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Journal of Food and Drug Analysis, Vol. 20, No. 1, 2012, Pages 34-40

Free Radical Scavenging and Antioxidant Activities of Dorema aitchisonii

SEYED MOHAMMAD NABAVI1,2, SEYED FAZEL NABAVI1,2 AND MOHAMMAD ALI EBRAHIMZADEH3*
1.

Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehram, Iran 2. National Elites Foundation of Iran, Tehran, Iran

3.

Pharmaceutical Sciences Research Center, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
(Received: November 29, 2010; Accepted: July 13, 2011)

ABSTRACT
The aim of this study was to investigate the antioxidant properties of the aerial parts of Dorema aitchisonii by employing various in vitro assay systems including 1,1-diphenyl-2-picryl hydrazyl radical, nitric oxide and hydrogen peroxide-scavenging activities, Fe2+ chelating ability, reducing power and hemoglobin-induced linoleic acid peroxidation. Antihemolytic activity was investigated against H2O2 induced hemolysis in rat erythrocytes. Gallic acid, rutin, quercetin and cumarin contents of the preparation were determined by HPLC with diode array detector (HPLC/DAD). The IC50 for DPPH radical-scavenging activity was 488 18 g/mL. The extract showed weak NO scavenging activity in the range of 0.4 and 3.2 mg/mL, as well as Fe2+ chelating ability (IC50 = 778 22 g/mL). The extract was effective in inhibiting hemoglobin-induced linoleic acid peroxidation, but its activity was less than Vitamin C (p < 0.01). The extract was capable of scavenging H2O2 in a concentration-dependent manner with an IC50 211 11 g/mL. The extract exhibited good antihemolytic activity with an IC50 385 15 g/mL (the corresponding value for Vitamin C being 235 9 g/mL). The total content of phenolic compounds was 41.2 1.9 mg gallic acid equivalents/g and the total flavonoid content was 13.8 0.8 mg quercetin equivalents/g. The extract contained 7.27 g rutin and 0.011 g gallic acid/mg extract, but cumarin and quercetin were present in trace amounts. In spite of presence of phenolic and flavonoid content, especially rutin, D. aitchisonii show weak antioxidant activity, but its antihemolytic activity was good. Key words: antihemolytic activity, antioxidant activity, Dorema aitchisonii, DPPH, phenolic contents

INTRODUCTION
There is increasing evidence that the consumption of phenolic compounds present in natural foods may lower the risk of serious health disorders such as atherosclerosis, inflammatory injury, cancer and cardiovascular disease as a result of their antioxidant activity(1). Tertiary-butylhydroquinone, butylated hydroxytoluene and butylated hydroxyanisole are the most commonly used primary synthetic antioxidants. However, many researchers have reported side effects of these antioxidants such as toxicity and carcinogenicity(2). Due to the safety and limitations of these synthetic antioxidants, natural antioxidants provide an interesting alternative(3). Among the various natural sources, endemic medicinal plant species are of particular interest because they may be used for producing raw materials or preparations containing phytochemicals with significant antioxidant capacities and possible health
* Author for correspondence. Tel: +98-151-3543081; Fax: +98-151-3543084; E-mail: zadeh20@yahoo.com

benefits(4). Genus Dorema (Apiaceae) contains 17 species, sub species, varieties, forms and cultivars(5). There is some Ethnomedicinal use for D. ammoniacum Oleo gum resin such as expectorant, stimulant and antispasmodic activities(6). A significant antimicrobial activity of D. ammoniacum has been reported(6). Three sesquiterpene derivatives, one prenylated coumarin and two steroid glucosides were isolated from the aerial parts of D. kopetdaghense(7). Agronomic characteristic of D. aitchisonii was briefly described previously(8). The presence of 2,6-Dihydroxy-4-methoxyacetophenone 2-O--D-gentiobioside in D. aitchisonii roots has been reported(5). This is the first umbelliferae plant found to produce exudate flavonoids(6). To the best of our knowledge, there have been no scientific reports regarding antioxidant and antihemolytic activities of this species. In this study, the antioxidant and antihemolytic activities of D. aitchisonii aerial parts at the flowering stage were examined by various in vitro assay systems in order to explore the potential usefulness of this plant.

