Garlic's ability to prevent <it>in vitro </it>Cu2+-induced lipoprotein oxidation in human serum is preserved in heated garlic: effect unrelated to Cu2+-chelation

1475-2891-3-10 1475-2891 Research Garlic's ability to prevent <it>in vitro </it>Cu2+-induced lipoprotein oxidation in human serum is preserved in heated garlic: effect unrelated to Cu2+-chelation Pedraza-Chaverrí José pedraza@servidor.unam.mx Gil-Ortiz Mariana mariana_80@yahoo.com Albarrán Gabriela rabbyt021@yahoo.com Barbachano-Esparza Laura lalibarbachano@yahoo.com.mx Menjívar Marta menjivar@servidor.unam.mx Medina-Campos N Omar mconoel@servidor.unam.mx

Departamento de Biología, Facultad de Química, Edificio B, Segundo Piso, Laboratorio 209, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, 04510, México, D.F., México

Nutrition Journal 1475-2891 2004 3 1 10 http://www.nutritionj.com/content/3/1/10 15341661 10.1186/1475-2891-3-10 07 7 2004 01 9 2004 01 9 2004 2004 Pedraza-Chaverrí et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

It has been shown that several extracts and compounds derived from garlic are able to inhibit Cu²⁺-induced low density lipoprotein oxidation. In this work we explored if the ability of aqueous garlic extract to prevent in vitro Cu²⁺-induced lipoprotein oxidation in human serum is affected by heating (a) aqueous garlic extracts or (b) garlic cloves. In the first case, aqueous extract of raw garlic and garlic powder were studied. In the second case, aqueous extract of boiled garlic cloves, microwave-treated garlic cloves, and pickled garlic were studied. It was also studied if the above mentioned preparations were able to chelate Cu²⁺.

Methods

Cu²⁺-induced lipoprotein oxidation in human serum was followed by the formation of conjugated dienes at 234 nm and 37°C by 240 min in a phosphate buffer 20 mM, pH 7.4. Blood serum and CuSO₄were added to a final concentration of 0.67% and 0.0125 mM, respectively. The lag time and the area under the curve from the oxidation curves were obtained. The Cu²⁺-chelating properties of garlic extracts were assessed using an approach based upon restoring the activity of xanthine oxidase inhibited in the presence of 0.050 mM Cu²⁺. The activity of xanthine oxidase was assessed by monitoring the production of superoxide anion at 560 nm and the formation of uric acid at 295 nm. Data were compared by parametric or non-parametric analysis of variance followed by a post hoc test.

Results

Extracts from garlic powder and raw garlic inhibited in a dose-dependent way Cu²⁺-induced lipoprotein oxidation. The heating of garlic extracts or garlic cloves was unable to alter significantly the increase in lag time and the decrease in the area under the curve observed with the unheated garlic extracts or raw garlic. In addition, it was found that the garlic extracts were unable to chelate Cu²⁺.

Conclusions

(a) the heating of aqueous extracts of raw garlic or garlic powder or the heating of garlic cloves by boiling, microwave or pickling do not affect garlic's ability to inhibit Cu²⁺-induced lipoprotein oxidation in human serum, and (b) this ability is not secondary to Cu²⁺-chelation.

Background

Garlic (Allium sativum) has been cultivated since ancient times and used as a spice and condiment for many centuries 1. During the past years, there has been a growing awareness of the potential medicinal uses of garlic 2345. The antioxidant properties of garlic are well documented 678. In particular, aqueous garlic extract 9 and aged garlic extract 101112 are able to prevent Cu²⁺-induced low density lipoprotein (LDL) oxidation. In addition, Ide et al. 10 and Ho et al. 13 have shown that some garlic compounds such as S-allylcysteine, N-acetyl-S-allylcysteine, S-allylmercaptocysteine, alliin, and allixin, are also able to prevent Cu²⁺-induced LDL oxidation. Ou et al. 14 showed that the garlic compounds S-ethylcysteine, N-acetylcysteine, diallyl sulfide, and diallyl disulfide inhibit amphotericin- and Cu²⁺-induced LDL oxidation. Huang et al. 15 showed that diallyl sulfide, diallyl disulfide, S-allylcysteine, S-ethylcysteine, S-methylcysteine, and S-propylcysteine are able to prevent glucose-induced lipid oxidation in isolated LDL. The protective effect of aged garlic extract 1011 and S-ethylcysteine, N-acetylcysteine, diallyl sulfide, and diallyl disulfide 14 on Cu²⁺-induced LDL oxidation may be explained, at least in part, for their ability to chelate Cu²⁺. Interestingly, the diethyl ether extract of aged garlic extract, which also inhibits Cu²⁺-induced LDL oxidation, is unable to chelate Cu²⁺11 indicating that its ability to prevent LDL oxidation is unrelated to Cu²⁺-chelation.

On the other hand, 95% of the sulfur in intact garlic cloves is found in two classes of compounds in similar abundance: the S-alkylcysteine sulfoxides and the γ-glutamyl-S-alkylcysteines 16. 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 16. 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 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 17. Due to the abundance of alliin, the main thiosulfinate formed upon crushing garlic is allicin 16. The half-life of allicin at room temperature is 2–16 hours; however, in crushed garlic (or in garlic juice) it is 2.4 days 16.

