1150 Eur. J. Lipid Sci. Technol.

2009, 111, 1150–1160

Research Paper
Phenolic compounds and antioxidant activity of extracts of
Ginkgo leaves

Joanna Kobus1, Ewa Flaczyk1, Aleksander Siger1, Małgorzata Nogala-Kałucka1, Józef Korczak1
and Ronald B. Pegg2

1
Faculty of Food Science and Nutrition, Poznań University of Life Sciences, Poznań, Poland
2
Department of Food Science and Technology, The University of Georgia, Athens, USA

The aim of the present study was to determine the flavonol contents of crude extracts from Ginkgo
(Ginkgo biloba L.) leaves, their antioxidant properties in model system studies, and their inhibitory action
of linoleic acid oxidation in an emulsion system and of triacylglycerols (TAG) in rapeseed oil. Extracts
were prepared from both green and yellow leaves of Ginkgo trees using water, aqueous acetone, and
ethanol. Extracts were analyzed by HPLC for their presence and content of selected flavonols; these
included myricetin, quercetin, kaempferol, isorhamnetin, and morin. The highest level of flavonols, espe-
cially quercetin, myricetin and kaempferol, were found in the aqueous acetonic extracts. Crude extracts
from yellow Ginkgo leaves contained greater amounts of flavonols. The best DPPH radical-scavenging
activity amongst the Ginkgo extracts examined was determined for aqueous acetonic extracts, while the
lowest was noted for the ethanolic extract of green leaves. Water infusion extracts exhibited the highest
iron(II) chelating activity. The reducing power of extracts from yellow leaves was 2 higher than that of
crude extracts from green leaves. Nevertheless, extracts from green Ginkgo leaves imparted a greater
protection factor against TAG oxidation, as assessed by the Rancimat method. Crude extracts from yellow
Ginkgo leaves were more efficacious than green ones at inhibiting oxidation of the linoleic acid emulsion.

Keywords: Antioxidant activity / Flavonols / Ginkgo biloba L. / Linoleic acid emulsion / Triacylglycerols
Received: December 23, 2008; accepted: April 15, 2009
DOI 10.1002/ejlt.200800299

1 Introduction The main classes of compounds responsible for the pur-

Currently, Ginkgo (Ginkgo biloba L.) is the only one living
species belonging to the phylum Gymnosperms (Gymno-
spermae) and class Ginkgopsida. Extracts prepared from green
Ginkgo leaves are considered attractive for their documented
multifaceted action in biological systems [1, 2]. The first
analyses on the chemical composition of the anatomical parts
of Ginkgo were conducted by Peschier in 1818 and later by
Schwarzenbach in 1857. Further investigations on this subject
continued in the next century by Kawamura and findings were
reported in print by 1928. Ten years later, the pioneering
studies on Ginkgo by Furkawa were published [3, 4].
Correspondence: Joanna Kobus, Faculty of Food Science and Nutrition,
Poznań University of Life Sciences, Poland Wojska Polskiego 31, 60-624
Poznań, Poland.
E-mail: joannak@up.poznan.pl
Fax: 148 61 8487431
ported health benefits afforded to man by Ginkgo prepara-
tions are the polyphenols. Polyphenols are ubiquitous
throughout the plant kingdom and include flavones, flavonol
glycosides, acylated flavonol glycosides, biflavonoids, flavan-
3-ols, and proanthocyanidins [1, 5, 6]. Of late, flavonoids have
received considerable attention in the literature, particularly
due to their widely recognized broad-spectrum free-radical-
scavenging effects and for being readily incorporated in redox
reactions [7, 8]. This pertains to all compounds in which
functional hydroxyl groups are located in the ortho- and para-
positions on aromatic rings [8, 9]. The transfer of hydrogen
atoms and electrons results in their oxidation; semi-quinones
and quinones are formed as a consequence of the reaction and
mediate in the oxidation of other compounds that do not react
directly with molecular oxygen. Thus, oxidation of poly-
phenols is a highly complex process and the rate of the reac-
tion is closely related to the type and location of substituents
on the molecule [8, 10]. Flavonoids exhibit the capacity of
inactivating the superoxide anion, peroxyl and hydroxyl radi-

