Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 38, Num. 4, 2006, pp. 276-280

Indian Journal of Pharmacology, Vol. 38, No. 4, July-August, 2006, pp. 276-280

Research Paper

In vitro antioxidant properties of Salvia verbenaca L. hydromethanolic extract

S. Khlifi, Y. El Hachimi, A. Khalil¹, N. Es-Safi², A. Belahyan, R. Tellal, A. El Abbouyi

Laboratoire de Biochimie Appliquée et Biotechnologie, Faculté des Sciences, El Jadida, Morocco, ¹ Research Center of Aging Sherbrooke Geriatric University INistitute, University fo Sherbrooke, Sherbrooke, Que. Canada²Laboratoire de Chimie Organique et d'Etudes Physico-Chiminiques Ecole Normale Supérieure Rabat Moroco
Correspondence Address:Laboratoire de Biochimie Appliquée et Biotechnologie, Faculté des Sciences, El Jadida, Morocco, elabbouyi@hotmail.com

Code Number: ph06072

Abstract

Keywords: Conjugated dienes, oxygen consumption, phenolic compounds

Introduction

The term 'antioxidant' refers to the activity of numerous vitamins, minerals and other phytochemicals to protect against the damage caused by reactive oxygen species (ROS). By their ability to react with and damage many structures in the body, ROS are involved in various related physiological processes and diseases such as aging,[1] cancer[2] and atherosclerosis.[3]

Several studies have demonstrated that plants produce potent antioxidants and represent an important source of natural antioxidants.[4],[5],[6] Among these, Salvia is an important and diversified genus of the Lamiaceae family, with more than 900 species. Commonly found in the Mediterranean regions, Salvia is widely cultivated[5] and used in flavouring and folk medicine for the treatment of coronary heart diseases,[7] cerebrovascular diseases,[8] hepatitis, hepatocirrhosis[9] and chronic renal failure.[10]

In Moroccan traditional medicine, Salvia is one of the most popular plant remedies. Decoctions of its aerial parts were reportedly used as a cholagogue, antiseptic, diuretic and astringent.[11] S. verbenaca , locally named Al-khyyata (healing), is particularly used in the healing of wounds.[12]

The aim of this work was to evaluate the in vitro antioxidant properties of the hydromethanolic extract of S. verbenaca and relate its antioxidant power to the total amount of polyphenolic compounds.

Materials and Methods

Plant material

S. verbenaca was collected from the region of El Jadida (Morocco) in April 2003. The botanical identification was performed by Dr. A. Belahyan (Laboratoire d'Ecologie Vιgιtale, Facultι des Sciences, El Jadida, Morocco). A voucher specimen (SV ₁ ) is kept in the herbarium of the Biology Department.

Preparation of the plant extract

Fresh aerial parts (stems and leaves) were washed and air-dried, in the shade, at room temperature (25°C). They were then mechanically powdered and sieved. One hundred grams of the obtained fine powder was macerated for 48 hours with 500 ml of a mixture of water-methanol (3/2 v/v), at room temperature, by occasional shaking. The obtained crude preparation was centrifuged and filtered. The supernatant was concentrated under reduced pressure at 25°C and the extract obtained (11.5g) was stored at -20°C until use.

Chemicals

Linoleic acid (99%), linolenic acid (99%), thiobarbituric acid (TBA), ethylenediaminetetra-acetic acid (EDTA), Tween 20 and butylated hydroxytoluene (BHT) were purchased from Sigma. All other unlabelled chemicals and reagents were of analytical grade.

