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-

ported health benefits afforded to man by Ginkgo prepara-
Currently, Ginkgo (Ginkgo biloba L.) is the only one living tions are the polyphenols. Polyphenols are ubiquitous
species belonging to the phylum Gymnosperms (Gymno- throughout the plant kingdom and include flavones, flavonol
spermae) and class Ginkgopsida. Extracts prepared from green glycosides, acylated flavonol glycosides, biflavonoids, flavan-
Ginkgo leaves are considered attractive for their documented 3-ols, and proanthocyanidins [1, 5, 6]. Of late, flavonoids have
multifaceted action in biological systems [1, 2]. The first received considerable attention in the literature, particularly
analyses on the chemical composition of the anatomical parts due to their widely recognized broad-spectrum free-radical-
of Ginkgo were conducted by Peschier in 1818 and later by scavenging effects and for being readily incorporated in redox
Schwarzenbach in 1857. Further investigations on this subject reactions [7, 8]. This pertains to all compounds in which
continued in the next century by Kawamura and findings were functional hydroxyl groups are located in the ortho- and para-
reported in print by 1928. Ten years later, the pioneering positions on aromatic rings [8, 9]. The transfer of hydrogen
studies on Ginkgo by Furkawa were published [3, 4]. 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-
Correspondence: Joanna Kobus, Faculty of Food Science and Nutrition,
Poznań University of Life Sciences, Poland Wojska Polskiego 31, 60-624
phenols is a highly complex process and the rate of the reac-
Poznań, Poland. tion is closely related to the type and location of substituents
E-mail: [email protected] on the molecule [8, 10]. Flavonoids exhibit the capacity of
Fax: 148 61 8487431 inactivating the superoxide anion, peroxyl and hydroxyl radi-

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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160 Antioxidant activity and phenolic components of Ginkgo 1151

cals, as well as oxygen in statu nascendi. Moreover, poly- methoxy-3,4’,5,7-tetrahydroxyflavone), quercetin

phenols are capable of forming complexes with metal ions (3,3’,4’,5,7-pentahydroxyflavone), kaempferol (3,4’,5,7-tet-
such as Fe(II) that can catalyze oxidation, and they can also rahydroxyflavone), myricetin (3,3’,4’,5,5’,7-hexahydroxy-
inhibit the activity of oxidizing enzymes like lipoxygenase flavone), and morin (2’,3,4’,5,7-pentahydroxyflavone). All
[11]. were of HPLC-grade purity and purchased from Sigma-
Flavonoids constitute the largest groups of bioactive sub- Aldrich Chemical (St. Louis, MO, USA), except for iso-
stances isolated from Ginkgo leaves [2]. A total of 33 flavo- rhamnetin which was acquired from Fluka (Buchs, Switzer-
noids were identified in Ginkgo leaves, and most of them were land). Fatty acid methyl ester (FAME) standards for reten-
in the form of mono-, di-, and triglycosides. It was determined tion time mapping during fatty acid profiling were obtained
that flavonol aglycones are contained in relatively significant from Supelco (Bellefonte, PA, USA). 2,2-Diphenyl-1-
quantities in extracts prepared from Ginkgo leaves [6]. For picrylhydrazyl. (DPPH.), 6-hydroxy-2,5,7,8-tetramethyl-
example, the presence of flavonoids determined in Egb 761 chroman-2-carboxylic acid (Trolox), Tween-40, and b-car-
extracts (see ref. [4] for more details) is reported to be any- otene were purchased from Sigma-Aldrich. Butylated hydro-
where from 22 to 27%, while in the leaves themselves their xytoluene (BHT) was acquired from Merck (Darmstadt,
content is only from 0.2 to 0.4 wt-% [4]. Germany).
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 2.1.1 Preparation of extracts
emphasized in numerous works. Many different herbs and
products therefrom have been investigated for their possible Of ground green (G) or yellow (Y) Ginkgo leaves, 20 g was
protective effect/action toward processed foodstuffs which are extracted with 1 L solvent at atmospheric pressure under the
sensitive to lipid oxidation [12, 13]. Indigenous antioxidative following conditions: deionized water at 95 7C during a 15-
compounds (e.g. phenolics) in the raw materials act by differ- min infusion (GW, YW); acetone/water (3 : 2, vol/vol) at
ent mechanisms, but their cumulative antioxidant efficacy is 40 7C during a 90-min extraction (GA, YA), or ethanol (96%,
difficult to predict and sometimes to estimate, because the vol/vol) at 18 7C during a 16-h maceration (GE, YE) [14].
total antioxidant capacity is affected not only by the properties Each solution was cooled to room temperature and filtered by
of the individual compounds, but also by their interactions or gravity through Whatman No. 1 filter paper. Organic solvents
lack thereof [8, 9]. were evaporated under vacuum at ,40 7C, and residual water
The aim of the present study is to investigate the content of (i.e. in the aqueous acetonic extract and the water infusion
flavonol aglycones and the mechanism(s) of the antioxidant extract) was removed by lyophilization. The crude extracts so
efficacy afforded by crude extracts from yellow and green prepared were stored in a dry, dark, and cool place until ana-
Ginkgo leaves, by determining the reducing power, chelating lyzed.
activity, and antiradical potential, together with antioxidant
properties in an anhydrous fat substrate, as well as a linoleic 2.1.2 Linoleic acid emulsion model system
acid emulsion.
A 10 mM linoleic acid emulsion was freshly prepared before
each assay using 0.1 mM phosphate buffer (pH 7.2) with
2 Materials and methods 0.3% (wt/vol) Tween-40 according to Flaczyk et al. [15].

