Protective Effect of Allium neapolitanum Cyr. Versus Allium sativum L. on Acute Ethanol-Induced Oxidative Stress in Rat Liver

Abstract

This study investigated the protective effect of Allium neapolitanum Cyr., a spontaneous species of the Italian flora, compared with garlic (Allium sativum L.) on liver injury induced by ethanol in rats. Male albino Wistar rats were orally treated with fresh Allium homogenates (leaves or bulbs, 250 mg/kg) daily for 5 days, whereas controls received vehicle only. At the end of the experimental 5-day period, the animals received an acute ethanol dose (6 mL/kg, i.p.) 2 hours before the last Allium administration and were sacrificed 6 hours after ethanol administration. The activities of catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) and the levels of malondialdehyde (MDA), ascorbic acid (AA), and reduced (GSH) and oxidized glutathione in liver tissue were determined. Administration of both Allium species for 5 days (leaves or bulbs) led to no statistical variation of nonenzymatic parameters versus the control group; otherwise Allium treatment caused an increase of GSH and AA levels compared with the ethanol group and a diminution of MDA levels, showing in addition that A. neapolitanum bulb had the best protective effect. Regarding to enzymatic parameters, GR and CAT activities were enhanced significantly compared with the ethanol group, whereas SOD activity showed a trend different from other parameters estimated. However, the treatment with both Allium species followed by acute ethanol administration reestablished the nonenzymatic parameters similar to control values and enhanced the activities of the enzymes measured. These results suggest that fresh Allium homogenates (leaves or bulbs) possess antioxidant properties and provide protection against ethanol-induced liver injury.

Introduction

G arlic (Allium sativum L.) has been used as a folk medicine for the treatment of many diseases, including infections, diabetes, and vascular disorders.¹ Recently, research has focused on the preventive and curative effects of garlic on cardiovascular disease and cancer.^2
–4

In addition, garlic protects against acetaminophen-induced hepatotoxicity,⁵ has a prophylactic action on the histological and histochemical patterns of gastric and hepatic tissues in rats injected with cobra snake venom,⁶ and seems to reduce the tissue lead (Pb) concentration.⁷

These properties are probably due to antioxidant activity. Administration of garlic extracts significantly lowered lipid peroxidation and enhanced the hepatic levels of glutathione and glutathione-dependent enzymes.⁸

All literature data concern the bulb of Allium sativum, but few details are available on the leaf of garlic and bulb and leaf of Allium neapolitanum, endemic among Italian flora.

An old pharmacological study on A. sativum leaf showed some effects of water and alcoholic extracts on isolated organs.⁹

We have previously demonstrated that bulb and leaf of A. neapolitanum have an in vitro antioxidant activity similar to A. sativum (bulb and leaf) and that this antioxidant activity is related to the polyphenol content.¹⁰

Two analytical studies have been performed with A. neapolitanum: the first reports the isolation of four new flavonoids that showed in vitro anti-aggregant activity toward human platelets,¹¹ and the second one characterizes three new compounds from the bulb extracts that showed in vitro antibacterial activity.¹²

In this study we investigate the protective effect of A. neapolitanum Cyr. (leaves and bulbs) compared with garlic (leaves and bulbs) on liver injury induced by ethanol in rats.

Ethanol has been shown to produce oxidative stress in tissues, and acute ingestion has been related to the formation of reactive oxygen species.^13,14 The liver is highly susceptible to the oxidative events associated with the toxicity of ethanol.¹⁵

The potential damage is controlled by the cellular antioxidant defense system. In effect, when the generation of reactive oxygen species in cells impairs antioxidant defenses or exceeds the ability of the antioxidant defense system to eliminate them, oxidative stress results. Reduced glutathione (GSH) is the predominant defense against reactive oxygen species/free radicals in different tissues of the body. In addition, antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), are essential in both scavenging reactive oxygen species/free radicals and maintaining cellular stability.^16,17

The hepatoprotective effects of both Allium species were evaluated in vivo using acute ethanol-intoxicated rats as an experimental model and measuring antioxidant defense systems in liver tissue, particularly GSH and oxidized glutathione (GSSG) levels, ascorbic acid (AA) levels, SOD, CAT, and GR activities, and malondialdehyde (MDA) levels as a marker of lipid peroxidation.

Materials and Methods

Plant collection

A. neapolitanum Cyr. was collected nearby Siena (Tuscany, Italy) in April 2008 during its blooming time (late spring) and identified by Prof. G.G. Franchi, University of Siena.

