lsevier.com/locate/lifescie

Life Sciences 78 (20

Reactive oxygen species scavenging capacity of different

cooked garlic preparations

Jose Pedraza-Chaverrı *, Omar N. Medina-Campos, Rosaura Avila-Lombardo,

Alma Berenice Zuniga-Bustos, Marisol Orozco-Ibarra

Facultad de Quımica, Edificio B, Segundo Piso, Laboratorio 209, Departamento de Biologıa, Universidad Nacional Autonoma de Mexico (UNAM),

Ciudad Universitaria, 04510, Mexico, D.F., Mexico

Received 4 March 2005; accepted 16 May 2005

Abstract

It was studied if the ability of aqueous garlic extracts to scavenge superoxide anion (O2&�), hydrogen peroxide (H2O2), and hydroxyl radical

(OH&) is altered in the following aqueous preparations: (a) extracts of boiled garlic cloves (BG), (b) extracts of microwave-treated garlic cloves

(MG), and (c) extracts of pickled garlic (PG), and heated extracts of (a) garlic powder (HGP) and (b) raw garlic (HRG). The data were compared

with the unheated raw garlic (RG) or with the unheated garlic powder (GP). Extracts of GP and RG scavenged O2&�, H2O2, and OH& in a

concentration-dependent way. The reactive oxygen species scavenging capacity was not decreased in the aqueous garlic extracts except in MG and

HRG (for O2&�) and in HGP and PG (for H2O2). The heating before or after garlic cutting was unable to eliminate the capacity of the extracts to

scavenge H2O2, O2&�, and OH&.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Raw garlic; Pickled garlic; Microwave-treated garlic; Boiled garlic; Superoxide anion; Hydrogen peroxide; Hydroxyl radical

Introduction

Garlic (Allium sativum) has been cultivated since ancient

times and used as a spice and condiment for many centuries

(Block, 1985). Ninety-five percent of the sulfur in intact garlic

cloves is found in two classes of compounds in similar

abundance: the S-alkylcysteine sulfoxides and the g-glu-

tamyl-S-alkylcysteines (Lawson, 1998). The most abundant

sulfur compound in garlic is alliin (S-allylcysteine sulfoxide),

which is present at 10 mg/g fresh garlic or 30 mg/g dry weight

(Lawson, 1998). When garlic cloves are cut, crushed, or

chopped (or when the powder of dried cloves becomes wet in a

non-acid solution), the cysteine sulfoxides, which are odorless,

are very rapidly converted to a new class of compounds, the

thiosulfinates, which are responsible for the odor of freshly

chopped garlic. The formation of thiosulfinates takes place

when the cysteine sulfoxides, which are located only in the

clove mesophyll storage cells, come in contact with the enzyme

allinase or alliin lyase (E.C. 4.4.1.4), which is located only in

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

doi:10.1016/j.lfs.2005.05.075

* Corresponding author. Tel./fax: +52 55 5622 3515.

E-mail address: pedraza@servidor.unam.mx (J. Pedraza-Chaverrı).

the vascular bundle sheath cells. Allinase is active at pH 4–5.8,

but is immediately inhibited at acidic pH values below 3.5 or

by cooking. Furthermore, microwave heating destroys allinase

activity in 1 min (Song and Milner, 1999). Due to the

abundance of alliin, the main thiosulfinate formed upon

crushing garlic is allicin (Lawson, 1998). The half-life of

allicin at room temperature is 2–16 h; however, in crushed

garlic (or in garlic juice) it is 2.4 days (Lawson, 1998).

During the past years, there has been a growing awareness of

the potential medicinal uses of garlic (Banerjee et al., 2002b;