Isolation of LDL

Isolation of human LDL (1.019 < d < 1.063) was performed, according to the Sattler method[13], 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 samples were dialysed overnight at 4°C with 10 ^-2 M sodium phosphate buffer (pH 7.0). For oxidation experiments, the LDL dialysed 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

The S. verbenaca extract (10 and 100 µg/ml) was added to 1.5 ml of a 7.5 mM linoleic acid (LA) emulsion in 10 mM aqueous phosphate buffer (pH 7.0) and 0.1 v/v per cent Tween 20 as emulsifier. To initiate the oxidation process at 100% of oxygen, the emulsion was air-saturated just before the addition of a freshly prepared solution of CuSO ₄ (10 µM). The measurement of oxygen consumption, on the 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 head space[14]. Oxygen consumption was measured with a Clark electrode and recorded for approximately 25 min, at time intervals of 30 sec, with a PC-based data collecting system. The oxymeter was calibrated at 0% of O ₂ , with a bisulphite sodium solution (3.85 M), and at 100% of O[2], with air saturated water solution.

The oxygen consumption rate V(O[2]) in µM.L ^-1 s ^-1 was calculated by the following equation:[15]

where the slope was calculated from the oxygen consumption curve, [O ₂ ] initial = 2.6 10 ^-4 M at 25°C.

The antioxidative index, relative to the rate of oxygen consumption (I _oxygen), was calculated by the following equation:[15]

Measurement of conjugated dienes

The CD were quantified by measuring the absorbance at 234 nm, according to Esterbauer.[16]

Linoleic acid oxidation

A mixture of linoleic acid (7.5 mM), emulsified with Tween 20 (0.1 v/v per cent), in phosphate buffer (pH 7), at a final concentration of 10 mM, was incubated alone (control) or with the S. verbenaca extract (10 and 100 µg/ml). The oxidation was initiated by the addition of freshly prepared CuSO ₄ , at a final concentration of 10 µM. The oxidation was stopped by cooling in an ice bath, in the presence of EDTA (100 µM) and BHT (20 µM). Oxidation kinetics were determined at 37°C, by measuring the absorbance at 234 nm, every 15 min, over 270 min.

LDL peroxidation

This was carried out by incubating LDL (100 µg/ml) alone (control) or in the presence of the S. verbenaca extract (10 and 100 µg/ml), at 37°C, in 10 mM sodium phosphate buffer (pH 7.0). LDL oxidation and the measure of CD formation were processed as described above. 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[17] of 29 500/M/cm.

The inhibition of lipid 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.

Measurement of TBARS

The quantification of TBARS was monitored, according to the Ohkawa method.[18] Briefly, the plant extract (10 and 100 µg/ml) was added to 1 ml of linolenic acid emulsion (10 mM) in PBS (10 mM, pH: 7.4) and Tween 20 (0.1 % v/v). The oxidation was initiated by freshly prepared CuSO ₄ solution, at a final concentration of 40 µM, in PBS. After incubation at 37°C for 3 h, in obscurity, the reaction was stopped by cooling and adding EDTA (10 µl at 20 mM). The BHT was used as a standard antioxidant (50 µg/ml).

The preparation, combined with 1 ml of 20% trichloroacetic acid (pH 3.5) and 1 ml of 0.78% thiobarbituric acid, was heated in boiling water (95°C), for 45 min and then cooled at room temperature. TBARS were extracted with n-butanol and the absorbance of the n-butanol layer was measured at 532 nm.

Determination of phenolic compounds

The amount of total phenolic compounds was measured by a method described by Taga,[19] using the Folin-Ciocalteu reagent. Briefly, samples and standards were prepared in acidified (0.3% Hcl) methanol-water (3:2, v/v). About 100 µl of this preparation was added to 2 ml of 0.2% Na ₂ CO ₃ . After 2 min, 100 µl of Folin-Ciocalteu/methanol (v/v) reagent was added and allowed to stand at room temperature (25°C) for 30 min. The blank consisted of all reagents and solvents, without test compounds or standard. The phenolic concentration, expressed as phenol equivalent (mg PE/g of dry matter), was determined 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 analysed, using one or two-way analysis of variance (ANOVA). Group means were compared, using the Duncan's multiple range test. P < 0.05 was considered statistically significant.

