Metal ion and antioxidant alterations in leaves between different sexes of Ginkgo biloba L

lsevier.com/locate/lifescie

Life Sciences 78 (200

Metal ion and antioxidant alterations in leaves between different sexes of

Ginkgo biloba L.

Eva Stefanovits-Banyai a, Klara Szentmihalyi b, Attila Hegedus a, Noemi Koczka c, Laszlo Vali d,

Gabriella Taba b,d, Anna Blazovics d,*

a Department of Applied Chemistry, Faculty of Food Science, Corvinus University of Budapest, P.O. Box 53, Budapest, Hungary, H-1518b Chemical Research Centre, Hungarian Academy, P.O. Box 17, Budapest, Hungary, H-1525

c Department of Horticultural Technology, Faculty of Agricultural and Environmental Sciences, Szent Istvan University, P.O. Box 53, Godollo, Hungary, H-2103d Department of Medicine, Semmelweis University of Medicine 2nd P.O. Box 277, Budapest, Hungary, H-1444

Received 10 January 2005; accepted 10 June 2005

Abstract

A comparative study was carried out to determine some valuable phytochemical components, macro- and microelement and redox parameters

in leaves of male and female Ginkgo biloba trees and in extracts made from them. G. biloba extracts have become more popular as a therapeutic

agent in the modern pharmacology in neurodegenerative diseases, in which increased brain metal levels can be observed and free radical reactions

are involved. Macro- and microelement components, total phenol content, H-donating activity and reducing power as well as total scavenger

capacity were determined in the samples. Well detectable differences were obtained for micro- and macroelement contents between male and

female samples, but no toxic elements could be detected in the extracts. Male extracts contained more hazardous metals (e.g. Fe) compared to the

female ones, while extracts from female leaves had higher levels of ions, which are known to have beneficial effects in neurodegenerative

diseases. The ethanolic extracts of male leaves showed the highest H-donating activity, reducing power and total phenol content, as well as the best

total scavenger activity. Ginkgo extracts due to the antioxidant properties may have favourable effects as dietary supplements in several

neurodegenerative diseases, but this study draws the attention that critical evaluation is required in view of the potential hazard induced by their

metal ion constitution. Our results lead us to the conclusion that although the aqueous extracts of female leaves are characterized by relatively

lower antioxidant properties, they may be more eligible for these purposes due to their favourable metal ion constitution.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Antioxidants; Ginkgo biloba; Metal elements; Neurodegenerative diseases; Phenol

Introduction

Ginkgo biloba L. is the most ancient living gymnosperm in

the world. As being the only representative of the Ginkgoaceae

family, it has often been called a ‘‘living fossil’’ by Charles

Darwin (Michel, 1986).

In the past few years G. biloba was one of the most

extensively studied species. Scientific attention is needed

because leaf-extract seems to have various curative effects

and excellent antioxidant capacity (Schilcher, 1988; Lugasi et

al., 1999; Ellnain-Wojtaszek et al., 2002).

0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.lfs.2005.06.012

* Corresponding author. Tel.: +36 1 266 0926; fax: +36 1 266 0926.

E-mail address: [email protected] (A. Blazovics).

Many data support the efficacy of G. biloba extracts in

biological systems, including in vitro and in vivo experiments

and its therapeutic efficacy was also observed in clinical trials

of elderly patients and patients with neurodegenerative diseases

(Droy-Lefaix, 1997; Akiba et al., 1998; Lugasi et al., 1999;

Wei et al., 2000; Kim, 2001; Bush, 2002; Maynard et al.,

2002). In ageing processes, G. biloba may ameliorate the

mitochondria respiratory chain function by quenching the

superoxide anion, and the hydroxyl and peroxyl radicals. It

protects the brain by facilitating the uptake of neurotransmitters

and by reducing ischemia–reperfusion episodes and level of

apoptosis (Droy-Lefaix, 1997). Some clinical data exist, that G.

biloba extracts might be used as an effective drug for the

treatment of neuronal diseases associated with the production

of peroxynitrite (Wei et al., 2000). G. biloba has a nonsteroidal

6) 1049 – 1056

www.e

Table 1

Ginkgo biloba (L.) samples and extracts

Marks of samples Sex Extract

0911 FW Female Aqueous

0911 MW Male Aqueous

0911 FA Female Ethanolic

0911 MA Male Ethanolic

E. Stefanovits-Banyai et al. / Life Sciences 78 (2006) 1049–10561050

anti-inflammatory property on Alzheimer’s disease (Dorais-

wamy, 2002).