Pedraza-Chaverri et al., 1998). The antioxidant properties of

garlic are well documented (Banerjee et al., 2002b; Dillon et al.,

2002; Rahman, 2003; Pedraza-Chaverri et al., 2000; Sener et

al., 2003, 2005; Gedik et al., 2005). Diet with 2% of garlic

powder decreased the ischemia and reperfusion-induced

arrhythmias (Rietz et al., 1993) and the acute renal failure and

oxidative stress induced by gentamicin (Pedraza-Chaverri et al.,

2000). Garlic feeding also decreased lipid peroxidation and

prevented the decrease in glutathione peroxidase activity in red

blood cells of mice treated with adriamycin (Thabrew et al.,

2000). In addition, chronic administration of raw garlic

homogenate protects heart against oxidative damage induced

06) 761 – 770

www.e

J. Pedraza-Chaverrı et al. / Life Sciences 78 (2006) 761–770762

by ischemia and reperfusion (Mukherjee et al., 2002). Fur-

thermore, it has been found that aqueous extract of raw garlic

scavenges hydroxyl radical (OH&) (Prasad et al., 1996; Kim et

al., 2001) and superoxide anion (O2&�) (Kim et al., 2001),

inhibits lipid peroxidation (Prasad et al., 1996; Yin and Cheng,

1998a), Cu2+-induced lipoprotein oxidation (Pedraza-Chaverri

et al., 2004b), and the formation of lipid hydroperoxides (Prasad

et al., 1996; Kim et al., 2001). Furthermore, aqueous extracts of

0

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0 0.3 0.7 2.6 3.1 84.1 101.5

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[GP], mg/mL

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[MG], mg/mL

0 0.1 0.4 1 2 4 8 15 20

[BG], mg/mL

0 0.1 0.3 0.6 1.5 3 15 30 60

[NDGA], µg/mL

A

C

E

G

O2.- SCAVENGIN

Fig. 1. Effect of extract of garlic powder (GP) (panel A), heated extract of garlic p

garlic cloves (BG) (panel E), heated extract of raw garlic (HRG) (panel F), extract of

(PG) (panel H), and of nordihydroguaiaretic acid (NDGA) (panel C) on O2&� scaveng

the assay system and was read at 560 nm (open bars). Xanthine oxidase activity was

percent of the values obtained in 0 mg/mL and are presented as meanTSEM, n =4–

mg/mL (uric acid production).

garlic powder are also able to inhibit lipoprotein oxidation

(Pedraza-Chaverri et al., 2004b) and to scavenge OH& (Lewin

and Popov, 1994) and O2&�(Torok et al., 1994).

Several studies have been performed to test the effect of

heating on several garlic properties. The OH& scavenging

properties of garlic was essentially preserved when garlic

extracts were heated at 100 -C by 20, 40 or 60 min (Prasad et

al., 1996). Yin and Cheng (1998a) found that heating treatment

0 0.3 0.7 2.6 3.1 84.1 101.5

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[HGP], mg/mL

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0 0.1 0.4 1 2 4 8 15 20

[RG], mg/mL

B

D

F

H

G CAPACITY

owder (HGP) (panel B), extract of raw garlic (RG) (panel D), extract of boiled

microwaved-treated garlic cloves (MG) (panel G), and extract of pickled garlic

ing capacity and uric acid production. Formazan was used as a measure of O2&�in

measured as uric acid production (closed bars) at 295 nm. Data are expressed as

7. ap <0.05, bp <0.001 vs. 0 mg/mL (O2&� in the assay system); cp <0.001 vs. 0

Table 1

O2&� scavenging capacity of nordihydroguaiaretic acid (NDGA) and extracts of

garlic powder (GP) and heated garlic powder (HGP)

Garlic extract or standard IC50, mg/mL

NDGA (7) 0.0031T0.00014

GP (4) 2.5160T0.4640a

HGP (4) 2.6610T0.3171a

Data are meanTSEM. ap <0.001 vs. NDGA. Number of determinations are in

parentheses.

Table 2

O2&� scavenging capacity of nordihydroguaiaretic acid (NDGA), extract of raw

garlic (RG), extract of boiled garlic cloves (BG), heated extract of raw garlic

(HRG), extract of microwave-treated garlic cloves (MG), and extract of pickled

garlic (PG)

Garlic extract or standard IC50, mg/mL

NDGA (7) 0.0031T0.00014

RG (7) 1.9270T0.5620a

BG (7) 2.4910T0.3000b

HRG (5) 7.8370T0.8088c,d

MG (6) 7.7100T0.7401c,d

PG (5) 2.8370T0.4422b

Data are meanTSEM. ap <0.05, bp <0.01, cp <0.001 vs. NDGA, dp <0.001 vs.

RG. Number of determinations are in parentheses.