2.1 Materials
2.1.3 Preparation of stripped triacylglycerols
Ginkgo (Ginkgo biloba L.) extracts were prepared from green
(harvested in August 2007 and marked with a ‘G’) and yellow Triacylglycerols (TAG) were collected from rapeseed oil by
leaves (harvested in October 2007 and marked with a ‘Y’) column chromatography according to Chimi et al. [16].
including leaf stalks coming from a plantation in Baranowo Rapeseed oil was purified in that endogenous antioxidants
belonging to the Poznań University of Life Sciences. Leaves were stripped from the oil before conducting the planned
were first dried in an incubation oven at 40 7C until a moisture experiments. Oil was dissolved in hexanes (1 : 3, vol/vol) and
content of ,8% was reached and then ground in a laboratory run through a glass column packed with activated carbon,
mill (ZBPP, type WŻ-1; Bydgoszcz, Poland). The degree of aluminum oxide (activated at 300 7C), and anhydrous sodium
comminution of plant material was determined by sieving sulfate. During the refining process, the column and receiver
using a mesh size of 0.8–0.03 mm. system were shielded from extraneous light using aluminum
Low-erucic acid rapeseed oil (produced in Kruszwica, foil. Next, the recovered hexanes were removed under
Poland) was purchased from a local supermarket in Poznań. vacuum at ,40 7C, after which the TAG free of endogenous
All solvents were of analytical grade unless otherwise antioxidants were sealed in an amber vial under a nitrogen
specified. Flavanol standards included isorhamnetin (3’- headspace.

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1152 J. Kobus et al. Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160

2.2 Methods were eluted with CH3OH and collected in a 10-mL volumetric
flask.
2.2.1 Fatty acid analysis
2.2.4 Composition of flavonols
After derivatizing the TAG with 6% (vol/vol) sulfuric acid in
anhydrous methanol, FAME were separated from one another Separation and identification of extracted flavonols were car-
using a Hewlett Packard 5890 GC gas chromatograph (Agi- ried out by high-performance liquid chromatography using a
lent, Wilmington, DE, USA) equipped with a Supelcowax-10 Nova-Pak C18 reversed-phase column (3.9 H 150 mm, 5 mm
fused-silica capillary column (30 m length625 mm i.d., particle size; both Waters, Milford, MA, USA). Solvent A was
0.25 mm film thickness; Supelco) and detected by FID. The 0.3% (vol/vol) HCOOH in H2O, whereas solvent B was 100%
oven temperature was initially set at 60 7C and then increased CH3CN. The flow rate was maintained at 1 mL/min. The
to 210 7C at 12 7C/min. The injector temperature was set at gradient profile was as follows: 85% A at 0 min and 25% A at
240 7C, split ratio at 1 : 25, and detector temperature at 40 min. Chromatograms were recorded with a UV-Vis detec-
260 7C. The carrier gas was ultra-high-purity helium at a flow tor at l = 370 nm. The identification of separated compounds
rate of 1 mL/min. Ultra-high-purity hydrogen and air were the was carried out by retention time mapping with a set of
fuel gases for the FID, with nitrogen being used as make-up standards. The quantity of each flavonol was determined
gas. Separated FAME were identified by comparing their using external and internal standards of individual flavonols.
retention times to those from commercially available stand-
ards [17]. 2.2.5 Chelating activity

2.2.2 Total phenolic content The chelating activities of crude extracts from green and yel-
low Ginkgo leaves were measured according to Tang et al.
The contents of total phenolics (TPC) in crude extracts of [19]. The colorimetric assay involves determining the quantity
green and yellow Ginkgo leaves were determined by visible of Fe21 which did not chelate with the crude Ginkgo extract
spectrophotometry based on a colorimetric oxidation/reduc- and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-tria-
tion reaction according to Cheung et al. [18]. Quercetin was the zine monosodium salt (i.e. ferrozine). To a test tube, 1 mL
standard employed in this work. The dry crude extracts were sample, 0.1 mL 2 mM FeCl2, and 0.2 mL ferrozine reagent
dissolved in CH3OH/H2O (1 : 1, vol/vol) and then 1 mL of this were added. The mixture was vortexed for ,60 s and left to
solution was added to a series of tubes. Next 1 mL of Folin- stand at room temperature for 20 min. Thereafter, absorb-
Ciocalteu’s phenol reagent was pipetted in and the contents ance readings were taken at l = 562 nm using the SPE-
were vortexed for ,60 s. Following the addition of 1 mL of a CORD® 40 (Analytik Jena). Deionized water was used instead
35% (wt/vol) NaHCO3 solution, the mixture was allowed to of an aliquot of the extract as a control and ferrozine was used
stand for 90 min in darkness to allow maximum color devel- as a reference. The chelating activity was calculated as follows:
opment. Just before measuring absorbances, the samples were Chelating activity = 1 – [(Abs of sample – Abs of refer-
centrifuged at 30006g (Sigma 204 laboratory centrifuge; St. ence)/Abs of control)]6 100
Louis, MO, USA). The absorbance of the supernatant was
measured at l = 725 nm using a SPECORD® 40 (Analytik 2.2.6 Reducing power
Jena AG, Germany). Based on the construction of a standard
curve, TPC data for the preparations from Ginkgo leaves were The reducing power of the crude extracts was determined
expressed as mg quercetin equivalents/g dry extract. according to Amarowicz et al. [20]. Briefly, an aliquot of dis-
solved extract in CH3OH was mixed with 2.5 mL 0.2 M
2.2.3 Extraction of flavonols 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.
Of crude extract, 1 g was dissolved in 10 mL 96% (vol/vol) Following incubation, 2.5 mL 10% (wt/vol) trichloroacetic
ethanol, and 2 mg ascorbic acid, as antioxidant, was added. acid was added and then the mixture was centrifuged for
Samples were hydrolyzed in 2 mL 2 M HCl at room temper- 10 min at 20006g. The 2.5-mL volume of the upper layer
ature for 60 min. Then, 2 mL 2 M NaOH was added. The aliquot was combined with 2.5 mL deionized water and
contents were transferred to a 25-mL volumetric flask and 0.5 mL of a 0.1% (wt/vol) FeCl3 reagent. The absorbance of
filled to the mark with ethanol. A Chromabond System the resultant chromophore was measured at l = 700 nm with
(Macherey-Nagel, Germany) with an SPE column filled with the SPECORD® 40 (Analytik Jena).
diol (Discovery® DSC-18 SPE tube, 500 mg, 50 mm, 70 Å
pore diameter; Supelco) was employed to recover the flavonol 2.2.7 Antiradical activity with DPPH.
fraction. The extraction process comprised three stages: First,
the column was conditioned with 5 mL CH3OH; second, The free-radical-scavenging potentials of crude extracts were
5 mL sample was loaded onto the column; and third, flavonols tested in a methanolic solution of DPPH. as described by

