Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 37, Num. 4, 2005, pp. 227-231

Indian Journal of Pharmacology, Vol. 37, No. 4, July-August, 2005, pp. 227-231

Research Paper

In vitro antioxidant effect of Globularia alypum L. hydromethanolic extract

Khlifi S., Hachimi Y.E.l., Khalil A., Es-Safi N., Abbouyi A.E.l.

Laboratoire de Biochimie Appliquée et Biotechnologie. Faculté des Sciences, EL Jadida

Correspondence Address: Laboratoire de Biochimie Appliquée et Biotechnologie. Faculté des Sciences, EL Jadida, elabbouyi@hotmail.com

Date of Submission: 05-Jan-2005
Date of Decision: 15-Jan-2005
Date of Acceptance: 22-Apr-2005

Code Number: ph05058

ABSTRACT

Keywords: Conjugated dienes, linoleic acid, lipid peroxidation

INTRODUCTION

MATERIALS AND METHODS

Plant material

Fresh G. alypum L. was collected during spring 2003 from the region of Taza, Morocco. Taxonomic identification was performed by Dr. R. Tellal (Laboratoire d′Ecologie Vιgιtale, Facultι des Sciences. El Jadida, Morocco). A voucher specimen (KS2) is kept in the herbarium of the biology department.

Preparation of plant extract

Fresh aerial parts (stems and leaves) were washed and air-dried in shade at room temperature. They were then mechanically powdered and sieved. One hundred grams of the obtained fine powder was macerated in a hermetically closed glass vessel for 48 h, at room temperature (25°C), under occasional shaking with 500 ml of a mixture of distilled water-methanol (3/2 v/v). The obtained crude preparation was centrifuged at 5000 g for 45 min (Sigma 2K15). After filtration, the supernatant filtrate was concentrated under reduced pressure at 25°C and the crude extract (10.75 g) stored at -20°C until use.

Chemicals

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

Isolation of low-density lipoproteins

Human low-density lipoproteins (LDL) (1.019 <1.063) were isolated according to the method of Sattler,[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 c 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 ( I_oxygen) 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 A_c is the absorbance of the control reaction and A_s 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

DISCUSSION

REFERENCES

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