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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160
cals, as well as oxygen in statu nascendi. Moreover, poly-
phenols are capable of forming complexes with metal ions
such as Fe(II) that can catalyze oxidation, and they can also
inhibit the activity of oxidizing enzymes like lipoxygenase
[11].
Flavonoids constitute the largest groups of bioactive sub-
stances isolated from Ginkgo leaves [2]. A total of 33 flavo-
noids were identified in Ginkgo leaves, and most of them were
in the form of mono-, di-, and triglycosides. It was determined
that flavonol aglycones are contained in relatively significant
quantities in extracts prepared from Ginkgo leaves [6]. For
example, the presence of flavonoids determined in Egb 761
extracts (see ref. [4] for more details) is reported to be any-
where from 22 to 27%, while in the leaves themselves their
content is only from 0.2 to 0.4 wt-% [4].
Flavonoids seem to play an important role in foods con-
taining lipids sensitive to oxidation and are therefore noted for
their marked antioxidant activity; this latter point has been
emphasized in numerous works. Many different herbs and
products therefrom have been investigated for their possible
protective effect/action toward processed foodstuffs which are
sensitive to lipid oxidation [12, 13]. Indigenous antioxidative
compounds (e.g. phenolics) in the raw materials act by differ-
ent mechanisms, but their cumulative antioxidant efficacy is
difficult to predict and sometimes to estimate, because the
total antioxidant capacity is affected not only by the properties
of the individual compounds, but also by their interactions or
lack thereof [8, 9].
The aim of the present study is to investigate the content of
flavonol aglycones and the mechanism(s) of the antioxidant
efficacy afforded by crude extracts from yellow and green
Ginkgo leaves, by determining the reducing power, chelating
activity, and antiradical potential, together with antioxidant
properties in an anhydrous fat substrate, as well as a linoleic
acid emulsion.
2 Materials and methods
2.1 Materials
Ginkgo (Ginkgo biloba L.) extracts were prepared from green
(harvested in August 2007 and marked with a ‘G’) and yellow
leaves (harvested in October 2007 and marked with a ‘Y’)
including leaf stalks coming from a plantation in Baranowo
belonging to the Poznań University of Life Sciences. Leaves
were first dried in an incubation oven at 40 7C until a moisture
content of ,8% was reached and then ground in a laboratory
mill (ZBPP, type WŻ-1; Bydgoszcz, Poland). The degree of
comminution of plant material was determined by sieving
using a mesh size of 0.8–0.03 mm.
Low-erucic acid rapeseed oil (produced in Kruszwica,
Poland) was purchased from a local supermarket in Poznań.
All solvents were of analytical grade unless otherwise
specified. Flavanol standards included isorhamnetin (3’-
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Antioxidant activity and phenolic components of Ginkgo 1151
methoxy-3,4’,5,7-tetrahydroxyflavone), quercetin
(3,3’,4’,5,7-pentahydroxyflavone), kaempferol (3,4’,5,7-tet-
rahydroxyflavone), myricetin (3,3’,4’,5,5’,7-hexahydroxy-
flavone), and morin (2’,3,4’,5,7-pentahydroxyflavone). All
were of HPLC-grade purity and purchased from Sigma-
Aldrich Chemical (St. Louis, MO, USA), except for iso-
rhamnetin which was acquired from Fluka (Buchs, Switzer-
land). Fatty acid methyl ester (FAME) standards for reten-
tion time mapping during fatty acid profiling were obtained
from Supelco (Bellefonte, PA, USA). 2,2-Diphenyl-1-
picrylhydrazyl. (DPPH.), 6-hydroxy-2,5,7,8-tetramethyl-
chroman-2-carboxylic acid (Trolox), Tween-40, and b-car-
otene were purchased from Sigma-Aldrich. Butylated hydro-
xytoluene (BHT) was acquired from Merck (Darmstadt,
Germany).
2.1.1 Preparation of extracts
Of ground green (G) or yellow (Y) Ginkgo leaves, 20 g was
extracted with 1 L solvent at atmospheric pressure under the
following conditions: deionized water at 95 7C during a 15-
min infusion (GW, YW); acetone/water (3 : 2, vol/vol) at
40 7C during a 90-min extraction (GA, YA), or ethanol (96%,
vol/vol) at 18 7C during a 16-h maceration (GE, YE) [14].
Each solution was cooled to room temperature and filtered by
gravity through Whatman No. 1 filter paper. Organic solvents
were evaporated under vacuum at ,40 7C, and residual water
(i.e. in the aqueous acetonic extract and the water infusion
extract) was removed by lyophilization. The crude extracts so
prepared were stored in a dry, dark, and cool place until ana-
lyzed.
2.1.2 Linoleic acid emulsion model system
A 10 mM linoleic acid emulsion was freshly prepared before
each assay using 0.1 mM phosphate buffer (pH 7.2) with
0.3% (wt/vol) Tween-40 according to Flaczyk et al. [15].
2.1.3 Preparation of stripped triacylglycerols
Triacylglycerols (TAG) were collected from rapeseed oil by
column chromatography according to Chimi et al. [16].
Rapeseed oil was purified in that endogenous antioxidants
were stripped from the oil before conducting the planned
experiments. Oil was dissolved in hexanes (1 : 3, vol/vol) and
run through a glass column packed with activated carbon,
aluminum oxide (activated at 300 7C), and anhydrous sodium
sulfate. During the refining process, the column and receiver
system were shielded from extraneous light using aluminum
foil. Next, the recovered hexanes were removed under
vacuum at ,40 7C, after which the TAG free of endogenous
antioxidants were sealed in an amber vial under a nitrogen
headspace.
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1152 J. Kobus et al.
2.2 Methods
2.2.1 Fatty acid analysis
After derivatizing the TAG with 6% (vol/vol) sulfuric acid in
anhydrous methanol, FAME were separated from one another
using a Hewlett Packard 5890 GC gas chromatograph (Agi-
lent, Wilmington, DE, USA) equipped with a Supelcowax-10
fused-silica capillary column (30 m length625 mm i.d.,
0.25 mm film thickness; Supelco) and detected by FID. The
oven temperature was initially set at 60 7C and then increased
to 210 7C at 12 7C/min. The injector temperature was set at
240 7C, split ratio at 1 : 25, and detector temperature at
260 7C. The carrier gas was ultra-high-purity helium at a flow
rate of 1 mL/min. Ultra-high-purity hydrogen and air were the
fuel gases for the FID, with nitrogen being used as make-up
gas. Separated FAME were identified by comparing their
retention times to those from commercially available stand-
ards [17].
2.2.2 Total phenolic content
The contents of total phenolics (TPC) in crude extracts of
green and yellow Ginkgo leaves were determined by visible
spectrophotometry based on a colorimetric oxidation/reduc-
tion reaction according to Cheung et al. [18]. Quercetin was the
standard employed in this work. The dry crude extracts were
dissolved in CH3OH/H2O (1 : 1, vol/vol) and then 1 mL of this
solution was added to a series of tubes. Next 1 mL of Folin-
Ciocalteu’s phenol reagent was pipetted in and the contents
were vortexed for ,60 s. Following the addition of 1 mL of a
35% (wt/vol) NaHCO3 solution, the mixture was allowed to
stand for 90 min in darkness to allow maximum color devel-
opment. Just before measuring absorbances, the samples were
centrifuged at 30006g (Sigma 204 laboratory centrifuge; St.
Louis, MO, USA). The absorbance of the supernatant was
measured at l = 725 nm using a SPECORD® 40 (Analytik
Jena AG, Germany). Based on the construction of a standard
curve, TPC data for the preparations from Ginkgo leaves were
expressed as mg quercetin equivalents/g dry extract.
2.2.3 Extraction of flavonols
Of crude extract, 1 g was dissolved in 10 mL 96% (vol/vol)
ethanol, and 2 mg ascorbic acid, as antioxidant, was added.
Samples were hydrolyzed in 2 mL 2 M HCl at room temper-
ature for 60 min. Then, 2 mL 2 M NaOH was added. The
contents were transferred to a 25-mL volumetric flask and
filled to the mark with ethanol. A Chromabond System
(Macherey-Nagel, Germany) with an SPE column filled with
diol (Discovery® DSC-18 SPE tube, 500 mg, 50 mm, 70 Å
pore diameter; Supelco) was employed to recover the flavonol