Results

Linoleic acid oxidation

To assess a possible protective action of S. verbenaca (Sv) against lipid oxidation, we initially examined its in vitro effect on linoleic acid. The obtained results, shown in [Figure - 1], indicated that the plant extract significantly inhibited the CD formation. The inhibition extents were 28% ( P < 0.001) and 74% ( P < 0.001) at 10 and 100 µg/ml, respectively, while the inhibition induced by BHT was 76% ( P < 0.001).

The kinetic parameters [Table - 1] showed that the plant extract (100 µg/ml) enhanced the lag time by 60±2.16 min ( P < 0.001) and reduced the maximum propagation rate (35%, P < 0.001) and the maximum amount of dienes production (74%, P < 0.001). These results did not differ significantly from those obtained with BHT (50 µM) used as a standard antioxidant.

The same protective effect of plant extract was shown, using the oxygen consumption method. [Figure - 2] The Sv extract (10 and 100 µg/ml) significantly reduced the rate of oxygen consumption by 73% ( P < 0.001), 92% ( P < 0.001), respectively. [Table - 2] In the same conditions, the extent of inhibition induced by BHT at 50 µg/ml was 76% ( P < 0.001).

Linolenic acid oxidation

The antioxidant activity of the S. verbenaca extract was also demonstrated by the quantification of TBARS [Table - 2], which were significantly reduced by 71% ( P < 0.001) and 93% ( P < 0.001), respectively, at 10 and 100 µg/ml, whereas the inhibition obtained with standard antioxidant BHT was 63% ( P < 0.001).

Human LDL peroxidation

As shown in [Figure - 3], the copper-catalysed oxidation of human LDL was significantly inhibited by the plant extract. The CD formation was inhibited by 76% ( P < 0.001) and 92% ( P < 0.001), respectively, at 10 and 100 µg/ml, while the inhibition induced by BHT reached 77% ( P < 0.001). [Table - 1] showed that both doses induced a significant modification of the kinetic peroxidation parameters. Thus, a prolongation of the lag phase and a reduction of the maximum rate and maximal amount of CD formation were observed. In the same conditions, BHT did not significantly influence the lag phase.

Discussion

In the present study, the antioxidant activity of the S. verbenaca hydromethanolic extract was monitored, by three different methods, toward two lipidic systems. The use of several methods is necessary for antioxidant activity assessment as it depends on the method and the lipid system used as previously described.[20],[21]

The obtained results showed that the plant extract exhibited different but effective degrees of in vitro antioxidant activity, with all the methods and lipid systems used. The quantification of CD formation showed that antioxidant activity of the S. verbenaca extract (100 µg/ml) was lesser (with linoleic acid) or higher (with human LDL) than BHT (50 µg/ml). Similar results were obtained with other antioxidants, namely probucol,[22] a synthesised antioxidant 2GBE43,[23] and the hydromethanolic extract of Globularia alypum .[21]

According to the kinetic parameters of CD formation, the plant extract (100 µg/ml) extended the lag time probably, by increasing the initiation stage of the chain reaction.

The uptake of oxygen and formation of lipid peroxides represent the second stage of the lipid peroxidation, after CD formation. In this way, the linoleic acid oxidation, in the presence of the S. verbenaca extract, exerted a significant decrease in the rate of oxygen uptake, which was higher than that induced by BHT. The plant extract can, probably, react with peroxyl radicals, inducing an inhibition of the propagation process.[20]

In conclusion, the ability of S. verbenaca to significantly inhibit, in vitro, the lipid peroxidation was shown in two different systems and by more than one assay. In an attempt to detect the compounds responsible for the observed activity, the quantitative determination of polyphenolic compounds was achieved. It showed that the S. verbenaca extract was rich in such compounds (172 mg/g extract). This high amount suggests that a part of the antioxidative activity of the plant extract may be due to the polyphenolic compounds in agreement with previously reported data.[4],[24],[25],[26] Further studies on the purification and chemical characterisation of these compounds are in progress and will be reported later.

References

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