A. sativum L. was obtained from plants cultivated for horticultural purposes nearby Siena.

Fresh Allium homogenates

Peeled bulbs and leaves were washed, cut into small pieces, and homogenized in deionized water (500 mg/mL) using a Turrax homogenizer (IKA-Werke GmbH & Co. KG, Staufen, Germany) on ice.

The fresh homogenate supernatant was made by centrifugation of fresh Allium homogenate at 12,000 g for 15 minutes at 4°C and used within 30 minutes of preparation.

Animal treatments

Male albino Wistar rats (weighing 175–200 g) were purchased from Charles River Laboratories Inc. (Calco, Lecco, Italy), and maintained under standard conditions of temperature and humidity and a 12-hour light-dark cycle with access to food and water for 8 days prior to experimentation.

All experimental procedures were performed in accordane with procedures of the local institutional animal ethics committee and conformed to the guide for the care and use of laboratory animals.

Animals were randomly divided into 10 groups of five rats each and treated for 5 days as follows:

Control group: vehicle (water) orally via an orogastric tube.

Ethanol group (EtOH group): ethanol (6 mL/kg i.p.) on day 5 at 6 hours before sacrifice.

A. sativum bulbs group (ASB group): 250 mg/kg fresh homogenate orally via an orogastric tube.

A. sativum leaves group (ASL group): 250 mg/kg fresh homogenate orally via an orogastric tube.

A. sativum bulbs + ethanol group (ASB + EtOH group): 250 mg/kg fresh homogenate orally via an orgogastric tube and on day 5 ethanol (6 mL/kg i.p.) 2 hours before the last Allium administration.

A. sativum leaves + ethanol group (ASL + EtOH group): 250 mg/kg fresh homogenate orally via an orogastric tube and on day 5 ethanol (6 mL/kg i.p.) 2 hours before the last Allium administration.

A. neapolitanum bulbs group (ANB group): 250 mg/kg fresh homogenate orally via an orogastric tube.

A. neapolitanum leaves group (ANL group): 250 mg/kg fresh homogenate orally via an orogastric tube.

A. neapolitanum bulbs + ethanol group (ANB + EtOH group): 250 mg/kg fresh homogenate orally via an orogastric tube and on day 5 ethanol (6 mL/kg i.p.) 2 hours before the last Allium administration.

A. neapolitanum leaves + ethanol group (ANL + EtOH group): 250 mg/kg fresh homogenate orally via an orogastric tube and on day 5 ethanol (6 mL/kg i.p.) 2 hours before the last Allium administration.

Animals were killed 6 hours after ethanol treatment by cervical dislocation and decapitation between 15:00 and 16:00 p.m. to minimize the possibility of diurnal variations in tissue GSH concentrations influencing the experimental results.¹⁸

The liver tissue was rapidly removed, rinsed with isotonic saline, and homogenized with a different method, as reported below.

Antioxidant defense system determinations

Glutathione by microassay procedure

Hepatic tissues were homogenized in ice-cold phosphate buffer (0.125 M, pH 7.4) containing 1 mM EDTA and then were added to an equal volume of 10% metaphosphoric acid. Samples were centrifuged at low speed (2,000 g) for 10 minutes at 0°C.

Total GSH and GSSG were quantified in supernatant using a microassay procedure previously described¹⁹ based on an enzymatic method [the GR-5,5'-dithiobis(2-nitrobenzoic acid) recycling assay] with reading at 415 nm.

Results were expressed in nmol of GSH or GSSG/mg of protein.

SOD activity

SOD activity in the liver was estimated by a spectrophotometric method based on the inhibition of superoxide-induced NADH oxidation, according to Paoletti et al.²⁰ Hepatic tissue was first homogenized in 25 nM triethanolamine–diethanolamine buffer (pH 7.4) and then centrifuged at 40,000 g for 60 minutes at 4°C.

One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of NADH oxidation of the control by 50%.

CAT activity

Liver samples were homogenized in ice-cold phosphate buffer (0.125 M, pH 7.4) containing 1 mM EDTA and then centrifuged at 4,000 g for 15 minutes at 4°C.

CAT activity was determined adapting the method described by Johansson and Borg²¹ using a microassay procedure. The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of hydrogen peroxide (H₂O₂). The formaldehyde production was measured spectrophotometrically at 540 nm with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald^®, Aldrich Chemical Co., Milwaukee, WI, USA) as a chromogen.