fraction. The extraction process comprised three stages: First,
the column was conditioned with 5 mL CH3OH; second,
5 mL sample was loaded onto the column; and third, flavonols
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160
were eluted with CH3OH and collected in a 10-mL volumetric
flask.
2.2.4 Composition of flavonols
Separation and identification of extracted flavonols were car-
ried out by high-performance liquid chromatography using a
Nova-Pak C18 reversed-phase column (3.9 H 150 mm, 5 mm
particle size; both Waters, Milford, MA, USA). Solvent A was
0.3% (vol/vol) HCOOH in H2O, whereas solvent B was 100%
CH3CN. The flow rate was maintained at 1 mL/min. The
gradient profile was as follows: 85% A at 0 min and 25% A at
40 min. Chromatograms were recorded with a UV-Vis detec-
tor at l = 370 nm. The identification of separated compounds
was carried out by retention time mapping with a set of
standards. The quantity of each flavonol was determined
using external and internal standards of individual flavonols.
2.2.5 Chelating activity
The chelating activities of crude extracts from green and yel-
low Ginkgo leaves were measured according to Tang et al.
[19]. The colorimetric assay involves determining the quantity
of Fe21 which did not chelate with the crude Ginkgo extract
and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-tria-
zine monosodium salt (i.e. ferrozine). To a test tube, 1 mL
sample, 0.1 mL 2 mM FeCl2, and 0.2 mL ferrozine reagent
were added. The mixture was vortexed for ,60 s and left to
stand at room temperature for 20 min. Thereafter, absorb-
ance readings were taken at l = 562 nm using the SPE-
CORD® 40 (Analytik Jena). Deionized water was used instead
of an aliquot of the extract as a control and ferrozine was used
as a reference. The chelating activity was calculated as follows:
Chelating activity = 1 – [(Abs of sample – Abs of refer-
ence)/Abs of control)]6 100
2.2.6 Reducing power
The reducing power of the crude extracts was determined
according to Amarowicz et al. [20]. Briefly, an aliquot of dis-
solved extract in CH3OH was mixed with 2.5 mL 0.2 M
phosphate buffer (pH 6.6) and 2.5 mL 1% (wt/vol)
K3Fe(CN)6. The mixture was incubated at 50 7C for 20 min.
Following incubation, 2.5 mL 10% (wt/vol) trichloroacetic
acid was added and then the mixture was centrifuged for
10 min at 20006g. The 2.5-mL volume of the upper layer
aliquot was combined with 2.5 mL deionized water and
0.5 mL of a 0.1% (wt/vol) FeCl3 reagent. The absorbance of
the resultant chromophore was measured at l = 700 nm with
the SPECORD® 40 (Analytik Jena).
2.2.7 Antiradical activity with DPPH.
The free-radical-scavenging potentials of crude extracts were
tested in a methanolic solution of DPPH. as described by
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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160
Amarowicz et al. [21]. The extent of discoloration of the so-
lution indicates the scavenging efficacy of the added sub-
stance. A 1-mL aliquot of extract solution was combined with
2 mL CH3OH and then 0.25 mL of a 1 mM ethanolic solu-
tion of DPPH.. The mixture was vortexed for ,60 s and then
left to stand at room temperature for 20 min. Absorbance was
measured at l = 517 nm (SPECORD® 40, Analytik Jena). A
reference sample was prepared with methanol instead of
DPPH. and the control instead of the extract sample. To con-
struct a calibration curve, absorbances were measured simul-
taneously of samples containing respective concentrations of
the standard, i.e. Trolox (0.5, 1.0, 1.5, and 2.0 mg/mL).
Results were expressed as mg Trolox equivalents/g extract.
Antioxidant activity was calculated as a percentage of DPPH.
decoloration using the following equation:
Antiradical activity = 100 – [(Abs of sample – Abs of
reference)/Abs of control)]6 100
2.2.8 â-Carotene-linoleate model system
The antioxidant activity of crude extracts of Ginkgo leaves
was determined according to the b-carotene bleaching method
of Liu et al. [22] with some modifications. Briefly, a stock so-
lution of the b-carotene-linoleic acid emulsion was prepared
as follows: 25 mg b-carotene was dissolved in ,50 mL
CHCl3. A 1-mL aliquot was transferred to a test tube and the
chloroform was removed using a vacuum evaporator. Next,
0.5 mL of the extract solution (100 ppm) and 4.5 mL of the
prepared linoleic acid emulsion were added. The sealed test
tubes were heated in a water bath at 50 7C and spectro-
photometric measurements (l = 470 nm) of the emulsion
were taken at 15-min intervals with the SPECORD® 40 for a
total of 90 min.
2.2.9 Rancimat test
Test samples, placed in the Rancimat (Methrom, Switzerland)
tubes, were exposed to a flow of air at an elevated temperature
and became oxidized (i.e. autoxidation). Oxygen was exposed
to a 2.5-g sample through hot oil at a rate of 20 L/h. During
thermal decomposition, volatile organic acids, notably
HCOOH, were produced. These organic acids were absorbed
in a measuring vessel filled with deionized H2O, and con-
tinuously monitored using a conductivity measuring cell. The
measuring curves so obtained were used to determine the
induction period. A protection factor (PF) was then calculated
as the ratio of the induction period of the samples with and
without the presence of an antioxidant.
2.2.10 Statistical analysis
All determinations were performed in at least three replica-
tions. Means and standard deviations were calculated with the
assistance of Microsoft Office Excel 2007 software. Pearson’s
correlation indexes were calculated with the use of STATIS-
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Antioxidant activity and phenolic components of Ginkgo 1153
TICA PL 7.0 StatSoft. The significance of differences be-
tween mean values was determined at p 0.05, after applying
a one-way analysis of variance (ANOVA) followed by Tukey’s
multiple range test.
3 Results and discussion
The FAME composition from the TAG is presented in
Table 1. The fatty acids found in the stripped rapeseed oil
were typical for such an edible oil and contained 19.32%
linoleic acid, based on the total fatty acid profile.
Table 1. Fatty acid composition of the TAG in rapeseed oil.
Fatty acid Content [%]
16:0 4.45 6 0.01
16:1n-7 0.22 6 0.01
18:0 1.70 6 0.00
18:1n-9 60.69 6 0.02
18:2n-6 19.32 6 0.01
18:3n-3 11.51 6 0.00
20:0 0.58 6 0.00
22:0 0.33 6 0.00
22:1n-9 1.20 6 0.00
Results are mean values of three determinations 6 standard devia-
tion.
In terms of antioxidant activity of the crude extract pre-
parations, the content of flavonol aglycones was found to be
the most important. For this study, the glycosidic forms of
isorhamnetin, quercetin, kaempferol, myricetin, and morin in
the extracts were first hydrolyzed before analysis. Qualita-
tively, the composition of flavonols in the aqueous acetonic,
ethanolic, and water infusion extracts was similar; however,
marked quantitative differences existed (Table 2). A signifi-
cantly (p ,0.05) higher content of flavonol aglycones was
found in extracts from yellow Ginkgo leaves. The highest
levels for these compounds were recorded in the aqueous
acetonic extract from yellow leaves (YA, 4938 mg/g d.m.) and
water infusion extract from yellow leaves (YW, 3146 mg/g
d.m.), while the lowest were noted in the water infusion
extract from green leaves (GW, 878 mg/g d.m.) and the etha-
nolic extract from green leaves (GE, 512 mg/g d.m.). Regard-
ing extract preparations from yellow Ginkgo leaves, the
dominant flavonol was myricetin accounting for 1683, 1116,
and 680 mg/g d.m. in the YA, YW, and YE extracts, respec-
tively. For extracts from green Ginkgo leaves, kaempferol,
myricetin, and isorhamnetin predominated in general and
ranged from 153 to 661 mg/g d.m. extract. The quantitative
differences in the flavonol aglycone profiles for the prepara-
tions likely stem from changes occurring within Ginkgo trees
during vegetation, i.e. primarily their accumulation of poly-
phenols over the course of a longer growth period, which is
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1154 J. Kobus et al. Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160