J. Pedraza-Chaverrı et al. / Life Sciences 78 (2006) 761–770 763

(100 -C for 15 min in an oven) of chopped garlic reduced the

ability to inhibit lipid peroxidation. In contrast, Shobana and

Naidu (2000) found that the capacity of garlic to inhibit lipid

peroxidation was not affected by boiling (30 min at 100 -C). We

recently showed that garlic’s ability to prevent in vitro Cu2+-

induced lipoprotein oxidation in human serum is preserved in

heated garlic (Pedraza-Chaverri et al., 2004b). However, to our

knowledge, there are no studies exploring if the heating of garlic

cloves affects its ability to scavenge O2&�, hydrogen peroxide

(H2O2), and OH&, or if the heating of aqueous extract of raw

garlic or garlic powder affects their ability to scavenge O2&� or

H2O2. In the present paper we studied if the ability of aqueous

garlic extracts to scavenge O2&�, H2O2, and OH

& is altered in the

following aqueous preparations: (a) extracts of boiled garlic

cloves (BG), (b) extracts of microwave-treated garlic cloves

(MG), and (c) extracts of pickled garlic (PG), and heated extract

of (a) garlic powder (HGP) or (b) of raw garlic (HRG). The data

were compared with the unheated raw garlic (RG) or with the

unheated garlic powder (GP).

Materials and methods

Materials and reagents

Bulbs of garlic were obtained in a local market. Garlic

powder was obtained from McCormick (Mexico City, Mexico).

Xanthine oxidase, xanthine, nitroblue tetrazolium (NBT),

xylenol orange, butylated hydroxytoluene, thiobarbituric acid,

and 2-deoxy-d-ribose, deferoxamine mesylate, nordihydro-

guaiaretic acid (NDGA), sodium pyruvate, dimethylthiourea

(DMTU), and tetramethoxypropane were purchased from

Sigma Chemical Co (St. Louis, Mo., USA).

Preparation of aqueous extracts of garlic

Extract of garlic powder (GP)

Garlic powder was weighted (0.3 g), dissolved, and stirred

with 3 mL of distilled water for 10 min. This solution was

centrifuged at 20,124 �g for 10 min at 4 -C. The supernatant

was recovered and used at the indicated final concentrations.

Heated extract of garlic powder (HGP)

The procedure was similar to the previous one except that

the mixture was boiled for 30 min before the centrifugation

step. The amount of water evaporated was replaced at the end

of the heating. The supernatant was recovered and used at the

indicated final concentrations.

Extract of raw garlic (RG)

Garlic cloves were peeled off, weighted, chopped, and

homogenized with distilled water in a Polytron (Model

PT2000, Brinkmann, Switzerland). This homogenate was

centrifuged at 20,124 �g for 10 min at 4 -C. The supernatant

was recovered and used at the indicated final concentrations.

Heated extract of raw garlic (HRG)

The procedure was similar to the previous one except that

the homogenate was boiled for 30 min before the centrifugation

step. The amount of water evaporated was replaced at the end

of the heating. The supernatant was recovered and used at the

indicated final concentration.

Extract of boiled garlic cloves (BG)

Unpeeled garlic cloves were boiled in water for 30 min.

After this time, garlic cloves were peeled off and the aqueous

extract was prepared as described before (extract of raw garlic).

The supernatant was recovered and used at the indicated final

concentrations.

Extract of microwave-treated garlic cloves (MG)

Unpeeled garlic cloves were submitted to microwave

heating for 30 s (1100 W). After this time, garlic cloves

were peeled off and the aqueous extract was prepared as

described before (extract of raw garlic). When allinase is

inactivated by heating, the cascade of thiosulfinate formation

is blocked from alliin, and allicin and its derivates can not be

formed. It has been shown that as little as 60 s of microwave

heating (600 W) can totally destroy allinase enzyme activity

whereas microwave heating for 30 s (600 W) inhibits 90% of

allinase activity compared with unheated garlic (3). The

supernatant was recovered and used at the indicated final

concentrations.

Extract of pickled garlic (PG)

Garlic cloves were peeled off carefully to avoid allinase

activation and put in an aqueous solution of vinegar (1 :1, v/v)

and then heated to the boiling point for 30 min. The

experiments with pickled garlic were performed the same

day. The aqueous extract was prepared as described before

(extract of raw garlic). The supernatant was recovered and used

at the indicated final concentrations.

J. Pedraza-Chaverrı et al. / Life Sciences 78 (2006) 761–770764

O2&� scavenging assay

Xanthine–xanthine oxidase system was used to determine

the O2&� scavenging capacity of the extracts. O2

&� in the assay

system and xanthine oxidase activity were measured as NBT

reduction and uric acid production, respectively (Bielski et al.,

1980), using a DU-64O series Beckman spectrophotometer.

