-CA, p-coumaric acid; FA, ferulic acid; 3-H-4-MBT,
3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone.

Vol. 79, Nr. 4, 2014 r Journal of Food Science C521

C: Food Chemistry
 Edible flowers phenolics and antioxidant . . .

(738.0 μg/g DW). P. suffruticosa and F. rosae rugosae were also rich Table 2–Relationship between total phenolic contents, total
in insoluble bound phenolic acids, at 684.2 and 604.5 μg/g DW, flavonoid contents, and antioxidant capacity.
respectively. The most abundant insoluble bound phenolic acids R DPPH ABTS ORAC FRAP
were gallic acid and caffeic acid, followed by p-hydroxybenzoic
SF
acid and ferulic acid. Caffeic acid was widely found in plants TPC 0.9978∗∗ 0.9961∗∗ 0.9061∗∗ 0.9562∗∗
because it was a key intermediate in lignin biosynthesis, one of TFC 0.3527 0.2623 0.2592 0.3548
the principal constituents of plant biomass. The insoluble bound ISBE
C: Food Chemistry

flavonoids were highest in P. suffruticosa (136.11 μg/g DW), but TPC 0.8768∗∗ 0.9042∗∗ 0.8837∗∗ 0.9748∗∗
TFC 0.5167 0.3477 0.6187 0.4784
we found no insoluble bound flavonoid compounds in R. chinensis ISBW
and F. lonicerae. Insoluble bound phenolic compounds were present TPC 0.8877∗∗ 0.8443∗∗ 0.8727∗∗ 0.9357∗∗
at lower concentrations than SF compounds, as shown in TPC and TFC 0.6353∗ 0.5649 0.9072∗∗ 0.7743∗∗
TFC values, which ranged from 0.56 to 12.18 mg/g DW. ∗
P < 0.05, ∗∗ P < 0.01.
SF, soluble free phenolic; ISBE, insoluble bound-ester phenolic; ISBW, insoluble
bound-water phenolic; TPC, total phenolic content; TFC, total flavonoid content.
Antioxidant activity
Radical scavenging activity (DPPH, ABTS, and
ORAC). The DPPH assay is a preliminary test for determin- various samples. The DPPH radical-scavenging activities (μmol
ing the antioxidant potential of edible flowers. This assay has TE/g DW) of the soluble free and insoluble bound phenolics of
been widely used to test the free radical scavenging ability of 10 edible flowers are presented in Figure 3A. Most of the DPPH

0.60
A 2 10

0.50

0.40

0.30
AU

0.20
9
0.10 1 3 4 5
6
7 8

0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
Minutes

259.6 1.20 326.3

0.14 0.050

B 0.12
226.5 1
1.00
2
0.040
3

0.10 0.80 0.030

0.08 294.1 240.7

0.020
AU

0.60
AU
AU

288.2
0.06 0.010 322.7
0.40
0.04 388.5
0.000
0.20
0.02
-0.010
368.0 0.00
0.00
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm nm nm
260.8 0.080 212.4
310.8
0.14 0.030 6
4 0.070 5
0.12
0.060 0.020 309.6
227.7
0.10 0.050
0.010
0.08 291.7 0.040 228.9
AU

387.3
AU

AU

0.030 0.000
0.06
0.020 -0.010
0.04
0.010
0.02 390.9 -0.020
0.000
339.5 376.4
0.00 -0.010
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm nm nm
1.176 Peak 1
2.40 204.4
224.2 254.9 365.6
0.060 0.050 254.9 0.40 10
2.20
7 8 9
0.050 2.00
322.7 355.1 0.35
0.040 1.80 362.1
0.040
226.5 1.60 265.9
0.30
0.030
0.030 1.40
AU
AU

AU

AU

0.020 0.25 1.20

0.010 1.00
0.020 0.20
392.1 0.80
0.000
0.60
-0.010 0.010 0.15
0.40
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm nm
nm nm

Figure 2–HPLC profile of Prunus persica (A) at 280 nm, and phenolic compounds detected are numbered 1 to 10, UV spectrograms of which are
presented below (B): protocatechuic acid (1), chlorogenic acid (2), caffeic acid (3), vanilic acid (4), syringic acid (5), p-coumaric acid (6), ferulic acid (7),
rutin (8), quercetin (9), kaempferol (10), correspondingly.