Table 2. Contents of total polyphenols and flavonol aglycones in extracts of Ginkgo (Ginkgo biloba L.) leaves prepared using different
solvents.

Component GW GA GE YW YA YE

Morin [mg/g d.m.] 16B 6 0 81E 6 8 0A 6 0 144F 6 7 58D 6 11 41C 6 5.5

Quercetin [mg/g d.m.] 64B 6 0 322D 6 5 0A 6 3.3 861E 6 20 1280F 6 7 145C 6 14.4
Myricetin [mg/g d.m.] 322B 6 24 569C 6 8 153A 6 5.5 1116E 6 6 1683F 6 29 680D 6 11.1
Kaempferol [mg/g d.m.] 295B 6 14 661E 6 21 235A 6 14 550D 6 23 1229F 6 76 478C 6 17
Isorhamnetin [mg/g d.m.] 154B 6 7 146B 6 5 123A 6 11 297C 6 9 382D 6 21 448E 6 25.5
Total flavonols [mg/g d.m.] 878B 6 22 1960C 6 32 513A 6 17 3146D 6 55 4998E 6 30 1812C 6 34.2
Total polyphenols [mg/g d.m.] 56.9 6 2 203.5 6 4 204.4 6 1 179.9 6 3 247.5 6 1 137.3 6 1

GW, Water infusion from green leaves of Ginkgo; GA, aqueous acetone extract from green leaves of Ginkgo; GE, ethanolic extract from green
leaves of Ginkgo; YW, water infusion from yellow leaves of Ginkgo; YA, aqueous acetone extract from yellow leaves of Ginkgo; YE, ethanolic
extract from yellow leaves of Ginkgo.
Results are mean values of three determinations 6 standard deviation.
Values sharing the same letter in a row are not significantly different (p ,0.05).

