The Antioxidant and Radical Scavenging Activities of Primrose (Primula Vulgaris)

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European Journal of Experimental Biology, 2014, 4(2):395-401

ISSN: 2248 –9215 CODEN (USA): EJEBAU

The antioxidant and radical scavenging activities of Primrose (Primula vulgaris )

Nazan Demir 1*, Azize Alayli Gungor 2, Hayrunnisa Nadaroglu 3 and Ya şar Demir 1,4

1Mugla University, Faculty of Sciences, Department of Chemistry, Mugla, Turkey 2Atatürk University, Erzurum Vocational School, Department of Chemical Technology, Erzurum, Turkey 3Atatürk University, Erzurum Vocational School, Department of Food Technology, Erzurum, Turkey 4University of Mehmet Akif Ersoy, Faculty of Science, Department of Chemistry, Burdur, Turkey ______

ABSTRACT

People have become more interested in Primrose (Primula vulgaris) in recent years as it has different areas to be applied especially within the food and cosmetic world. This study evaluated the in vitro antioxidant activity of water and ethanol extracts of Primrose (Primula vulgaris). The antioxidant properties of the Primrose (Primula vulgaris) were evaluated the in vitro by antioxidant assays such as 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) scavenging, superoxide anion radical scavenging, and metal chelating activities. α-Tocopherol, butylated hydroxyanisole (BHA), and ascorbic acid were used as the reference antioxidant compounds. The concentration of 45 µg/mL of water and ethanol extracts of Primrose (Primula vulgaris) showed 43,0 and 39,4% reducing power, respectively. On the other hand, 45 µg/mL of standard antioxidant such as α-tocopherol indicated 38,4% on reducing power. In addition primrose is an effective DPPH• scavenging, superoxide anion radical scavenging and metal chelating on ferrous ions activities.

Keywords: Primrose (Primula vulgaris ), antioxidant activity; metal chelating; reducing power; radical scavenging. ______

INTRODUCTION

The botanical name of the order, Primulaceae, is based on that of the genus Primula , to which belong not only those favorite spring flowers of the countryside such as the primrose, primrose and their less common relative, the oxlip, but also the delicately tinted greenhouse species that are such welcome pot plants for our rooms in midwinter. The crinkled green leaves are covered on both sides with a fine layer of downy hairs; they form rosettes, which tend to lie close to the ground. A downy flower stalk rises from the center of this rosette and is topped with a cluster of 1–30 yellow or buff-colored flowers. The flowers are funnel-shaped and have characteristic orange spots at the base of the lobes [1,2].

The harmful intervention of free radicals in normal metabolic processes leading to pathologic changes is a consequence of their interaction with various biological compounds inside and outside cells. To protect bio molecules against the attack of free radicals and/or to suppress the resultant damage, numerous natural and synthetic free radical scavengers and antioxidants have been developed and studied. Among them, flavonoids, natural polyphenolic compounds, have attracted significant interest [3]. Actually, the beneficial clinical and curative effects of flavonoids in the treatment of virus infection, inflammation, diabetes mellitus, headache, etc. in humans have long been shown, but they are “to a large extent based on empirism since this praxis is much older than the science of chemistry” [4]. However, at present, many studies show the importance of the antiradical activity of flavonoids.

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Thus, it has been shown that flavonoids are the effective inhibitors of lipid peroxidation, oxygen radical overproduction by inflammatory cells, free radical-mediated cytotoxicity, and chromosome damage [5-11].

Flavonoids (or bioflavonoids) are a group of about 4000 naturally occurring compounds that are ubiquitous in all vascular plants [4]. They are pigments responsible for the autumnal burst of hues and the many shades of yellow, orange, and red in flowers [12. They are also important for the normal growth, development and defense of plants [13]. Flavonoids, important constituents of the human diet, are found in fruits (in citrus fruit they may represent up to 1% of fresh material) and vegetables. Beverages like red wine, tea, coffee, and beer also contain large amounts of flavonoids: on the average, the daily diet contains approximately 1 g of flavonoids per day [14]. Flavonoids are also found in several medicinal plants, and herbal remedies containing flavonoids have been used in folk medicine around the world.