C522 Journal of Food Science r Vol. 79, Nr. 4, 2014

Edible flowers phenolics and antioxidant . . .

Table 3–Percentage distribution profile of antioxidants in 10 ed- ABTS radical-scavenging activities (μmol TE/g DW) of the sol-
ible flowers. uble free and insoluble bound phenolics from 10 edible flowers
Flowers AAI SF% ISBE% ISBW% are presented in Figure 3B. As in the DPPH assay, P. suffruti-
cosa scavenged the most ABTS radicals (2065 μmol TE/g DW),
Paeonia suffruticosa 2.589 ± 0.048a 96.10 2.03 1.87
Rosa chinensis 2.282 ± 0.011b 96.44 1.62 1.94
followed by R. chinensis at 1954 μmol TE/g DW. The ABTS
Flos rosae rugosae 1.313 ± 0.003c 91.12 4.71 4.17 radical-scavenging activity of insoluble bound phenolics ranged
Flos lonicerae 1.092 ± 0.058d 96.70 1.47 1.83 from 2.13 to 43.67 μmol TE/g DW.

C: Food Chemistry
Lavandula pedunculata 0.759 ± 0.030e 80.10 8.96 10.94 The ORAC assay is based on the scavenging of peroxyl radicals
Prunus persica 0.497 ± 0.013f 87.80 5.08 7.12 generated by AAPH, which prevent the degradation of the fluo-
Lilium brownii var.viridulum 0.449 ± 0.009g 92.20 2.23 5.57
rescein probe and, consequently, prevent the loss of fluorescence
Hibiscus sabdariffa 0.410 ± 0.003gh 96.31 1.50 2.19
Chrysanthemum morifolium 0.369 ± 0.022h 82.66 14.23 3.10 of the probe. The oxygen radical absorbance capacities (μmol
Flos carthami 0.239 ± 0.005i 95.06 1.35 3.58 TE/g DW) of the soluble free and insoluble bound phenolics from
10 edible flowers are presented in Figure 3C. In accordance with
Values are shown as the mean ± SD (n = 3).
Different superscript letters indicate that the values are statistically different from each the previous 2 assays, SF of P. suffruticosa showed remarkable oxy-
other at the level P < 0.05 and values marked by the same letter within a column are not gen radical absorbance capacity (990 μmol TE/g DW), and SFs of
statistically different.
AAI, average antioxidant index; SF, soluble free phenolic; ISBE, insoluble bound-ester C. morifolium was the least effective in deactivating oxygen radicals
phenolic; ISBW, insoluble bound-water phenolic.
(225 μmol TE/g DW). The ORAC of insoluble bound phenolics
ranged from 2.12 (L. brownii var. viridulum ISBE) to 56.88 μmol
radical-scavenging activity was contributed by the SFs of the flow- TE/g DW (L. pedunculata ISBW).
ers because they contained most of the bioactive compounds. The Ferric reducing/antioxidant power (FRAP). The FRAP
SFs of P. suffruticosa had the strongest DPPH radical-scavenging ac- assay measures the reducing potential of an antioxidant, which
tivity (1028 μmol TE/g DW), followed by R. chinensis (854 μmol reacts with a ferric–tripyridyltriazine (Fe3+ –TPTZ) complex
TE/g DW). and produces a colored ferrous tripyridyltriazine (Fe2+ –TPTZ).
The ABTS+ radical, formed from ABTS−e reacts quickly with The FRAPs of the soluble free and insoluble bound phenolics
the electron/hydrogen donors to form a colorless substance. The from 10 edible flowers are presented in Figure 3D. The SFs of

Figure 3–DPPH radical-scavenging activity (A), ABTS radical-scavenging activity (B), oxygen radical absorption capacity (C), and ferric reduc-
ing/antioxidant power (D) of soluble free phenolic (SF), insoluble bound-ester phenolic (ISBE), and insoluble bound-water phenolic (ISBW) of 10
edible flowers.

Vol. 79, Nr. 4, 2014 r Journal of Food Science C523

 Edible flowers phenolics and antioxidant . . .

Table 4–Cellular antioxidant activity (CAA) of 10 edible flowers.