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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160 Antioxidant activity and phenolic components of Ginkgo 1153

Amarowicz et al. [21]. The extent of discoloration of the so- TICA PL 7.0 StatSoft. The significance of differences be-
lution indicates the scavenging efficacy of the added sub- tween mean values was determined at p 0.05, after applying
stance. A 1-mL aliquot of extract solution was combined with a one-way analysis of variance (ANOVA) followed by Tukey’s
2 mL CH3OH and then 0.25 mL of a 1 mM ethanolic solu- multiple range test.
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 3 Results and discussion
reference sample was prepared with methanol instead of
DPPH. and the control instead of the extract sample. To con- The FAME composition from the TAG is presented in
struct a calibration curve, absorbances were measured simul- Table 1. The fatty acids found in the stripped rapeseed oil
taneously of samples containing respective concentrations of were typical for such an edible oil and contained 19.32%
the standard, i.e. Trolox (0.5, 1.0, 1.5, and 2.0 mg/mL). linoleic acid, based on the total fatty acid profile.
Results were expressed as mg Trolox equivalents/g extract.
Antioxidant activity was calculated as a percentage of DPPH. Table 1. Fatty acid composition of the TAG in rapeseed oil.
decoloration using the following equation:
Antiradical activity = 100 – [(Abs of sample – Abs of Fatty acid Content [%]
reference)/Abs of control)]6 100
16:0 4.45 6 0.01
16:1n-7 0.22 6 0.01
2.2.8 â-Carotene-linoleate model system 18:0 1.70 6 0.00
18:1n-9 60.69 6 0.02
The antioxidant activity of crude extracts of Ginkgo leaves 18:2n-6 19.32 6 0.01
was determined according to the b-carotene bleaching method 18:3n-3 11.51 6 0.00
of Liu et al. [22] with some modifications. Briefly, a stock so- 20:0 0.58 6 0.00
lution of the b-carotene-linoleic acid emulsion was prepared 22:0 0.33 6 0.00
as follows: 25 mg b-carotene was dissolved in ,50 mL 22:1n-9 1.20 6 0.00
CHCl3. A 1-mL aliquot was transferred to a test tube and the
chloroform was removed using a vacuum evaporator. Next, Results are mean values of three determinations 6 standard devia-
0.5 mL of the extract solution (100 ppm) and 4.5 mL of the tion.
prepared linoleic acid emulsion were added. The sealed test
tubes were heated in a water bath at 50 7C and spectro- In terms of antioxidant activity of the crude extract pre-
photometric measurements (l = 470 nm) of the emulsion parations, the content of flavonol aglycones was found to be
were taken at 15-min intervals with the SPECORD® 40 for a the most important. For this study, the glycosidic forms of
total of 90 min. isorhamnetin, quercetin, kaempferol, myricetin, and morin in
the extracts were first hydrolyzed before analysis. Qualita-
2.2.9 Rancimat test tively, the composition of flavonols in the aqueous acetonic,
ethanolic, and water infusion extracts was similar; however,
Test samples, placed in the Rancimat (Methrom, Switzerland) marked quantitative differences existed (Table 2). A signifi-
tubes, were exposed to a flow of air at an elevated temperature cantly (p ,0.05) higher content of flavonol aglycones was
and became oxidized (i.e. autoxidation). Oxygen was exposed found in extracts from yellow Ginkgo leaves. The highest
to a 2.5-g sample through hot oil at a rate of 20 L/h. During levels for these compounds were recorded in the aqueous
thermal decomposition, volatile organic acids, notably acetonic extract from yellow leaves (YA, 4938 mg/g d.m.) and
HCOOH, were produced. These organic acids were absorbed water infusion extract from yellow leaves (YW, 3146 mg/g
in a measuring vessel filled with deionized H2O, and con- d.m.), while the lowest were noted in the water infusion
tinuously monitored using a conductivity measuring cell. The extract from green leaves (GW, 878 mg/g d.m.) and the etha-
measuring curves so obtained were used to determine the nolic extract from green leaves (GE, 512 mg/g d.m.). Regard-
induction period. A protection factor (PF) was then calculated ing extract preparations from yellow Ginkgo leaves, the
as the ratio of the induction period of the samples with and dominant flavonol was myricetin accounting for 1683, 1116,
without the presence of an antioxidant. and 680 mg/g d.m. in the YA, YW, and YE extracts, respec-
tively. For extracts from green Ginkgo leaves, kaempferol,
2.2.10 Statistical analysis myricetin, and isorhamnetin predominated in general and
ranged from 153 to 661 mg/g d.m. extract. The quantitative
All determinations were performed in at least three replica- differences in the flavonol aglycone profiles for the prepara-
tions. Means and standard deviations were calculated with the tions likely stem from changes occurring within Ginkgo trees
assistance of Microsoft Office Excel 2007 software. Pearson’s during vegetation, i.e. primarily their accumulation of poly-
correlation indexes were calculated with the use of STATIS- 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 extracts. From their study of five different pharmacological
extraction has a pronounced effect on the quantity of flavonols preparations, Dubber and Kanfer [5] found that rutin was
that can be recovered, with the total amount in aqueous present in all analyzed preparations, but the contents of quer-
acetonic extracts being from 1.5 to 2.2H greater than those cetin, kaempferol, and isorhamnetin varied depending on the
from water infusions or ethanol. The highest amounts of extract preparation. In this study, it is believed that rutin was
kaempferol, quercetin, and myricetin were determined in the hydrolyzed during the acid hydrolysis step to quercetin and
aqueous acetonic preparations. Generally, the total amount of rhamnose. Hasler et al. [26] investigated dried Ginkgo leaves
flavonol aglycones was proportional to the total polyphenols and found aglycone contents of 0.2–1.4 wt-%, which corre-
estimated using the assay with Folin-Ciocalteu’s phenol sponded to a calculated Ginkgo flavonol glycoside content of
reagent. 0.5–3.5 wt-%. In this study, the content of flavonol aglycones
As mentioned above, the flavonol content is positively in extracts from yellow Ginkgo leaves was 2.5 to 3.5H greater