As a result of wide ranging research, flavonol-glycosides,

terpene lactones, ginkgolides A, B, C, bilobalide, amino acids

in G. biloba leaves were identified (Hodisan et al., 1998; Lolla

et al., 1998; Yuping and Fengchang, 2000). The G. biloba

extract showed copper-binding property (Lugasi et al., 1999).

Ginkgolic acids, the toxic phenolic compounds are also present

in the fruits and leaves of G. biloba L. Polyphenol and

flavonoid contents are responsible for radical scavenger

activity (Lugasi et al., 1999). Antioxidant properties of

polyphenols and flavonoids were identified on the apoptotic

processes in hippocampal cell cultures (Barkats et al., 1995;

Bastianetto et al., 2000). Ginkgolide B and bilobalide but not

its terpenoid constituents may play a beneficial role in

oxidative stress, to protect against free radicals through actions

on heme oxygenase gene expression and activity, and also

significantly increased GPx gene expression and GPx enzyme

activity (Chen et al., 2001).

However, meagre number of data is known about the metal

ion composition of G. biloba. Supraoptimal concentration of

several metals e.g. Fe, Zn, Cu, Al or Mn have toxic actions on

nerve cells and neurobehavioral functioning, which can be

expressed either as developmental effects or as an increased

risk of neurodegenerative diseases in old age. Tissue injury,

e.g. by ischemia or trauma, can cause increased metal ion

availability and accelerate free radical reactions, highly reactive

hydroxyl radical and other oxidants in the presence of

Fcatalytic_ Fe or Cu ions. Free radical reactions are involved

in the neurotoxicity of Al and in damage to the substantia nigra

in patients with Parkinson’s disease and beta amyloid

accumulation in the plaques are reviewed (Halliwell, 1992;

Bush, 2002). Mn neurotoxicity could be related to the

capability of this metal to increase catechol autooxidation in

catecholaminergic neurons, therefore, increasing the formation

of toxic compounds such as peroxides, superoxides, free

radicals, and semi-orthoquinones (Vescovi et al., 1989). Beta

amyloid aggregation is not spontaneous. The process is age

dependent and belongs to the metal elements, which induces

the protein to precipitate into metal-enriched masses. In this

precipitating process hydrogen peroxide, superoxide anion and

hydroxyl radicals take part (Bush, 2002). Beta amyloid is a

result of misprocessing of amyloid precursor protein, and

gamma-secetase is involved in the process (Torp et al., 2003).

Amyloid beta /Fe2+ can induce apoptotic death in neuronal

cells. Metal ion chelation and inhibitors of pro-apoptotic kinase

cascades may be beneficial in therapy of Alzheimer’s disease

(Kuperstein and Yavin, 2003). The ability of Al to alter the

degradation of beta amyloid suggests a way in which Al and

amyloid neurotoxic substances might interact (Banks et al.,

1996). The hippocampal Zn is decreased in Alzheimer disease,

and amyloid is formed within the walls of capillaries, disturb

the blood brain barrier and toxic metals may enter the cerebral

cortex, where they displace the zinc in Zn enzymes (Deloncle

and Guillard, 1990).

There is some epidemiological evidence for elevated risk of

Alzheimer’s disease in areas where there is high concentration

of aluminium in drinking water. The impacts of Al on humans

and its impact on major physiological systems, such as

neurological-system, musculoskeletal-, respiratory-, cardiovas-

cular-, hepatobiliary-, endocrine-, urinary-, and reproductive

system are well known (Nayak, 2002).

