Food Chemistry 155 (2014) 112–119

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In vitro antioxidant properties and anthocyanin compositions of elderberry extracts Hale Gamze Duymusß a,⇑, Fatih Göger a, K. Hüsnü Can Basßer a,b,c a

Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, 26470 Eskisehir, Turkey King Saud University, College of Science, Botany and Microbiology Department, P.O. Box 2455, Riyadh 11451, Saudi Arabia c _ Turkey Technology Transfer Office, Bahcesehir University, 34354 Besiktas-Istanbul, b

a r t i c l e

i n f o

Article history: Received 9 September 2013 Received in revised form 5 December 2013 Accepted 13 January 2014 Available online 23 January 2014 Keywords: Sambucus nigra Elderberry HPLC LC/MS–MS Radical scavenging activity Antioxidant activity

a b s t r a c t In this study, dried elderberry fruits growing wild in Turkey were macerated using different solvents and an infusion was prepared according to traditional methods. All extracts were investigated for their total phenolic content, total monomeric anthocyanins, qualitative–quantitative determination of cyanidin3-glucoside (by HPLC–UV analysis), anthocyanin compositions (by LC/MS–MS), free radical scavenging activity (DPPH and ABTS) and inhibition of b-carotene/linoleic acid co-oxidation. An extract with 70% ethanol was found to be richer in cyanidin-3-glucoside when compared to the other extracts. The infusion was found to be as rich as the 70% ethanol extract. Ethanol and acetone extracts (both 70%) were found to be more active in the free radical activity and b-carotene bleaching assays. Water extract showed good ABTS radical scavenging activity when compared with ascorbic acid. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Current research into free radicals has confirmed that foods rich in antioxidants play an essential role in the prevention of cardiovascular diseases, cancers and neurodegenerative diseases (Hamid, Aiyelaagbe, Usman, Ameen, & Lawal, 2010; Sharma, Jha, Dubey, & Pessarakli, 2012). Therefore, natural antioxidants and colourants present in foods have attracted interest because of their safety and potential nutritional and therapeutic effects. These natural compounds can also be alternatives to synthetic dyes (Espin, Soler-Rivas, Wichers, & Garcia-Viguera, 2000). Anthocyanins belong to the most common class of phenolic compounds which occur naturally in fruits and vegetables as glycosides, containing glucose, galactose, rhamnose, xylose or arabinose attached to an aglycon nucleus (Wang & Stoner, 2008). Nowadays, the potential health benefits associated with consumption of anthocyanins are the focus of much research. Intake of anthocyanin is increasing because extracts and juices with high anthocyanin contents from fruits and vegetables such as grape, various berries and red carbage are becoming more and more popular. The anthocyanin content of berries from Sambucus, Lonicera and Viburnum species have received great attention. Especially juices and extracts from the elderberry, known as Sambucus nigra L., have ⇑ Corresponding author. Tel.: +90 (222) 335 0580; fax: +90 (222) 335 3616. E-mail address: [email protected] (H.G. Duymusß). http://dx.doi.org/10.1016/j.foodchem.2014.01.028 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

been used in biological studies (Abuja, Murkovic, & Pfannhauser, 1998; Bitsch et al., 2004; Bratu, Guiu, Samarineanu, Gaidargiu, & Porta, 2003; Murkovic, Adam, & Pfannhauser, 2000; Netzel et al., 2005; Roschek, Fink, McMichael, Li, & Alberte, 2009; Wu, Gu, Prior, & McKay, 2004). Elderberries general grow wild in several countries in Europe and are also cultivated on a small scale in some northern European countries (Akbulut et al., 2009). Elderberry has also been used to colour jams, jellies and wines in Europe (Inami, Tamura, Kikuzaki, & Nakatani, 1996). The dried ripe or fresh berries are recommended for the treatment of constipation, as a diuretic and diaphoretic in upper respiratory tract infections, and to alleviate pain. For the treatment of these complaints, juice or tea are consumed. The infusion, prepared from 10 g dried berries standing in cold water for several minutes, is then slowly heated up and boiled briefly. Before filtering, a drawing-time of 5–10 min is recommended (Vlachojannis, Cameron, & Chrubasik, 2009). In Turkey, S. nigra occurs sporadically in western and eastern parts, particularly in the northern coastal strip. Elderberry bark, root, stem and fruits have been used particularly by the rural population as medicine and food (Baytop, 1984). But the consumption of elderberry is not very common and only a few studies have been reported (Akbulut, Ercisßli, & Tosun, 2009; Yenen & Özgen, 1997). However, none of these studies focus on elderberry anthocyanin composition and identification by liquid chromatography. The aim of this study was to determine the anthocyanin composition and to evaluate the antioxidant capacity of elderberry

