In vitro antioxidant effect of Globularia alypum L. hydromethanolic extract

S Khlifi¹, Y El Hachimi¹, A Khalil², N Es-Safi³, A El Abbouyi^1
1 Laboratoire de Biochimie Appliquée et Biotechnologie. Faculté des Sciences, EL Jadida, Morocco
² Research Center on Aging, Sherbrooke Geriatric University Institute, University of Sherbrooke, Sherbrooke, Que, Canada
³ Laboratoire de Chimie Organique et d'Etudes Physico-Chimiques, Ecole Normale Supérieure, Rabat, Morocco

Correspondence Address:
A El Abbouyi
Laboratoire de Biochimie Appliquée et Biotechnologie. Faculté des Sciences, EL Jadida
Morocco

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0253-7613.16568

Objective : To investigate the in vitro antioxidant activity of the hydromethanolic extract of aerial parts (leaves and stems) of Globularia alypum L. toward linoleic acid emulsion and human low-density lipoproteins (LDL) peroxidation. Materials and Methods : Lipid peroxidation was carried out in the presence of G. alypum hydromethanolic extract (10 and 100 µg of extract/ml). CuSO4 (10 µM) was used as the oxidation initiator. Conjugated dienes (CD) formation and oxygen consumption were assessed for monitoring the antioxidant properties of the plant extract. Butylated hydroxytoluene at 50 µg/ml was used as standard antioxidant. Quantification of total polyphenolic compounds was carried out according to the Folin-Ciocalteu method. Results : The hydromethanolic extract of G. alypum exhibited significant antioxidant effect. There was a significant inhibition of CD formation in copper ions-mediated linoleic acid emulsion as well as human LDL peroxidation. Analysis of the plant extract revealed a high amount of polyphenols, suggesting a possible role of these compounds in the antioxidant properties. Conclusion : The obtained results suggested that G. alypum could be a potential source of antioxidants. Further investigations are in progress to determine the active constituent(s).

Keywords: Conjugated dienes, linoleic acid, lipid peroxidation

» Introduction

» Materials and Methods

[10] using a Beckman Optima TLX ultracentrifuge equipped with a TLA 100.4 rotor, in the presence of EDTA (0.4 g/l). After separation, LDL was dialyzed overnight at 4°C with 1/102 M sodium phosphate buffer (pH 7.0). For oxidation experiments, the LDL dialyzed solutions were adjusted by dilution to 100 µg/ml, and proteins were measured by commercial assay (Pierce method, Rockford III, USA).

Measurement of oxygen consumption

Globularia alypum (Ga) extract (10 and 100 µg/ml) was added to 1.5 ml of a 7.5 mM linoleic acid emulsion in 10 mM aqueous phosphate buffer (pH 7.0) and 0.1% of Tween 20 (v/v) as emulsifier. The emulsion was air saturated. A freshly prepared solution of CuSO4 was added, at a final concentration of 10 µM, to initiate the oxidation process. Measurement of the oxygen consumption, according to the modified technique of Genot[11] using Strathkelvin Instruments Oxymeter 949 was started by injection of the sample into a thermostated (25.0±0.1°C) 2 ml measuring cell with no headspace. Oxygen consumption was measured with a Clark electrode and recorded for approximately 25 min at time intervals of 30 s with a computer-based data collecting system. The oxymeter was standardized at 0% of O2 with bisulfite sodium solution (3.85 M) and at 100% of O2 with air-saturated water solution.

The oxygen consumption rate V (O2) in µM/l/s was calculated by the following equation:

where the slope was calculated from the oxygen consumption curve, [O2]initial = 2.6 10-4 M at 25°C Moller.[12]

The antioxidant index relative to the rate of oxygen consumption ( Ioxygen) was calculated by the following equation:[12]

Measurement of conjugated dienes

The conjugated dienes (CD) were determined by measuring the absorbance at 234 nm according to the modified technique described by Esterbauer.[13] Briefly, linoleic acid (7.5 mM) emulsified with Tween 20 (0.1%, v/v) or human LDL (100 µg/ml) mixture, in phosphate buffer (pH 7.0, 10 mM), was incubated alone (control) or with Ga extract (10 and 100 µg/ml). Oxidation was initiated by addition of freshly prepared CuSO4 (10 µM) and stopped by cooling in an ice bath in the presence of EDTA (100 µM) and BHT (20 µM). The absorbance readings were taken every 15 min over 270 min at 37°C in a Beckman UV-Vis spectrophotometer.

The lipid peroxidation kinetic parameters: lag time (min), maximal rate of oxidation (nM/min), and maximal amount of CD formation (µM) were calculated using a molar extinction coefficient of 29 500/M/cm.

The inhibition of linoleic acid and LDL peroxidation was calculated by the following equation:

where Ac is the absorbance of the control reaction and As is the absorbance of the treated sample.

Determination of total polyphenolic compounds (PPC)

The amount of total PPC was measured by the method described by Taga[14] using the Folin-Ciocalteu reagent. Briefly, samples and standards were prepared in acidified (0.3% HCl) methanol-water solution (60/40 v/v). One hundred microliter of this preparation was added to 2 ml of 0.2% Na2CO3. After 2 min, 100 µl of Folin-Ciocalteu/methanol (v/v) reagent was added to start reaction at room temperature (25°C) during 30 min. The control mixture consisted of all reagents and solvent without extract. The phenolic concentrations were expressed as phenol equivalent by comparison with a standard calibration curve using phenol solution (0.01-1 mg/ml).