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MATERIALS AND METHODS

I. Materials D. aitchisonii aerial parts were collected from Veresk, north of Iran, in July 2008 at the full flowering stage and were identified by Dr. B. Eslami. A voucher specimen (number 1477) has been deposited at the Sari school of pharmacy. Ferrozine, Linoleic acid, Trichloroacetic acid (TCA), 1,1-diphenyl-2-picryl hydrazyl (DPPH), potassium ferricyanide and hydrogen peroxide were purchased from Sigma Chemicals Co. (St Louis, MO, USA). Gallic acid, Quercetin, Butylated hydroxyanisole (BHA), Vitamin C, Sulfanilamide, N-(1-naphthyl)ethylenediamine dihydrochloride, EDTA and Ferric chloride were purchased from Merck (Darmstadt, Germany). All other chemicals were of analytical grade or pure. II. Sample Preparation The collected plant material was dried at room temperature (r. t.) and coarsely ground before extraction. One hundred grams of dried sample was extracted by maceration method using ethanol for 24 h at r. t. The extract was then separated from the sample residue by filtration through Whatman No. 1 filter paper. This procedure was repeated three times. The resultant extracts were combined and concentrated in a rotary evaporator until a crude solid material obtained; this was then freeze-dried for complete solvent removal. Yield was 12%. III. Determination of Total Phenolic Compounds and Flavonoid Content Total phenolic compound content was determined by the Folin-Ciocalteau method(7,8). An aliquot of extract (0.5 mL, 1.6 mg/mL) was mixed with 2.5 mL of 0.2 N Folin-Ciocalteau reagent for 5 min and 2.0 mL of 75 g/L sodium carbonate was then added. The absorbance of the reaction was measured spectrophotometrically (Perkin Elmer Wellesley, MA, USA) at 760 nm after 2 h of incubation at r. t. Results were expressed as gallic acid equivalents. Total flavonoid content was estimated as previously described(7,8). Briefly, 0.5 mL solution of extract in methanol (1.6 mg/mL) was mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water and left at r. t. for 30 min. The absorbance of the reaction mixture was measured at 415 nm with a double beam spectrophotometer. Total flavonoid content was calculated as quercetin equivalents from a calibration curve. IV. DPPH Radical-Scavenging Activity The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of the free radical scavenging activity of the extract(9,10). Different concentrations of extract were added equal volume to a methanolic solution

of DPPH (100 M). After 15 min at r. t., the absorbance was recorded at 517 nm. The experiment was done in triplicate. Vitamin C, BHA and quercetin were used as standard controls. IC50 values denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals. V. Determination of Metal Chelating Activity The ability of the D. aitchisonii extract to chelate ferrous ions was estimated by a method described in our recently published paper(11). Briefly, different amounts of extract were added to a solution of FeCl2 (0.05 mL). The reaction was initiated by the addition of ferrozine (0.2 mL) and the mixtures was then shaken vigorously and left to stand at r. t. for 10 min. The absorbance of the mixture was measured at 562 nm. The percentage inhibition of ferrozine-Fe2+ complex formation was calculated as [(A0 A1)/A0] 100, where A0 was the absorbance of the control and A1 was the absorbance of extract or a standard solution. EDTA was used as a standard. VI. Assay of Nitric Oxide-Scavenging Activity For this measurement, sodium nitroprusside (1 mL), in phosphate-buffered saline, was mixed with different amounts of extract dissolved in water and incubated at r. t. for 150 min. An identical reaction mixture, in which the extract was replaced by an equivalent amount of water, served as control. After the incubation period, 0.5 mL of Griess reagent was added. The absorbance of the chromophore formed was at 546 nm. Quercetin was used as positive control(12). VII. Scavenging of Hydrogen Peroxide The ability of the extract to scavenge hydrogen peroxide was determined as described in our recently published paper(8). One point four milliliter of extract (0.1 - 3.2 mg/mL) in distilled water was added to a solution of H2O2 (0.6 mL, 40 mM in phosphate buffer, pH 7.4). The absorbance of H2O2 at 230 nm was determined after 10 minutes against a blank solution containing phosphate buffer without H2O2. The percentage of H2O2 scavenge by the extract or standard compounds was calculated as follows: % Scavenged [H2O2] = [(A0 A1)/A0] 100 where A0 was the absorbance of the control and A1 was the absorbance in the presence of the sample of extract and standard. Ascorbic acid and BHA were used as standard. VIII. Determination of Reducing Power The reducing power of extract was determined according to recently published paper(10). Briefly, 2.5 mL of sample (25 - 800 g/mL) in water was mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1%). The mixture was incubated at 50C for 20 min. A portion of trichloroacetic acid (10%) was added to the mixture to stop the reaction, which was then centrifuged at 3000 rpm for 10 min. The upper layer of