This system is useful to test for O2&� scavenging capacity only

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H2O2 SCAVENGING CAP

A

C

E

G

B

D

F

H

Fig. 2. Effect of extract of garlic powder (GP) (panel A), heated extracts garlic powd

cloves (BG) (panel E), heated raw garlic (HRG) (panel F), microwave-treated garli

pyruvate (panel C) on H2O2 scavenging capacity. Data are expressed as percent oap <0.05, bp <0.001 vs. 0 mg/mL.

when the extracts or compounds used do not interfere with the

xanthine oxidase activity. A compound with O2&� scavenging

capacity should decrease NBT reduction without interfering

with xanthine oxidase activity measured as uric acid produc-

tion. Eight hundred microliters of the following reaction

mixture: 90 AM xanthine, 16 mM Na2CO3, 22.8 AM NBT, and

18 mM phosphate buffer (pH 7.0) were mixed with 100 AL of

phosphate buffer 50 mM (pH 7.0) (0 % scavenging tube) or

0 0.1 0.3 0.8 1.5 2.6 4.6

0 0.1 0.3 0.8 1.5 2.6 4.6

0 0.1 0.3 0.8 1.5 2.6 4.6

0 0.1 0.3 0.8 1.5 2.6 4.6

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b

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[PG], mg/mL

ACITY

er (HGP) (panel B), extract of raw garlic (RG) (panel D), extract of boiled garlic

c cloves (MG) (panel G), and extracts of pickled garlic (PG) (panel H), and of

f the values obtained in 0 mg/mL and are presented as meanTSEM, n =4–6.

Table 3

H2O2 scavenging capacity of pyruvate and of garlic powder (GP) and heated

extract of garlic powder (HGP)

Garlic extract or standard IC50, mg/mL

Pyruvate (6) 0.4856T0.0222

GP (4) 0.8509T0.0067a

HGP (4) 1.7646T0.0974a,b

Data are meanTSEM. ap <0.001 vs. pyruvate; bp <0.001 vs. GP. Number of

determinations are in parentheses.

Table 4

H2O2 scavenging capacity of pyruvate and extract of raw garlic (RG), extract of

boiled garlic cloves (BG), heated extract of raw garlic (HRG), extract of

microwave-treated garlic cloves (MG), and extract of pickled garlic (PG)

Garlic extract or standard IC50, mg/mL

Pyruvate (6) 0.4856T0.0222

RG (4) 0.6416T0.0421BG (4) 0.8226T0.0772a

HRG (4) 0.7330T0.0596a

MG (6) 0.4592T0.0263

PG (4) 0.9618T0.0175a,b

Data are meanTSEM. ap <0.01 vs. pyruvate, bp <0.001 vs. RG. Number of

determinations are in parentheses.

J. Pedraza-Chaverrı et al. / Life Sciences 78 (2006) 761–770 765

with 100 AL of different concentrations of the garlic extracts.

The reaction was started by the addition of 100 AL of xanthine

oxidase (168 U/L). Optical density was registered both at 295

nm (for uric acid production) and 560 nm (for O2&� in the

assay system). Scavenging percent was obtained from the

optical densities at 560 nm. NDGA was used as standard for

O2&� scavenging in this assay (unpublished data from our

laboratory).

Determination of H2O2 by the ferrous ion oxidation-xylenol

orange (FOX) assay

A solution of 75 AM H2O2 was mixed (1 :1 v/v) with

water (0% scavenging tube) or with different concentrations

of garlic extracts and incubated for 30 min at room temper-

ature. After this, H2O2 was measured by the method

described by Long et al. (1999). Briefly, 9 volumes of 4.4

mM butylated hydroxytoluene in HPLC-grade methanol were

mixed with 1 volume of 1 mM xylenol orange and 2.56 mM

ammonium ferrous sulfate in 0.25 M H2SO4 to give the

‘‘working’’ FOX reagent. Ninety microliters of the H2O2-

sample solutions were pippeted in 1.5 mL Eppendorf tubes

and mixed with 0.01 mL of HPLC-grade methanol immedi-

ately followed by the addition of 0.9 mL of FOX reagent,

vortexed for 5 s and then incubated at room temperature for

10 min. The tubes were centrifuged for 15,000 �g for 10 min

and absorbance at 560 nm was read against methanol blank.

