Antioxidant Potential of Different Dill (Anethum Graveolens L.) Leaf Extracts

Abstract

Dill (Anethum graveolens L.) have been extensively used in salads, soups, and pickles for its aromatic odor and flavor. Recently, interest in plant-derived food additives has grown. In this study, the possible antioxidant properties of water, ethanol, and acetone extracts of dill leaves were investigated. In order to evaluate antioxidant activities of all extracts, different antioxidant tests were used, such as total antioxidant activity by ferric thiocyanate method, reducing power, DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging, hydrogen peroxide scavenging, and ferrous ions chelating activities. The content of phenolic compounds was also determined to be the gallic acid equivalent. Among the three extracts, the water extract of dill leaf showed the most potent antioxidative capacity in each assay, showing 79.66% (at 1 mg/mL) in the DPPH^• radical scavenging activity, 63% (at 800 μg/mL) in the metal chelating effect, 60% (at 400 μg/mL) in the H₂O₂ scavenging activity, and 0.61 absorbance (at 1 mg/mL) in the reducing power.

Keywords:

• Anethum graveolens L.
• Dill leaf
• Antioxidant activity
• Radical scavenging
• Phenolic compound

INTRODUCTION

In living organisms, free radicals can occur in different ways as in normal physiological conditions or exogenous factors. They can easily react with cellular components, especially lipids, DNA (deoxyribonucleic acid), and carbohydrates, to cause many disorders in humans, including cancer, atherosclerosis, carciovascular diseases, and aging.[1,2] On the other hand, free radicals can also cause lipid peroxidation in food products that contain unsaturated fatty acid. Lipid peroxidation can lead to the development of unpleasant rancid or off flavours as well as nutritional loss.[3] Therefore, much attention has been focused on the use of antioxidants, especially plant-derived natural antioxidants to inhibit lipid peroxidation.

Antioxidants are capable of stabilizing or deactivating free radicals before they attack cells, and have the ability to protect the body from damage caused by free radical-induced oxidative stress.[4] There is a growing interest for aromatic and medicinal plants because of their antioxidative properties in both the scientific research and the industry. The importance of aromatic plants as natural antioxidants is well established. These properties of herbs and plants are coming from their content of phytochemicals, including flavonoids, carotenoids, terpenoids, phenolic acids, ascorbic acids, and so on.[5]

Anethum graveolens L. (Umbelliferae), commonly known as dill, is native to Mediterranean countries and southeastern Europe. Dill (Anethum graveolens L.) is an important aromatic herb, used for flavoring of various foods, such as salads, sauces, soups, sea foods, and especially in pickles. The leaves of the dill contain phosphorus, potassium, and magnesium minerals.[6] Additionally, dill leaves are also used as dried. In the traditional herbal medicine, the dill is used as a diuretic and also to solve some gastrointestinal problems, such as flatulence, intestinal spasms, and various digestive problems.[7] The aroma composition of the dill has been investigated in numerous studies and was reported that the main components were calvone, limonene, and dill adipole.[8–10] Moreover, there are many reports on antioxidant activity of dill seed and flower.[11,12] There is, however, no detailed report on evaluation of the antioxidant potential of dill leaves, which are the most consumed type of dill. The goal of this study was to evaluate the antioxidative activity of dill leaves and to find a new potential source of natural antioxidants. Therefore, water, ethanol, and acetone extracts of dill leaves were prepared, and their total antioxidant activity, free radical scavenging activity, reducing power ability, metal chelating capacity, and H₂O₂ scavenging activity were determined. Total phenolic compounds were determined as well.

MATERIAL AND METHODS

Chemicals

All solvents and reagents used for this study were of analytical grade and obtained from various suppliers, including Merck, Sigma, and Riedel-de Haen.

Sample Preparation

The plant was purchased from a local market and the leaves were dried in the shade at room temperature. The plant was taxonomically identified by botanical experts at the Department of Biology, Faculty of Science and Art, Trakya University. For water extraction, 15 g of dried dill sample was extracted in 300 mL of boiling distilled water with a magnetic stirrer for 30 min. The extract was filtrated and then freeze-dried. For ethanol and acetone extractions, 15 g of dried dill sample was extracted twice with both 300 mL of ethanol and acetone at room temperature for 3 h in a shaking water bath. After the filtration, each extracts, ethanol and acetone extracts were combined separately. The solvent of the fractions was removed using a rotary evaporator at 35°C. All samples were stored at 4°C until used for analysis.