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Journal of Food and Drug Analysis, Vol. 20, No. 1, 2012

solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%), and the absorbance was measured at 700 nm. Vitamin C was used as positive control. IX. Antioxidant Activity in a Hemoglobin-Induced Linoleic Acid Peroxidation Test The antioxidant activity of the extract was determined by a modified photometry assay(10). Reaction mixtures (200 L) containing 40 L of extract (10 - 400 mg/mL in ethanol), 40 L of linoleic acid emulsion (1 mmol/L), 70 L of phosphate buffer 40 mmol/L (pH 6.5), and 50 L hemoglobin (0.0016%), were incubated at 37C for 45 min. After incubation, 2.5 mL of 0.6% HCl in ethanol was added to stop the lipid peroxidation. The amount of peroxide in the test solution was measured in triplicate using the thiocyanate method by reading the absorbance at 480 nm after coloring with 100 mL of 0.02 mol/L of FeCl2 and 50 mL of ammonium thiocyanate (30 g/100 mL). Vitamin C was used as a positive control. X. Antihemolytic Activity of the Extract Against H2O2 Induced Hemolysis The inhibition of rat erythrocyte hemolysis by the extract was evaluated as described in our previous paper(13). Hemolysis was induced using H2O2 as free radical initiator. To 100 L of a 5% (v/v) suspension of washed erythrocytes in PBS, aliquots of 50 L containing different amounts of extract (50 - 250 g/mL in PBS pH 7.4), which corresponds to 100 - 3200 g of extract, was added. To this, 100 L of H2O2 (in PBS pH 7.4) was added. The reaction mixtures were shaken gently during incubation at 37C for 3 h. The reaction mixtures were diluted with 8 mL of PBS and centrifuged at 2000 g for 10 min. The absorbance of the resulting supernatant was measured at 540 nm by spectrophotometer to determine the extent of hemolysis. Likewise, the erythrocytes were treated with 100 M of H 2O2 without plant extract to obtain complete hemolysis. The absorbance of the supernatants was measured under the same conditions. The inhibitory effect of the extract was compared with that of the standard antioxidant, Vitamin C. To evaluate the hemolysis induced by the extract, erythrocytes were pre-incubated with 50 L of extract for 1 h and the hemolysis was determined. Percentage of hemolysis was calculated by taking hemolysis caused by 100 M H2O2 as 100%. The IC50 values were determined graphically as the antioxidant concentration required to produce a 50% inhibition of hemolysis. XI. Assay of Putative Biologically Active Components (I) Gallic Acid A Knauer series liquid chromatograph system with a DAD was used for these analyses. The column used was a C18 reversed phase (Perfecsil Target ODS-3, 250 4.6 mm). The mobile phase eventually adopted for this study was

methanol : water : orthophosphoric acid (20 : 79.9 : 0.1) and the flow rate was 1 mL/min. The absorption wavelength was chosen at 210 nm. The column was operated at 30C and the injection volume was 20 L(14). The content of gallic acid was calculated on the basis of a calibration curve constructed using authentic reference gallic acid concentration range 25 - 100 g/mL. (II) Quercetin and Rutin The mobile phase was methanol : acetonitrile : water (40 : 15 : 45) containing 1% acetic acid. Quercetin and rutin were quantified by DAD following HPLC separation from absorption at 368 nm and at 257 nm, respectively. Flow rate was 1 mL/min and injection volume was l0 L. The chromatographic peaks of the analytes were confirmed by comparing their retention time and UV spectra with those of the reference standards(15). Quantification was carried out by the integration of the peak area using an external standard. (III) Coumarin Separations were done in the isocratic mode, using acetonitrile : water (40 : 60) at a flow rate of 1 mL/min with an injection volume of 20 L detection 274 nm(16). The assay of coumarin in plant material was performed using external standard method, in which pure coumarin was used as reference. XII. Statistical Analysis Experimental results are expressed as mean SD. All measurements were performed in triplicate. The data were analyzed by an analysis of variance (p < 0.05) and the means separated by Duncans multiple range test.

RESULTS
The total phenolic content of D. aitchisonii aerial parts was 41.2 1.9 mg gallic acid equivalents/g of extract (estimated from a standard curve, y = 0.0054x + 0.0628, r 2 = 0.987). The total flavonoid content was 13.8 0.8 mg quercetin equivalents/g of extract, estimated from a standard curve (y = 0.0063x, r 2 = 0.999). The IC50 for DPPH radical-scavenging activity was 488.1 18 g/mL, as shown in Table 1. The corresponding IC50 values for ascorbic acid, quercetin and BHA were 5.05 0.1, 5.28 0.2 and 53.96 3.1 g/mL, respectively. The extract showed weak iron chelating activity with an IC50 = 778.2 22 g/mL. EDTA showed very powerful iron chelating activity (IC50 = 18 1.5 g/mL). In terms of nitric oxide scavenging activity, the percent inhibition increased with increasing concentrations of the extract but the activity was very weak. The IC50 was 1.3 0.01 mg/mL. Quercetin with an IC50 20 1 g/mL. The extract was capable of scavenging hydrogen peroxide in a concentration-dependent manner with an IC50