Several studies have been performed to test the effect of heating on several garlic properties. It has been shown that the boiling of garlic cloves by 15 min impairs significantly its ability to inhibit cyclooxygenase activity 18 and thromboxane B₂synthesis 19. In addition, heating of garlic cloves by 60 seconds in microwave reduces its anticancer properties 17. Interestingly when microwave heating was applied 10 minutes after garlic crushing the anticancer properties were preserved indicating that allinase activation is necessary to generate anticancer compounds which are heat stable 17. In a similar way, the hydroxyl scavenging properties of garlic were essentially preserved when garlic extracts were heated at 100°C by 20, 40 or 60 min 20. In contrast, heating of garlic extracts by 10 min at 100°C reduced the bactericidal activity against Helicobacter pylori 21 and the ability to inhibit platelet aggregation 22. However, to our knowledge, there are no studies exploring if the heating of garlic cloves or aqueous extract of raw garlic or garlic powder are able to inhibit Cu²⁺-induced lipoprotein oxidation in human serum. In the present paper we studied if the ability of aqueous garlic extracts to inhibit in vitro Cu²⁺-induced lipoprotein oxidation in human serum is altered in the following aqueous preparations: (a) heated extract of garlic powder, (b) heated extract of raw garlic, (c) extract of boiled garlic cloves, (d) extract of microwave-treated garlic cloves, and (e) extract of pickled garlic. In addition it was studied if the above mentioned preparations are able to chelate Cu²⁺.

It was found that (a) the heating of garlic extracts or garlic cloves had no influence on the ability of garlic extracts to prevent in vitro Cu²⁺-induced lipoprotein oxidation in human serum, and (b) this protective effect was unrelated to Cu²⁺-chelation.

Methods

Materials and reagents

Bulbs of garlic were from a local market. Garlic powder was from McCormick (Mexico City, Mexico). Copper sulfate, Na₂EDTA, Na₂CO₃, KH₂PO₄, and Na₂HPO₄were from JT Baker (Mexico City, Mexico). Copper sulfate was dissolved in distilled water. Xanthine oxidase, xanthine, and nitroblue tetrazolium (NBT) were from Sigma Chemical Co. (St. Louis, MO., USA).

Preparation of aqueous extracts of garlic

Extract of garlic powder (GP)

Garlic powder was weighted (0.6 g), dissolved, and stirred with 6 mL of distilled water for 20 min. This solution was centrifuged at 20,124 × g for 5 min at 4°C. The supernatant was recovered and used at the final concentration of 0.05, 0.075, 0.10, and 0.25 mg/mL.

Heated extract of garlic powder (HGP)

The procedure was similar to the previous one except that the mixture was boiled for 20 min before the centrifugation. The supernatant was recovered and used at the final concentration of 0.05 and 0.10 mg/mL.

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 1,277 × g for 10 min and the supernatant was centrifuged at 20,124 × g for 5 min at 4°C. The supernatant was recovered and used at the final concentration of 0.125, 0.25, 0.5, and 0.75 mg/mL.

Heated extract of raw garlic (HRG)

The procedure was similar to the previous one except that the homogenate was boiled for 20 min before the second centrifugation step. The amount of water evaporated was replaced at the end of the heating. The supernatant was recovered and used at the final concentration of 0.25 and 0.5 mg/mL.

Extract of boiled garlic (BG)

Unpeeled garlic cloves were boiled in water for 10 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 final concentration of 0.25 and 0.5 mg/mL.

Extract of garlic cloves submitted to microwave heating (MG)

Unpeeled garlic cloves were submitted to microwave heating for 1 min (1100 watts). 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 seconds of microwave heating can totally destroy allinase enzyme activity whereas microwave heating for 30 seconds inhibits 90% of allinase activity compared with unheated garlic 17. The supernatant was recovered and used at the final concentration of 0.25 and 0.5 mg/mL.

Preparation 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. Garlic cloves were put into jars with same solution and then pasteurized for 5 min at 72°C. The jars were closed immediately and stored at 4°C. The experiments with pickled garlic were performed five weeks after. The aqueous extract was prepared as described before (extract of raw garlic). The supernatant was recovered and used at the final concentration of 0.25 and 0.5 mg/mL.

Blood collection

Blood samples were obtained from 2 male and 3 female healthy volunteers aged 24 to 46 years. The experimental protocol is in compliance with the Helsinki Declaration and written informed consent was obtained from all subjects. A fasting blood sample was drawn from the antecubital fossa vein into glass tubes, allowed to clot, and then centrifuged at 2,000 × g for 10 min. The serum removed was aliquoted and frozen at -80°C until assayed. The concentration of glucose, cholesterol and triglycerides in serum was measured by an autoanalyzer (Hitachi 917 Automatic Analyzer, Boheringer Mannheim Corporation, Indianapolis, IN, USA).

Cu²⁺-induced lipoprotein oxidation in human serum

A modification of the serum oxidation method described by Regnstrom et al. 23 was used. This method provides an indication of conjugated dienes formation in lipoprotein fatty acids present in serum exposed to Cu²⁺, assessed by measuring changes in absorbance at 234 nm. The formation of conjugated dienes in lipoprotein deficient serum exposed to Cu²⁺is absent, indicating that diene formation in lipoprotein fatty acids is primarily responsible for the increase in absorbance 23. The oxidation curves have three phases: lag, propagation, and decomposition. This method has been used previously by others 2425262728. Serum was diluted to a final concentration of 0.67% in 20 mM phosphate buffer, pH 7.4 and saturated with O₂. Oxidation was initiated by the addition of CuSO₄to a final concentration of 0.0125 mM. The formation of conjugated dienes was followed by monitoring the change in absorbance at 234 nm at 37°C on a Beckman DU-64 spectrophotometer equipped with a six position automatic sample changer (Fullerton, CA, USA). Absorbance readings were taken every 10 min over 240 min. The aqueous garlic extracts were added at the indicated concentrations (ranging from 0.05 to 0.75 mg/mL). Control tubes consisted of identical assays conditions but without the garlic extract. Since the transition from lag phase to propagation phase was continuous, lag time was defined as the intercept of the tangents of the propagation and lag phases and expressed in minutes.