One unit of CAT activity is defined as the amount of enzyme that will cause the formation of 1 nmol of formaldehyde/minute at 25°C. Results were expressed as KU/mg of protein (μmoL/minute/mg of protein).

GR activity

To measure enzyme activity, liver samples were homogenized in 6 volumes of cold 0.25 M sucrose in 0. 1 M phosphate buffer, pH 7.4. The homogenates were centrifuged at 40,000 g for 20 minutes at 4°C, and the supernatants were used for GR assays.

GR activity was analyzed as described by Cribb et al.²² The method is based on the increase in absorbance at 412 nm when 5,5'-dithiobis(2-nitrobenzoic acid) is reduced by GSH generated from an excess of GSSG.

Samples were prepared in 96-well plates, and absorbance, measured with a programmable microplate reader, was determined every 30 seconds for 3 minutes. The rate of increase in absorbance is directly proportional to the amount of GR in the sample.

AA

Hepatic tissue was homogenized in phosphate buffer (pH 7.4) (1:3, wt/vol) at 0°C, and the samples were added to an equal volume of 10% metaphosphoric acid. The samples were immediately centrifuged at 2,000 g and 0°C for 10 minutes. The supernatants were filtered (Anotop 0.2 Am, Merck, Darmstadt, Germany), and 20 μL was injected into the high-performance liquid chromatography column and analyzed as described by Ross.²³

MDA

Lipid peroxidation in the rat liver was estimated by calculation of MDA levels. Samples were homogenized in 0.04 M Tris-HCl (pH 7.4) containing 0.01% butylhydroxytoluene (1:3 wt/vol, 0°C) to prevent artificial oxidation of polyunsaturated free fatty acids during assay. Homogenates were deproteinized with acetonitrile (1:1 vol/vol) and then centrifuged at 3,000 g for 15 minutes at 0°C. The supernatant was used for MDA analysis by high-performance liquid chromatography according to Shara et al.²⁴

Protein assay

Protein concentrations were determined by the method of Lowry et al. ²⁵ calibrated with bovine serum albumin.

Statistical analysis

Results were expressed as mean ± SD values. Student's t test was applied for detecting the significance of difference between different groups. Values of P ≤ .05 were considered significant.

Analyses were performed using the SPSS for Windows computer program (SPSS Inc., Chicago, IL, USA). Differences between the means of two groups were evaluated by a two-tailed t test for independent samples.

Results

Effects of Allium bulbs on oxidative status

Values of nonenzymatic parameters measured and the statistical significances after treatment with Allium bulbs are shown in Table 1. The liver concentration of GSH and AA were significantly decreased (P < .01 vs. control group) by acute ethanol treatment, and GSSG and MDA levels were increased (P < .01 and P < .001, respectively, vs. control group), whereas no significant variations occurred in the other treatment groups versus control. Levels of GSH and AA significantly were increased in rats treated with Allium bulbs compared with the EtOH group, whereas GSSG and MDA levels were diminished; in particular, the GSH concentration in rats treated with A. neapolitanum bulbs significantly increased compared with A. sativum bulbs (ANB group vs. ASB group, P < .001; ANB + EtOH group vs. ASB + EtOH group, P < .01), showing a better protective effect of this wild Allium species.

Table 1.

Nonenzymatic Parameters of the Antioxidant Defense System After Treatment with Allium Bulbs

Group	GSH (nmol/mg of protein)	GSSG (nmol/mg of protein)	AA (μmol/g of tissue)	MDA (nmol/g of tissue)
Control	26.53 ± 3.83	1.64 ± 0.23	1.30 ± 0.17	31.91 ± 4.60
EtOH	17.85 ± 1.56^aa	2.24 ± 0.33^aa	1.02 ± 0.06^aa	58.86 ± 4.64^aaa
ASB	24.23 ± 1.61^bbb	1.29 ± 0.36^bb	1.12 ± 0.07^b	36.66 ± 5.53^bbb
ANB	29.37 ± 1.77^{bbb ccc}	1.37 ± 0.34^bb	1.19 ± 0.12^b	38.34 ± 11.22^bb
ASB + EtOH	21.89 ± 3.17^b	1.66 ± 0.33^b	1.49 ± 0.16^bb	37.06 ± 5.43^bbb
ANB + EtOH	29.59 ± 4.08^{bbb dd}	1.69 ± 0.20^bb	1.58 ± 0.35^bbb	33.74 ± 6.85^bbb

Data are mean ± SD values.