37
Journal of Food and Drug Analysis, Vol. 20, No. 1, 2012 Table 1. Antioxidant activity of D. aitchisonii extract in diphenyl2-picryl hydrazyl (DPPH) radical scavenging test. Ascorbic acid, quercetin and BHA used as positive control Sample D. aitchisonii Ascorbic acid Quercetin BHA * Values are mean SD (n = 3). IC50 (mg/mL)* 488.1 18 5.05 0.1 5.28 0.2 53.96 3.1

2.8

Absorbance at 700 nm

D. aitchisonii 1.8 Vitamin C

0.8

-0.2 0

100

200

400

800

210.6 11 g/mL. The IC50 values for vitamin C and quercetin were 21.4 1.1 and 52 2.6 g/mL, respectively. Figure 1 shows the dose-response curves for the reducing power of extract. It was found that the reducing power of extract increased with the increase of its concentration. There was significant difference among the extract and Vitamin C in reducing power (p < 0.01). The extract show good activity in hemoglobin-induced lipid peroxidation test but there was a significant difference between the activity of the extract and that of Vitamin C (p < 0.01) (Figure 2). The extract show a rapid and concentration-dependent increase of antioxidant activity. The extract was found to have no harmful effect on erythrocytes, in fact, the extract exhibited potent antihemolytic activity with an IC50 of 384.6 15 g/mL as compared with 235 9 g/mL for Vitamin C which served as a positive control. HPLC/DAD analysis of the extract indicated rutin as a major component (7.27 g/mg extract) as shown in Figure 3. Amount of gallic acid (0.011 g/mg extract), cumarin and quercetin (< 0.01 g/mg extract) were low and trace. Their retention times were 7.15, 12.63 and 10.08 min, respectively.

Concentration (g/mL)

Figure 1. Reducing power of D. aitchisonii extract. Vitamin C used as positive control.

100 80 60 40 20 0
D. aitchisonii Vitamin C

Percentage of Inhibition

125

250

500

1000

Concentration (g/mL)

Figure 2. Antioxidant activity of D. aitchisonii extract against hemoglobin-induced lipid peroxidation. Vitamin C used as positive control.

DISCUSSION
The total phenolic content of the aerial portion of D. aitchisonii was 41.2 1.9 mg gallic acid equivalents/g of extract and the total flavonoid content was 13.8 0.8 mg quercetin equivalents/g of extract. The extract showed high levels of total phenolic and flavonoid components. Phenols and polyphenolic compounds, such as flavonoids, are widely found in food products derived from plant sources and they have been shown to possess significant antioxidant activities(17). Detailed HPLC/DAD analysis of the extract indicated that it contained 7.27 g rutin and 0.011 g gallic acid/ mg extract whereas cumarin and quercetin were present in only trace amounts (< 0.01 g/mg extract). DPPH is a stable nitrogen-centered free radical which its color changes from violet to yellow upon reduction by reactions involving either hydrogen- or electron-donation. Substances involved in such processes can be considered as antioxidants and therefore radical scavengers(18). The phenolic and flavonoid components of this plant may determine or at least contribute to this DPPH-scavenging activity(19). The correlation between total phenolic content
100 80
2.683

mAU

4.333

7.183

40 20 0 0 1 2 3 4

5.483 5.833

60

8.183

Minutes

Figure 3. HPLC chromatogram of D. aitchisonii extract.

and antioxidant activity has been widely studied in various foodstuffs such as fruit and vegetables(20). Iron chelators mobilize tissue iron by forming soluble, stable complexes that are then excreted in the feces and/or urine. Chelation therapy reduces iron-related complications in humans and thereby improves quality of life and overall