Other metals, especially Pb, Hg, Mn and Cu have been

implicated in amyotrophic lateral sclerosis and Parkinson’s

disease, as well (Carpenter, 2001). The activated transcellular

transport of Cu complexed with plant flavonoids and phenolic

carboxylic acids is ensured by a carrier protein, divalent cation

transporter (DCT1). Menkes protein transports it from the

endothelial cells of the brain-barrier to the brain. Free Cu(I) and

Cu(II) occur in the body in a small amount only; their

concentration is 10–18 mol/L in the plasma. These ions are

very reactive and catalyze the formation of oxygen free radicals

(Ferruzza et al., 1999). Ceruloplasmin can also be found in the

brain and has an oxidase function. Cu occurs in beta amyloid

protein and in prion caused Creutzfeld–Jacob disease (Siegel

and Siegel, 1981).

The metabolism of Zn is regulated by DCT1 carrier protein,

Zn transporters and metallothioneins. Zn is a key element in

superoxide dismutase. Zn-metallothionein has hydroxyl scav-

enging ability (Brando-Neto and Bell, 1994). Zn plays role in

the hippocampal neurons for memory, intellect, behaviour and

neuropsychological function (Sandstead et al., 2000).

Our purpose was to determine the metal ion content and

antioxidant/scavenger bioactive components in extracts of

female and male G. biloba leaves growing in Hungary to

prove their efficacy in vitro.

Materials and methods

Plant material

About 50 leaves from a similar position within the canopy

were collected in September (code number: 0911) 2003

severally from 3 male and 3 female G. biloba (L.) trees growing

at the Botanical Garden of Eotvos Lorand University, Budapest.

Preparation of G. biloba extracts

Extracts of G. biloba (L.) were prepared from young fresh

leaves. Leaves were cut into small pieces, and 5 g of fresh

leaves were infused with 100 ml boiling water or hot aqueous

ethanol (25 -C; water / ethanol 80 /20, v/v). The ethanolic and

aqueous extracts were stored at room temperature for 24 h.

After centrifugation (13,000 rpm, 10 min) the supernatant were

stored in a refrigerator until the analyses. Table 1 summarizes

the leaf samples and the extracts made.

Table 2

Element concentrationT standard deviation (Ag/g, n =3) of male and female

Ginkgo biloba leaves

Elements Male Female

Ala 50.18T0.9 74.17T1.5

Ba 167.8T2.0 147.6T1.5Baa 43.53T0.5 41.31T0.3

Caa 44,223T28 35,720T190

Cd 0.168T0.017 0.150T0.150

Fea 144.7T2.0 177.5T2.1Ka 11, 062T100 21,144T180

Mga 6369T50 6026T58

Mna 16.33T0.20 18.51T0.29

Naa 124.4T4.2 142.8T5.3Pa 2822T30 6265T45

Sra 173.6T1.3 127.3T1.5

Tia 1.807T0.101 2.547T0.001Zna 8.93T0.21 18.43T0.32a Significant differences between concentrations of elements of groups

(P <0.05).

E. Stefanovits-Banyai et al. / Life Sciences 78 (2006) 1049–1056 1051

Determination of element concentration

Dried leave samples (0.2 g) were digested in amixture of 2 ml

HNO3 and 2 ml H2O2 in teflon bomb (PTFE) for ICP

analysis. The digested samples were filled up with deionised

water to 10ml. The following elements were determined by ICP-

OES (Thermo Jarrell Ash Co, ICAP 61): Al, As, B, Ba, Ca, Cd,

Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Ni, P, Sr, Ti, V, Zn.

Determination of total phenol content

Total amount of soluble phenols were determined using

Folin–Ciocalteu’s reagent according to the method of Singleton

and Rossi (1965). The content of soluble phenols was calculated

from a standard curve obtained by different concentrations of

gallic acid.