correlated with the antioxidant capacity of the extracts. Etha- than for extracts from green Ginkgo leaves. The choice of the
nol is commonly employed to extract tannins, polyphenols, two growing periods before Ginkgo leaf collection was not
and flavonols, and ethanolic extracts typically exhibit a pro- accidental because Ginkgo leaves used in medicinal and
nounced antioxidant capacity. During water infusion, mainly nutraceutical preparations are collected in August when the
anthocyanins and tannins leached into the polar solvent with leaves are young and also in October at the end of the growing
only a small proportion of flavonols; hence, such preparations season.
have lower antiradical-scavenging potentials [23]. The most All analyzed extracts exhibited an Fe(II)-chelating ability,
effective solvents that extract the greatest quantities of phe- which depended both on the solvent employed for extraction
nolic compounds with antioxidant activity are mixtures of ac- and the leaf type (Fig. 1). Water infusions demonstrated the
etone and water [24]. Qualitative analysis of flavonols in greatest chelating ability of ferrous iron ranging from 33 to
Ginkgo extracts and leaves was initiated by Pietta et al. [25]. 42%, with aqueous acetonic extracts possessing a 5–41% ca-
These authors developed an HPLC-PDA method with a gra- pacity and ethanolic extracts demonstrating the weakest ca-
dient system for the purpose of flavonol analysis. The HPLC pacity at 4–17%. It was also determined that constituents
method was employed to separate flavonols endogenous to contained in extracts from green Ginkgo leaves formed che-
Ginkgo leaves, i.e. quercetin and kaempferol. Hasler et al. lates with Fe21 more readily than those prepared from yellow
[26], however, first applied acid hydrolysis to release flavonol leaves. An exception, however, was noted in the assay for
aglycones from their bound forms (i.e. esterified and glycosi- extracts prepared by water infusion of both leaf types at a
dic), and using this approach they were able to isolate and concentration of 400 ppm and for aqueous acetonic extracts
identify 21 different flavonols in Ginkgo leaves. The most of both leaf types at a concentration of 800 ppm: The chelat-
recent findings published by Ding et al. [27], who applied LC/ ing ability did not differ significantly (p .0.05) in these pre-
ESI-MS/MS to characterize the composition of Ginkgo pre- parations. It was also shown that an increase in the con-
parations, reported the presence of over 70 compounds, centration of extracts from 400 to 800 ppm and then to
including aglycone and biflavonoid forms, as well as glycosidic 2000 ppm resulted in an increase in chelating activity of
forms of flavonoids and terpenoids. According to Dubber and aqueous acetone extracts by 4 and 7.2H in the GA samples
Kanfer [5], the glycosidic forms are predominant in Ginkgo 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 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].
Figure 1. Chelating activity of various crude extracts of green and
yellow Ginkgo (Ginkgo biloba L.) leaves. Results are mean values of
From in vitro studies, it has been shown that flavonoids chelate
three determinations 6 standard deviation. Abbreviations are ions of copper and other intermediate metals that catalyzed
defined in Table 2. ascorbic acid oxidation and significantly inhibited the trans-
formation of ascorbate to dehydroascorbate. Most phenolic
compounds in vitro exhibit considerable antioxidative and
infusions and ethanolic extracts, a proportional increase of antiradical activities. Like the action of vitamin C, plant phe-
chelating activity was observed when the sample concentra- nols sometimes stimulate processes of oxidative character by
tion increased from 400 to 800 ppm. However, such a de- reducing intermediate metal contents. What is more, certain
pendence was not found in these extracts at concentrations flavonoids in the presence of nitrogen oxides (NOx) can ex-
greater than 2000 ppm; in other words, the chelating activity hibit pro-oxidative activity [8, 34].
at higher concentrations was not significantly (p .0.05) dif- All crude extracts analyzed exhibited reducing power to a
ferent from that at the 2000-ppm addition level. degree (Fig. 2). The highest optical density readings, thereby
Statistical analysis of the data revealed a positive correla- indicating a greater capacity to reduce Fe31, were recorded for
tion between the chelating activity of the crude extracts and the YA extract (0.740), followed by the water infusion YW
their contents of myricetin and quercetin. Logically, the extent extract (0.682), when assayed at a 0.5-mg level. Both these
to which flavonols can form metal ion complexes depends on extracts reduced iron the most when tested at other con-
their chemical nature/structure, i.e. primarily on the number centrations. Moreover, leaf type had a marked impact on the
and position of hydroxyl moieties attached to the B-ring. observed reducing power: Lower optical densities were
Chelating properties of compounds containing a larger num- recorded for crude extract preparations from green rather
ber of hydroxyl substituents are correspondingly stronger. than yellow leaves, with the lowest reducing capacity being
The sites of metal binding to flavonoid molecules are the observed in the GE extract, for which absorbance readings of
ortho-diphenol groups at positions 3’ and 4’ on the B-ring as 0.264 and 0.133 were found at 0.5- and 0.1-mg levels,
well as the 4-keto and 3-hydroxyl groups or 4-keto and 5-hy- respectively. Furthermore, the type of solvent used in the
droxyl groups on the flavanoid’s C-ring. Moreover, some extract preparation impacted on the extent of the crude
authors suggest that a double bond between C-2 and C-3 of extract’s reducing power. Amongst the preparations analyzed,
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 Figure 2. Reducing power of various crude extracts of green and
chelate Fe21. Fernandez et al. [31] reported that flavonoids yellow Ginkgo (Ginkgo biloba L.) leaves. Results are mean values of
exhibited chelating properties towards metal ions. Results of three determinations 6 standard deviation. Abbreviations are
experiments conducted by these authors suggested that a defined in Table 2.