The interest in polyphenolic antioxidants has increased remarkably in the last decade because of their elevated capacity in scavenging free radicals associated with various diseases. This property has been evidenced by a large number of tests measuring the antioxidant activity in vitro [15-19]. In vivo , the antioxidant protection attributed to the polyphenols can be checked through the level of biomarkers, such as malondialdehyde, which is associated with lipid peroxidation [20], or 8-oxo-7,8-dihydroguanine, which indicates oxidative damage to the DNA bases [21]. The protective capacity of polyphenols is also supported by a number of studies indicating an effect of dietary polyphenols on coronary heart disease (CHD) [22], gene regulation [25], and neurodegenerative diseases [26].

However, there is no information about antioxidant activity of aqueous extract of Primrose ( Primula vulgaris ) In our investigation, we wanted to describe the antioxidant effects of Primrose and to compare their antioxidant effects with those commonly used as food antioxidants, such as BHT, BHA, and α-tocopherol. In addition to this, the components responsible for the antioxidative ability of Primrose ( Primula vulgaris ), are currently unclear. Hence, it is suggested that further work could be performed on the isolation and identification of the antioxidative components in Primrose ( Primula vulgaris ).

The present study investigated the antioxidant properties of Primrose ( Primula vulgaris ) in order to evaluate its medicinal value and to point to an easily accessible source of natural antioxidants that could be used as a possible food supplement or in the pharmaceutical, cosmetic and perfume industries.

MATERIALS AND METHODS

Chemicals Ammonium thiocyanate was purchased from E. Merck. Ferrous chloride, polyoxyethylenesorbitan monolaurate (Tween-20), α-tocopherol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH •), 3-(2-Pyridyl)-5,6-bis (4-phenyl-sulfonic acid)- 1,2,4-triazine (Ferrozine), nicotinamide adenine dinucleotide (NADH), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and trichloracetic acid (TCA) were obtained from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany). All other chemicals used were of analytical grade and were obtained from Sigma (Sigma-Aldrich GmbH, Sternheim, USA).

Plant Material and Extraction Primroses ( Primula veris ) were collected from the flora of Mersin in May. Then, they were kept in deep-freeze until they were used. The taxonoomic identification of plant material was confirmed by plant taxonomist Meryem Sengül (Atatürk University, Faculty of Art and Sciences, Department of Biology). A voucher specimen was deposited in the Herbarium of Faculty of Biology, Atatürk University. For water extraction, 25 g sample was put into a fine powder in a mill and was mixed with 500 ml boiling water by magnetic stirrer for 15 min. Then, the extract was filtered over Whatman No. 1 paper. The filtrates were frozen and lyophilized used a lyophilizator at 5 µm-Hg pressure at −50°C (Labconco, Freezone 1L). For ethanol extraction 25 g sample was put into a fine powder in a mill and was mixed with 500 ml ethanol. The residue was re-extracted until extraction solvents became colorless. The obtained extracts were filtered over Whatman No. 1 paper and the filtrate was collected, and then ethanol was removed by a rotary evaporator (RE 100 Bibby, Stone, Staffordshire England, ST15 OSA). Both extracts were placed in a plastic bottle, and then stored at −20 oC until used.

Free Radical Scavenging Activity The free radical scavenging activity of primrose extracts was measured by the 1,1-diphenyl-2-picryl-hydrazil (DPPH •) method proposed by Blois [27]. Briefly, 0.1 mM solution of DPPH in ethanol was prepared and 1 ml of this solution was added 3 ml of primrose extracts solution in water at different concentrations of 10 µg/ml. Thirty minutes later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher

396 Pelagia Research Library Nazan Demir et al Euro. J. Exp. Bio., 2014, 4(2): 395-401 ______free radical scavenging activity. The DPPH concentration in the reaction medium was calculated from the following calibration curve, determined by linear regression ( R2: 0.9545):

The capability to scavenge the DPPH • radical was calculated using the following equation:

Where Ao is the absorbance of the control reaction and A1 is the absorbance in the presence of the sample of primrose extracts.