H-donating activity

H-donating activity was determined by the method of Blois

(1958) modified by Hatano et al. (1988) in the presence of a

Table 3

Element concentration (Ag/100 ml) of Ginkgo biloba extracts

Elements Aqueous extract Eth

0911MW 0911 FW 09

Al 5.69T1.86 <dl 1

B 28.86T4.35 52.9T5.09 9

Ba <dl 8.87T0.89Ca 1172T69 2492T121 1

Fe 19.98T2.32 14.27T2.25 3

K 9279T289 7833T247 19

Mg 1696T211 3133T32 5

Mn 37.50T1.02 5.13T1.14

P 1161T38 2668T103 3

Sr 6.17T1.08 16.93T2.08 1

Zn 51.2T0.99 3.31T1.20 1

<dl under detection limit.

Significant difference ( P <0.05) between concentration of elements of four groups

1,1-diphenyl-2-picryl-hydrasyl radical (DPPH). Absorbance of

the methanolic DPPH-dye was assessed spectrophotometrically

at 517 nm. For characterization of the activity, inhibition

percent was given to the DPPH degradation.

Reducing power property

Oyaizu’s method (1986) was adopted for analysis of the

reducing property. The change in absorbance was measured

which accompanied Fe3+YFe2+ transformation at 700 nm, and

the reducing power property was compared to that of ascorbic

acid.

Total scavenger capacity

Recently developed chemiluminescense assay was adopted

(Blazovics et al., 1999). A Berthold Lumat 9501 instrument was

applied for the determination of the total scavenger capacity of

different G. biloba extracts. The emitted light was measured in

390–620 nm range by scanning. Reaction mixture contained

H2O2 (0.30 ml, 10�4 dilution of 33% H2O2), microperoxidase

(0.30 ml of 1 mM) as a catalyst and alkaline luminol solution

(pH 9.8) (in 0.050 ml of 0.07 mM). The emitted photons were

accumulated during a 30 s exposure time and are expressed in

relative light unit (RLU). The volumes of biological samples

were 0.050–0.200 ml, which were added to luminol solution

and mixed with vortex (10 s) before the tube was placed in a

holder. Each measuring point represents four parallel data in

luminol dependent chemiluminescence experiments.

Statistical analysis

Mean and SD and c.v. percent were calculated from the data.

For comparison of the results, Student’s t-test or ANOVA was

applied. Statistica 6.0 program was used.

Results

Table 2 demonstrates the element concentrations in dry

leaves of G. biloba in both sexes. Since the concentrations of

anolic extract Significance ( P <0.05)

11 MA 0911 FA

0.63T0.42 16.21T0.44 *,**,***

2.43T7.46 76.22T6.25 *,**,***

8.48T1.16 13.26T2.19 *,**,***

767T82 3410T166 *,**,***

2.26T2.83 26.80T2.05 *,**

,520T64 15,860T486 *,**,***

037T39 6324T52 *,**,***

8.69T1.85 12.01T3.01 *,**

032T118 5739T238 *,**,***

0.43T1.97 18.78T2.88 *,**,***

4.61T3.04 16.21T3.78 *,**

(*), of MW and FW (**), and MA and FA (***).

Table 4

Total polyphenol content of Ginkgo biloba extractsT standard deviation in mg/

100 ml solvent

Sample Total polyphenol content

0911 MW 52.49T2.77 mg/100ml

0911 FW 50.67T4.16 mg/100ml

0911 MA 74.57T3.25 mg/100ml

0911 FA 51.97T2.86 mg/100ml

Significance ( P <0.05) **, ***

Significant difference ( P <0.05) between contents of male and female leaves

(*), of four groups (**), and MA and FA (***).

E. Stefanovits-Banyai et al. / Life Sciences 78 (2006) 1049–10561052

As, Co, Cr, Cu, Hg, Li, Mo and Pb were below the detection

limit in each sample, therefore, these elements were omitted

from the table. In general the element concentration in leaves is

in a good agreement with data in the literature for average

element contents in other plant samples (Kabata-Pendias and

Pendias, 1992), although the levels of Na in all samples are less

enough and the levels of B in samples are higher. Significantly

higher (P <0.05) concentration was found for Al, Fe, K, Mn,

Na, P, Ti and Zn in female leaves and for B, Ba, Ca, Mg and Sr

in male samples. The concentrations of elements in extracts

varied depending on the sample and the solvent of extraction

(Table 3). In contrary to the other plant materials the ethanolic

extracts contain elements in higher concentrations compared to

the aqueous extracts, and the female ethanolic extract is

especially rich in elements, while the antioxidant Mn and Zn

in the highest amount were found in the aqueous extract of

male leaves.