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1156 J. Kobus et al. Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160

the aqueous acetone extracts exhibited the highest absorbance Antiradical activities of extracts from Ginkgo leaves are
values, and the ethanolic ones the lowest. It was also observed presented in Table 3. The values ranged from 0.69 for sample
that an increase in the concentration of extracts in the assay to YE to 4.89 mM/g d.m. for sample GA. Aqueous acetonic
0.5 mg resulted in an increased absorbance ranging from 34% extracts (i.e. GA and YA) scavenged radicals to the greatest
in GE to 246% in the YE extract. Statistical analysis revealed degree, amounting to 4.89 and 3.37 mM Trolox/g d.m. A
that there is a positive correlation between the content of fla- slightly weaker activity in relation to scavenging DPPH. was
vonols and reducing power of extracts (r = 0.64). observed for water infusion preparations. It was found that 1 g
The reducing power of extracts prepared from Ginkgo GW extract quenches radicals corresponding to 2.22 mM
leaves has also been investigated by Stefanovits-Banyai et al. Trolox, while the YW analog preparation exhibited an activity
[35]. These authors took into consideration many factors that equivalent to 2.91 mM Trolox. The activities of ethanolic
might possibly affect the absorbance value; that is, they com- extracts were the poorest, amounting to 1.02 mM Trolox/g
pared the reducing power of water infusion and ethanolic d.m. for GE and 0.69 mM for the YE extract. From the
extracts from leaves of individual Ginkgo specimens. The experiment, it was determined that both the types of solvent
aqueous extracts reduced iron to a lesser degree than the etha- and leaves employed had a marked effect on the observed
nolic counterparts. In their studies, however, the authors used antiradical activity. Aqueous acetonic and ethanolic extracts
80% (vol/vol) ethanol solutions, which – as noted by the from green leaves scavenged DPPH radicals to a greater
authors – extracts more polyphenols. It is the polyphenols that degree compared to those from yellow Ginkgo leaves, but the
are responsible for the enhanced reducing power compared water infusion extract from yellow leaves was more active than
with the water infusion preparations. In our study, 96% (vol/ that of its green counterpart. Statistical analysis revealed a
vol) ethanol was employed and it had a pronounced effect on significant positive correlation between the total flavonols
both the qualitative and quantitative composition of the content and activity towards DPPH. (r = 0.54). The presence
extracts so prepared. The ethanolic extracts contained the of flavonols such as quercetin (r = 0.78), kaempferol
smallest quantities of the bioactives in comparison to the water (r = 0.62), and morin (r = 0.55), contributed to the greatest
infusion and aqueous acetonic extracts. The recorded absorb- antioxidant activity towards DPPH radicals.
ance values from the experiment were comparable to results Ellnain-Wojtaszek et al. [41] studied the activity of etha-
obtained for other raw plant materials rich in polyphenols, nolic, methanolic and aqueous extracts from green Ginkgo
characterized by a high reducing activity and thus also anti- leaves by electron paramagnetic resonance (EPR) spectros-
oxidant activity. Yen and Chen [36], for example, determined copy in a model system with DPPH radicals. The EPR signal
the reducing power of extracts from different types of tea. The is based on the Zeeman effect, which consisted of splitting
greatest absorbance was observed for an infusion of oolong tea, energy levels of paramagnetic substances in the outer mag-
which at an addition level of 0.2 mg in the assay achieved an netic field. In the applied magnetic field, the spins of unpaired
absorbance of 0.6. When the level of the oolong tea extract was electrons are oriented either parallel or anti-parallel to the
increased to 1 mg in the assay, it yielded an absorbance reading field’s direction. Following absorption of microwave radiation
of 2.75 after correction of the dilution factor. Ningappa et al. from a klystron at a frequency matching the difference in
[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 Table 3. The scavenging effect of the DPPH radical by extracts of
curry leaves reduced iron ions to the same degree as that of Ginkgo (Ginkgo biloba L.) leaves prepared using different solvents.
BHT. Water infusion was characterized by a ,20% smaller
reducing power in comparison to the ethanolic extract prepa- Sample [mM Trolox/g d.m.]
ration. Based on a study conducted by Amarowicz et al. [38], it
GW 2.22C 6 0.04
was shown that the application of methanol and acetone as a GA 4.89E 6 0.03
solvent did not have any pronounced effect on absorbance GE 1.02B 6 0.07
values denoting reducing power. Absorbance values of 0.8 YW 2.91D 6 0.11
were reported for methanolic and acetonic extracts at a 0.5 mg/ YA 3.37C 6 0.02
assay addition. Similar observations were made and conclu- YE 0.69A 6 0.04
sions drawn by Naczk et al. [39] for ethanolic and acetonic
extracts from blueberry leaves. Moreover, Farhoosh et al. [40] GW, Water infusion from green leaves of Ginkgo; GA, aqueous ace-
reported that the resulting antioxidative potential of extracts tone extract from green leaves of Ginkgo; GE, ethanolic extract from
depended not only on the concentration but also the propor- green leaves of Ginkgo; YW, water infusion from yellow leaves of
tions between individual active compounds. Based on the Ginkgo; YA, aqueous acetone extract from yellow leaves of Ginkgo;
YE, ethanolic extract from yellow leaves of Ginkgo.
examples of black tea leaves and tea dust, as well as green tea, Results are mean values of three determinations 6 standard devia-
these authors showed that the reducing power did not always tion.
correlate with antioxidative properties determined in free- Values sharing the same letter in a row are not significantly different
radical in vitro tests. (p ,0.05).