Reducing Power The reducing power of primrose extracts was determined by the method of Oyaizu [29] . Different concentrations of primrose extracts (2.7–13.4 µg/ml) in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH o 6.6) and potassium ferricyanide [K 3Fe(CN) 6] (2.5 ml, 1%). The mixture was incub ated at 50 C for 20 min. Aliquots (2.5 ml) of trichloroacetic acid (10%) were added to the mixture, which was then centrifuged for 10 min at 1036× g (MSE Mistral 2000, UK). The upper laye r of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl 3 (0.5 ml, 0.1%), and the absorbance was measured at 700 nm in a spectrophotometer. Increased absorbance of the reaction mixture indicates increased reducing power.

Superoxide Anion S cavenging Activity Measurement of superoxide anion scavenging activity of primrose extracts was based on the method described by Liu et al., [29] with slight modification. Superoxide radicals are generated in PMS –NADH systems by oxidation of NADH and assayed by the reduction of nitroblue tetrazolium (NBT). In this experiments, the superoxide radicals were generated in 3 ml of Tris–HCl buffer (16 mM, pH 8.0) containing 1 ml of NBT (50 µM) solution, 1 ml NADH (78 µM) solution and sample solution of primrose extract of 10 µg/ml in water. The reaction started by adding 1 ml of phenazine methosulphate (PMS) solution (10 µM) to the mixture. The react ion mixture was incubated at 25 °C for 5 min, and the absorbance at 560 nm was measured against blank samples. L-Ascorbic acid was used as a control. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the following formula:

Where Ao is the absorbance of the control, and A1 is the absorbance of primrose extracts or standards [30].

Ferrous Ions Chelating Activity The chelating of ferrous ions by the primrose extracts and standards was estimated by the method of Dinis et al. [31]. Briefly, extracts of 15 µg/ml were added to a solution of 2 mM FeCl 2 (0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml) and the mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm. All test and analyses were run in triplicate and averaged. The percen tage of inhibition of ferrozine –Fe 2+ complex formation was calculated using the formula given bellow:

Where Ao is the absorbance of the control, and A1 is the absorbance in the presence of the sample of primrose extracts or standards. The control does not contain FeCl 2 and ferrozine, complex formation molecules.

Scavenging of Hydrogen Peroxide The ability of the primrose extracts to scavenge hydrogen peroxide was determined according to the method of Ruch et al. [32]. A solut ion of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically measuring absorption with extinction coefficient for H2O2 of 81 M −1 cm −1 . Extracts (12.5–62.5 µg/ml) in distilled water were added to a hydrogen peroxide solution (0.6 ml, 40 mM). Absorbance of hydrogen peroxide at 230 nm was determined 10 min later against a blank solution containing the phosphate buffer wit hout hydrogen peroxide. The percentage of hydrogen peroxide scavenging of both primrose extracts and standard compounds was calculated:

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Where Ao is the absorbance of the control, and A1 is the absorbance in the presence of the sample of primrose extracts or standards.

Determination of Total Phenolic Compounds Total soluble phenolics in the primrose extracts were determined with Folin-Ciocalteu reagent according to the method of Slinkard and Single ton [33] using gallic acid as a standard phenolic compound. Briefly, 1.0 ml of extract solution containing 1.0 g extracts in a volumetric flask was diluted with distilled water (46 ml). One millitre of Folin-Ciocalteau reagent was added and the content of the fl ask mixed thoroughly. Three minutes later, 3 ml of Na 2CO 3 (2%) was added, and the mixture was allowed to stand for 2 h with intermittent shaking. The absorbance was measured at 760 nm. The concentration of total phenolic compounds in the primrose extracts was determined as microgram of gallic acid equivalent using an equation obtained from the standard gallic acid graph:

Statistical analysis Experimental results were mentioned as mean±S.D. of three parallel measurements. P values < 0.05 were regarded as significant and P values <0.01 as very significant.