P < .01, ^aaa P < .001 versus control group.

P < .05, ^bb P < .01, ^bbb P < .001, versus EtOH group.

ccc

P < .001 versus ASB group.

P < .01 versus ASB + EtOH group.

In Table 2, SOD, GR, and CAT activities and the statistical significances after treatment with Allium bulbs are reported. Treatment of animals with ethanol decreased significantly SOD, GR, and CAT activities compared with the control group. On the whole, treatment with both Allium spp. bulbs caused an increase of CAT activity versus control and ethanol-treated groups (with P < .001 in all cases) and an increase of GR activity versus ethanol-treated groups (P < .05 for ASB and ANB groups and P < .001 for ASB + EtOH and ANB + EtOH); SOD activity was increased in the ASB group with respect to the control group (P < .001) and in all groups treated with Allium with respect to ethanol-treated groups (P < .001 for ASB and ANB and P < .01 for ASB + EtOH and ANB + EtOH).

Table 2.

Enzymatic Parameters of the Antioxidant Defense System After Treatment with Allium Bulbs

Group	SOD (U/mg of protein)	GR (U/mg of protein)	CAT (KU/mg of protein)
Control	31.31 ± 2.56	64.71 ± 6.62	190.84 ± 9.67
EtOH	16.11 ± 2.97^aaa	54.10 ± 7.91^a	172.99 ± 12.53^a
ASB	44.47 ± 2.16^{aaa bbb}	64.27 ± 7.58^b	237.44 ± 5.70^{aaa bbb}
ANB	36.88 ± 5.34^{bbb c}	66.85 ± 8.46^b	426.16 ± 16.09^{aaa bbb ccc}
ASB + EtOH	22.31 ± 2.26^{aaa bb}	89.12 ± 6.59^{aaa bbb}	292.63 ± 14.27^{aaa bbb}
ANB + EtOH	24.49 ± 4.63^{a bb}	75.08 ± 7.31^{aaa bbb}	322.68 ± 27.79^{aaa bbb}

Data are mean ± SD values.

P < .05, ^aaa P < .001 versus control group.

P < .05, ^bb P < .01, ^bbb P < .001 versus EtOH group.

P < .05, ^ccc P < .001 versus ASB group.

Effects of Allium leaves on oxidative status

Values of nonenzymatic and enzymatic parameters measured and the statistical significances after treatment with Allium leaves are shown in Tables 3 and 4, respectively.

Table 3.

Nonenzymatic Parameters of the Antioxidant Defense System After Treatment with Allium Leaves

Group	GSH (nmol/mg of protein)	GSSG (nmol/mg of protein)	AA (μmol/g of tissue)	MDA (nmol/g of tissue)
Control	26.53 ± 3.83	1.64 ± 0.23	1.30 ± 0.17	31.91 ± 4.60
EtOH	17.85 ± 1.56^aa	2.24 ± 0.33^aa	1.02 ± 0.06^aa	58.86 ± 4.64^aaa
ASL	29.80 ± 3.223^bbb	1.32 ± 0.35^bb	1.07 ± 0.21	37.46 ± 6.09^bbb
ANL	32.56 ± 3.721^{a bbb}	1.35 ± 0.28^bb	1.10 ± 0.14	35.34 ± 5.97^bbb
ASL + EtOH	29.16 ± 4.26^bbb	2.03 ± 0.47	1.29 ± 0.11^bbb	37.46 ± 3.39^bbb
ANL + EtOH	26.65 ± 3.42^bbb	1.86 ± 0.50	1.40 ± 0.28^b	35.01 ± 4.31^bbb

Data are mean ± SD values.

P < .05, ^aa P < .01, ^aaa P < .001 versus control group.

P < .05, ^bb P < .01, ^bbb P < .001 versus EtOH group.

Table 4.