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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160 Antioxidant activity and phenolic components of Ginkgo 1157

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-
Figure 3. Antioxidant activity based on the average b-carotene
erties of Ginkgo extracts, which come chiefly from the flavo-
bleaching. Results are mean values of three determinations
noid fraction, may inactivate radicals such as the superoxide
6 standard deviation. Abbreviations are defined in Table 2.
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 b-carotene bleaching. The performance of extracts from yel-
closely related to their accumulation levels over the course of low Ginkgo leaves was better than that of green ones, which
the growing season; during this time, both the proportions and was in direct contrast to the results afforded by the DPPH
forms of the bioactives vary [42–44]. radical-scavenging assay. Based on further correlation analy-
Shi and Niki [45] calculated stoichiometrically the anti- ses, it was found that a positive relationship existed between
radical activity of an aqueous acetone extract of Ginkgo leaves antioxidant activity as assessed by the emulsion system assay
(Egb 761). Following quantitative analysis of the active com- and isorhamnetin (r = 0.76) and myricetin (r = 0.81) con-
pounds, the authors selected two bioactives present in the tents. According to Gramza et al. [46], hydrophilic anti-
largest quantities (i.e. quercetin and kaempferol) and com- oxidants such as flavonols are very effective in emulsions. In
pared the results to that of a standard of a-tocopherol, which is their study, Roedig-Penman and Gordon [47] reported a
commonly considered as an effective natural antioxidant. strong antioxidant action of myricetin and quercetin in an oil-
Based on the chemistry of the molecules, the mathematical in-water emulsion. They concluded that the antiradical prop-
calculations revealed that a-tocopherol can inactivate one erties of myricetin and its ability to chelate metals accounted
molecule of the free radical whereas quercetin and kaempferol for the lion’s share of the observed antioxidant effect, while in
can inactivate 4.0 and 1.9, respectively. The authors con- the case of quercetin, it was only its metal-chelating capacity
firmed their preliminary assumptions by analyzing the kinetics that participated because of its orientation in the o/w layer.
of inactivation reactions of free radicals using Ginkgo com- Investigations on the effect of crude Ginkgo extracts to
ponents with bioactivity. stabilize the TAG of rapeseed oil using the accelerated oxida-
Antioxidant activities of extracts were investigated using a tion test with a Rancimat system were performed and the
peroxidating linoleic system in an emulsion model with b- findings are indicated in Table 4. Induction periods were
carotene; the findings are depicted in Fig. 3. In this model, established for the analyzed samples based on the increase in
free radicals generated from linoleic acid attack highly unsa- water conductivity caused by the dissociation of volatile car-
turated b-carotene, resulting in a loss of color of b-carotene boxylic acids formed in the course of oil oxidation. It was
molecules (i.e. bleaching). The presence of various anti- found that all analyzed Ginkgo extracts extended the induc-
oxidants can hinder the extent of b-carotene bleaching by tion periods of TAG, devoid of native antioxidants, by as
neutralizing the linoleate free radicals formed during oxidation much as 40%. The longest induction period for the analyzed
[22]. All Ginkgo extracts exhibited antioxidant properties in extracts was recorded for GA and GE (i.e. at a concentration
relation to the linoleic acid/b-carotene emulsion system. of 500 ppm), accounting for 10.90 and 10.15 h, respectively.
Similar to the findings from the DPPH. assay, aqueous aceto- On the other hand, the lowest PF was afforded by water infu-
nic extracts showed the highest degree of inhibition of linoleic sion extracts. It was also noted that an increase in the addition
acid oxidation. After 90 min of incubation at 50 7C, the level of extracts to 500 ppm (i.e. more antioxidant com-
absorbances of the GA and YA extracts were 0.561 and 0.657, pounds) gave larger values of PF for all analyzed samples,
respectively. A weaker antioxidant activity was found for the ranging from 1.21 for YE to 1.34 for the GA extract. The best
GW and YW water infusion preparations; after 90 min, the antioxidant properties under the adopted testing conditions
absorbance readings were 0.445 and 0.496, respectively. were, however, recorded for the synthetic antioxidant BHT,
Ethanolic extracts demonstrated the weakest activity against which gave an induction period of 11.21 h. No correlation of

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

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

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Eur. J. Lipid Sci. Technol. 2009, 111, 1150–1160 Antioxidant activity and phenolic components of Ginkgo 1159

References tivity of selected plant species from the Canadian prairies.