Determination of Cu²⁺chelation of garlic extracts

The Cu²⁺chelating properties of aqueous garlic extracts were assessed using an approach based upon restoring the activity of xanthine oxidase described previously 11. This enzyme is inhibited in the presence of 0.050 mM CuSO₄29. The activity of xanthine oxidase can be assessed by monitoring either the production of superoxide anion or the formation of uric acid. Xanthine oxidase activity would be restored if garlic extracts were able to chelate Cu²⁺.

Xanthine oxidase activity was measured by NBT reduction and uric acid production. The following concentrated solutions were prepared in distilled water: xanthine oxidase 168 U/L, xanthine 0.30 mM, NBT 0.15 mM, and Na₂CO₃0.4 M. Superoxide anions were generated in a reaction volume of 1 mL containing in a final concentration: xanthine 0.087 mM, Na₂CO₃15 mM, NBT 0.022 mM, and 50 mM phosphate buffer pH 7.4 or garlic extract in a volume of 0.1 mL for control or experimental tube, respectively. In additional tubes with or without garlic extract, CuSO₄was added in a final concentration of 0.050 mM. The reaction was initiated by the addition of 0.025 U of xanthine oxidase, and superoxide anion production was monitored at 560 nm. In the same experiment, xanthine oxidase activity was measured by following the uric acid production at 295 nm 30. Absorbance at 560 and 295 nm was obtained every minute for 3 minutes and the results were expressed as change of absorbance/min. The garlic extracts were added at the higher concentration used in the experiments for Cu²⁺-induced lipoprotein oxidation along with (+CuSO₄) or without (-CuSO₄) 0.050 mM CuSO₄. Control tubes contained all the reagents but without garlic extracts and they considered as 100% production of uric acid and superoxide anion. In a separate tube with 0.050 mM CuSO₄, 0.060 mM EDTA was added as an additional control in which was expected the restoration of xanthine oxidase since EDTA is a metal-chelating agent.

Statistics

Data are expressed as mean ± SEM of five determinations using different serum samples. The variables used to describe the difference between the oxidation curves were lag time, area under the oxidation curve (AUC), and slope of the propagation phase. These parameters were obtained using the GraphPad Prism software v. 3.02 (San Diego, CA, USA). All parameters were compared using either one way analyses of variance followed by Bonferroni's t test or Kruskall-Wallis analysis of variance followed by Dunn's test. p < 0.05 was considered significant.

Results

Glucose, cholesterol, and triglycerides in human serum

The concentration, in mmol/L, of glucose, cholesterol, and triglycerides in serum, of the subjects involved in this study was 4.56 ± 0.15, 4.24 ± 0.42, and 1.05 ± 0.11, respectively. These data show that the human beings from whom the blood serum samples were obtained for this study had no alterations in the circulating levels of glucose, cholesterol, and triglycerides.

Effect of garlic extracts on Cu²⁺-induced lipoprotein oxidation in human serum

Unheated garlic extracts from raw garlic or from garlic powder inhibited lipoprotein oxidation in a dose-dependent way. Figure 1 shows a representative graph obtained from a single subject. Panel A shows the effect of increasing concentrations of garlic powder extract (0.05 to 0.25 mg/mL) and panel B shows the effect of increasing concentrations of raw garlic extract (0.125 to 0.75 mg/mL) on Cu²⁺-induced lipoprotein oxidation in human serum. The increasing concentrations of garlic extracts displaced the curve to the right, compared to the control curve obtained without garlic extract, indicating that the inhibition of Cu²⁺-induced lipoprotein oxidation is dose-dependent. Lag time, AUC, and slope were obtained from these oxidation curves. Figure 2 shows the lag time (panels A and C) and AUC (panels B and D) and Table 1 shows the slopes of the propagation phase from the five subjects studied. Panels A and B show the effect of garlic powder extract and panels C and D show the effect of raw garlic extract. It can be seen that garlic extracts dose-dependently increased lag time and decreased AUC and slopes. Based on these curves the concentrations of all the extracts studied were chosen: 0.25 and 0.5 mg/mL for HRG, BG, MG, and PG; and 0.05 and 0.1 mg/mL for GP and HGP.

Figure 1

Representative curves showing the effect of aqueous garlic extracts on Cu²⁺-induced lipoprotein oxidation in human serum

Representative curves showing the effect of aqueous garlic extracts on Cu²⁺-induced lipoprotein oxidation in human serum. Panel A shows the effect of garlic powder extract and panel B shows the effect of raw garlic extract. Cu²⁺-induced oxidation was followed at 234 nm. Readings were taken every 10 min. Oxidation was started by the addition of CuSO₄at a final concentration of 0.0125 mM in 20 mM phosphate buffer, pH 7.4 saturated with O₂and the readings were followed by 240 min at 37°C.

Figure 2

Dose-dependent effect of aqueous garlic extracts on lag time and AUC

Dose-dependent effect of aqueous garlic extracts on lag time and AUC. Panels A and B shows the effect of garlic powder extract and panels C and D shows the effect of raw garlic extract. Lag time is shown in panels A and C and AUC is shown on panels B and D. ^ap < 0.01 (panel A) and p < 0.001 (panels B, C, and D) vs. 0 mg/mL. Data are mean ± SEM of five determinations using independent samples.

Table 1

Effect of extract of garlic powder (GP) and raw garlic (RG) on the slopes of oxidation curves.