Enzymatic Parameters of the Antioxidant Defense System After Treatment with Allium Leaves

Group	SOD (U/mg of protein)	GR (U/mg of protein)	CAT (KU/mg of protein)
Control	31.31 ± 2.56	64.71 ± 6.62	190.84 ± 9.67
EtOH	16.11 ± 2.97^aaa	54.10 ± 7.91^a	172.99 ± 12.53^a
ASL	24.12 ± 1.93^{aaa bbb}	67.63 ± 6.81^b	304.79 ± 15.61^{aaa bbb}
ANL	26.01 ± 1.67^{aa bbb ccc}	65.56 ± 6.84^b	463.31 ± 14.82^{aaa bbb ccc}
ASL + EtOH	17.26 ± 0.98^aaa	90.13 ± 3.08^{aaa bbb}	351.07 ± 24.64^{aaa bbb}
ANL + EtOH	22.99 ± 1.56^{aaa bb ddd}	89.20 ± 5.73^{aaa bbb}	293.51 ± 29.04^{aaa bbb dd}

Data are mean ± SD values.

P < .05, ^aa P < .01, ^aaa P < .001 versus control group.

P < .05, ^bb P < .01, ^bbb P < .001 versus EtOH group.

ccc

P < .001 versus ASL group.

P < .01, ^ddd P < .001 versus ASL + EtOH group.

In general, treatment with Allium leaves led to no statistical variation of nonenzymatic parameters versus control (except for GSH levels, where P < .05 for ANL vs. control) but showed a protective effect with respect to ethanol-treated groups, particularly significant for GSH and MDA levels (P < .001). With regard to enzymatic parameters GR and CAT activities were enhanced significantly compared with ethanol-treated groups and the control group (except for GR activity in ANL and ASL groups), whereas SOD activity decreased significantly in all groups treated with both Allium leaves with respect to the control and increased significantly compared to ethanol-treated groups (except for the ASL + EtOH group).

Discussion

In the present study the rat hepatotoxicity model was successfully produced by administration of ethanol (6 mL/kg i.p.).

Acute ingestion of ethanol has been related to the formation of several reactive oxygen species,^13,14 considered to be a major factor in oxidative cell injury, with consequent development of a state of oxidative stress, due to both ethanol and its oxidation products.

Without a doubt the liver is the main site of ethanol biotransformation (to acetaldehyde and then oxidized to acetate) and at the same time the main site of free radical formation.^26,27

In our study, we found that acute ethanol administration causes a significant rise in hepatic MDA and GSSG levels and a decrease of GSH and AA concentrations and SOD, GR, and CAT activities.

In effect, our data show that ethanol administration significantly alters the enzymatic and nonenzymatic antioxidant defense systems of the liver and in consequence enhances lipid peroxidation, according to literature data.^28
–30

The increase of MDA level might be a consequence of enhanced formation of free radicals as well as inhibition of SOD and CAT activities.

SOD and CAT are important antioxidant enzymes that protect from lipid peroxidation via elimination of reactive oxygen species. SOD catalyzes the reaction of superoxide anion radical (O₂ ^•−) dismutation to H₂O₂, produced during ethanol metabolism, whereas CAT degrades H₂O₂ into oxygen and water and prevents generation of hydroxyl radicals.³¹

We also observed a significant decrease of GR activity after ethanol treatment, which suggests a limitation in GSH availability in the liver. Because the redox cycle of glutathione is important for the efficiency of glutathione-utilizing detoxification, the inhibition of the glutathione recycling enzyme activity would exacerbate the ethanol-induced oxidative stress.

In fact, in our study a diminution of GSH and an increase of GSSG are observed, after treatment with ethanol. These variations can be attributed both to the production of reactive oxygen species³² during the metabolism of ethanol and to the production of acetaldehyde, which reacts with hepatic GSH causing depletion.³⁰

It is well understood that GSH is an important cellular antioxidant capable of direct or indirect conjugation with reactive oxygen species such as lipid hyperperoxides and H₂O₂ ³³; in consequence, depletion of cellular GSH impairs cellular defense against chemical-induced cytotoxicity. There is a general agreement that in oxidative stress, the GSH level is depleted below a certain critical threshold.³⁴

AA decreases in several liver diseases, particularly in alcoholics.³⁵ AA, like GSH, is a naturally occurring free radical scavenger³⁶ and plays an important protective role in the process of lipid peroxidation. Thus it can be assumed that the reduced AA concentration in the animals exposed to ethanol may be a result of its management in reactions involving radicals generated during ethanol biotransformation.³⁷ The reduced AA levels may also result from the deficient amounts of GSH, noted in the rats exposed to ethanol.³⁸ GSH is necessary for the vitamin regeneration.³⁹

In this study administration of both Allium species reversed generally the changes induced by ethanol with some differences between A. sativum and A. neapolitanum and between bulbs and leaves.