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extracts. Five extracts were prepared with solvents of different polarities and an infusion of elderberry fruits was investigated for its antioxidant activity in in vitro test systems. Furthermore, the total phenol and total monomeric anthocyanin were also analysed using spectrophotometric techniques. Anthocyanin contents of the extracts were also characterized by HPLC–UV and LC–MS/MS analyses.

2.1. Plant material and reagents Air-dried mature fruits of elderberry were obtained from ‘Evçay Company’, Yalova, Turkey in 2009. Chromatographic standards were purchased from Sigma Chemical Company. Ultra-pure water was used throughout and was prepared using a Millipore Milli-RO 12 plus system (Millipore Corp., MA, USA). All remaining reagents were of the highest purity available and obtained from the Sigma Chemical Company (St. Louis, MO, USA). 2.2. Preparation of the extracts Ground elderberries (5 g) were macerated with 100 ml of water, 70% ethanol, 70% acetone and methanol at room temperature by using a shaker 3 times (48, 48, 24 h) during 5 days in the dark. HCl (0.1%) in methanol extracts were prepared by using an ultrasonic bath at room temperature for 30 min, 3 times. The infusion was prepared according to traditional methods (Vlachojannis et al., 2009) and freeze dried. The extracts were evaporated to dryness at 35 °C by a rotary evaporater and stored at 18 °C. Prior to analysis, an aliquot of each extract was dissolved and filtered through a 0.45 lm membrane (Whatman, UK) and used in all the test systems. 2.3. Total phenols Total phenols were estimated as gallic acid equivalents (GAE), expressed as mg gallic acid/100 g extract (Singleton, Orthofer, & Lamuela-Raventos, 1999). To ca. 6.0 ml H2O, 100 ll of sample was transferred into a 10.0 ml volumetric flask, to which 500 ll undiluted Folin–Ciocalteu reagent was added subsequently. After 1 min, 1.5 ml of a 20% (w/v) Na2CO3 solution was added and the volume was made up to 10.0 ml with H2O. After 2 h incubation at 25 °C, the absorbance was measured at 760 nm and compared to a gallic acid calibration curve. The data are presented as the average of triplicate analyses. 2.4. Total monomeric anthocyanins Total monomeric anthocyanins were measured according to the ‘pH differential method’ in triplicate (Giusti & Wrolstad, 2000). All extracts were dissolved in suitable solvents and used as stock solutions. Appropriate dilution factors for each sample were determined by diluting with 0.025 M potassium chloride buffer (pH 1.0) until the absorbance of the sample at 510 nm was within the linear range of the spectrophotometer. For each sample, two dilutions were prepared, both with 0.025 M potassium chloride buffer (pH 1.0) and with 0.4 M sodium acetate buffer (pH 4.5) diluting each by the previously determined dilution factor. These dilutions were equilibrated for 15 min. The absorbance of each dilution was measured at 510 nm and at 700 nm (to correct for haze), against a blank cell filled with distilled water. The absorbance of the diluted sample (A) was calculated as follows: 1:0

 ðA510  A700 ÞpH

Monomeric anthocyanin pigment ðmg=lÞ ¼ ðA  MW  DF  1000Þ=ðe  1Þ:

ð2Þ

Anthocyanin content was calculated as cyanidin-3-glucoside, where MW = 449.2 and e = 26,900. 2.5. Qualitative–quantitative chromatographic analysis

2. Materials and methods

A ¼ ðA510  A700 ÞpH

113

4:5 :

ð1Þ

The monomeric anthocyanin concentration in the original sample was calculated using the following formula:

2.5.1. HPLC–UV analysis The liquid chromatographic apparatus (Shimadzu LC 10Avp, Ant Ltd. Sß ti., Istanbul, Turkey) consisted of an in-line degasser, pump and controller coupled to a Shimadzu UV–Vis Spectrophotometer equipped with an automatic injector interfaced to Class VP chromatography manager software (Shimadzu, Japan). Separations were performed on a 250  4.6 mm i.d., 5 lm particle size, octadecyl silica gel analytical column (Supelco, PA, USA) operating at 40 °C at a flow rate of 0.4 ml/min. Detection was carried out at 520 nm. Elution was carried out using a binary gradient of formic acid/water (8.5/91.5, v/v) (solvent A) and tetrahydrofuran/formic acid/acetonitrile/methanol/water (5/8.5/22.5/22.5/41.5, v/v/v/v/v) (solvent B). The composition of B was increased from 15% to 30% in 10 min, increased to 40% in 10 min and increased to 100% in 5 min, then the composition was decreased to 15% in 5 min. Cyanidin-3-glucoside in all extracts was identified by comparison of its retention time to that of authentic standards under identical analysis conditions and UV spectra. A 10 min equilibrium time was allowed between injections. All standard and sample solutions were injected in triplicate. 2.5.2. HPLC–UV–MS–MS analysis The identification of some of the anthocyanins present in the elderberry extracts was carried out by means of their molecular weight and their fragments. For this, HPLC with UV detection and tandem mass spectrometry was used (HPLC–UV–MS–MS). The analytical conditions used were those described by Bermudez-Soto and Thomas-Barberan (2004), with some modifications. Samples of each elderberry extract (5 ll) were analysed using an HPLC system equipped with a model LC20AD Shimadzu pump and a model SPD20A Shimadzu UV Detector. The samples were injected by means of a model SIL20A Shimadzu Autosampler. Separations were carried out using an Intersil ODS-3 column (4.6  250 mm, 5 lm particle size). The CTO20A Shimadzu Column Oven was kept at 40 °C. The mobile phase was water with 1% formic acid (v/v) (solvent A) and HPLC grade methanol (solvent B) at a flow rate of 1 ml min1. The linear gradient started with 3% solvent B reaching 5% at 5 min, 8% solvent B at 10 min, 13% solvent B at 15 min, 15% solvent B at 19 min, 40% solvent B at 47 min, 65% solvent B at 64 min, 80% solvent B at 65 min and 98% solvent B, followed by 66 min at isocratic elution until 69 min. At 70 min the gradient reached the initial conditions again. HPLC chromatograms were recorded at 380 and 520 nm. The Shimadzu HPLC–UV system was connected directly to a 3200 Q TRAP mass spectrometer (MS) (AB Sciex, Toronto, Canada). The software used for data acquisition and analysis was Analyst 1.5. The chromatographic conditions were used as described above. For enhanced mass scan (EMS), the MS was operated in positive polarity at a scan rate of 1000 Da/s within the mass range of 200–800 amu. Mass scan (MS) and daughter (MS–MS) spectra were measured from m/z 100 up to m/z 800. Collision-induced fragmentation experiments were performed in the ion trap using nitrogen as the collision gas, with the collision energy set at 30–100. The parameters were as follows: Collision Energy Spread (CES) = 15, Declostiring Potential (DP) = 45, Enterance Potential (EP) = 10, Curtain gas (CUR) = 10 and Temperature (TEM) = 600. For the IDA experiment, the criteria were arranged for ions greater than 200.000 m/z and smaller than

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Table 1 Extract yield, total phenols, monomeric anthocyanins, and HPLC qualitative and quantitative data for elderberry. Yielda

Sample

Spectrophotometric results b

(A) (B) (C) (D) (E) (F)

