Evaluation of Antioxidant Activity of Ruta Graveolens L. Extract On
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Research Journal of Pharmacognosy (RJP) 1, 2014: 45-50 Received: July 2013 Accepted: Oct 2013 Original article Evaluation of antioxidant activity of Ruta graveolens L. extract on inhibition of lipid peroxidation and DPPH radicals and the effects of some external factors on plant extract's potency S. Mohammadi Motamed*, S. Shahidi Motlagh, H. Bagherzadeh, S. Azad Forouz, H. Tafazoli Department of Pharmacognosy, Pharmaceutical Sciences Branch, Islamic Azad University (IAU), Tehran, Iran. Abstract The antioxidant properties of Ruta graveolens L. were evaluated by two different methods; free radical scavenging using DPPH and inhibition of lipid peroxidation by the ferric thiocyanate method. The IC50 value of the methanol extract in DPPH inhibition was 200.5 μg/mL which was acceptable in comparison with BHT (41.8 μg/mL). In thiocyanate method, the plant extract demonstrated activity as much as BHT in prevention of lipid peroxidation. Increasing the temperature during extraction, significantly decreased the extract power in inhibition of DPPH radicals. The storage time and temperature had no effect on lipid peroxidation inhibition. Keywords: Antioxidant, extract, Ruta graveolens L., storage time, temperature Introduction In our body, free radicals and other reactive cause food rancidity [7] and some diseases oxygen species (ROS) are produced by such as cancer [6]. Therefore, antioxidants different natural mechanisms including play an important role in prevention of such phagocytosis [1], fatty acid beta oxidation [2] conditions. Toxicity and carcinogenicity of and arachidonate oxidation [3]. Excessive artificial antioxidants such as BHT and BHA ROS, beyond the antioxidant defense capacity [8] has made the consumption of natural of the body can cause oxidative stress [4]. antioxidants safer. - Superoxide anion radical (O2° ) is one of the Rue (Ruta graveolens L.), which is native to strongest reactive oxygen species among the Northern Iran, is used as a tonic and anti- free radicals that is generated when oxygen is bleeding agent in folklore Iranian medicine - taken into living cells [5]. O2° changes to other [9]. The anticancer activity of this plant harmful reactive oxygen species and free [10,11] and the high content of rutin [12], radicals such as hydrogen peroxide and drew our attention to select rue for evaluation hydroxyl radical [4]. Hydroxyl radical can of its antioxidant activity. then attack macromolecules in the body, Since antioxidants act by different including lipids [6]. Lipid peroxidation can mechanisms, in this research we decided to Available at: www.rjpharmacognosy.ir Copy right© 2014 by the Iranian Society of Pharmacognosy *Corresponding author: [email protected], Tel: +9821-22640051, Fax: +9821-22602059 Mohammadi Motamed S. et al. evaluate the effect of the plant extract on control absorbance sample absorbance ×100 radical scavenging and lipid peroxidation by control absorbance two different methods; 2,2-diphenyl-1- The DPPH solution plus methanol was used as picrylhydrazyl (DPPH) and thiocyanate, control. BHT was used as the positive control. respectively. Moreover, the role of some external factors such as temperature and Inhibition of lipid peroxidation by ferric storage time on the quality of the extract has thiocyanate method also been evaluated. The antioxidant activity of the extracts was determined according to the ferric thiocyanate Experimental method as reported by Kikuzaki and Nakatani Chemicals [14]. A mixture containing the extract (4 mL) Trichloroacetic acid (TCA) and iron (II) in absolute ethanol, final concentration: 200 chloride were purchased from Aldrich (USA). μg/mL, 2.51% linoleic acid in absolute ethanol 2-Thiobarbituric acid (TBA), linoleic acid, (4.1 mL), 0.05 M phosphate buffer pH 7 (8 ammonium thiocyanate, 2,2-diphenyl-1- mL) and distilled water (3.9 mL), was placed picrylhydrazyl (DPPH), and butylated in a vial with a screw cap, and then placed in hydroxytoluene (BHT) were purchased from an oven at 40 °C in the dark. 75% ethanol (9.7 Sigma (USA). Iron (III) chloride, absolute mL) and 30% ammonium thiocyanate (0.1 ethanol and methanol were purchased from mL) were added to 0.1 mL of the sample. Merck (Germany). All chemicals and reagents Three minutes after adding of 20 mM ferrous were of analytical grade. chloride in 3.5% hydrochloric acid (0.1 mL) to the reaction mixture, the absorbance of the red Plant material color was measured at 500 nm each 24 h until Plant sample was obtained from the one day after the absorbance of the control herboratum of Faculty of Pharmacy, Tehran (without sample) reached maximum. BHT was University of Medical Sciences and was used as the positive control. identified by Dr Gholam Reza Amin. %Inhibition of lipid peroxidation was calculated by the following equation: Preparation of the plant extract Ac As The specimen was dried at room