It is known that garlic is effective in preventing or ameliorating oxidative stress probably through its intrinsic antioxidant properties widely documented in vivo ^40
–42 and in vitro ^43
–45 and/or through its ability to modify antioxidant enzyme expression.^40,42,46,47 Besides, we have demonstrated A. neapolitanum shows an in vitro antioxidant activity similar to that of A. sativum.¹⁰

Garlic extract increased SOD^43,44 and CAT⁴⁶ activities in vascular endothelial cells in culture, and S-allyl-cysteine sulfoxide (alliin), a garlic compound, prevented the decrease in hepatic SOD and CAT activities observed in diabetic rats.⁴²

There is substantial experimental evidence of the ability of garlic and its constituent to offer protection against oxidative stress induced by drugs or diseases in hepatocytes.⁴⁸

In addition, garlic compounds exert their protective effects by influencing a key endogenous antioxidant defense tool in tissues: glutathione and glutathione-dependent enzymes. Two main mechanisms can be described for the postulated hepatoprotective role of garlic organosulfur components: the first mechanism is the prevention of GSH depletion,^8,49,50 and the second one is the alteration of GSH-dependent enzymes activity and/or their gene expression.^51,52

Our results show that administration of Allium (bulbs or leaves) alone has no important effects on stress oxidative parameters with values close to control levels with the exception of A. neapolitanum leaves, which increased GSH levels significantly with respect to control rats. On the other hand, the administration of fresh Allium homogenate followed by ethanol demonstrates a protective effect; indeed, the measured parameters are significantly greater than for ethanol-treated groups, and in particular GR and CAT activities are also higher than in the control group.

The distinctive feature of our experiment concerns the enzyme SOD. Ethanol administration reduces the enzyme activity in accord with the literature,^28
–30 and also when Allium bulbs or leaves are administered in advance for 5 consecutive days and subsequently rats are treated with ethanol, the enzymatic activity is not normalized. This underlines a greater sensitivity of SOD to the ethanol damage,⁵³ although the administration of A. sativum bulb produces a stimulation of the enzymatic activity with an increase of SOD levels.

In this article we confirm not only the in vivo antioxidant activity of garlic bulb, but we found that also garlic leaves, a part of the plant not usually studied, possess this activity stimulating the antioxidant defense system.

Besides, we validated the data obtained in our precedent study, which demonstrated the in vitro antioxidant activity of leaves and bulb of A. neapolitanum,¹⁰ an Italian Allium spontaneous species generally poorly studied. A. neapolitanum has a behavior analogous to that of A. sativum in the rat group treated with Allium homogenate followed by ethanol; instead, when it is administered alone it shows a better ability than A. sativum to influence the antioxidant defense system. In particular, A. neapolitanum bulbs produced a significant increase of GSH levels (versus A. sativum), and A. neapolitanum bulbs and leaves enhanced CAT activity.

In summary, ethanol administration induces oxidative stress, and the fresh Allium homogenates possess antioxidant properties and provide protection against ethanol-induced liver injury. In particular, the bulb of A. sativum, which represents the part largely consumed and abundantly studied, does not evidence the best antioxidant power. This study opens the way to more extensive research about leaves of garlic and spontaneous Allium species that are less investigated and surely interesting.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Essman

. The medicinal uses of herbs. Fitoterapia, 1984; 55:279–289.

Gardner

, Messina

, Lawson

, Farquhar

. Soy, garlic, and Ginkgo biloba: their potential role in cardiovascular disease prevention and treatment. Curr Atheroscler Rep, 2003; 5:468–475.

Khanum

, Anilakumar

, Viswanathan

. Anticarcinogenic properties of garlic: a review. Crit Rev Food Sci Nutr, 2004; 44:479–488.

Tattelman

. Health effects of garlic. Am Fam Physician, 2005; 72:103–106.

Wang

, Li

, Lin

, Chen

, Stein

, Reuhl

, Yang

. Protective effects of garlic and related organosulfur compounds on acetaminophen-induced hepatotoxicity in mice. Toxicol Appl Pharmacol, 1996; 136:146–154.

Rahmy

, Hemmaid

. Prophylactic action of garlic on the histological and histochemical patterns of hepatic and gastric tissues in rats injected with a snake venom. J Nat Toxins, 2001; 10:137–165.

Senapatiat

, Dey

, Dwivedi

, Swarup

. Effect of garlic (Allium sativum L.) extract on tissue lead level in rats. J Ethnopharmacol, 2001; 76:229–232.