260 432 404 328 602 161

HPLC results

Total phenols

Total anthocyanins

8974 ± 37 7594 ± 3 4917 ± 63 8206 ± 167 6399 ± 80 6715 ± 21

878.5 ± 15 1066.6 ± 55 408.6 ± 85 651.1 ± 26 600 ± 16 734.2 ± 33

c

Cyanidin-3-glucosided

Total anthocyaninse

100.5 ± 0.72 254.3 ± 5.03 42.4 ± 0.02 175.4 ± 0.63 68.1 ± 0.07 202.9 ± 1.87

1247 ± 53 1326 ± 48 285 ± 20 890 ± 50 650 ± 15 951 ± 40

(A) Water extract; (B) 70% ethanol extract; (C) methanol extract; (D) 70% acetone extract; (E) acidified methanol; (F) infusion. a Extract yields expressed as milligrams of extract per gram (dry weight) of fruits. b Total phenols expressed as gallic acid equivs. milligrams of gallic acid per 100 gram (dry weight) of extract. c Total monomeric anthocyanins expressed as cyanidin-3-glucoside equivs. milligrams of cyanidin-3-glucoside per 100 gram (dry weight) of extract. d Values (mg/100 g extract) are expressed as means ± standard error. e Total anthocyanins expressed as cyanidin-3-glucoside equivs. milligrams of cyanidin-3-glucoside per 100 g of extract by HPLC method.

uV 700000 600000

B

500000 400000

A

300000 200000 100000 0 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

32.5

min

Fig. 1. (A) The HPLC–UV chromatogram of elderberry extract with absorbance at 520 nm; (B) the HPLC–UV chromatogram of cyanidin-3-glucoside standard.

800.000 m/z, and excluded former target ions after 3.0 occurrence(s) for 3.000 s. 2.6. The antioxidant activity 2.6.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity Serial dilutions were carried out with the stock solutions (10 mg/ml1) of the extracts to obtain the concentrations of 10, 5, 25  101, 125  102, 625  103, 3125  104 mg/ml1. Diluted solutions were mixed with DPPH (equal amounts) and allowed to stand for 30 min for any reaction to occur. The UV absorbance was recorded at 517 nm. The experiment was performed in triplicate and the average absorption was noted for each concentration. The same procedure was followed for the positive control, ascorbic acid. The percentage inhibition was calculated using Eq(3). The IC50 value, which is the concentration of the test material that inhibits 50% of the free radical concentration, was calculated as mg/ml1 (Kumarasamy et al., 2007):

Percentage inhibition ¼



Abscontrol  Abssample Abscontrol



 100:

ð3Þ

2.6.2. TEAC assay (Trolox Equivalent Antioxidant Capacity) This assay assesses the capacity of a compound to scavenge the stable ABTS radical in comparison to the antioxidant activity of Trolox, a water-soluble form of vitamin E that is used as a standard. The blue–green ABTS was produced through the reaction of 7 mM ABTS with 2.5 mM sodium persulfate (Na2S2O8) (final

concentrations) in the dark at room temperature for 12–16 h before use. The concentrated ABTS solution was diluted with ethanol to a final absorbance of 0.7–0.8 at 734 nm. A 10-ll portion of sample (concentrations of 0.6, 0.3 and 0.1 mg/ml1) was added to 990 ll of ABTS solution, and the reduction in absorbance was measured 1 min after addition of Trolox (final concentration 1–20 lM) and up to 40 min after addition of the each extract. The stock solution of Trolox (2.5 mM) was prepared in ethanol. Absorbance was measured on a UV/spectrophotometer (Papandreou et al., 2006). 2.6.3. Determination of inhibition of b-carotene/linoleic acid cooxidation Antioxidant activity of the elderberry extracts was determined according to b-carotene bleaching methods (Oomah & Mazza, 1996; Velioglu, Mazza, Gao, & Oomah, 1998). Briefly, 1 ml of b-carotene (0.2 mg/ml1 dissolved in chloroform; Sigma Chemical Co., St. Louis, MO) was added to a flask containing linoleic acid (40 mg) and Tween 80 (400 mg). Chloroform was evaporated under a stream of nitrogen. Distilled water (50 ml) was added and shaken vigorously. A control was prepared without sample or standards with same procedure. Blanks of control and sample were also prepared without b-carotene. Their absorbance were measured on a spectrophotometer at 470 nm. The samples were then subjected to thermal autoxidation by keeping them in a constant temperature water bath at 50 °C for 2 h. The rate of bleaching of b-carotene was monitored by taking the absorbance at 15 min intervals. Antioxidative activity was calculated according to Eq.(4):    AA% ¼ 1  Ab0sample  Abs120sample =ðAb0control  Abs120control Þ  100: ð4Þ