temperature %inhibition = × 100 and powdered. A sample (10 g) of the dried Ac powder was extracted thrice with 100 mL As is the absorbance of the sample on the day methanol for 24 h on each occasion through when the absorbance of the control is maceration. The extract was filtered and the maximum, and Ac is the absorbance of the combined filtrates were concentrated. For control on the day when the absorbance of the evaluation of the temperature effect during control is maximum. extraction on DPPH scavenging activity of the The effect of the extract storage time on the plant, extraction was carried out by soxhlet antioxidant activity of the plant in prevention apparatus as well. of lipid peroxidation was evaluated by the thiocyanate method every three months for six DPPH free radical scavenging activity months; for understanding the effect of the Experiments were carried out according to the storage temperature of the extract on the method of Blois [13] with a slight antioxidant activity of the plant in prevention modification. Briefly, a 0.1 mM DPPH radical of lipid peroxidation, the extract was kept in 4, solution in methanol was prepared and 1 mL 25 and 100 °C and the antioxidant activity was of this solution was mixed with the sample measured by thiocyanate method. solution (3 mL) in methanol. After 30 min, the absorbance was measured at 517 nm. Results and Discussion Decreasing the DPPH solution absorbance Free radical scavenging by using DPPH indicates an increase of the DPPH radical Inhibition effect of different concentrations of scavenging activity. This activity is given as % the plant extract and BHT in scavenging of DPPH radical inhibition calculated as: DPPH was evaluated. To determine the IC50 value of the extract and BHT, regression 46 Evaluation of antioxidant activity of Ruta graveolens extract equations of calibration curves were calculated For evaluating the effect of the storage time on using MS Excel software (y = 0.05x + 38.11, inhibition of lipid peroxidation, the test was R2 = 0.98 for the plant extract and y = 0.85x + carried out 3 and 6 months after the first 14.02, R2 = 0.98 for BHT). From these experiment. The results are presented in equations the IC50 value for the extract and figures 2 and 3. BHT were calculated as 200.5 and 41.8 To evaluate the effect of the extract storage μg/mL, respectively. temperature on lipid peroxidation inhibition, The IC50 value for the extract obtained by instead of keeping the extract in refrigerator (4 soxhlet apparatus was 378.3 μg/mL. °C ), one part of the extract was kept in room temperature (25 °C ) for 24 h and one part was The inhibitory activity of the extract against placed in an oven (100 °C ) for 60 min. the linoleic acid peroxidation results of the effects of these extracts on lipid The extract significantly decreased the peroxidation have been shown in figures 4 and absorbance and showed inhibitory activity 5. against linoleic acid peroxidation. This inhibitory effect was as much as BHT (figure 1). Figure 4. Total antioxidant activity of R. graveolens extract (final conc. 200 ug/mL), control and BHT, the first month kept for 24 h (25 °C). Figure 1. Total antioxidant activity of R. graveolens extract (final conc. 200 ug/mL), control and BHT, after the first month storage in refrigerator. Figure 5. Total antioxidant activity of R. graveolens extract (final conc. 200 ug/mL), control and BHT, the Figure 2. Total antioxidant activity of R. graveolens first month kept for 60 min (100 °C). extract (final conc. 200 ug/mL), control and BHT, after 3 months storage in refrigerator Table 1. Inhibition of lipid peroxidation (%) of rue extracts on the day when the absorbance of the control was maximum Inhibition of lipid Sample peroxidation (%) PE*, the first month, storage in refrigerator 81.66 PE, 3 months later, storage in refrigerator 87.22 PE, 6 months later, storage in refrigerator 84.18 PE, the first month, at room temperature (25°C) 86.97 for 24 h PE, the first month, oven (100°C) for 60 min 82.95 BHT 88.54 *plant extract Figure 3. Total antioxidant activity of R. graveolens extract (final conc. 200 ug/mL), control and BHT, after 6 months storage in refrigerator. 47 Mohammadi Motamed S. et al. Inhibition of lipid peroxidation (%) of the Flavonoids [26-28] and coumarines [29] are extracts on the day when the absorbance of the heat stable while phenolics are less stable [23- control was maximum has been shown in table 26] and may have structural changes and the 1. decrease in inhibition of DPPH radicals might Free radicals can attack different be due to the thermal deterioration of macromolecules including lipids and therefore phenolics during the extraction. can cause many diseases, aging process and Linoleic acid, an unsaturated fatty acid, can be food rancidity [15]. Consumption of natural easily peroxidased and produces various antioxidants such as plants [16] can prevent compounds such as aldehydes and epoxides the occurrence of such events or postpone [30].