The concentration of H2O2 was calculated from a standard

curve prepared with increasing H2O2 concentrations. Pyruvate

was used as standard for H2O2 scavenging (Salahudeen et al.,

1991).

OH & scavenging assay

The ability of garlic extracts to scavenge OH& was

conducted in the Fe3+–EDTA–H2O2-deoxyribose system

(Halliwell et al., 1987). Nine hundred microliters of the

following reaction mixture: 0.2 mM ascorbic acid, 0.2 mM

FeCl3, 0.208 mM EDTA, 1 mM H2O2, 0.56 mM 2-deoxy-d-

ribose, and 20 mM phosphate buffer (pH 7.4) were mixed

with 100 AL of distilled water (0% scavenging tube) or with

100 AL of different concentrations of garlic extracts.

Hydroxyl radicals were generated by incubating the mixture

at 37 -C for 60 min. The iron salt (FeCl3) was mixed with

EDTA before addition to the reaction mixture. The extent of

2-deoxy-d-ribose degradation by the formed OH& was

measured directly in the aqueous phase by the thiobarbituric

acid test. DMTU was used as standard for OH& scavenging

(Walker and Shah, 1988).

Statistics

Data are expressed as meanTSEM. The data were compared

against 0% scavenging tube using one way analyses of variance

and Dunnett post-test. O2&� and H2O2 scavenging capacity was

expressed as 50% of inhibitory concentration (IC50) value,

which denotes the concentration of the extract and standard

(mg/mL) required to give a 50% reduction in scavenging

capacity relative to the tube without extract or standard. The

lower the IC50 value the higher is the scavenging capacity of the

compound. OH& scavenging capacity was expressed as percent

of the tube without extract. The IC50 values and the percent data

were compared by one way analysis of variance and Bonferroni

t post-test. P <0.05 was considered significant.

Results

O2&� scavenging capacity of garlic extracts

Fig. 1 shows the effect of garlic extracts (panels A, B, and

D–H) and of NDGA (panel C) on O2&� in the assay system and

uric acid production. Data are expressed as percent of O2&� in

the assay system and uric acid production, and were compared

against the tube without garlic extract or NDGA (0 Ag/mL).

Garlic extracts and NDGA decreased concentration-depend-

ently O2&� in the assay system. GP and HGP decreased

concentration-dependently O2&� in the assay system starting at

0.7 and 1.5 mg/mL, respectively (Panels A and B). GP was

unable to affect uric acid production indicating that the

decrease in O2&� in the assay system was not secondary to the

inhibition of xanthine oxidase. In contrast, HGP decreased uric

acid production at 4.1, 8 and 10 mg/mL suggesting that the

decrease in O2&� in the assay system is affected partially (at 4.1

mg/mL) or completely (at 8 and 10 mg/mL) by the decrease in

xanthine oxidase activity. However, it is clear that at 1.6–3.1

mg/mL, HGP decreases O2&� in the assay system without

affecting uric acid production. Panel C shows that the standard

NDGA decreased O2&� in the assay system starting on 0.6 Ag/

mL without affecting uric acid production. Table 1 shows that

the IC50 values for GP and HGP were not different between

them but they were higher than that of NDGA indicating that

J. Pedraza-Chaverrı et al. / Life Sciences 78 (2006) 761–770766

(1) the heating was unable to affect the scavenging capacity of

garlic extract and (2) NDGA is more effective to scavenge O2&�

than GP and HGP. Panels D–H show the effect of RG, BG,

HRG, MG, and PG on the above mentioned parameters. The

decrease in O2&� in the assay system was significative at 0.1

mg/mL for RG and BG, at 1 mg/mL for PG, at 2 mg/mL for

MG, and at 8 mg/mL for HRG. PG decreased uric acid

production at the higher concentrations: 8–20 mg/mL, how-

ever the decrease in O2&� in the assay system was clearly higher

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[MG], mg/mL

OH. SCAVENGING

A

C

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G

B

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F

H

Fig. 3. Effect of extract of garlic powder (GP) (panel A), heated extract of garlic po

garlic cloves (BG) (panel E), heated extract of raw garlic (HRG) (panel F), microwav

dimethylthiourea (DMTU) (panel C) on OH& scavenging capacity. Data are expressed

n =6–7. ap <0.001 vs. 0 mg/mL.

than the decrease in uric acid production indicating that PG

truly scavenges O2&�. The IC50 values of HRG and MG were

significantly higher than that of RG indicating that HRG and

MG are less effective to scavenge O2&� than RG (Table 2). In

contrast the IC50 values of BG and PG were similar compared

to RG (Table 2) ( p <0.001). In addition, the IC50 value for

NDGA was significantly lower than those of garlic extracts

indicating that NDGA is more effective to scavenge O2&˙� than

the garlic extracts (Table 2).