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H.G. Duymusß et al. / Food Chemistry 155 (2014) 112–119 Table 2 HPLC–MS–MS data for elderberry extracts. Peak

HPLC Rt (min)

Molecular ion (MS) m/z+

Fragment (MS–MS) m/z+

Structure

A

B

C

D

E

F

X

26.70

Y

33.08

Z

52.64

611 743 449 581 633

449, 581, 287 449, 487,

Cyanidin-3,5-diglucoside Cyanidin-3-sambubioside-5-glucoside Cyanidin-3-glucoside Cyanidin-3-sambubioside Quercetin-3-rutinoside Na+ adduct

+ + + + +

+ + + + +

+ + + + +

+ + + + +

+ + +  

+ + + + +

287 449, 287 287 331

(A), water extract; (B), 70% ethanol extract; (C), methanol extract; (D), 70% acetone extract; (E), Acid. methanol extract; (F), infusion.

Fig. 2. LC chromatogram at 520 nm and EMS–EPI spectra of the X peak at tR 26.7 min.

2.7. Statistical analysis

3. Results and discussion

Data are presented as mean values ± standard deviation. All the statistical analyses were carried out using SPSS 10.0.1. (SPSS Inc., Chicago, IL). Analysis of variance (ANOVA) was performed by ANOVA procedures. Significant differences between means were determined by Tukey’s pairwise comparison test at a level of p < 0.05. IC50 values were estimated using a non-linear regression algorithm.

3.1. Fraction yields, total phenols, total monomeric anthocyanins and compositional analysis Ground elderberries were macerated with water, 70% ethanol, 70% acetone, ethanol and methanol at room temperature. Acidified methanol (0.1% HCl Inc.) extracts were prepared using an ultrasonic bath at room temperature for 30 min in triplicate. The

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Fig. 3. LC chromatogram at 520 nm and EMS-EPI spectra of the Y peak at tR 33.08 min.

infusion was prepared according to traditional methods. The results of fractionation, total phenols, total monomeric anthocyanins and the amount of cyanidin-3-glucoside of extracts are presented in Table 1. According to the data presented in Table 1, water, 70% acetone, 70% ethanol, infusion, acidified methanol and methanol extracts contained the highest amount of total phenol content, respectively. The highest yields were obtained also from the acidified methanol, 70% ethanol and methanol extracts. The results of the qualitative–quantitative analyses of the extracts, carried out using an HPLC apparatus coupled with a UV detector, are presented in Table 1, with a selected chromatogram shown in Fig. 1. Cyanidin-3-glucoside was identified and quantified at 520 nm. Cyanidin-3-glucoside was identified by comparison of the retention time and UV spectra of authentic standard, while quantitative data were calculated from their calibration curves. The 70% ethanol, infusion and 70% acetone extracts were found to be the richest in cyanidin-3-glucoside, as measured by HPLC. In addition, total anthocyanins of each extract obtained from chromatogram at 520 nm were quantified as cyanidin-3-glucoside equivalents (Table 1). When the spectroscopic total anthocyanin analysis data was compared with that from the HPLC method, the total anthocyanins measured by HPLC were higher than those observed in the spectroscopic method, except for the methanol extract. Correlation was determined on the anthocyanin values

obtained by the pH differential method and HPLC. The correlation between these two methods was significant (r2 = 0.9302). All extracts were determined for their total phenolic and anthocyanin contents by spectrophotometric methods. Cyanidin3-glucoside was also identified and calculated in the extracts by HPLC. Other anthocyanins in the extracts were identified by LC– MS–MS. In order to achieve ionisation of the anthocyanins, HPLC conditions reported by Bermudez-Soto and Thomas-Barberan (2004) were employed with some modifications. Four different cyanidin glycosides, such as cyanidin-3,5-diglucoside, cyanidin-3sambubioside-5-glucoside, cyanidin-3-sambubioside and cyanidin-3-glucoside were detected in the 520 nm chromatogram, while quercetin-3-rutinoside Na+ adduct was detected in the 380 nm chromatogram. These compounds were identified by their UV spectra, their molecular weights and their fragmentation patterns. At 520 nm, MS fragmentation ions at m/z 287 were attributed to cyanidin derivatives (Table 2). The X peak (Fig. 2) was confirmed as cyanidin-3,5-diglucoside (m/z+ 611, MS2 fragments at 449, 287) and cyanidin-3-sambubioside-5-glucoside (m/z+ 743, MS2 fragments at 581, 449, 287). The Y peak (Fig. 3) was characterized as cyanidin-3-sambubioside (m/z+ 581, MS2 fragments 449, 287) and cyanidin-3-glucoside (m/z+ 449, MS2 fragment 287). The Z peak (Fig. 4) obtained from the 380 nm chromatogram was identified as quercetin-3-rutinoside Na+ adduct (m/z+ 633, MS2