GP, [mg/mL]

0.05

0.075

0.1

0.25

slope

0.0025 ± 0.0002

0.0017 ± 0.0003

0.0014 ± 0.0002

0.0009 ± 0.0003

0.0003 ± 0.00003^a

RG, [mg/mL]

0.125

0.25

0.5

0.75

slope

0.0027 ± 0.0002

0.0018 ± 0.0002

0.0022 ± 0.0002

0.0017 ± 0.0002^b

0.0007 ± 0.0003^a

Data are mean ± SEM of five determinations using independent samples. ^ap < 0.001, ^bp < 0.05 vs. 0 mg/mL.

Effect of different treatments of garlic on Cu²⁺-induced lipoprotein oxidation in human serum

The effect of HGP, HRG, BG, MG, and PG on the lag time and AUC is shown on Figs. 3,4,5,6,7 respectively. The effects of these extracts on the slopes of oxidation curves are shown in Tables 2 and 3. GP and HGP were studied at 0.05 and 0.1 mg/mL and HRG, BG, MG, and PG were studied at 0.25 and 0.5 mg/mL. The data of HGP and HRG were compared with those of unheated extracts, GP and RG, respectively (Figs. 3 and 4 and Tables 2 and 3). The data of BG, MG, and PG were compared with those of RG (Figs. 5,6,7 and Table 3). It can be seen that the extracts increased lag time and decreased AUC at both concentrations studied indicating that they inhibit Cu²⁺-induced lipoprotein oxidation. The decrease in the slope was significant only at the higher concentration for GP and HGP (Table 2) and for RG and HRG, BG, and MG (Table 3). The decrease in the slope in PG was not significant (Table 3). Interestingly, the treatments (heating of extracts of garlic powder or raw garlic or heating garlic cloves by boiling, microwave or pickling) had no significative effect on lag time, AUC, and slope. All the comparisons between unheated and heated extracts were not different. Our data show that the antioxidant ability of garlic on Cu²⁺-induced lipoprotein oxidation in human serum is not significantly affected by the above mentioned treatments.

Figure 3

Effect of heated extract of garlic powder (HGP) on lag time and AUC

Effect of heated extract of garlic powder (HGP) on lag time and AUC. Aqueous extract of garlic powder was heated at 100°C by 10 min. Aqueous extracts of unheated (GP) and HGP were added to the system at two concentrations (0.25 and 0.5 mg/mL). Cu²⁺-induced oxidation was followed at 234 nm. Readings were taken every 10 min. Oxidation was started by the addition of Cu²⁺at a final concentration of 0.0125 mM in 20 mM phosphate buffer, pH 7.4 saturated with O₂and the readings were followed by 240 min at 37°C. ^ap < 0.001, ^bp < 0.01, and ^cp < 0.05 vs. 0 mg/mL. Δ= HGP. Data are mean ± SEM of five determinations using independent samples.

Figure 4

Effect of heated extract of raw garlic (HRG) on lag time and AUC

Effect of heated extract of raw garlic (HRG) on lag time and AUC. Aqueous extract of raw garlic was heated at 100°C by 10 min. Aqueous extracts of unheated (RG) or HRG were added to the system at two concentrations (0.25 and 0.5 mg/mL). Cu²⁺-induced oxidation was followed at 234 nm. Readings were taken every 10 min. Oxidation was started by the addition of Cu²⁺at a final concentration of 0.0125 mM in 20 mM phosphate buffer, pH 7.4 saturated with O₂and the readings were followed by 240 min at 37°C. ^ap < 0.05 vs. 0 mg/mL. Δ= HRG. Data are mean ± SEM of five determinations using independent samples.

Figure 5

Effect of aqueous extract of boiled garlic (BG) cloves on lag time and AUC

Effect of aqueous extract of boiled garlic (BG) cloves on lag time and AUC. Comparison was made with aqueous extract of raw garlic (RG). Aqueous extracts of BG and RG were added to the system at two concentrations (0.25 and 0.5 mg/mL). Cu²⁺-induced oxidation was followed at 234 nm. Readings were taken every 10 min. Oxidation was started by the addition of Cu²⁺at a final concentration of 0.0125 mM in 20 mM phosphate buffer, pH 7.4 saturated with O₂and the readings were followed by 240 min at 37°C. ^ap < 0.001, ^bp < 0.01, and ^cp < 0.05 vs. 0 mg/mL. Δ= BG cloves. Data are mean ± SEM of five determinations using independent samples.

Figure 6

Effect of aqueous extract of garlic cloves submitted to microwave heating (MG) on lag time and AUC

Effect of aqueous extract of garlic cloves submitted to microwave heating (MG) on lag time and AUC. Comparison was made with aqueous extract of raw garlic (RG). Aqueous extracts of microwave-treated garlic cloves (MG) and RG were added to the system at two concentrations (0.25 and 0.5 mg/mL). Cu²⁺-induced oxidation was followed at 234 nm. Readings were taken every 10 min. Oxidation was started by the addition of Cu²⁺at a final concentration of 0.0125 mM in 20 mM phosphate buffer, pH 7.4 saturated with O₂and the readings were followed by 240 min at 37°C. ^ap < 0.001 and ^bp < 0.01 vs. 0 mg/mL. Δ= MG. Data are mean ± SEM of five determinations using independent samples.

Figure 7

Effect of aqueous extract of pickled garlic (PG) on lag time and AUC

Effect of aqueous extract of pickled garlic (PG) on lag time and AUC. Comparison was made with aqueous extract of raw garlic (RG). Aqueous extracts of PG and RG were added to the system at two concentrations (0.25 and 0.5 mg/mL). Cu²⁺-induced oxidation was followed at 234 nm. Readings were taken every 10 min. Oxidation was started by the addition of Cu²⁺at a final concentration of 0.0125 mM in 20 mM phosphate buffer, pH 7.4 saturated with O₂and the readings were followed by 240 min at 37°C. ^ap < 0.05, ^bp < 0.001 (panel A), and ^ap < 0.01 (panel B) vs. 0 mg/mL. Δ= PG. Data are mean ± SEM of five determinations using independent samples.