0 0.1 0.3 0.7 1.5 2.6

0 0.1 0.3 0.7 1.5 2.6

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0 0.1 0.3 0.7 1.5 2.6

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CAPACITY

wder (HGP) (panel B), extract of raw garlic (RG) (panel D), extracts of boiled

e-treated garlic cloves (MG) (panel G), and pickled garlic (PG) (panel H), and of

as percent of the values obtained in 0 mg/mL and are presented as meanTSEM,

Table 6

OH& scavenging capacity of dimethylthiourea (DMTU) and extract of raw

garlic (RG), extract of boiled garlic cloves (BG), heated extract of raw garlic

(HRG), extract of microwave-treated garlic cloves (MG), and extract of pickled

garlic (PG)

Extract or DMTU (2.61 mg/mL) OH& scavenging capacity (%)

DMTU (6) 95.93T0.29

RG (6) 42.94T1.22a

BG (7) 41.37T3.65a

HRG (7) 46.04T1.32a

MG (6) 44.89T0.76a

PG (6) 48.21T2.15a

Data are meanTSEM. ap <0.001 vs. DMTU. Number of determinations are in

parentheses.

J. Pedraza-Chaverrı et al. / Life Sciences 78 (2006) 761–770 767

H2O2 scavenging capacity of garlic extracts

Fig. 2 shows the ability of garlic extracts (Panels A, B, and

D–H) and pyruvate (panel C) to scavenge H2O2. Data were

expressed as percent of H2O2 in the assay mixture and were

compared against tube 0 mg/mL which does not contain

garlic extract or pyruvate. Garlic extracts and pyruvate

scavenged H2O2 in a concentration-dependent way. GP and

HGP scavenged H2O2 starting at 0.3 and 0.8 mg/mL,

respectively (Panels A and B). Panel C shows the effect of

the standard pyruvate which decreased H2O2 concentration

starting at 0.1 mg/mL. The IC50 value for HGP was

significantly higher than that of RG (Table 3) indicating that

heating decreased the H2O2 scavenging capacity of garlic

extract. IC50 for pyruvate was significantly lower that those

for GP and HGP indicating that pyruvate is more effective to

scavenge H2O2 than GP and HGP. Panels D–H show the

H2O2 scavenging capacity of RG, BG, HRG, MG, and PG.

The H2O2 scavenging capacity was significative at 0.1 mg/

mL for RG, MG, and PG, and at 0.3 mg/mL for BG and

HRG. The IC50 value of PG was significantly higher than that

of RG indicating that PG is less effective than RG to

scavenge H2O2. In contrast, the IC50 values of BG, HRG, and

MG were not different from that of RG (Table 4) ( p <0.001).

The IC50 value of pyruvate was similar than those of RG and

MG but lower than those of BG, HRG and PG indicating that

pyruvate is more effective to scavenge H2O2 than these three

last extracts.

Hydroxyl radical scavenging capacity of garlic extracts

Fig. 3 shows the ability of garlic extracts (Panels A, B, and

D–H) and DMTU (Panel C) to scavenge OH&. Data are

expressed as percent of OH& in the assay mixture and were

compared against the tube without garlic extract (0 mg/mL).

The garlic extracts and the standard DMTU scavenged OH& in a

concentration-dependent way. The OH& scavenging capacity of

GP and HGP was significative at 0.3 mg/mL (Panel A and B)

and that of DMTU was significative at 0.002 mg/mL (Panel C).