No PBS wash PBS wash

Cytotoxicity
EC50 CAA (μmol EC50 CAA (μmol CC50
Flowers (mg DW/mL) QE/100 g DW) (mg DW/mL) QE/100 g DW) (mg DW/mL)
Paeonia suffruticosa 5.3 ± 0.5a 153.0 ± 17.0a 22.7 ± 2.4a 26.7 ± 3.0a 20
Rosa chinensis 5.7 ± 0.5a 134.0 ± 12.0a 30.5 ± 2.6b 19.1 ± 1.9b 20
C: Food Chemistry

Flos Rosae Rugosae 9.5 ± 1.4b 85.1 ± 12.5b 38.5 ± 5.8c 16.6 ± 2.4c 20
Flos Lonicerae 15.8 ± 1.9c 51.7 ± 5.9c 63.9 ± 9.3d 10.1 ± 1.2d 20
Prunus persica 19.5 ± 3.1cd 41.5 ± 6.2cd 101 ± 13e 6.4 ± 1.0e 20
Flos Carthami 24.5 ± 3.4d 32.9 ± 4.6d 107 ± 14.8e 5.9 ± 0.8e 20
Lilium brownii var. viridulum 26.3 ± 3.9d 30.9 ± 4.7d 96.2 ± 14.3e 6.6 ± 1.0 e 20
Hibiscus sabdariffa 28.8 ± 2.7de 28.6 ± 2.8d 117.0 ± 10.4ef 5.5 ± 0.5e 20
Lavandula pedunculata 30.7 ± 3.9e 26.4 ± 3.4d 125.0 ± 15.9f 5.3 ± 0.7e 20
Chrysanthemum morifolium 65.6 ± 6.6f 12.5 ± 1.3e 214.0 ± 22.6g 3.2 ± 0.3f 20
Values are shown as the mean ± SD (n = 3).
Different superscript letters indicate that the values are statistically different from each other at the level P < 0.05 and values marked by the same letter within a column are not
statistically different.