Relatively high polyphenol contents were measured both

in leaves (ranging from 104 to 137 mg/g dry weight) and

extracts (Table 4). Significant differences have been measured

between the male and female leaves and between the

alcoholic extracts of dry male and female samples, while no

0

10

20

30

40

50

60

70

80

90

0 50 100 150

inh

ibit

ion

(%

)

volume (µl)

Fig. 1. H-donating activity. Alcoholic (A) and aqueous (W) extracts were prepared fr

each measuring point c.v. percent <5.

difference could be detected in the total phenol contents of

aqueous extracts.

Regarding the quality of plant extracts, it must be

considered that very different extracts can be pronounced.

Depending upon the ratio of water and alcohol, both hydro-

soluble and liposoluble components can be extracted. Poly-

phenols have an antioxidant or scavenger characteristic.

Elevated element concentrations in water phase may induce

free radical reactions. Therefore, we applied three independent

measuring systems (H-donating ability, reducing power prop-

erty, total scavenger capacity) to evaluate the antioxidant

ability of ethanolic (ethanol /water: 20 /80, v/v) and aqueous

extracts of G. biloba.

Figs. 1 and 2 show H-donating ability and reducing power

of the aqueous and alcoholic extracts of leaves. The strongest

antioxidant activity was determined in sample, 0911 MA,

which was prepared with 20% alcoholic solution from leaves of

a male tree, the alcoholic extract from leaves of a female tree

(0911 FA) had only significant effect on DPPH stable free

radical. Aqueous extracts showed smaller antioxidant effects in

this system and no difference was observed between two sexes

(Fig. 1).

Reducing power property (Fig. 2) was also better in 0911

MA than in 0911 FA, and the reducing power was lower in

both aqueous extracts. These results were in accordance with

the luminometric measurements, since the total scavenger

capacity was significantly better in 0911 MA extract compared

to the other samples.

G. biloba extracts were able to scavenge free radicals in

H2O2/OH microperoxidase system dependent of the dose, but it

can be seen that the extracts in lower concentrations increased

the light emission of the system (Fig. 3). Water extract of male

sample had higher antioxidant capacity (Fig. 4) than that of

y = 0,2803x + 5,0652R2 = 0,9962

y = 0,2113x + 2,2453R2 = 0,9928

y = 0,2088x + 1,2069R2 = 0,9952

y = 0,351x + 11,725R2 = 0,9904

200 250

0911 MW

0911 FW

0911 MA

0911 FA

om young fresh leaves of male (M) and female (F) Ginkgo biloba (L.) plants. In

y = 0,0055x + 0,0231R2 = 0,9943

y = 0,0036x + 0,0244R2 = 0,9986

y = 0,0021x + 0,0118R2 = 0,9914

y = 0,0025x + 0,0042R2 = 0,9956

0

0,2

0,4

0,6

0,8

1

1,2

0 50 100 150 200 250

(µm

ol a

sco

rbic

aci

d/m

l)

0911 MW

0911 FW

0911 MA

0911 FA

volume (µl)

Fig. 2. Reducing power of alcoholic (A) and aqueous (W) extractions prepared from young fresh leaves of male (M) and female (F) Ginkgo biloba (L.) plants. In

each measuring point c.v. percent <5.

E. Stefanovits-Banyai et al. / Life Sciences 78 (2006) 1049–1056 1053

female. This measuring system can be induced with trace of

metal ions; therefore, probably this catalytic effect can be seen

in the figures.

Discussion

Active compounds of G. biloba were evidenced to produce

physiological effects against the pathogenesis of some neuro-

degenerative diseases (Wei et al., 2000; Bush, 2002; Maynard

et al., 2002; Gong et al., 2005). Since differences between the

inner contents of leaves from trees of different sexes may occur

as well as extraction methods may differ in the dissolution

efficiency of significant compounds, we compared the aqueous

and ethanolic extracts of leaves from male and female trees in

this respect.