characteristic of the yellow leaves. The type of solvent used for
extraction has a pronounced effect on the quantity of flavonols
that can be recovered, with the total amount in aqueous
acetonic extracts being from 1.5 to 2.2H greater than those
from water infusions or ethanol. The highest amounts of
kaempferol, quercetin, and myricetin were determined in the
aqueous acetonic preparations. Generally, the total amount of
flavonol aglycones was proportional to the total polyphenols
estimated using the assay with Folin-Ciocalteu’s phenol
reagent.
As mentioned above, the flavonol content is positively
correlated with the antioxidant capacity of the extracts. Etha-
nol is commonly employed to extract tannins, polyphenols,
and flavonols, and ethanolic extracts typically exhibit a pro-
nounced antioxidant capacity. During water infusion, mainly
anthocyanins and tannins leached into the polar solvent with
only a small proportion of flavonols; hence, such preparations
have lower antiradical-scavenging potentials [23]. The most
effective solvents that extract the greatest quantities of phe-
nolic compounds with antioxidant activity are mixtures of ac-
etone and water [24]. Qualitative analysis of flavonols in
Ginkgo extracts and leaves was initiated by Pietta et al. [25].
These authors developed an HPLC-PDA method with a gra-
dient system for the purpose of flavonol analysis. The HPLC
method was employed to separate flavonols endogenous to
Ginkgo leaves, i.e. quercetin and kaempferol. Hasler et al.
[26], however, first applied acid hydrolysis to release flavonol
aglycones from their bound forms (i.e. esterified and glycosi-
dic), and using this approach they were able to isolate and
identify 21 different flavonols in Ginkgo leaves. The most
recent findings published by Ding et al. [27], who applied LC/
ESI-MS/MS to characterize the composition of Ginkgo pre-
parations, reported the presence of over 70 compounds,
including aglycone and biflavonoid forms, as well as glycosidic
forms of flavonoids and terpenoids. According to Dubber and
Kanfer [5], the glycosidic forms are predominant in Ginkgo
extracts. From their study of five different pharmacological
preparations, Dubber and Kanfer [5] found that rutin was
present in all analyzed preparations, but the contents of quer-
cetin, kaempferol, and isorhamnetin varied depending on the
extract preparation. In this study, it is believed that rutin was
hydrolyzed during the acid hydrolysis step to quercetin and
rhamnose. Hasler et al. [26] investigated dried Ginkgo leaves
and found aglycone contents of 0.2–1.4 wt-%, which corre-
sponded to a calculated Ginkgo flavonol glycoside content of
0.5–3.5 wt-%. In this study, the content of flavonol aglycones
in extracts from yellow Ginkgo leaves was 2.5 to 3.5H greater
than for extracts from green Ginkgo leaves. The choice of the
two growing periods before Ginkgo leaf collection was not
accidental because Ginkgo leaves used in medicinal and
nutraceutical preparations are collected in August when the
leaves are young and also in October at the end of the growing
season.
All analyzed extracts exhibited an Fe(II)-chelating ability,
which depended both on the solvent employed for extraction
and the leaf type (Fig. 1). Water infusions demonstrated the
greatest chelating ability of ferrous iron ranging from 33 to
42%, with aqueous acetonic extracts possessing a 5–41% ca-
pacity and ethanolic extracts demonstrating the weakest ca-
pacity at 4–17%. It was also determined that constituents
contained in extracts from green Ginkgo leaves formed che-
lates with Fe21 more readily than those prepared from yellow
leaves. An exception, however, was noted in the assay for
extracts prepared by water infusion of both leaf types at a
concentration of 400 ppm and for aqueous acetonic extracts
of both leaf types at a concentration of 800 ppm: The chelat-
ing ability did not differ significantly (p .0.05) in these pre-
parations. It was also shown that an increase in the con-
centration of extracts from 400 to 800 ppm and then to
2000 ppm resulted in an increase in chelating activity of
aqueous acetone extracts by 4 and 7.2H in the GA samples
and by 4.6 and 7H in the YA samples. In the case of water