Statistical analysis

The results are presented as the mean±SD of five replicates. Data were analyzed using one-way analysis of variance (ANOVA) and group means were compared using Duncan's multiple range test. P values <0.05 were considered significant.

» Results

Linoleic acid oxidation

As shown in [Figure - 1], Ga hydromethanolic extract exhibited a significant inhibition of linoleic acid oxidation as assessed by CD formation. The inhibition extents were 24% ( P <0.01) and 64% ( P <0.001), respectively, at 10 and 100 µg/ml, while BHT, used as standard antioxidant at 50 µg/ml, gave 76% ( P <0.001).

The kinetic parameters of oxidation [Table - 1] showed that at a low concentration (10 µg/ml), the plant extract did not influence the lag time and the maximal propagation rate of linoleic acid oxidation, but induced a low and significant reduction (24%) ( P <0.05) in the maximal amount of CD formation. At a high concentration (100 µg/ml), the extract caused a mean increase in lag time of 52±2.5 ( P <0.001) and inhibited both propagation rate (35%, P <0.001) and maximal amount of CD formation (64%, P <0.001).

The antioxidant properties of G. alypum were also demonstrated by the oxygen consumption method where the plant extract induced significant effect [Figure - 2]. Thus, the Ga extract (10 and 100 µg/ml) reduced the rate of oxygen consumption by 63% ( P <0.001) and 84% ( P <0.001), respectively. It must be noted that the effect observed with a concentration of 100 µg/ml was more efficient than that obtained with BHT at 50 µg/ml (77%, P < 0.001) [Table - 2].

Human LDL peroxidation

In order to confirm the protective action of Ga extract on linoleic acid oxidation, we tested its effect on human LDL oxidation by quantifying the CD formation.

The obtained results [Figure - 3] showed that copper-induced LDL peroxidation was significantly inhibited by Ga extract. The extent of inhibition of CD formation was 61%, ( P <0.001), 83% ( P <0.001) and 76% ( P <0.001), respectively, at 10 and 100 µg/ml of plant extract and BHT at 50 µg/ml.

The effects on kinetic parameters showed that the plant extract prolonged the lag time, diminished the propagation rate, and inhibited the maximal amount of CD formation [Table - 1]. Standard antioxidant BHT (50 µg/ml) did not affect the lag time but inhibited to a lesser extent the propagation rate and the maximal amount of CD formation.

» Discussion

The present investigation carried out on the antioxidant properties of hydromethanolic extract of G. alypum clearly showed the protective activity on lipid peroxidation. However, as previously described,[15],[16] the efficiency of antioxidant activity was dependant on the used lipid system and the method of assessment.

Globally, in the presence of linoleic acid (simple lipid system), the antioxidant effect produced by the extract was lesser or higher than BHT, respectively, when assessed by CD formation or oxygen consumption. According to the kinetic parameters of linoleic acid oxidation, the plant extract (100 µg/ml) extended the lag time probably by increasing the initiation stage of the chain reaction. The plant extract also inhibited both the propagation rate and the maximal amount of CD formation.

After the CD formation, one of the first events of lipid peroxidation was the uptake of oxygen and formation of lipid peroxides.[17] In the presence of plant extract, linoleic acid oxidation showed significant decrease in the rate of oxygen uptake, which was higher than that induced by BHT. Probably, the plant extract reacts with peroxyl radicals inducing an inhibition of the lipid peroxidation chain propagation.[16]

The antioxidant effect exerted by the plant extract seems to be more efficient in protecting human LDL (complex lipid system) against peroxidation than linoleic acid. The extract increased the lag time and decreased the propagation rate and the maximal amount of CD formation, whereas BHT did not affect the lag phase and weakly decreased the propagation rate. Similar results were obtained with probucol[18] and with a synthesized antioxidant 4GBE43.[19]

Otherwise, the high amount of total phenolic content (120 mg of phenol equivalent per gram of extract) led us to suggest that these substances could be responsible of the antioxidant properties of the extract. Polyphenols were reported to have an important role in stabilizing lipid peroxidation[20] and are associated with a wide range of biological activities including antioxidant properties [21],[22],[23] due to their redox properties, as reducing agent or hydrogen atom donors.[4]

The implication of the polyphenolic compounds in the antioxidant activity of G. alypum is supported by previous results obtained with other Globularia species. Thus phenylethanoid glycosides extracted from Globularia Trichosantha ,[24] Globularia davisiana ,[25] and Globularitol obtained from Globularia orientalis[26] have been reported to possess scavenging properties.

In conclusion, the protective effect of Ga hydromethanolic extract toward linoleic acid emulsion and human LDL peroxidation was demonstrated by highly significant diminution of both oxygen consumption and CD formation. Further investigations are in progress to characterize the active compounds and to evaluate the usefulness in the treatment of disorders that involve oxidative stress.



 

» References

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Figures

[Figure - 1], [Figure - 2], [Figure - 3]

Tables

[Table - 1], [Table - 2]

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