Arivazhagan

, Balasenthil

, Nagini

. Modulatory effects of garlic and neem leaf extracts on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced oxidative stress in Wistar rats. Cell Biochem Funct, 2000; 18:17–21.

Abdo

, al-Kafawi

. Biological activities of Allium sativum. Jpn J Pharmacol, 1969; 19:1–4.

10.

Nencini

, Cavallo

, Capasso

, Franchi

, Giorgi

, Micheli

. Evaluation of antioxidative properties of spontaneous Allium species from Italy. Phytother Res, 2007; 21:874–878.

11.

Carotenuto

, Fattorusso

, Lanzotti

, Magno

, De Feo

, Cicala

. The flavonoids of Allium neapolitanum. Phytochemistry, 1996; 41:531–536.

12.

O'Donnel

, Gibbons

. Antibacterial activity of two canthin-6-one alkaloids from Allium neapolitanum. Phytother Res, 2007; 21:653–657.

13.

Reinke

, Kotake

, McCoy

, Janzen

. Spin trapping studies of hepatic free radicals formed following acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN. Free Radic Biol Med, 1991; 11:31–39.

14.

Bondy

, Orozco

. Treatment upon sources of reactive oxygen species in brain and liver. Alcohol Alcohol, 1994; 29:375–383.

15.

Husain

, Somani

. Interaction of exercise training and chronic ethanol ingestion on hepatic and plasma antioxidant system in rat. J Appl Toxicol, 1997; 17:189–194.

16.

Halliwell

, Gutteridge

JMC

. Free Radicals in Biology and Medicine, 2 ^nd. Clarendon Press: Oxford, UK, 1989.

17.

Somani

. Exercise, drugs and tissue specific antioxidant system. Pharmacology in Exercise and Sports. Somani

. CRC Press: Boca Raton, FL, 1996; 57–95.

18.

Giorgi

, Micheli

, Segre

. A diurnal variation of the glutathione content in rat liver and stomach. IRCS Med Sci, 1984; 12:560–561.

19.

Nencini

, Giorgi

, Micheli

. Protective effect of silymarin on oxidative stress in rat brain. Phytomedicine, 2007; 14:129–135.

20.

Paoletti

, Aldinucci

, Mocali

, Caparrini

. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal Biochem, 1986; 154:536–541.

21.

Johansson

, Borg

LAH

. A spectrophotometric method for determination of catalase activity in small tissue samples. Anal Biochem, 1988; 174:331–336.

22.

Cribb

, Leeder

, Spielberg

. Use of a microplate reader in an assay of glutathione reductase using 5,5'-dithiobis(2-nitrobenzoic acid) Anal Biochem, 1989; 183:195–196.

23.

Ross

. Determination of ascorbic acid and uric acid in plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl, 1994; 657:197–200.

24.

Shara

, Dickson

, Bagchi

, Stohs

. Excretion of formaldehyde, malondialdehyde, acetaldehyde and acetone in the urine of rats in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin, paraquat, endrin and carbon tetrachloride. J Chromatogr, 1992; 576:221–233.

25.

Lowry

, Rosebrough

, Farr

, Randall

. Protein measurement with the Folin phenol reagent. J Biol Chem, 1951; 193:265–275.

26.

Lindros

. Alcoholic liver disease: pathobiological aspects. J Hepatol, 1995; 23:7–15.

27.

Balkan

, Dogru-Abbasoglu

, Kanbagli

, Çevikbaþ

, Aykaç-Toker

, Uysal

. Taurine has a protective effect against thioacetamide-induced liver cirrhosis by decreasing oxidative stress. Hum Exp Toxicol, 2001; 20:251–254.

28.

D'Almedia

, Menteiro

, Oliveria

, Pomarico

, Bueno

. Long lasting effects of ethanol administration on the antioxidant enzymes. J Biochem Toxicol, 1994; 9:141–143.

29.

Jaya

, Menon

. Role of lipid peroxides, glutathione and antiperoxidative enzymes in alcohol and drug toxicity. Indian J Exp Biol, 1993; 31:453–459.

30.

Lieber

. Alcohol and the liver: metabolism of alcohol and its role in hepatic and extrahepatic diseases. Mt Sinai J Med, 2000; 67:84–94.

31.