Table 2

Effect of extract of garlic powder (GP) and heated garlic powder (HGP) on the slopes of oxidation curves.

Extract [mg/mL]

0.05

0.05 Δ

0.1

0.1 Δ

GP or HGP

0.0024 ± 0.0002

0.0017 ± 0.0002

0.0018 ± 0.0002

0.0013 ± 0.0001^a

Data are mean ± SEM of five determinations using independent samples. ^ap < 0.01 vs. 0 mg/mL. Δ = HGP.

Table 3

Effect of different extracts of garlic on the slopes of oxidation curves.

Extract, [mg/mL]

0.25

0.25 Δ

0.5

0.5 Δ

RG or HRG

0.0023 ± 0.0003

0.0020 ± 0.0001

0.0014 ± 0.0001^a

0.0013 ± 0.002^b

0.0011 ± 0.0002^b

RG or BG

0.0021 ± 0.0001

0.0016 ± 0.0002

0.0008 ± 0.0002^c

0.0007 ± 0.0004^c

0.0005 ± 0.0001^c

RG or MG

0.0024 ± 0.0002

0.0018 ± 0.0002

0.0015 ± 0.0002

0.0011 ± 0.0003^b

0.0005 ± 0.0002^c

RG or PG

0.0026 ± 0.0002

0.0023 ± 0.0004

0.0021 ± 0.0004

0.0016 ± 0.0003

0.0019 ± 0.0003

Data are mean ± SEM of five determinations using independent samples. ^ap < 0.05, ^bp < 0.01, ^cp < 0.001, vs. 0 mg/mL. Δ = HRG, BG, MG or PG.

Cu²⁺-chelation studies

To investigate if the ability of garlic extracts to inhibit Cu²⁺-induced lipoprotein oxidation in human serum was secondary to Cu²⁺-chelation, these extracts were tested in an in vitro system (see material and methods) to know if they were able to chelate Cu²⁺. The results for each extract at the higher concentration are presented in Fig. 8. Panel A shows uric acid production measured at 295 nm and panel B shows superoxide production measured by the NBT reduction at 560 nm. Cu²⁺-induced a decrease in xanthine oxidase activity measured both by uric acid production at 295 nm which decreased 71% and by NBT reduction at 560 nm which decreased 96% (Fig. 8). Uric acid production and NBT reduction were restored by EDTA. The extracts were added at the following concentrations: GP and HGP = 0.1 mg/mL, and RG, HRG, BG, MG, and PG = 0.5 mg/mL. We have previously shown that at these concentrations, the extracts increased lag time and decreased AUC indicating that they were able to inhibit Cu²⁺-induced lipoprotein oxidation (Figs. 3,4,5,6). In absence of Cu²⁺(-CuSO₄) the extracts were unable to modify uric acid production indicating that they were unable to modify xanthine oxidase activity. In absence of Cu²⁺, NBT reduction was decreased significantly by RG indicating that this extract quenched superoxide anion. In presence of Cu²⁺(+CuSO₄) the extracts were unable to restore (a) uric acid production (p > 0.05 vs. CT without Cu²⁺), with the exception of RG which was able to restore it partially (p < 0.05 vs. CT without Cu²⁺), and (b) NBT reduction (p > 0.05 vs. CT without Cu²⁺). In summary, the extracts were unable to restore xanthine oxidase activity indicating that they do not chelate Cu²⁺. Only RG showed a weak Cu²⁺-chelating activity.

Figure 8

Effect of garlic extracts on Cu²⁺-chelation

The Cu²⁺-chelating properties of aqueous garlic extracts were assessed using an approach based upon restoring the activity of xanthine oxidase which has been inhibited in the presence of 0.050 mM CuSO₄. The activity of xanthine oxidase can be assessed by monitoring either the production of superoxide anion (measuring the reduction of NBT at 560 nm) or the formation of uric acid (following absorbance at 295 nm). Xanthine oxidase activity would be restored if the garlic extracts were able to chelate Cu²⁺. Panel A shows xanthine oxidase activity measuring uric acid production at 295 nm. ^ap < 0.0001 vs. CT (-Cu²⁺), ^bp < 0.0001 vs. CT (+Cu²⁺), ^cp < 0.0001 vs. its respective group without Cu²⁺. Panel B shows xanthine oxidase activity measuring superoxide production by NBT reduction at 560 nm. ^ap < 0.0001 vs. CT (-Cu²⁺); ^bp < 0.0001 and ^cp < 0.05 vs. CT (+Cu²⁺); ^dp < 0.05 vs. its respective group without Cu²⁺. CT = Control (-Cu²⁺, +Cu²⁺), GP = aqueous extracts of garlic powder, HGP = heated aqueous extracts of garlic powder, RG = aqueous extracts of raw garlic, HRG = heated aqueous extracts of raw garlic, BG = aqueous extracts of boiled garlic, MG = aqueous extracts of microwave-treated garlic, and PG = aqueous extracts of pickled garlic. GP and HGP = 0.1 mg/mL, RG, HRG, BG, MG, and PG = 0.5 mg/mL. Data are mean ± SEM of 3 determinations, except for both CT groups and EDTA group in which n = 7.