Increasingly, the etiology of some neurodegenerative dis-

eases has been linked to exposures to environmental toxicants.

Transition metals (e.g. Fe, Cu) as well as Zn or Al were shown to

be involved in the pathogenesis of Parkinson’s disease, tardive

0,00E+00

5,00E+06

1,00E+07

1,50E+07

2,00E+07

2,50E+07

3,00E+07

0 50 100 1

chem

ilum

ines

cen

ce (

RL

U)

1,

1,

1,

1,

1,

(RL

U)

volume (µ

Fig. 3. Total chemiluminescent intensity of alcoholic (A) extractions made from you

measuring point c.v. percent <5.

dyskinesia, metal intoxication syndromes, Down’s syndrome,

and possibly also in schizophrenia, Huntington’s disease, where

free radicals have been implicated, as well. The abnormal

combination of trace elements with beta amyloid takes part in

the free radical formation (Bush, 2002; Maynard et al., 2002).

Well detectable differences were obtained between the

element compositions of the two sexes. Element content of

plant tissues on the one hand may vary according to weather,

soil or environmental conditions, which highlights the fact that

extracts for curative purposes must be carefully checked and

only those possessing favourable element constitution and free

from harmful substances must be allowed for use. In a recent

comprehensive study it was mentioned that in most countries

the standard quality control of medicinal plants is not always

enforced (Caldas and Machado, 2004). On the other hand, the

extraction methods used in this study highly influenced the

element content of extracts.

Aluminium concentration was significantly higher in

ethanolic extracts, especially in case of female leaves. Fe

50 200 250

0911 MA

0911 FA

00E+00

00E+02

00E+04

00E+06

00E+08

0 100 200 (µl)

l)

ng fresh leaves of male (M) and female (F) Ginkgo biloba (L.) plants. In each

0,00E+00

5,00E+06

1,00E+07

1,50E+07

2,00E+07

2,50E+07

3,00E+07

3,50E+07

0 50 100 150 200 250

chem

ilum

ines

cen

ce (

RL

U)

0911 MW

0911 FW

1,00E+00

1,00E+02

1,00E+04

1,00E+06

1,00E+08

0 100 200 (µl)

(RL

U)

volume (µl)

Fig. 4. Total chemiluminescent intensity of aqueous (W) extractions made from young fresh leaves of male (M) and female (F) Ginkgo biloba (L.) plants. In each

measuring point c.v. percent <5.

E. Stefanovits-Banyai et al. / Life Sciences 78 (2006) 1049–10561054

content of male leaf-extracts was slightly higher than that of

female leaf-extracts. Fe and Al are two metal ions, which

concentration is increased in the substantia nigra destruction of

neurons, in the substantia nigra pars compacta of the basal

ganglia of patients with Parkinson’s disease, and both metal

elements are suspected to be involved in the pathophysiology

of Alzheimer’s disease. Al induced brain dysfunction was

improved by Ginkgo extracts (Gong et al., 2005). Total Fe is

increased and ferritin is reduced in the zona compacta in

patients with Parkinson’s disease. Transition metals, as Fe and

Cu, may reduce dioxygen molecule to form superoxide radical

and catalyze Fenton reaction under aerobic circumstances and

lead to a membrane and cell damage (Siegel and Siegel,

1999a).

Mn is essential in the antioxidant defence system, since

superoxide dismutase enzyme, which contains Mn, scavenges

superoxide anions. Mn and Zn contents were 4.3- and 3.5-

times higher in aqueous extracts of male leaves compared to

those in the ethanolic extracts. Mn(II) is an antioxidant, since in

fast reaction it exterminates the alkyl peroxyl radicals formed

by peroxidation of fatty acids, while Fe(II) ions generate

alkoxy and hydroxyl radicals and continue the chain reaction

(Kies, 1987; Siegel and Siegel, 1999a). Mn(II) ions, similarly

to Zn ions, are able to decrease the formation of superoxide

radicals (Schramm and Wedler, 1986).