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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160
Figure 1. Chelating activity of various crude extracts of green and
yellow Ginkgo (Ginkgo biloba L.) leaves. Results are mean values of
three determinations 6 standard deviation. Abbreviations are
defined in Table 2.
infusions and ethanolic extracts, a proportional increase of
chelating activity was observed when the sample concentra-
tion increased from 400 to 800 ppm. However, such a de-
pendence was not found in these extracts at concentrations
greater than 2000 ppm; in other words, the chelating activity
at higher concentrations was not significantly (p .0.05) dif-
ferent from that at the 2000-ppm addition level.
Statistical analysis of the data revealed a positive correla-
tion between the chelating activity of the crude extracts and
their contents of myricetin and quercetin. Logically, the extent
to which flavonols can form metal ion complexes depends on
their chemical nature/structure, i.e. primarily on the number
and position of hydroxyl moieties attached to the B-ring.
Chelating properties of compounds containing a larger num-
ber of hydroxyl substituents are correspondingly stronger.
The sites of metal binding to flavonoid molecules are the
ortho-diphenol groups at positions 3’ and 4’ on the B-ring as
well as the 4-keto and 3-hydroxyl groups or 4-keto and 5-hy-
droxyl groups on the flavanoid’s C-ring. Moreover, some
authors suggest that a double bond between C-2 and C-3 of
the C-ring enhances the flavonoid’s capacity to chelate ferrous
ions. After metal ion complexes are formed, flavonoids also
retain the ability to quench free radicals [28, 29].
The chelating properties of extracts from Ginkgo leaves
might be due to the presence of myricetin on account of the six
hydroxyl groups in its structure. In turn, quercetin contains
four –OH moieties. Mira et al. [30] stated that the chelating
properties of flavonols resulted in a reduced formation of
intermediate products in the cycle of free-radical reactions,
when metal ions participated. As a consequence, the result
was a reduction in the production of free radicals. Conclusions
drawn by the authors are comparable to findings presented in
this study; i.e., an important correlation was noted for the
ability of myricetin (r = 0.73) and quercetin (r = 0.60) to
chelate Fe21. Fernandez et al. [31] reported that flavonoids
exhibited chelating properties towards metal ions. Results of
experiments conducted by these authors suggested that a
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Antioxidant activity and phenolic components of Ginkgo 1155
“metal/flavonoid” ratio of 1 : 2 contributed to the formation of
at least 20% or more chelates than the quantity measured at
1 : 1, 2 : 1, and 2 : 3 ratios of the aforementioned components.
This fact may explain data obtained in the case of water infu-
sions, which showed that an increase in concentration did not
result in an enhancement of chelating capacity. The reaction
was likely stabilized, and an increase in concentration did not
have any additional effect. It has been proven that complexes
formed by flavonoids with Fe31, Cu21, and to a lesser degree
with Fe21, resulted in an antiradical activity enhanced in bio-
logical systems compared with the unchelated forms. Due to
their chemical nature, polyphenols are more active anti-
oxidants in vitro than some vitamin preparations [32, 33].
From in vitro studies, it has been shown that flavonoids chelate
ions of copper and other intermediate metals that catalyzed
ascorbic acid oxidation and significantly inhibited the trans-
formation of ascorbate to dehydroascorbate. Most phenolic
compounds in vitro exhibit considerable antioxidative and
antiradical activities. Like the action of vitamin C, plant phe-
nols sometimes stimulate processes of oxidative character by
reducing intermediate metal contents. What is more, certain
flavonoids in the presence of nitrogen oxides (NOx) can ex-
hibit pro-oxidative activity [8, 34].
All crude extracts analyzed exhibited reducing power to a
degree (Fig. 2). The highest optical density readings, thereby
indicating a greater capacity to reduce Fe31, were recorded for
the YA extract (0.740), followed by the water infusion YW
extract (0.682), when assayed at a 0.5-mg level. Both these
extracts reduced iron the most when tested at other con-
centrations. Moreover, leaf type had a marked impact on the
observed reducing power: Lower optical densities were
recorded for crude extract preparations from green rather
than yellow leaves, with the lowest reducing capacity being
observed in the GE extract, for which absorbance readings of
0.264 and 0.133 were found at 0.5- and 0.1-mg levels,
respectively. Furthermore, the type of solvent used in the
extract preparation impacted on the extent of the crude
extract’s reducing power. Amongst the preparations analyzed,
Figure 2. Reducing power of various crude extracts of green and
yellow Ginkgo (Ginkgo biloba L.) leaves. Results are mean values of
three determinations 6 standard deviation. Abbreviations are
defined in Table 2.
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1156 J. Kobus et al.
the aqueous acetone extracts exhibited the highest absorbance
values, and the ethanolic ones the lowest. It was also observed
that an increase in the concentration of extracts in the assay to
0.5 mg resulted in an increased absorbance ranging from 34%
in GE to 246% in the YE extract. Statistical analysis revealed
that there is a positive correlation between the content of fla-
vonols and reducing power of extracts (r = 0.64).
The reducing power of extracts prepared from Ginkgo
leaves has also been investigated by Stefanovits-Banyai et al.
[35]. These authors took into consideration many factors that
might possibly affect the absorbance value; that is, they com-
pared the reducing power of water infusion and ethanolic
extracts from leaves of individual Ginkgo specimens. The
aqueous extracts reduced iron to a lesser degree than the etha-
nolic counterparts. In their studies, however, the authors used
80% (vol/vol) ethanol solutions, which – as noted by the
authors – extracts more polyphenols. It is the polyphenols that
are responsible for the enhanced reducing power compared
with the water infusion preparations. In our study, 96% (vol/
vol) ethanol was employed and it had a pronounced effect on
both the qualitative and quantitative composition of the
extracts so prepared. The ethanolic extracts contained the
smallest quantities of the bioactives in comparison to the water
infusion and aqueous acetonic extracts. The recorded absorb-
ance values from the experiment were comparable to results
obtained for other raw plant materials rich in polyphenols,
characterized by a high reducing activity and thus also anti-
oxidant activity. Yen and Chen [36], for example, determined
the reducing power of extracts from different types of tea. The
greatest absorbance was observed for an infusion of oolong tea,
which at an addition level of 0.2 mg in the assay achieved an
absorbance of 0.6. When the level of the oolong tea extract was
increased to 1 mg in the assay, it yielded an absorbance reading
of 2.75 after correction of the dilution factor. Ningappa et al.
[37] characterized extracts from curry leaves prepared by
water infusion and ethanol: These authors reported that the
reducing power of 100 mg/mL of an ethanolic extract from
curry leaves reduced iron ions to the same degree as that of
BHT. Water infusion was characterized by a ,20% smaller
reducing power in comparison to the ethanolic extract prepa-
ration. Based on a study conducted by Amarowicz et al. [38], it
was shown that the application of methanol and acetone as a
solvent did not have any pronounced effect on absorbance
values denoting reducing power. Absorbance values of 0.8
were reported for methanolic and acetonic extracts at a 0.5 mg/
assay addition. Similar observations were made and conclu-
sions drawn by Naczk et al. [39] for ethanolic and acetonic
extracts from blueberry leaves. Moreover, Farhoosh et al. [40]
reported that the resulting antioxidative potential of extracts
depended not only on the concentration but also the propor-
tions between individual active compounds. Based on the
examples of black tea leaves and tea dust, as well as green tea,
these authors showed that the reducing power did not always
correlate with antioxidative properties determined in free-
radical in vitro tests.
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160
Antiradical activities of extracts from Ginkgo leaves are