Discussion

Garlic has been used for millennia in folk medicine of many cultures to treat cardiovascular diseases and other disorders 12345678. It has been shown in many cases that the protective effect of garlic is associated with its antioxidant properties 78. The antioxidant properties of some garlic extracts used in this work have been studied. It has been found that aqueous extract of raw garlic scavenges hydroxyl radicals 203132 and superoxide anion 32, inhibits lipid peroxidation 20, LDL oxidation 9, the formation of lipid hydroperoxides 203132, and in vivo enhances endogenous antioxidant system 33 and prevents oxidative stress to the heart 3435. Chronic administration of raw garlic homogenate increases catalase and superoxide dismutase in rat heart 33 and protects heart against oxidative damage induced by adriamycin 34 or ischemia and reperfusion 35. Aqueous extract of garlic powder are also able to scavenge hydroxyl radicals 36 and superoxide anion 37. The heated aqueous extract of garlic powder maintains its ability to scavenge hydroxyl radicals 20. In addition the ability of the aqueous extracts from boiled garlic cloves to scavenge hydroxyl radicals, superoxide anion, and hydrogen peroxide is not altered (unpublished results from our group). To our knowledge, additional antioxidant properties of the extracts from microwave-treated garlic cloves or from pickled garlic have not been studied.

Furthermore, the antioxidant properties of some isolated garlic compounds also have been studied. Allicin, the main component in aqueous extract from raw garlic and garlic powder, scavenges hydroxyl radicals and inhibit lipid peroxidation 38 and prevents the lung damage induced by ischemia-reperfusion 39. The antioxidant properties of allicin may explain, at least in part, the ability of these extracts (from raw garlic or garlic powder) to inhibit Cu²⁺-induced lipoprotein oxidation in human serum. Alliin, the main component in extracts from boiled garlic cloves, microwave-treated garlic cloves and pickled garlic, scavenges hydroxyl radicals 40, hydrogen peroxide 41, and inhibits lipid peroxidation 41 and LDL oxidation 42. This may explain, at least in part, the ability of boiled garlic, microwave-treated garlic, or picked garlic to inhibit Cu²⁺-induced lipoprotein oxidation in human serum. Interestingly, it has been shown that another garlic compounds such as S-allylcysteine 1013, N-acetyl-S-allylcysteine 10, S-allylmercaptocysteine 10, alliin 10, allixin 10, and S-ethylcysteine, N-acetylcysteine, diallyl sulfide, and diallyl disulfide 14 are able to inhibit Cu²⁺-induced LDL oxidation. The antioxidant properties of S-allylcysteine 43, S-allylmercaptocysteine 44, diallyl sulfide 45, and diallyl disulfide 46 also have been seen in vivo in an experimental model of nephrotoxicity induced by gentamicin.

Our data strongly suggest that the ability of garlic to prevent Cu²⁺-induced lipoprotein oxidation in human serum is preserved in spite of inactivation of allinase by boiling, microwave or pickling or by the heating of garlic extracts and that the compound(s) involved in the inhibition of Cu²⁺-induced lipoprotein oxidation are heat stable. Our data are in contrast with previous studies in the literature showing that the heating may impair significantly several garlic properties. For example, microwave-treatment for 1 min impaired the anticancer properties of garlic 17 and the heating of garlic cloves by 15 min impairs significantly its ability to inhibit thromboxane B₂synthesis 19, and platelet aggregation 22, and the cyclooxygenase activity 18. The heating by 10 min at 100°C reduced the bactericidal activity against Helicobacter pylori 21. Interestingly, Kasuga et al. 47 have found that garlic extracts, prepared from boiled cloves, show efficacy in the following three experimental models: testicular hypogonadism induced by warm water treatment, intoxication of acetaldehyde, and growth of inoculated tumor cells, and Prasad et al. 20 found that the heating did not modify the ability of garlic extract to scavenge hydroxyl radicals. The data from Prasad et al. 20 and Kasuga et al. 47 strongly suggest that some garlic properties may remain unmodified after heating. Our data are in agreement with those of Prasad et al. 20 suggesting that the ability to inhibit Cu²⁺-induced lipoprotein oxidation is also preserved after the heating of garlic.

In addition, it was found that the garlic extracts used in our study were unable to chelate Cu²⁺suggesting that the ability of these extracts to inhibit Cu²⁺-induced lipoprotein oxidation is not secondary to Cu²⁺-chelation. Only RG showed a weak Cu²⁺-chelation activity, which was more evident at 295 nm. Based on previous data with aged garlic extracts 11 and some individual garlic compounds such as S-ethylcysteine, N-acetylcysteine, diallyl sulfide, and diallyl disulfide 14, we expected that our garlic extracts had Cu²⁺-chelating activity. The discrepancy with our data may simply reflect differences in composition in each garlic extract. This is additionally supported by the fact that the diethyl ether extract from aged garlic extract has no Cu²⁺-chelating activity 11. The precise mechanism by which our extracts inhibit Cu²⁺-induced lipoprotein oxidation remains to be studied.

Conclusions

Authors's contributions

JPCH conceived, designed and coordinated the study and drafted the manuscript. MGO performed the studies on Cu²⁺-chelation and studied the effect of boiled garlic, picked garlic and garlic submitted to microwave heating on Cu²⁺-induced lipoprotein oxidation in human serum. GA developed in our laboratory the method to oxidize lipoproteins by Cu²⁺and performed studies with garlic extracts of raw garlic and garlic powder. LBE participated in the studies on Cu²⁺-chelation. MM participated in the blood obtention and clinical chemistry analyses of blood serum. ONMC participated in the statistical analyses and developed the method to analyze the Cu²⁺chelating properties of garlic extracts. All authors read and approved the final manuscript.

Competing interests

None declared.

Acknowledgements

Financial support. This work was supported by CONACYT (Grant No. 40009-M).