RESULTS AND DISCUSSION

DPPH • is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [34-36]. The reduction capability of DPPH • radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. Hence, DPPH is often used as a substrate to evaluate antioxidative activity of antioxidants [37]. Fig. 1 il lustrates a significant ( P<0.05) decrease in the concentration of DPPH radical due to the scavenging ability of the extracts of primrose and standards. We used BHA, BHT and α-tocopherol as standards. The scavenging effect of water and ethanol extracts of p rimrose and standards on the DPPH radical decreased in that order: Primrose water extract> primrose ethanol extract>BHA> a -tocopherol>BHT which were 99.5, 99.4, 71.4, 64.0 and 41.0%, respecti vely, at the concentration of 45 µg/ml. This result indicates tha t both primrose extracts have a noticeable effect on scavenging free radical. Free radical scavenging activity also increased with increasing concentration (Fig. 1)

The reductive capabilities of Primrose extracts compared with BHA, BHT and α-tocopherol. For the measurements of the reductive ability, we investigated the Fe 3+ - Fe 2+ transformation in the presence of Primrose extracts using the method of Oyaizu [28]. Like the antioxidant activity, the reducing power of primrose extracts increased with increasing amount of sample. All of the amounts of both Primrose extracts showed higher activities than control and these differences were statistically significant ( P<0 .01). Reducing power of water and ethanol extracts of Primrose and standard compounds exhibited the following or der: water extract of primrose> BHT> ethanol extract of primrose=BHA > α-tocopherol which were 43.0, 39.9, 39.4, 39.4 and 38,4%, respectively, a t the concentration of 45 µg/ml.

In the PMS–NADH–NBT system, superoxide anion derived from dissolved oxygen by PMS –NADH coupling reaction reduces NBT. The decrease of absorbance at 560 nm with antioxidants indicates the consumption of superoxide anion in the reaction mixture. Fig. 2 show the % inhibition of superoxide radical generation by 10 µg/ml of water and ethanol extracts of Primrose and comparison with same concentrations of BHA, BHT, and α- tocopherol. Both extracts of primrose had strong superoxide radical scavenging activity and exhibited higher superoxide radical scavenging activity than and BHT and α-tocopherol. The results were found statistically significant ( P<0.05). As seen in Fig. 2, the percentage inhibition of superoxide generation by 10 µg/ml concentration of BHA, BHT, α-tocopherol, water and ethanol extracts of Primrose was found as 75.6, 47.5, 75.6, 82.1 and 79.4 respectively. Superoxide radical scavenging activity of those samples showed the followin g order: water extract of primrose >ethanol extract of primrose > α-tocopherol=BHA>BHT

The chelating of ferrous ions by the extracts of primrose was estimated by the method of Dinis et al. [31]. Ferrozine can quantitatively form complexes with Fe 2+ . In the presence of chelating agents, the complex formation is disrupted, resulting in a decrease in the red colour of the complex. Measurement of color reduction therefore allows estimating the metal chelating activity of the coexisting chelator . As shown in Fig. 4, the formation of the Fe 2+ – ferrozine complex is not complete in the presence of water and ethanol extracts of primrose , indicating that both extracts of primrose chelate with the iron. The absorbance of Fe 2+ -ferrozine complex was linearly decreased dose 398 Pelagia Research Library Nazan Demir et al Euro. J. Exp. Bio., 2014, 4(2):395-401 ______dependently 10 µg/ml. The difference between both extracts of primrose and the control was statistically significant (P<0.05). The percentages of metal scavenging capacity of 10 µg/ml concentration of water and ethanol extracts of primrose, BHA, BHT and α-tocopherol, were found as 59.2, 81.5, 72.5, 61.5, and 66.96%, respectively. The metal scavenging effect of both extracts of primrose and standards decreased in the order of primrose water extract>BHA> BHT> α-tocopherol >primrose-ethanol extract.