presented in Table 3. The values ranged from 0.69 for sample
YE to 4.89 mM/g d.m. for sample GA. Aqueous acetonic
extracts (i.e. GA and YA) scavenged radicals to the greatest
degree, amounting to 4.89 and 3.37 mM Trolox/g d.m. A
slightly weaker activity in relation to scavenging DPPH. was
observed for water infusion preparations. It was found that 1 g
GW extract quenches radicals corresponding to 2.22 mM
Trolox, while the YW analog preparation exhibited an activity
equivalent to 2.91 mM Trolox. The activities of ethanolic
extracts were the poorest, amounting to 1.02 mM Trolox/g
d.m. for GE and 0.69 mM for the YE extract. From the
experiment, it was determined that both the types of solvent
and leaves employed had a marked effect on the observed
antiradical activity. Aqueous acetonic and ethanolic extracts
from green leaves scavenged DPPH radicals to a greater
degree compared to those from yellow Ginkgo leaves, but the
water infusion extract from yellow leaves was more active than
that of its green counterpart. Statistical analysis revealed a
significant positive correlation between the total flavonols
content and activity towards DPPH. (r = 0.54). The presence
of flavonols such as quercetin (r = 0.78), kaempferol
(r = 0.62), and morin (r = 0.55), contributed to the greatest
antioxidant activity towards DPPH radicals.
Ellnain-Wojtaszek et al. [41] studied the activity of etha-
nolic, methanolic and aqueous extracts from green Ginkgo
leaves by electron paramagnetic resonance (EPR) spectros-
copy in a model system with DPPH radicals. The EPR signal
is based on the Zeeman effect, which consisted of splitting
energy levels of paramagnetic substances in the outer mag-
netic field. In the applied magnetic field, the spins of unpaired
electrons are oriented either parallel or anti-parallel to the
field’s direction. Following absorption of microwave radiation
from a klystron at a frequency matching the difference in
Table 3. The scavenging effect of the DPPH radical by extracts of
Ginkgo (Ginkgo biloba L.) leaves prepared using different solvents.
Sample [mM Trolox/g d.m.]
GW 2.22C 6 0.04
GA 4.89E 6 0.03
GE 1.02B 6 0.07
YW 2.91D 6 0.11
YA 3.37C 6 0.02
YE 0.69A 6 0.04
GW, Water infusion from green leaves of Ginkgo; GA, aqueous ace-
tone extract from green leaves of Ginkgo; GE, ethanolic extract from
green leaves of Ginkgo; YW, water infusion from yellow leaves of
Ginkgo; YA, aqueous acetone extract from yellow leaves of Ginkgo;
YE, ethanolic extract from yellow leaves of Ginkgo.
Results are mean values of three determinations 6 standard devia-
tion.
Values sharing the same letter in a row are not significantly different
(p ,0.05).
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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160
energy between the two orientations, electrons parallel to the
magnetic field flip their direction. During the experiment,
changes in signal amplitude of tracers (i.e. DPPH.) under the
influence of antioxidants present in samples, in successive
time intervals (1–60 min), were measured. It was found that
the radical-scavenging potential of extracts decreased in the
following order: methanol . water . ethanol. The authors
stated that the antioxidant activity was affected by the pres-
ence of quercetin in extracts to the greatest degree. Further-
more, rutin, detected in considerable amounts and being a
glycoside of quercetin, exhibited a slightly lower activity in this
experiment, which is believed to be due to the bound glucose
moiety. It appears that the scavenging of DPPH radicals most
likely results from the proportions of individual components,
as has been confirmed in other studies. The antiradical prop-
erties of Ginkgo extracts, which come chiefly from the flavo-
noid fraction, may inactivate radicals such as the superoxide
anion radical (O2.–), the hydroxyl radical (HO.), hydrogen
peroxide (H2O2), and reactive singlet oxygen (1O2). Further-
more, the contents of individual classes of bioactives are
closely related to their accumulation levels over the course of
the growing season; during this time, both the proportions and
forms of the bioactives vary [42–44].
Shi and Niki [45] calculated stoichiometrically the anti-
radical activity of an aqueous acetone extract of Ginkgo leaves
(Egb 761). Following quantitative analysis of the active com-
pounds, the authors selected two bioactives present in the
largest quantities (i.e. quercetin and kaempferol) and com-
pared the results to that of a standard of a-tocopherol, which is
commonly considered as an effective natural antioxidant.
Based on the chemistry of the molecules, the mathematical
calculations revealed that a-tocopherol can inactivate one
molecule of the free radical whereas quercetin and kaempferol
can inactivate 4.0 and 1.9, respectively. The authors con-
firmed their preliminary assumptions by analyzing the kinetics
of inactivation reactions of free radicals using Ginkgo com-
ponents with bioactivity.
Antioxidant activities of extracts were investigated using a
peroxidating linoleic system in an emulsion model with b-
carotene; the findings are depicted in Fig. 3. In this model,
free radicals generated from linoleic acid attack highly unsa-
turated b-carotene, resulting in a loss of color of b-carotene
molecules (i.e. bleaching). The presence of various anti-
oxidants can hinder the extent of b-carotene bleaching by
neutralizing the linoleate free radicals formed during oxidation
[22]. All Ginkgo extracts exhibited antioxidant properties in
relation to the linoleic acid/b-carotene emulsion system.
Similar to the findings from the DPPH. assay, aqueous aceto-
nic extracts showed the highest degree of inhibition of linoleic
acid oxidation. After 90 min of incubation at 50 7C, the
absorbances of the GA and YA extracts were 0.561 and 0.657,
respectively. A weaker antioxidant activity was found for the
GW and YW water infusion preparations; after 90 min, the
absorbance readings were 0.445 and 0.496, respectively.
Ethanolic extracts demonstrated the weakest activity against
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Antioxidant activity and phenolic components of Ginkgo 1157
Figure 3. Antioxidant activity based on the average b-carotene
bleaching. Results are mean values of three determinations
6 standard deviation. Abbreviations are defined in Table 2.
b-carotene bleaching. The performance of extracts from yel-
low Ginkgo leaves was better than that of green ones, which
was in direct contrast to the results afforded by the DPPH
radical-scavenging assay. Based on further correlation analy-
ses, it was found that a positive relationship existed between
antioxidant activity as assessed by the emulsion system assay
and isorhamnetin (r = 0.76) and myricetin (r = 0.81) con-
tents. According to Gramza et al. [46], hydrophilic anti-
oxidants such as flavonols are very effective in emulsions. In
their study, Roedig-Penman and Gordon [47] reported a
strong antioxidant action of myricetin and quercetin in an oil-
in-water emulsion. They concluded that the antiradical prop-
erties of myricetin and its ability to chelate metals accounted
for the lion’s share of the observed antioxidant effect, while in
the case of quercetin, it was only its metal-chelating capacity
that participated because of its orientation in the o/w layer.
Investigations on the effect of crude Ginkgo extracts to
stabilize the TAG of rapeseed oil using the accelerated oxida-
tion test with a Rancimat system were performed and the
findings are indicated in Table 4. Induction periods were
established for the analyzed samples based on the increase in
water conductivity caused by the dissociation of volatile car-
boxylic acids formed in the course of oil oxidation. It was
found that all analyzed Ginkgo extracts extended the induc-
tion periods of TAG, devoid of native antioxidants, by as
much as 40%. The longest induction period for the analyzed
extracts was recorded for GA and GE (i.e. at a concentration
of 500 ppm), accounting for 10.90 and 10.15 h, respectively.
On the other hand, the lowest PF was afforded by water infu-
sion extracts. It was also noted that an increase in the addition
level of extracts to 500 ppm (i.e. more antioxidant com-
pounds) gave larger values of PF for all analyzed samples,
ranging from 1.21 for YE to 1.34 for the GA extract. The best
antioxidant properties under the adopted testing conditions
were, however, recorded for the synthetic antioxidant BHT,
which gave an induction period of 11.21 h. No correlation of
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1158 J. Kobus et al. Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160