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Journal of Food and Drug Analysis, Vol. 20, No. 1, 2012

survival in some diseases such as Thalassemia(21,22). Deferoxamine and Deferiprone are clinically useful iron chelators; however, many adverse effects can occur after their administration(23). There remains an urgent need to identify other chelators with an acceptable degree of tolerability(23). Therefore, much research has focused on natural product particularly flavonoid that can directly influence iron levels within tissues(21). Ferrozine can quantitatively form complexes with iron. In this assay, both the extract and EDTA interfered with the complexation of iron with ferrozine, although the iron chelating activity of the extract was weak. Activity in the metal chelating test indicated that some compounds in the extract are electron donors and can react with free radicals to convert them into more stable products and terminate radical chain reactions. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates NO which can interact with oxygen to produce nitrite ions that can be estimated using Griess reagent. Scavengers of NO compete with oxygen, leading to reduced production of nitrite ions. NO has been associated with a variety of physiologic processes in the human body since it was identified as a novel signalling molecule. It also participates in pathways underlying a large group of disorders such as muscle diseases, inflammatory bowel disease, primary headaches, stroke and neurodegenerative disorders such as Alzheimer disease(24,25). In such situations, the use of herbal remediation a NO scavenger may prove useful. In scavengers of nitric oxide, the degree of inhibition increased with increasing concentrations of the extract but the activity was very weak. Scavenging of H2O2 by the extract may be related to its phenolic content as well as other active components such as anthocyanins, tannins and flavonoids - all of which can donate electrons to H2O2, thereby neutralizing it to water(26,22). Although H2O2 itself is not very reactive, it can cause cytotoxicity by giving rise to hydroxyl radicals in the cell. Thus, removal H2O2 is very important throughout food systems(8). The extract was capable of scavenging H2O2 in a concentration-dependent manner with an IC50 210.6 11 g/mL. The IC50 values for vitamin C and quercetin were 21.4 1.1 and 52 2.6 g/mL, respectively. In the reducing power assay, the presence of reductants (antioxidants) in the sample would result in the reduction of Fe3+ to Fe2+ by donating an electron. Amount of Fe2+ complex can then be monitored by measuring the formation of Perls Prussian blue at 700 nm. Increasing absorbance at 700 nm indicates an increase in reductive ability(8). It was found that the reducing power of extract increased with the increase of its concentration. Extract showed weak activity (Figure 1). Membrane lipids are rich in unsaturated fatty acids which are most susceptible to oxidative processes. Specifically, linoleic acid and arachidonic acid are targets of lipid peroxidation(27). Since polyphenolic compounds appear to function as good electron and hydrogen atom donors and therefore are able to terminate radical chain reaction by converting free radicals and reactive oxygen species to more stable products, the reducing potential of the extract may

be attributed to this mode of action. A similar observation has been reported for several plant extracts(28). The hemoglobin-induced linoleic acid peroxidation test yielded results following only 1 h of oxidation time. Generally, antioxidant assays with linoleic acid need a long time for auto-oxidation (the order of 5 - 6 days)(28). The extract show activity in this test but there was a significant difference between extract and vitamin C (p < 0.01) (Figure 2). The extract showed a rapid and concentration-dependent increase in antioxidant activity. This effect may be the result of its good flavonoid contents, especially rutin. Flavonoid interactions with cell membranes, which generally serve as targets for lipid peroxidation (LP), constitute an important area of research(29). Various model membrane systems such as LDL and red blood cell (RBC) membranes offer physiologically relevant and relatively simple systems for studying LP(30). The RBC has been chosen as an in vitro model to study oxidant/antioxidant interactions since its membrane is rich in polyunsaturated fatty acids, which are extremely susceptible to peroxidation(29). Recent years, a few interesting studies have been reported, indicating the protective effects of some plant extracts against oxidative damage in intact RBC membranes(31,32). Hemolysis has a long history of use in measuring free radical damage and its inhibition by antioxidants. There are many published studies involving oxidative challenge of intact erythrocytes in whole blood. In this study, we used a biological test based on free radical-induced erythrocyte lysis in rat blood. This assay is useful for screening studies for various molecules and their metabolites, especially those having an oxidizing or antioxidizing activity and/or having a long-term action(33). Lipid oxidation of rat RBC membranes mediated by H2O2 induces membrane damage and subsequent hemolysis. The effect of the extract was tested and found that they did not show any harmful effects on erythrocytes in fact, it exhibited potent antihemolytic activity with an IC50 of 384.6 15 g/mL compared with 235 9 g/mL for Vitamin C which served as a positive control. Antihemolytic activity and the relationship between iron chelating activity and the protective activity against oxidative damage to erythrocyte membranes by quercetin have been reported previously(29,34). It seems high total phenolic and flavonoid contents, especially rutin, in extract are responsible the observed antihemolytic activity.

CONCLUSIONS
Our study demonstrated the presence of weak antioxidant and antihemolytic activities in a hydroalcoholic extract of D. aitchisonii for the first time. Future biochemical experiments will focus on evaluating the mechanism underlying these effects.

ACKNOWLEDGMENTS
This research was partially supported by a grant from Pharmaceutical Sciences Research Center, Iran. This paper

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