Total Antioxidant Activity Assay

The antioxidant activity of dill leaf extracts were determined according to the thiocyanate method.[13] Linoleic acid emulsion was prepared with linoleic acid and Tween 20 in phosphate buffer (0.04 M, pH 7.0). A reaction solution containing extracts, linoleic acid emulsion, and phosphate buffer (0.04 M, pH 7.0) was placed in a test tube. The mixed solution was incubated in darkness at 37°C. The amount of peroxide was determined by reading the absorbance at 500 nm, after reaction with FeCl₂ (0.1 mL, 20 mM in 3.5% HCl) and thiocyanate (0.1 mL, 30%) at intervals during incubation. The solution without added extract was used as a blank sample. The inhibition of lipid peroxidation in percent was calculated by the equation: Inhibition % = [(A ₀ – A ₁)/A ₀] × 100, where A ₀ is the absorbance of the control reaction and A ₁ is the absorbance of the sample dill leaf extracts.

DPPH^• Radical Scavenging Activity

Free radical scavenging activity of dill leaf extracts was evaluated with 1,1-diphenyl-2-picryl-hydrazil (DPPH^•) using the Blois method.[14] Briefly, one milliliter of the sample solution was added to 4 mL of ethanol solution of DPPH^• (0.1 mM), and the mixture was kept at room temperature. After 30 min, the absorbance was measured at 517 nm with a spectrophotometer (Shimadzu UV-1601, Japan). The antiradical activity was calculated using the ratio: (A_blank – A_sample /A_blank ) × 100, where A_blank is the absorption of the DPPH^• solution and A_sample is the absorption of the DPPH^• solution after the addition of the sample.

Reducing Power

The reducing power of the extracts was determined according to the method of Oyaizu.[15] One mL of extracts was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide [K₃Fe(CN)₆] (1%), and then the mixture was incubated at 50°C for 20 min. Afterward, 2.5 mL of trichloroacetic acid (10%) were added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer solution was mixed with distilled water and FeCl₃ (0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated reducing power.

Hydrogen Peroxide Scavenging Activity

The ability of the extracts to scavenge hydrogen peroxide was determined according to the method of Ruch.[16] A solution of H₂O₂ (40 mM) was prepared in phosphate buffer (0.1 M, pH 7.4). Extracts in phosphate buffer were added to 0.6 mL of H₂O₂ solution. After 30 min at room temperature, the absorbance value of the reaction mixture was measured at 230 nm. Blank solution was containing the phosphate buffer without H₂O₂. The percentage of H₂O₂ scavenging of extracts and standard compounds was calculated as: [1 – (Absorbance of the sample at 230 nm/Absorbance of the blank at 230 nm)] × 100.

Metal Chelating Activity on Fe⁺²

The chelating activity of ferrous ions by dill leaf extracts was measured according to Dinis et al.[17] Dill leaf extracts were added to 0.1 mL of 2 mM FeCl₂ and the solution was incubated at room temperature for 30 min. After incubation, 0.2 mL of 5 mM ferrozine was added, and total volume was adjusted to 5 mL with deionized water. Then, the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was measured at 562 nm against the same mixture, without the sample, as a blank. EDTA served as the possitive control. The percentage of inhibition of ferrozine-Fe⁺² complex formation was calculated using the equation: metal chelating activity % = [1 – (A ₁/A ₀)] × 100, where A ₀ is the absorbance of the blank and A ₁ is the absorbance of the sample.

Determination of Total Phenolic Compounds

Total phenolic content was determined using the Folin−Ciocalteu colorimetric method, described by Singleton and Rossi.[18] One mL of extract solution was transferred to an Erlenmeyer flask and the volume was adjusted to 46 mL by addition of distilled water. Then, 1 mL of Folin-Ciocalteu reagent was added to the mixture, followed after 3 min by 3 mL of Na₂CO₃ solution (2%). The mixture was shaken and the absorbance was measured at 760 nm with a spectrophotometer after incubation for 2 h at room temperature. Quantification was done on the basis of the standard curve of gallic acid concentration range between 50 to 500 μg/mL (r ² = 0.9961). The amount of total phenolic compounds was expressed as mg of gallic acid equivalent (GAE)/g dry weight of the plant material.

Statistical Analysis

All determinations were conducted in triplicate, and all results were expressed as mean ± standard deviation (SD) in the study. The correlation coefficient (r ²) between the parameters tested was established by regression analysis. Statistical comparisons were performed with Student's t-test. Differences were considered significant at p < 0.05.

RESULTS AND DISCUSSION

Solvent extraction is frequently used for isolation of antioxidants and both extraction yield and antioxidant activity of extracts are dependent on the solvent. Table 1 shows the amount of extractable compounds from dried plant material and antioxidant activity of water, ethanol, and acetone extracts of dill leaf. The extractions with acetone and water resulted in the highest amount of total extractable compounds.