Table 4. The effect of extracts of Ginkgo (Ginkgo biloba L.) leaves prepared using different solvents on the
oxidative stability of stripped TAG of rapeseed oil according to the Rancimat test.

Sample Induction period [h] Protection factor, PF

200 ppm 500 ppm 200 ppm 500 ppm

GW 8.86B 6 0.3 8.84B 6 0.95 1.09B 6 0.04 1.09B 6 0.12

GA 9.67D 6 0.09 10.90F 6 0.35 1.19D 6 0.01 1.34F 6 0.01
GE 10.04E 6 0.07 10.15E 6 0.03 1.23E 6 0.01 1.25E 6 0.04
YW 8.11A 6 0.72 8.44A 6 0.58 1.00A 6 0.09 1.04A 6 0.07
YA 9.55D 6 0.08 9.63C 6 0.11 1.17D 6 0.01 1.18C 6 0.03
YE 9.38C 6 0.18 9.82D 6 0.23 1.15C 6 0.01 1.21D 6 0.01
BHT 11.2F 6 0.1 nd 1.38F 6 0.01 nd
Control 8.14A 6 1.34 – – –

GW, Water infusion from green leaves of Ginkgo; GA, aqueous acetone extract from green leaves of Ginkgo; GE,
ethanolic extract from green leaves of Ginkgo; YW, water infusion from yellow leaves of Ginkgo; YA, aqueous
acetone extract from yellow leaves of Ginkgo; YE, ethanolic extract from yellow leaves of Ginkgo; BHT, butylated
hydroxytoluene; nd, not determined.
Results are mean values of three determinations 6 standard deviation.
Values sharing the same letter in a row are not significantly different (p ,0.05).

inhibition of oxidation was determined between the content of 4 Conclusions

flavonol aglycones and the extent of TAG oxidation.
The total antioxidant effect of extracts, being a composite
of many bioactives, is very difficult to forecast. Numerous
studies have shown that the total antioxidant capacity is
determined not only by the properties of the individual com-
pounds, but also, and perhaps most importantly, by their
interactions within the system [48]. Roedig-Penman and
Gordon [47] studied the effect of myricetin and quercetin on
the stability of TAG of sunflower oil at 30 and 60 7C. Myr-
icetin imparted a strong antioxidant activity in the TAG,
regardless of the presence of endogenous tocopherols or citric
acid. In contrast, quercetin showed a similar antioxidant effect
in TAG, but it did not bestow antioxidant properties in the
presence of tocopherols or citric acid. Nogala-Kałucka et al.
[49] reported that the PF of an ethanolic rosemary extract at
500 ppm towards TAG of rapeseed oil was 1.6H that afforded
by BHTat 100 ppm concentration and 2.2H that for a mixture
of a-, b-, and g-tocopherol also at 100 ppm concentration.
Additionally, Beddows et al. [50] showed a positive synergistic
effect between extracts of oregano, marjoram, rosemary, and
ascorbic acid as stabilizers of sunflower oil.
Due to the fact that the Ginkgo extract preparations from
this study are mixtures of many bioactive compounds, their
interactions could have affected the inhibition of TAG oxida-
tion, even though no correlations were determined between
the flavonol content and the PF. On the other hand, a lack of
dependence between the flavonol content and the degree of
inhibition of TAG oxidation may be explained by poor solu-
bility of these compounds in the bulk oil. It is likely that other
compounds such as proteins also contained in the extracts
could have affected the inhibition of oxidation processes; this
topic warrants further investigation.
The results of this study have demonstrated that extracts from
yellow and green Ginkgo (Ginkgo biloba) leaves, especially the
aqueous acetonic preparations, demonstrated a strong anti-
oxidant activity in various in vitro model systems. Amongst the
analyzed Ginkgo preparations, the yellow ones were richer in
flavonol aglycones. Positive correlations were found between
the flavonol content and chelating activity, reducing power, and
DPPH. scavenging activity. The aqueous acetonic extracts,
with the highest flavonol content, showed excellent activity
against linoleic oxidation in the b-carotene-linoleate model
system. In turn, extracts prepared from green Ginkgo leaves,
despite less flavonols relative to their yellow counterparts,
exhibited a greater inhibition of lipid oxidation according to the
accelerated Rancimat test. The results of this study suggest that
extracts prepared from both yellow and green Ginkgo leaves
may constitute a good source of natural antioxidants to be used
by industries in functional food formulations. It should be
stressed, however, that no harm results to Ginkgo trees in later
stages of the growing period (i.e. October) when the yellow
leaves, as a source of raw material, are collected.
Acknowledgments
The study was financed by the Ministry of Education and Science,
Poland, under Project No. N N312 2857 33.
Conflict of interest statement
The authors have declared no conflict of interest.

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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160
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