R. chinensis showed the strongest ferric ion–reducing activity others 2012). The significance of bound phytochemicals in flow-
(2645 μmol Fe2+ /g DW), followed by P. suffruticosa (1960 μmol ers to human health is unclear. However, different flowers with
Fe2+ /g DW), F. rosae rugosae (1454 μmol Fe2+ /g DW). By con- different amounts of bound phytochemicals can be digested and
trast, ISBW phenolics of F. rosae rugosae had the highest FRAP absorbed at different sites of the gastrointestinal tract to provide
(57.04 μmol Fe2+ /g DW). their unique health benefits. Bound phytochemicals, mainly in the
form of β-glycosides, cannot be digested by human enzymes and
Average antioxidant index (AAI) reach the colon to provide site specific health benefits (Nayaka
The antioxidant capacities from 4 different methods were incon- and others 2010). Therefore, aside from using flowers to make
sistent. Thus, we determined the correlations of the antioxidant tea, people should incorporate them in their diet so that insoluble
capacities from different methods with the corresponding TPCs bound phenolics would be ingested.
and TFCs (Table 2). TPC was strongly correlated with antioxidant
capacity, with R values ranging from 0.8443 to 0.9978 (P < 0.01). CAA
By contrast, TFC was poorly correlated with antioxidant capacity, The distribution profile of antioxidant capacity showed that SFs
which could be due to that the antioxidant capacity observed was in flowers contributed most to their antioxidant activity. Thus, we
not only from the flavonoid content, but could be affected by investigated further the SFs using CAA assay. The CAA assay is a
other nonflavonoid phenolics such as phenolic acids. ISBW TFC more biologically relevant method than the popular chemical an-
exhibited a high correlation with DPPH (R = 0.6353, P < 0.05), tioxidant activity assays in vitro because it accounts for factors such
ORAC (R = 0.9072, P < 0.01) and FRAP (R = 0.7743, P < as uptake, metabolism, and localization of antioxidant compounds
0.01). Among the 4 assays, DPPH and ABTS are based on within cells. The EC50 and CAAs of the flowers, along with their
reactions with stable free radicals, whereas ORAC is based on median cytotoxic doses (CC50 ), are listed in Table 4. The cellular
competition, expressed as Trolox equivalents. FRAP is based on antioxidant activities were measured using 2 protocols: the PBS
the reduction of metal ions and is expressed in Fe2+ equivalents. wash protocol and the no PBS wash protocol. Both protocols were
These values represent relative capacities in 1 aspect, however, the used because the difference between them provides some insight
value rank of 10 edible flowers varied in 4 methods; in order to into how the antioxidants interact with the cells. The EC50 values
evaluated the rank of total antioxidant capacity, we calculated the of all flowers were significantly lower and their cellular antioxi-
AAI (Formula 1, 2) according to Morales and others (2009) with dant activities were higher in the no PBS wash protocol than in
minor modifications, which had weighed the 4 results together. the PBS wash protocol. The results implied that the edible flower
The AAI is just used to compare the antioxidant capacity of the extracts were partly taken up by cells or closely associated with
10 edible flowers but limited to indicate the absolute antioxi- the cell membrane. The CAA values of the flowers in the no
dant capacity. Furthermore, we investigated the antioxidant ca- PBS protocol ranged from 12.5 μmol QE/100 g DW (C. mori-
pacity distribution profiles of the soluble free and insoluble bound folium) to 153 μmol QE/100 g DW (P. suffruticosa). The CAA
phenolics in the 10 edible flowers based on DPPH, ABTS, ORAC, values in the PBS wash protocol ranged from 3.2 μmol QE/100 g
and FRAP values. AAI and the distribution profiles of antioxidant DW (C. morifolium) to 26.7 μmol QE/100 g DW (P. suffruticosa).
capacity of the 10 edible flowers are summarized in Table 3. P. suf- P. suffruticosa had the highest CAA value in both protocols, fol-
fruticosa and R. chinensis had the highest AAI, followed by F. rosae lowed by R. chinensis, F. rosae rugosae, which also exhibited strong
rugosae, F. lonicerae, L. pedunculata, P. persica, H. sabdariffa, L. brownii in vitro antioxidant capacity. In addition, L. brownii var. viridulum,
var. viridulum, C. morifolium, and F. carthami. Most of flowers exhib- H. sabdariffa, L. pedunculata, and C. morifolium had relatively lower
ited significant differences (P < 0.05). The antioxidant capacity of CAAs. The results are almost consistent with the results of the in
the flowers was mainly from the SFs in flowers (80.1% to 96.7%), vitro antioxidant capacity assessment assays.
1.4% to 14.2% of the antioxidant capacity was from ISBE pheno-
lics, and 1.8% to 10.9% was from ISBW phenolics. It may be due Conclusions
to the following reasons: the SFs content was much higher than This study indicates that the 10 edible flowers in China are
other fractions. In addition, the synergistic effects among some rich sources of phytochemicals, with high levels of pheno-
phenolics may help elevating the antioxidant capacity (Jaiswal and lic compounds and antioxidant capacity. There were significant

C524 Journal of Food Science r Vol. 79, Nr. 4, 2014

Edible flowers phenolics and antioxidant . . .
differences in different flowers in phenolic compounds and an-
tioxidant capacity. P. suffruticosa was the richest in phenolics and
highest in antioxidant capacity among 10 edible flowers. Gallic
acid, chlorogenic acid, and rutin were the most abundant among
the phenolic compounds in 10 edible flowers. TPC was strongly
correlated with antioxidant capacity, which indicated that pheno-
lics were the major contributors to the antioxidant activity of the
selected edible flowers. This study not only provides useful infor-
mation about the health benefit of edible flowers for consumers,
but also encourages researchers to utilize edible flowers as sources
of phytochemicals. In addition, the flower extracts are potential to
be used as addictive in food to prevent the chronic disease, help the
health promotion, and prevent the food oxidization. However, the
antioxidant mechanisms, the antitumor, anti-inflammation, and
anti-aging activity of the edible flower extracts should be further
studied to develop more applications as natural antioxidants.
Acknowledgments
This work was supported by Foundation of Fuli Inst. of Food
Science, Zhejiang Univ., and the Natl. Natural Science Founda-
tion of China (Nr. 31271847) for financial support.
Author Contributions
B. Lu guided the study designing. L. Xiong participated study
designing, executed the whole study, and interpreted the results.
J. Yang helped the part of total phenolic content. Y. Jiang helped
the material treatment. Y. Hu helped the RP-HPLC analysis of
phenolic compounds. F. Zhou, S. Mao, and C. Shen collected test
data.
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