Table 1 Yield, total phenolic content, and total antioxidant activities in different extracts from Anethum graveolens L. leaves

	Yield (mg extract/g dried plant)	Total phenolics (mg GAE/g dried plant)	Inhibition of lipid peroxidation (%) ^a
Water extract	58.66	65.05 ± 2.06	40.72 ± 5.81
Ethanol extract	44.03	71.29 ± 1.05	87.62 ± 1.15
Aceton extract	60.70	55.46 ± 1.90	79.25 ± 0.47
Data are presented as mean ± SD.
^aThe total antioxidant activities of extracts (250 μg/mL) were determined by the thiocyanate method.

The total phenolic compound contents in the extracts were determined by a colorimetric assay, using the Folin-Ciocalteu reagent. As shown in Table 1, the amount of total phenolic compounds was the highest in the ethanol and water extracts and lowest in the acetone extract. These amounts were higher than the results described in the literature for other extracts of dill herb.[10,19] This discrepancy may be linked to the different extraction conditions and using solvent,[20] since in the other papers it was reported high values to our results for dill seed extracts[11] and ethanolic dill leaf extract.[12] It was also reported that different phenolic compounds showed different antioxidant activities, depending on their chemical structures.[21] Based on this, the different antioxidant activities of the present extracts may be due to their extractable phenol contents and to their chemical structures.

Total Antioxidant Activity

The measuring of antioxidant activity in food is dependent on several factors, including the method adopted and type of system (lipidic or aqueous) used, properties of the substrates, the condition of oxidation. Hence, different methods have been developed to evaluate the antioxidative potential of plant extracts. This generally covers total antioxidant activity, free radical scavenging capacity, reductive capacity, metal chelating, and scavenging the active oxygen species, such as H₂O₂, O₂ ^•−, and OH^•.[22]

The antioxidative activity of leaf extracts of Anethum graveolens L. was determined by the ferric thiocyanate method using linoleic acid emulsion system, and BHT (butylated hydroxytoluene) was used as standart compound. The inhibitory effects of various concentrations of extracts from dill leaf on the lipid peroxidation of linoleic acid emulsion are shown in Fig. 1A. Concentration-dependent inhibition of lipid peroxidation was observed. All concentration of the ethanol and acetone extracts of dill leaf showed high antioxidant activities and inhibition percent on lipid peroxidation of linoleic acid system. However, there was no significant difference between ethanol extract and acetone extract (p > 0.05). It was reported that water and alcohol extracts from dill seed were the most effective inhibitors of linoleic acid peroxidation.[11]

Figure 1 Antioxidant activities of dill (Anethum graveolens L.) leaf extracts and standard compounds with different antioxidant assays: (A) inhibition (%) of lipid peroxidation in the linoleic acid emulsion, (B) DPPH scavenging activities, (C) reductive potential, and (D) metal chelating effects.

DPPH^• Scavenging Activity

One of the most frequently used tools for testing the radical scavenging activity of antioxidants is 1,1-diphenyl-2-picryl-hydrazil radical (DPPH^•) assay. It has been widely used to evaluate the antioxidant action as the DPPH^• is relatively a stable free radical.[23,24] When DPPH^• reacted with an antioxidant compound, it was reduced. The change in colour was determined spectrophotometrically.

The scavenging effect of the water, ethanol and acetone extracts of dill leaf was evaluated and compared with BHA (butylated hydroxyanisole) and α-tocopherol (Fig. 1B). Free radical scavenging activities increased with increasing concentration. The water and ethanol extracts showed similar DPPH^• radical scavenging activity, while the acetone extract was no respectable activity in contrast to the reported one for dill seed by Singh et al.[9] The scavenging effects of water and ethanol extracts at concentration of 800 μg/mL (79.73 ± 0.63 and 87.22 ± 0.97, respectively) may be comparable to those of BHA (89.28 ± 1.64) and α-tocopherol (89.07 ± 1.06).

EC₅₀ value is the effective concentration to inhibits 50% of DPPH^• radicals. A lower EC₅₀ value is resulted in a stronger DPPH^• radical scavenging activity. According to effective concentration of the extracts; it was seen that water extract (1.93 ± 0.53 mg/mL) had the highest DPPH^• radical scavenging activity, followed by the ethanol extract (4.75 ± 1.35 mg/mL), while the acetone extract had the lowest activity (8.95 ± 1.41 mg/mL). EC₅₀ values were significantly different (p < 0.05) from those obtained for BHA (0.71 ± 0.006 mg/mL) and α-tocopherol (0.56 ± 0.009 mg/mL). It was reported that[25] IC₅₀ values (inhibitory concentration to inhibit 50% of free radical scavenging activity) have been reduced in the aqueous and methanolic extracts of soup mix, including also dill leaf powder.