The chemistry of garlic and onions Block E Sci Am 1985 252 114 119 3975593 Effect of garlic on cardiovascular disorders: a review Banerjee SK Maulik SK Nutr J 2002 1 4 139960 12537594 10.1186/1475-2891-1-4 Garlic as an antioxidant: the good, the bad and the ugly Banerjee SK Mukherjee PK Maulik SK Phytother Res 2003 17 97 106 12601669 10.1002/ptr.1281 Therapeutic actions of garlic constituents Agarwal KC Med Res Rev 1996 16 111 124 10.1002/(SICI)1098-1128(199601)16:1<111::AID-MED4>3.0.CO;2-5 8788216 Garlic prevents hypertension induced by chronic inhibition of nitric oxide synthesis Pedraza-Chaverri J Tapia E Medina-Campos ON de los Angeles Granados M Franco M Life Sci 1998 62 PL71 77 10.1016/S0024-3205(97)01155-7 Dietary supplementation with aged garlic extract reduces plasma and urine concentrations of 8-iso-prostaglandin F(2 alpha) in smoking and nonsmoking men and women Dillon SA Lowe G Billington D Rahman K J Nutr 2002 132 168 171 11823573 Garlic and aging: new insights into an old remedy Rahman K Ageing Res Rev 2003 2 39 56 10.1016/S1568-1637(02)00049-1 12437995 Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes Pedraza-Chaverri J Maldonado PD Medina-Campos ON Olivares-Corichi IM Granados-Silvestre MA Hernandez-Pando R Ibarra-Rubio ME Free Radic Biol Med 2000 29 602 611 10.1016/S0891-5849(00)00354-3 11033412 Antioxidant effects of aqueous extract. 2nd communication: Inhibition of the Cu(2+)-initiated oxidation of low density lipoproteins Lewin G Popov I Arzneimittelforschung 1994 44 604 607 8024630 Aged garlic extract and its constituents inhibit Cu(2+)-induced oxidative modification of low density lipoprotein Ide N Nelson AB Lau BH Planta Med 1997 63 263 264 9225610 Antioxidant properties of aged garlic extract: an in vitro study incorporating human low density lipoprotein Dillon SA Burmi RS Lowe GM Billington D Rahman K Life Sci 2003 72 1583 1594 10.1016/S0024-3205(02)02475-X 12551747 Daily supplementation with aged garlic extract, but not raw garlic, protects low density lipoprotein against in vitro oxidation Munday JS James KA Fray LM Kirkwood SW Thompson KG Atherosclerosis 1999 143 399 404 10.1016/S0021-9150(98)00293-7 10217370 S-allyl cysteine reduces oxidant load in cells involved in the atherogenic process Ho SE Ide N Lau BH Phytomedicine 2001 8 39 46 11292238 Protective action on human LDL against oxidation and glycation by four organosulfur compounds derived from garlic Ou CC Tsao SM Lin MC Yin MC Lipids 2003 38 219 224 12784861 Antioxidative and antiglycative effects of six organosulfur compounds in low-density lipoprotein and plasma Huang CN Horng JS Yin MC J Agric Food Chem 2004 52 3674 3678 10.1021/jf0307292 15161248 Garlic: A review of its medicinal effects and indicated active compounds Lawson LD In Phytomedicines of Europe: chemistry and biological activity Washington, DC: American Chemical Society Lawson LS, Bauer R 1998 176 209 ACS Symposium series 691 Heating garlic inhibits its ability to suppress 7, 12-dimethylbenz(a)anthracene-induced DNA adduct formation in rat mammary tissue Song K Milner JA J Nutr 1999 129 657 661 10082770 Mechanism by which garlic (Allium sativum) inhibits cyclooxygenase activity. Effect of raw versus boiled garlic extract on the synthesis of prostanoids Ali M Prostaglandins Leukot Essent Fatty Acids 1995 53 397 400 10.1016/0952-3278(95)90102-7 8821119 An evaluation of garlic and onion as antithrombotic agents Bordia T Mohammed N Thomson M Ali M Prostaglandins Leukot Essent Fatty Acids 1996 54 183 186 10.1016/S0952-3278(96)90014-9 8860105 Evaluation of hydroxyl radical-scavenging property of garlic Prasad K Laxdal VA Yu M Raney BL Mol Cell Biochem 1996 154 55 63 8717417 Inhibition of Helicobacter pylori by garlic extract (Allium sativum) Cellini L Di Campli E Masulli M Di Bartolomeo S Allocati N FEMS Immunol Med Microbiol 1996 13 273 277 10.1016/0928-8244(95)00120-4 8739190 Effect of raw versus boiled aqueous extract of garlic and onion on platelet aggregation Ali M Bordia T Mustafa T Prostaglandins Leukot Essent Fatty Acids 1999 60 43 47 10.1054/plef.1998.0006 10319916 Analysis of lipoprotein diene formation in human serum exposed to copper Regnstrom J Strom K Moldeus P Nilsson J Free Radic Res Commun 1993 19 267 278 8294049 Soybean isoflavonoids and their metabolic products inhibit in vitro lipoprotein oxidation in serum Hodgson JM Kroft KD Puddey IB Mori TA Beilin LJ J Nutr Biochem 1996 7 664 669 10.1016/S0955-2863(96)00133-7 Increased lipoprotein oxidation in Alzheimer's disease Schippling S Kontush A Arlt S Buhmann C Sturenburg HJ Mann U Muller-Thomsen T Beisiegel U Free Radic Biol Med 2000 28 351 360 10.1016/S0891-5849(99)00247-6 10699746 Long-term