Stohs

, Bagchi

. Oxidative mechanism in the toxicity of metal ions. Free Radic Biol Med, 1995; 18:321–336.

32.

, Kim

, Chun

, Lee

, Park

. Glutathione recycling is attenuated by acute ethanol feeding in rat liver. J Korean Med Sci, 1997; 12:316–321.

33.

Meister

. Selective modification of glutathione metabolism. Science, 1983; 220:472–477.

34.

Kaplowitz

, Tsukamoto

. Oxidative stress and liver disease. Prog Liver Dis, 1996; 14:131–159.

35.

Bjørneboe

, Johnsen

, Bjørneboe

, Mørland

, Drevon

. Effect of heavy alcohol consumption on serum concentrations of fat-soluble vitamins and selenium. Alcohol Alcohol Suppl, 1987; 1:533–537.

36.

. Cellular defences against damage from reactive oxygen species. Physiol Rev, 1994; 74:139–162.

37.

Zloch

, Ginter

. Moderate alcohol consumption and vitamin C status in the guinea-pig and the rat. Physiol Res, 1995; 44:173–178.

38.

Jurczuk

, Moniuszko-Jakoniuk

, Brzóska

. Involvement of some low-molecular thiols in the peroxidative mechanisms of lead and ethanol action on rat liver and kidney. Toxicology, 2006; 219:11–21.

39.

Jacob

. The integrated antioxidant system. Nutr Res, 1995; 15:755–766.

40.

Iqbal

, Athar

. Attenuation of iron-nitrilotriacetate (Fe-NTA)-mediated renal oxidative stress, toxicity and hyperproliferative response by the prophylactic treatment of rats with garlic oil. Food Chem Toxicol, 1998; 36:485–495.

41.

Singh

, Abraham

, Kesavan

. In vivo radioprotection with garlic extract. Mutat Res, 1995; 345:147–153.

42.

Augusti

, Sheela

. Antiperoxide effect of S-allyl cysteine sulfoxide, an insulin secretagogue, in diabetic rats. Experientia, 1996; 52:115–119.

43.

Prasad

, Laxdal

, Yu

, Raney

. Evaluation of hydroxyl radical-scavenging property of garlic. Mol Cell Biochem, 1996; 154:55–63.

44.

Török

, Belágyi

, Rietz

, Jacob

. Effectiveness of garlic on the radical activity in radical generating systems. Arzneimittelforschung, 1994; 44:608–611.

45.

Rabinkov

, Miron

, Konstantinovski

, Wilchek

, Mirelman

, Weiner

. The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins. Biochim Biophys Acta, 1998; 1379:233–244.

46.

Wei

, Lau

BHS

. Garlic inhibits free radical generation and augments antioxidant enzyme activity in vascular endothelial cells. Nutr Res, 1998; 18:61–70.

47.

Geng

, Lau

BHS

. Aged garlic extract modulates glutathione redox cycle and superoxide dismutase activity in vascular endothelial cells. Phytother Res, 1997; 11:54–56.

48.

Banerjee

, Mukherjee

, Maulik

. Garlic as an antioxidant: the good, the bad and the ugly. Phytother Res, 2003; 17:97–106.

49.

Gedik

, Kabasakal

, Sehirli

, Ercan

, Sirvanci

, Keyer-Uysal

, Sener

. Long-term administration of aqueous garlic extract (AGE) alleviates liver fibrosis and oxidative damage induced by biliary obstruction in rats. Life Sci, 2005; 76:2593–2606.

50.

Sener

, Satyroglu

, Ozer Sehirli

, Kacmaz

. Protective effect of aqueous garlic extract against oxidative organ damage in a rat model of thermal injury. Life Sci, 2003; 73:81–91.

51.

Tsai

, Yang

, Chen

, Sheen

, Lii

. Garlic organosulfur compounds upregulate the expression of the pi class of glutathione S-transferase in rat primary hepatocytes. J Nutr, 2005; 135:2560–2565.

52.

Fukao

, Hosono

, Misawa

, Seki

, Ariga

. The effects of allyl sulfides on the induction of phase II detoxification enzymes and liver injury by carbon tetrachloride. Food Chem Toxicol, 2004; 42:743–749.

53.

Santiard

, Ribiére

, Nordmann

, Houee-Levin

. Inactivation of Cu,Zn-superoxide dismutase by free radicals derived from ethanol metabolism: a gamma radiolysis study. Free Radic Biol Med, 1995; 19:121–127.