Food Chem. 2004, 84, 551–562.
[1] T. A. van Beek: Ginkgolides and bilobalide: Their physical, [21] R. Amarowicz, M. Naczk, F. Shahidi: Antioxidant activity of
chromatographic and spectroscopic properties. Biorg Med crude tannins of canola and rapeseed hulls. J Am Oil Chem
Chem. 2005, 13, 5001–5012. Soc. 2000, 77, 957–961.
[2] L. M. L. Goh, P. J. Barlow: Flavonoid recovery and stability [22] X. Liu, M. Dong, X. Chen, M. Jiang, X. Lv, G. Yan: Anti-
from Ginkgo biloba subjected to a simulated digestion process. oxidant activity and phenolics of an endophytic Xylaria sp.
Food Chem. 2004, 86, 195–202. from Ginkgo biloba. Food Chem. 2007, 105, 548–554.
[3] P. Del Tredici: Ginkgos and people – A thousand years of [23] M. M. Cowan: Plant products as antimicrobial agents. Clin
interaction. Arnoldia. 1991, 51, 2–15. Microbiol Rev. 1999, 12, 564–582.
[4] T. A. van Beek: Chemical analysis of Ginkgo biloba leaves and [24] N. Turkmen, F. Sari, Y. S. Velioglu: Effects of extraction sol-
extracts. J Chromatogr A. 2002, 21, 21–51. vents on concentration and antioxidant activity of black and
[5] M.-J. Dubber, I. Kanfer: High-performance liquid chromato- black mate tea polyphenols determined by ferrous tartrate and
graphic determination of selected flavonols in Ginkgo biloba Folin-Ciocalteu methods. Food Chem. 2006, 99, 835–841.
solid oral dosage forms. J Pharm Pharm Sci. 2004, 7, 303–309. [25] P. Pietta, P. Mauri, A. Bruno, A. Rava, E. Manera, P. Ceva:
[6] B. Singh, P. Kaur, D. Gopichan, R. D. Singh, P. S. Ahuja: Bi- Identification of flavonoids from Gingko biloba L., Anthemis
ology and chemistry of Ginkgo biloba. Fitoterapia. 2008, 79, nobilis L., Equisetum arvense L. by high-performance liquid
401–418. chromatography with diode-array UV detection. J Chroma-
togr. 1991, 553, 223–231.
[7] M. Krauze-Baranowska, T. Ba˛czek, D. Głód, R. Kaliszan, E.
Wollenweber: HPLC separation of O-acylated flavonoids and [26] A. Hasler, O. Sticher, B. Meier: Identification and determina-
biflavones from some species of Gymnospermae. Chromato- tion of the flavonoids from Gingko biloba by high-performance
graphia. 2004, 60, 9–15. liquid chromatography. J Chromatogr A. 1992, 605, 41–48.
[8] E. Sikora, E. Cieślik, K. Topolska: The sources of natural [27] S. Ding, E. Dudley, S. Plummer, J. Tang, R. P. Newton, A. G.
antioxidants. Acta Sci Pol Technol Alim. 2008, 7, 5–17. Brenton: Fingerprint profile of Ginkgo biloba nutritional sup-
plements by LC/ESI-MS/MS. Phytochemistry. 2008, 69,
[9] N. J. Miller, M. B. Ruiz-Larrea: Flavonoids and other plant 1555–1564.
phenols in the diet: Their significance as antioxidants. J Nutr
Environ Med. 2002, 12, 39–51. [28] S. A. B. E. van Acker, G. P. van Balen, D.-J. van den Berg, A.
Bast, W. J. F. van der Vijgh: Influence of iron chelation on the
[10] C. Manach, A. Scalbert, C. Morand, C. Rémésy, L. Jiménez: antioxidant activity of flavonoids. Biochem Pharmacol. 1998,
Polyphenols: Food sources and bioavailability. Am J Clin Nutr. 56, 935–943.
2004, 79, 727–747.
[29] J. Yu, M. Ahmedna, I. Goktepe: Effects of processing methods
[11] M. Škerget, P. Kotnik, M. Hadolin, A. R. Hraš, M. Simonič, and extraction solvents on concentration and antioxidant ac-
Ž. Knez: Phenols, proanthocyanidins, flavones and flavonols tivity of peanut skin phenolics. Food Chem. 2005, 90, 199–206.
in some plant materials and their antioxidant activities. Food
Chem. 2005, 89, 191–198. [30] L. Mira, M. T. Fernandez, M. Santos, R. Rocha, M. H. Flor-
êncio, K. R. Jennings: Interactions of flavonoids with iron and
[12] J. G. Sebranek, V. J. H. Sewalt, K. L. Robbins, T. A. Houser: copper ions: A mechanism for their antioxidant activity. Free
Comparison of a natural rosemary extract and BHA/BHT for Radic Res. 2002, 36, 1199–1208.
relative antioxidant effectiveness in pork sausage. Meat Sci.
2005, 69, 289–296. [31] T. M. Fernandez, M. M. Lurdes, H. M. Florêncio, K. R. Jen-
nings: Iron and copper chelation by flavonoids: An electro-
[13] S. Burda, W. Oleszek: Antioxidant and antiradical activities of spray mass spectrometry study. J Inorg Biochem. 2002, 92,
flavonoids. J Agric Food Chem. 2001, 49, 2774–2779. 105–111.
[14] J. Kobus, E. Flaczyk: Optimization of acetone-water extrac- [32] M. Y. Moridani, J. Pourahmad, H. Bui, A. Siraki, P. J. O’Brien:
tion of natural antioxidants of Ginkgo biloba leaves. Pol J Hum Dietary flavonoid iron complexes as cytoprotective super-
Nutr Metab. 2007, 34, 1430–1434. oxide radical scavengers. Free Radic Biol Med. 2003, 34, 243–
[15] E. Flaczyk, R. Amarowicz, J. Korczak: Antioxidant activity of 253.
protein hydrolyzates from by-products of the food industry. J [33] R. F. V. de Souza, W. F. De Giovanni: Antioxidant properties
Food Lipids. 2003, 10, 129–140. of complexes of flavonoids with metal ions. Redox Rep. 2004,
[16] H. Chimi, J. Cillard, P. Cillard: Autoxidation of argan oil 9, 97–104.
Argania spinoza L. from Morocco. Sci Aliments. 1994, 14, [34] K. Schwarz, S.-W. Huang, J. B. German, B. Tiersch, J. Hart-
117–124. mann, E. N. Frankel: Activities of antioxidants are affected by
[17] E. Flaczyk, M. Rudzińska, E. Wa˛sowicz, J. Korczak, R. colloidal properties of oil-in-water and water-in-oil emulsions
Amarowicz: Effect of cracklings hydrolysates on oxidative and bulk oils. J Agric Food Chem. 2000, 48, 4874–4882.
stability of pork meatballs fat. Food Res Int. 2006, 39, 924–931. [35] É. Stefanovits-Bányai, K. Szentmihályi, A. Hegedűs, N.
[18] L. M. Cheung, P. C. K. Cheung, V. E. C. Ooi: Antioxidant Koczka, L. Váli, G. Taba, A. Blázovics: Metal ion and anti-
activity and total phenolics of edible mushrooms extracts. Food oxidant alterations in leaves between different sexes of Ginkgo
Chem. 2003, 81, 249–255. biloba L. Life Sci. 2006, 78, 1049–1056.
[19] S. Z Tang, J. P. Kerry, D. Sheehan, D. J. Buckley: Anti- [36] G.-C. Yen, H.-Y. Chen: Antioxidant activity of various tea
oxidative mechanisms of tea catechins in chicken meat sys- extracts in relation to their antimutagenicity. J Agric Food
tems. Food Chem. 2002, 76, 45–51. Chem. 1995, 43, 27–32.
[20] R. Amarowicz, R. B. Pegg, P. Rahimi-Moghddam, B. Barl, J. [37] M. B. Ningappa, R. Dinesha, L. Srinivas: Antioxidant and
A. Weil: Free-radical scavenging capacity and antioxidant ac- free radical scavenging activities of polyphenol-enriched curry