Reducing Power

The Fe³⁺-Fe²⁺ transformation is often used as an indicator of electron donating activity and can be correlated with other antioxidant properties.[26] Figure 1C shows the reductive capabilities of dill leaf extracts compared with BHT and α-tocopherol. The water and ethanol extracts with higher concentration showed a higher reducing power than the acetone extract. The maximum absorbance for the acetone extract was 0.14, compared to 0.62 and 0.51 for the water and ethanol extracts, respectively. At a dosage of 50–600 μg/mL α-tocopherol and 60–400 μg/mL BHT showed high reducing values of 0.29–1.22 and 0.36–1.45, respectively. This suggests that the dill leaf extracts have not exhibited transition metal ion chelating activity when incubated with Fe³⁺ in the present study.

Although the reducing power of a compound is related to its electron transfer ability and may serve as a indicator of its potential antioxidant activity,[27] we have found that this might not be in this case. However, our results were in accordance with other investigators, having also reported the same case.[28]

Metal Chelating Activity on Fe²⁺

Transition metals can stimulate lipid peroxidation by the Fenton reaction. Iron (II) ions may react with H₂O₂ to give free hydroxyl radicals, which are highly reactive, and also accelerate peroxidation.[29] Ferrozine can quantitatively form complexes with Fe²⁺ ions. In the presence of chelating agents, formation of the complex is disrupted and, as a result, the absorbance of the complex (dark pink colour) is decreased. Measurement of colour of the complex, therefore, allows estimation of the chelating capacity of the extracts. In this study, the water, ethanol, and acetone extracts of dill leaf were assessed for their chelating ability to compare to ferrozine for Fe²⁺ ions. As shown in Fig. 1D, the ability to chelate Fe²⁺ ions was in a concentration dependent manner (0.1–1 mg/mL) in the presence of the water and ethanol extracts of dill leaf. The water and ethanol extracts chelated ferrous ions by 68.67 ± 2.47% and 54.84 ± 1.74% at 1 mg/mL, whereas the acetone extract showed 37.16 ± 2.5% at the same concentration. But, none of the extracts appeared to be better chelators of Fe²⁺ ions than the reference compound EDTA, an excellent chelator, in this system.

H₂O₂ Scavenging Activity

Hydrogen peroxide can be formed in vivo by many oxidizing enzymes, such as superoxide dismutase. Hydrogen peroxide itself is not very reactive, but it can cross biological membranes and sometimes be toxic to cells.[29] Extracts from dill leaf were not very well capable of scavenging hydrogen peroxide when compared with standard compounds. The water extract of dill leaf showed the scavenging activity of 44.13 ± 0.81% and 60.96 ± 2.78% at concentrations of 250 and 400 μg/mL, respectively, while the ethanol extracts of dill leaf have scavenging activity of 16.60 ± 0.84% and 25.05 ± 0.35% at same doses. BHT and BHA were showed the strongest scavenging activity of 95.9 ± 0.42% and 84.85 ± 0.49%, respectively, at a concentration of 400 μg/mL.

Statistical Analysis

The antioxidant activity of plants is mostly due to the presence of phenolic compounds.[30,31] Several studies have been reported on the relationships between phenolic content and antioxidant activity on vegetables and fruits. Some authors found a correlation between the phenolic content and antioxidant activity,[20,32,33] while others found no such relationship.[34] However, the findings of this study showed a relationship between some of antioxidant activity assays and total phenolic contents. The content of total phenolics show a correlation with the antioxidant assays, such as DPPH^• scavenging activity (r ² = 0.553, p < 0.05), reducing power (r ² = 0.519, p < 0.05) and iron chelation (r ² = 0.620, p < 0.05). No significant correlation can be found between total phenolics and inhibition of lipid peroxidation.

CONCLUSIONS

Dill is cultivated in many countries and consumed in the daily diet. Fresh dill leaves are eaten as salad, and used for flavoring of sauces, sea foods, and especially pickles. In the present study, the antioxidative potential of water, ethanol, and acetone extracts of dill (Anethum graveolens L.) leaf was examined with different antioxidant methods. The water extract showed the best antioxidative activity. The water extracts of dill leaf had high reducing power, metal chelating, DPPH^• radical scavenging, and H₂O₂ scavenging activities, when compared with the other two extracts. Total antioxidant activity of ethanol and acetone extracts of dill leaf exhibited effective antioxidant activity compared with BHT, but the water extract showed lower levels of inhibitory activity towards lipid peroxidation. In the studied other antioxidant assays the ethanol extract showed close antioxidant potential to the water extract, but the acetone extract exhibited lower antioxidant potential than other extracts. As it can be seen in these results, the dill may be also used as an easily accessible source of natural antioxidants and as a possible food supplement. Nevertheless it is suggested that further work may be done on the isolation and identification of the antioxidative components in dill leaf.

ACKNOWLEDGMENT

The authors are grateful to Necmettin Guler for the taxonomic identity of the plant and F. Nesrin Turan for statistical analysis. This work was supported by the Research Fund of Trakya University, Project number: TUBAP-660.