Ferrous ion chelating capacity was significant, since it reduced the concentration of the catalyzing transition metal in lipid peroxidation [37]. It was reported that chelating agents, which form σ-bonds with a metal, are effective as secondary antioxidants because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion. The data obtained from Fig. 2 reveal that both extracts of primrose demonstrate a marked capacity for iron binding, suggesting that their action as peroxidation protector may be related to its iron binding capacity.

The ability of the both extracts of primrose to scavenge hydrogen peroxide was determined according to the method of Ruch et al. [32]. The scavenging ability of water and ethanol extracts of primrose on hydrogen peroxide is shown in Fig. 3. They were compared with BHA, BHT and α-tocopherol as standards. Primrose extracts were capable of scavenging hydrogen peroxide in a concentration-dependent manner. The water and ethanol extracts 10 µg/ml of primrose exhibited 77.5 and 78.3% scavenging activity on hydrogen peroxide, respectively. In the other hand, BHA, BHT, and α-tocopherol exhibited 37.5, 86, and 57% hydrogen peroxide scavenging activity at the same dose. These results showed that both primrose extracts had stronger hydrogen peroxide scavenging activity. These values are close to that of BHA, but lower than that of BHT and α-tocopherol. There was statically significant correlation between those values and control ( P<0.05). The hydrogen peroxide scavenging effect of 10 µg/ml concentration of the both extracts of primrose and standards decreased in the order of BHT >ethanol extract of primrose> water extract of primrose> α-tocopherol> BHA. Hydrogen peroxide itself is not very reactive, but it can sometimes be .− toxic to cell because it may give rise to hydroxyl radical in the cells [35]. Thus, removing H 2O2 as well as O 2 is very important for protection of food systems.

Phenols are very important plant constituents because of their radical scavenging ability due to their hydroxyl groups [38]. In water and ethanol extracts of primrose (1 mg), 89.6 and 122.8 µg gallic acid equivalent of phenols was detected. There was no relationship between total phenols and total antioxidant activity in primrose extracts. The phenolic compounds may contribute directly to the antioxidative action [37]. It is suggested that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans, when up to 1.0 g daily ingested from a diet primrose in fruits and vegetables [36, 39-45].

BHA 150 BHT a-tocopherol Caper-Water Extract

Caper-Ethanol Extract 100

DPPH% scavenging

0 0 10 20 30 40 50

Concentration ( µg/mL)

Figure 1. Free radical scavenging activity of water and ethanol extracts of primrose, BHA, BHT, and α-tocopherol. Primrose (primula vulgaris), BHA: buthylated hydroxyanisole, BHT: Buthylated hydroxytoluene)

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Superoxide scavenging effect Metal chelating effect 100

0 BHA BHT a-tocopherol Primrose-Water Primrose-Ethanol Extract Extract .

Figure 2. Superoxide anion radical scavenging activityand metal chelating effect of water and ethanol extracts of Primrose, BHA, BHT, and α-tocopherol

100

ScaveningofH2O2 (%) 25

0 BHA BHT a-Tocopherol Cowslip-Water Cowslip Ethanol Extract Extract .

Figure 3. Hydrogen peroxide scavenging activities of water and ethanol extracts of primrose, BHA, BHT, and α-tocopherol. Primrose (Primula vulgaris ), BHA: buthylated hydroxyanisole, BHT: buthylated hydroxytoluene)

CONCLUSION

Both extracts of primrose showed strong antioxidant activity, reducing power, DPPH radical and superoxide anion scavenging, hydrogen peroxide scavenging, and metal chelating activities when compared with different standards such as BHA, BHT, and α-tocopherol. The results of this study show that the extract of primrose can be used as an

400 Pelagia Research Library Nazan Demir et al Euro. J. Exp. Bio., 2014, 4(2):395-401 ______easily accessible source of natural antioxidants and as a possible food supplement or in the pharmaceutical and cosmetic industry.

Acknowledgement The authors would like to thank the University of Ataturk Project Research Fund 2007/175 for supporting this research.

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