effects of vitamin E, vitamin C, and combined supplementation on urinary 7-hydro-8-oxo-2'-deoxyguanosine, serum cholesterol oxidation products, and oxidation resistance of lipids in nondepleted men Porkkala-Sarataho E Salonen JT Nyyssonen K Kaikkonen J Salonen R Ristonmaa U Diczfalusy U Brigelius-Flohe R Loft S Poulsen HE Arterioscler Thromb Vasc Biol 2000 20 2087 2093 10978253 Influence of vitamin E and C supplementation on lipoprotein oxidation in patients with Alzheimer's disease Kontush A Mann U Arlt S Ujeyl A Luhrs C Muller-Thomsen T Beisiegel U Free Radic Biol Med 2001 31 345 354 10.1016/S0891-5849(01)00595-0 11461772 Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples Gheldof N Engeseth NJ J Agric Food Chem 2002 50 3050 3055 10.1021/jf0114637 11982440 Free radical stimulation of tyrosine kinase and phosphatase activity in human peripheral blood mononuclear cells Lowe GM Hulley CE Rhodes ES Young AJ Bilton RF Biochem Biophys Res Commun 1998 245 17 22 10.1006/bbrc.1998.8370 9535775 The inhibition of bovine xanthine oxidase activity by Hg2+ and other metal ions Mondal MS Mitra S J Inorg Biochem 1996 62 271 279 10.1016/0162-0134(95)00160-3 8676104 Garlic as antioxidant and free radical scavenger Yang GC Yasaei MP Page SW J Food Drug Anal 1993 1 357 364 Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine Kim KM Chun SB Koo MS Choi WJ Kim TW Kwon YG Chung HT Billiar TR Kim YM Free Radic Biol Med 2001 30 747 756 10.1016/S0891-5849(01)00460-9 11275474 Dose-dependent induction of endogenous antioxidants in rat heart by chronic administration of garlic Banerjee SK Maulik M Mancahanda SC Dinda AK Gupta SK Maulik SK Life Sci 2002 70 1509 1518 10.1016/S0024-3205(01)01514-4 11895102 Protection against acute adriamycin-induced cardiotoxicity by garlic: role of endogenous antioxidants and inhibition of TNF-alpha expression Banerjee SK Maulik M Dinda AK Talwar KK Maulik SK BMC Pharmacol 2003 3 16 324401 14687418 10.1186/1471-2210-3-16 Chronic garlic administration protects rat heart against oxidative stress induced by ischemic reperfusion injury Mukherjee S Banerjee SK Maulik M Dinda AK Talwar KK Maulik SK BMC Pharmacol 2002 2 16 126262 12182765 10.1186/1471-2210-2-16 Efectiveness of garlic on the radical activity in radical generating systems Torok B Belagyi J Rietz B Jacob R Arzneimittelforschung 1994 44 608 611 8024631 Effects of garlic preparations on superoxide production by phorbol ester activated granulocytes Siegers CP Robke A Pentz R Phytomedicine 1999 6 13 16 10228606 Antioxidant activity of allicin, an active principle in garlic Prasad D Laxdal VA Yu M Raney BL Mol Cell Biochem 1995 148 183 189 8594422 Intravenous allicin improves pulmonary blood flow after ischemia-reperfusion injury in rats Batirel HF Naka Y Kayano K Okada K Vural K Pinsky DJ Oz MC J Cardiovasc Surg (Torino) 2002 43 175 179 11887050 Effect on active oxygen species of alliin and Allium sativum (garlic) powder Kourounakis PN Rekka EA Res Commun Chem Pathol Pharmacol 1991 74 249 252 1667340 Scavenging effect of aged garlic extract and its constituents on active oxygen species Ide N Matsuura H Itakura Y Phytother Res 1996 10 340 341 10.1002/(SICI)1099-1573(199606)10:4<340::AID-PTR831>3.3.CO;2-W S-allyl cysteine reduces oxidant load in cells involved in the atherogenic process Ho SE Ide N Lau BH Phytomedicine 2001 8 39 46 11292238 Antioxidant S-allylcysteine prevents gentamicin-induced oxidative stress and renal damage Maldonado PD Barrera D Rivero I Mata R Medina-Campos ON Hernández-Pando R Pedraza-Chaverrí J Free Radic Biol Med 2003 35 317 324 10.1016/S0891-5849(03)00312-5 12885594 S-allylmercaptocysteine scavenges hydroxyl radical and singlet oxygen <it>in vitro </it>and attenuates gentamicin-induced oxidative and nitrosative stress and renal damage <it>in vivo</it> Pedraza-Chaverrí J Barrera D Maldonado PD Chirino YI Macías-Ruvalcaba NA Medina-Campos ON Castro L Salcedo MI Hernández-Pando R BMC Clin Pharmacol 2004 4 5 419708 15119956 10.1186/1472-6904-4-5 Protective effect of diallyl sulfide on oxidative stress and nephrotoxicity induced by gentamicin in rats Pedraza-Chaverrí J Maldonado PD Barrera D Cerón A Medina-Campos ON Hernández-Pando R Mol Cell Biochem 2003 254 125 130 10.1023/A:1027372102135 14674690 Diallyl disulfide ameliorates gentamicin-induced oxidative stress and nephropathy Pedraza-Chaverrí J González-Orozco AE Maldonado PD Barrera D Medina-Campos ON Hernández-Pando R Eur J Pharmacol 2003 473 71 78 10.1016/S0014-2999(03)01948-4 12877940 Pharmacologic activities of aged garlic extract in comparison with other garlic preparations Kasuga S Uda N Kyo E Ushijima M Morihara N Itakura Y J Nutr 2001 131 1080S 1084S 11238821