Concentrations of GP and HGP higher than 1.5 mg/mL

interfered with the assay (Panels A and B). Concentrations of

RG, BG, HRG, MG, and PG higher than 2.6 mg/mL interfered

with the assay (data not shown). Therefore, we were unable to

test higher concentrations of garlic extracts in our system and

thus to reach 50% of OH& scavenging capacity (with the

exception of PG) which prevented us to calculate IC50. Then,

the comparisons among the OH& scavenging capacity were

Table 5

OH& scavenging capacity of dimethylthiourea (DMTU) and extracts of garlic

powder (GP) and heated garlic powder (HGP)

Extract or DMTU (1.53 mg/mL) OH& scavenging capacity (%)

DMTU (6) 95.61T0.47

GP (6) 27.59T0.49a

HGP (6) 30.57T1.75a

Data are meanTSEM. ap <0.001 vs. DMTU. Number of determinations are in

parentheses.

made at the maximum concentration tested. Table 5 shows that

the OH& scavenging capacity was unaffected by the heating

(GP vs. HGP, p >0.05). In addition, the OH& scavenging

capacity of DMTU was significantly higher than those of GP

and HGP (Table 5). The OH& scavenging capacity of RG, BG,

HRG, MG and PG was significative at 0.1 mg/mL (Panels D–

H). The OH& scavenging capacity was not significantly

different among the extracts (Table 6) ( p >0.05). However,

the OH& scavenging capacity of DMTU was significantly

higher than those of RG, BG, HRG, MG, and PG (Table 6)

( p <0.001).

Discussion

Garlic is among the oldest of all cultivated plants and has

been used as a medicinal agent for thousands of years. Many

recent studies have demonstrated several pharmacologic effects

of garlic, such as antibacterial, antifungal, hypolipidemic,

hypoglycemic, antithrombotic, antihypertensive, and anti-

cancer properties (Block, 1985; Lawson, 1998; Song and

Milner, 1999; Banerjee et al., 2002a; Pedraza-Chaverri et al.,

1998; Dillon et al., 2002). It has been shown in many cases that

the protective effect of garlic is associated with its antioxidant

properties (Banerjee et al., 2002b). The method of garlic’s

preparation may influence its medicinal properties (Banerjee et

al., 2002a,b; Dillon et al., 2002; Rahman, 2003; Pedraza-

Chaverri et al., 2000; Rietz et al., 1993; Thabrew et al., 2000;

Mukherjee et al., 2002). In this context, several studies have

been performed to test the effect of heating on several garlic

properties including the antioxidant properties. The boiling of

garlic cloves by 15 min impairs significantly its ability to

inhibit cyclooxygenase activity (Ali, 1995) and thromboxane

B2 synthesis (Bordia et al., 1996). In addition, heating of garlic

cloves by 60 s in microwave reduces its anticancer properties

(Song and Milner, 1999). Interestingly when microwave

heating was applied 10 min after garlic crushing the anticancer

properties were preserved indicating that allinase activation is

necessary to generate anticancer compounds which are thermo-

stable (Song and Milner, 1999). In a similar way, the OH&

scavenging properties of garlic were essentially preserved

when garlic extracts were heated (Prasad et al., 1996). In

contrast, heating of garlic extracts by 10 min at 100 -C reduced

the bactericidal activity against Helicobacter pylori (Cellini et

J. Pedraza-Chaverrı et al. / Life Sciences 78 (2006) 761–770768

al., 1996), the antifungal activity (Yin and Cheng, 1998b), and

the ability to inhibit platelet aggregation (Ali et al., 1999). In

addition, it was recently shown that the ability of garlic extracts

to inhibit Cu2+-induced lipoprotein oxidation in human serum

was not affected by heating garlic cloves before cutting (by

boiling, microwaving, and pickling) or by the heating of

extracts of garlic powder or raw garlic showing that this

antioxidant property is not affected by the heating before or

after garlic cutting (Pedraza-Chaverrı et al., 2004b). In this

work it was studied the O2&�, OH&, and H2O2 scavenging

capacity of heated extracts of garlic powder and raw garlic and

aqueous extracts of boiled garlic cloves, microwave-treated

garlic cloves, and pickled garlic to test (a) if the scavenging

compound(s) are thermostable and (b) if allinase activity is

related with the antioxidant capacity of garlic extracts.

Chopping or crushing of garlic releases alliinase which rapidly

converts alliin (S(+)-alkyl-l-cysteine sulfoxide) to allicin

(dialkyl thiosulfinate) (Lawson, 1998). Allicin is the major

thiosulfinate compound found in crushed garlic but is quite

unstable and quickly is converted to several other sulfur

compounds, such as diallyl sulfide, diallyl disulfide, diallyl

trisulfide, ajoene, and polysulfide compounds (Block, 1985).