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

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

leaf (Murraya koenigii L.) extracts. Food Chem. 2008, 106, [44] A. Torres de Pinedo, P. Peñalver, J. C. Morales: Synthesis and
720–728. evaluation of new phenolic-based antioxidants: Structure-ac-
[38] R. Amarowicz, O. Narolewska, M. Karamać, A. Kosińska, S. tivity relationship. Food Chem. 2007, 103, 55–61.
Weidner: Grapevine leaves as a source of natural antioxidants. [45] H. Shi, E. Niki: Stoichiometric and kinetic studies on Ginkgo
Pol J Food Nutr Sci. 2008, 58, 73–78. biloba extract and related antioxidants. Lipids. 1998, 33, 365–
370.
[39] M. Naczk, R. Amarowicz, R. Zadernowski, R. Pegg, F. Sha-
hidi: Antioxidant activity of crude phenolic extracts from wild [46] A. Gramza-Michałowska, M. He˛ś, J. Korczak: Tea extracts
blueberry leaves. Pol J Food Nutr. Sci. 2003, 12/53, 166–169. antioxidative potential in emulsified lipid systems. Acta Sci Pol
Technol Alim. 2008, 7, 29–34.
[40] R. Farhoosh, G. A. Golmovahhed, M. H. H. Khodaparast:
[47] A. Roedig-Penman, M. H. Gordon: Antioxidant properties of
Antioxidant activity of various extracts of old tea leaves and
myricetin and quercetin in oil and emulsions. J Am Oil Chem
black tea wastes (Camellia sinensis L.). Food Chem. 2007, 100,
Soc. 1998, 75, 169–180.
231–236.
[48] I. Medina, I. Tombo, M. T. Satué-Gracia, J. B. German, E. N.
[41] M. Ellnain-Wojtaszek, Z. Kruczyński, J. Kasprzak: Investiga- Frankel: Effects of natural phenolic compounds on the anti-
tion of free radical scavenging activity of Gingko biloba L. oxidant activity of lactoferrin in liposomes and oil-in-water
leaves. Fitoterapia. 2003, 74, 1–6. emulsions. J Agric Food Chem. 2002, 50, 2392–2399.
[42] M. Ellnain-Wojtaszek, Z. Kruczyński, J. Kasprzak: Analysis of [49] M. Nogala-Kałucka, J. Korczak, M. Dratwia, E. Lampart-
the content of flavonoids, phenolic AIDS as well as free radical Szczapa, A. Siger, M. Buchowski: Changes in antioxidant ac-
from Ginkgo biloba L. leaves during the vegetative cycle. Acta tivity and free radical scavenging potential of rosemary extract
Pol Pharm. 2001, 58, 205–209. and tocopherols in isolated rapeseed oil triacylglycerols during
[43] M. Ellnain-Wojtaszek, G. Zgórka: High-performance liquid accelerated tests. Food Chem. 2005, 93, 227–235.
chromatography and thin-layer chromatography of phenolic [50] C. G. Beddows, C. Jagait, M. J. Kelly: Preservation of a-toco-
acids from Ginkgo biloba L. leaves collected within vegetative pherol in sunflower oil by herbs and spices. Int J Food Sci Nutr.
period. J Liq Chromatogr Relat Technol. 1999, 22, 1457–1471. 2000, 51, 327–339.

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com