In the blood, Mn(II) ions are in the form of free aqua-

complexes or bounded to glycoproteins, but when oxidized,

Mn(III) ions change apotransferrine to transmanganine. Mn can

be transported through the blood–brain barrier (Siegel and

Siegel, 1999b), therefore in a supraoptimal concentration it

primarily damages the globus pallidus and substantia nigra pars

reticularis and relatively spares the nigrostriatal dopaminergic

system (Olanow et al., 1996).

It must be highlighted that our results revealed clearly

higher Mg content in extracts from female leaves compared to

male leaf-extracts in case of both extraction methods. Mg-rich

Ginkgo extract may contribute to the regeneration of brain or

brain barrier and change the generally low concentrations of

Mg and other elements (such as Ca, K, Na, P, S and Zn)

favourable in neurological diseases (Yasui et al., 1992).

Calcium, magnesium, sodium, and potassium are involved in

maintaining balance of sympathetic and parasympathetic

systems of the autonomic nervous systems. Mg takes place in

several enzyme functions and it is one of the most important

factors in the homeostasis of elements.

Antioxidant defence mechanisms appear to be reduced in

the parkinsonian substantia nigra with the findings of decreased

activities of glutathione peroxidase and catalase (Fahn and

Cohen, 1992). Since free radical induced oxidative reactions

are also involved in the pathogenesis of several neurodegen-

erative diseases (Halliwell, 1992), antioxidant properties of

curative agents may be of great consideration. Chen et al.

(2001) described that the extracts of G. biloba indeed induces

several antioxidant responses in human cells. We demonstrated

that total content of the antioxidant phenols involving poly-

phenols, flavonoids etc. is outstanding in case of ethanolic

extracts from male leaves. Significantly higher total phenol

content occurred in leaves of male trees and their ethanolic

extracts, while in aqueous extracts no difference could be

detected between the two sexes, which indicate that the

phenolic compounds particularly accumulating in male leaves

are characterized by a preferably liposoluble nature. Similar

deviations in the dissolution efficiency of several phenolic

compounds in ethanolic and aqueous extracts were reported by

Goh and Barlow (2004).

All the measured antioxidant parameters were in a good

accordance with the phenol content of samples. Ethanolic

extracts of male leaves were shown to possess the highest H-

donating ability, which represents the chain-breaking property

of extracts, a feature of how efficiently can an antioxidant

molecule serve H to free radicals and neutralize them

(Blazovics et al., 2003).

Ethanolic male extracts also produced the highest values in

case of the reducing power, which is an index of secondary

E. Stefanovits-Banyai et al. / Life Sciences 78 (2006) 1049–1056 1055

antioxidation. Secondary antioxidants can reduce the rate of

chain reaction initiation in the lipid peroxidation process or can

convert the lipid peroxidation end products to non-radical

compounds. In accordance with it, our chemiluminescent assay

also verified the strong total scavenger capacity of male

extracts. Several lines of evidence confirm that Ginkgo extracts

proved to be useful against many diseases coupled with

oxidative challenge of tissues (Droy-Lefaix, 1997; Akiba et

al., 1998; Mahady, 2001).

Conclusion

Our results lead us to the conclusion that due to the

antioxidant properties Ginkgo extracts may have favourable

effects as dietary supplements in several neurodegenerative

diseases, but critical evaluation is required in view of the

potential hazard induced by their metal ion constitution. Hence,

although the aqueous extracts of female leaves are character-

ized by relatively lower antioxidant properties, due to their

favourable metal ion constitution they may be more eligible for

these purposes.

Acknowledgements

This study was supported by 1/016 Szechenyi Project, ETT

002/2003 and by the Hungarian National Scientific Research

Fund (OTKA T030165, T043063). The authors would like to

express their thanks to Mrs. Edina Pinter, Mrs. Sarolta

Barkovits and Mrs. Katalin Kertesz for their excellent technical

assistance.

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