In this work we have confirmed the observation of Prasad

et al. (1996) who found that the OH& scavenging properties of

garlic were essentially preserved when extracts of garlic

powder were heated at 100 -C by 20, 40 or 60 min. In

addition, we found that the O2&� scavenging capacity of the

heated extract of garlic powder (HGP) is preserved (Fig. 1)

and that the H2O2 scavenging capacity of the above extract is

decreased, but no eliminated, compared to the unheated extract

of garlic powder (GP) (Fig. 2). Furthermore, the heating of

raw garlic extracts does not affect the OH& and H2O2

scavenging properties but it decreases the O2&� scavenging

properties. The above data suggest that the heating is not able

to eliminate the scavenging properties of aqueous extract of

garlic powder or raw garlic suggesting that the compound(s)

involved in these properties are thermostable. The in vitro

scavenging properties of extracts of garlic powder or raw

garlic are in agreement with the in vivo antioxidant properties

of these extracts found in several experimental models such as

gentamicin nephrotoxicity (Pedraza-Chaverri et al., 2000),

heart ischemia and reperfusion (Rietz et al., 1993; Mukherjee

et al., 2002), adriamycin toxicity (Thabrew et al., 2000),

quinolinic acid toxicity (Perez-Severiano et al., 2004a),

amyloid-beta peptide-induced neurotoxicity (Perez-Severiano

et al., 2004b), and cancer (reviewed in Khanum et al., 2004).

The H2O2 scavenging ability of garlic observed in the in vitro

assay in this work agrees with the decrease in H2O2

production in the kidney and liver of garlic-fed rats

(Pedraza-Chaverri et al., 2001).

We studied additional extracts in which the thermolabile

enzyme allinase was inhibited before garlic cutting (boiled

garlic, microwave-treated garlic cloves and pickled garlic). The

ability of these extracts to scavenge O2&�, H2O2, and OH& was

completely preserved with the following exceptions in which

the scavenging capacity was decreased but no eliminated: the

ability of microwave-treated garlic (MG) to scavenge O2&� (Fig.

1), and the ability of pickled garlic (PG) to scavenge H2O2

(Fig. 2). These data strongly suggest that the antioxidant

properties of garlic are not dependent of allinase activation.

Our data are essentially in agreement with previous findings in

which it was observed that the allinase activation is not related

to the ability of garlic to inhibit Cu2+-induced lipoprotein

oxidation in human serum (Pedraza-Chaverri et al., 2004b).

The antioxidant properties of allicin, the main component in

aqueous extract from raw garlic and garlic powder, may

explain, at least in part, the reactive oxygen species scavenging

capacity of extracts of raw garlic and garlic. It has been found

that allicin scavenges OH& and inhibits lipid peroxidation

(Prasad et al., 1995) and prevents the lung damage induced by

ischemia-reperfusion (Batirel et al., 2002). Furthermore, alliin,

the main component in extracts from boiled garlic cloves,

microwave-treated garlic cloves and pickled garlic, scavenges

OH& (Kourounakis and Rekka, 1991), H2O2 (Ide et al., 1996),

and inhibits lipid peroxidation (Ide et al., 1996) and LDL

oxidation (Ho et al., 2001). This may explain, at least in part,

the ability of boiled garlic, microwave-treated garlic, or pickled

garlic to scavenge reactive oxygen species. Interestingly, it has

been shown that another garlic compounds, such as S-allyl-

cysteine (Ide et al., 1997; Kim et al., 2001), N-acetyl-S-allyl-

cysteine (Ide et al., 1997), allixin (Ide et al., 1997), and S-

ethylcysteine, N-acetylcysteine, diallyl sulfide (Ou et al., 2003;

Pedraza-Chaverri et al., 2003b), diallyl disulfide (Ou et al.,

2003; Pedraza-Chaverri et al., 2003a; Koh et al., 2005), and S-

allylmercaptocysteine (Pedraza-Chaverrı et al., 2004a), which

may be present in some of the garlic extracts used in this work,

also have antioxidant properties.

Conclusion

In conclusion, the heating before or after garlic cutting was

unable to eliminate the capacity of the extracts to scavenge

O2&�, H2O2, and OH&. These data suggest that (a) the

compound(s) involved in the scavenging capacity of garlic

extracts are essentially heat stable and (b) the scavenging

capacity of the garlic extracts is not related to allinase activity.

Acknowledgements

Thisworkwas supported byCONACYT (GrantNo. 40009-M).

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