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117

Fig. 4. LC chromatogram at 380 nm and EMS–EPI spectra of the Z peak at tR 52.64 min.

fragments at 487, 331). The results are given in Table 2. Cyanidin-3,5-diglucoside, cyanidin-3-sambubioside-5-glucoside and cyanidin-3-glucoside were identified in all extracts. Cyanidin-3-sambubioside and quercetin-3-rutinoside Na+ adduct were detected in all the extracts except for the acidified methanol extract. These compounds were evaluated according to LC/MS–MS results of Bermudez-Soto and Thomas-Barberan (2004).

acetone > water > 70% ethanol > infusion > acidified methanol > methanol. Also cyanidin-3-glucoside at 1 mg/ml1 concentration showed 80% inhibition, whereas ascorbic acid at the same concentration exhibited 94% inhibition on DPPH radical. These phenolics have antioxidant activity and they are considered to be responsible for the antioxidant activity. 3.3. Inhibition of b-carotene/linoleic acid co-oxidation

3.2. DPPH radical scavenging activity The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical is a stable radical, with an absorption maximum at 517 nm. When reduced to the hydrazine derivative by an antioxidant via electron or hydrogen atom transfer reactions, this absorption maximum decreases (Lu & Foo, 2001). IC50 values, defined as the concentration required to scavenge 50% of the available free radicals, were estimated by nonlinear regression for all the extracts. All the extracts showed free radical scavenging activity in this test. According to the results, the 70% acetone extract was the most active free radical scavenger of them all (IC50 value 117 lg/ml1). The water extract was the second active free radical scavenger with an IC50 value of 123 lg/ml1 However, none of the extracts were as active as the positive control, ascorbic acid (IC50 value of 8 lg/ml1). The order of DPPH radical scavenging ability for the fractions was as follows: 70%

Food lipids and cell membranes contain unsaturated fatty acids, linoleic and arachidonic acids, which can be easily oxidised with oxidative agents. Therefore, unsaturated fatty acid–base medium in antioxidant activity tests are important to determine the activities of test samples. The b-carotene–linoleic acid bleaching assay is such a model, widely used to investigate the oxidation of unsaturated fatty acids, especially in the cell wall and food products (Kosar, Goger, & Baser, 2008). The b-carotene/linoleic acid oxidation method evaluates the inhibitory activity of free radicals generated during the peroxidation of linoleic acid. The method is based on spectrophotometric discolouration measurements or oxidation of b-carotene-induced oxidative degradation products of linoleic acid. In the b-carotene/ linoleic acid co-oxidation assay, the degree of lipid peroxidation is measured. The inhibition percentages of all extracts of

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elderberry are calculated. According to results, some extracts could inhibit the oxidation of linoleic acid. The 70% ethanol extract had the best activity (58% inhibition). The hierarchy of the extracts were 70% ethanol > infusion > 70% acetone > methanol > water > acidified methanol. None of the extracts of elderberry were found to be as active as the positive control BHT. Cyanidin-3-glucoside at 1 mg/ml1 concentration had 43% inhibition on the oxidation of linoleic acid. According to these results, cyanidin-3glucoside is thought to be responsible for this activity. 3.4. TEAC assay (Trolox Equivalent Antioxidant Capacity) In this assay, the capacity of elderberry extracts to scavenge the ABTS radical (ABTS+) was assessed. This assay is based on the scavenging of the relatively stable blue/green ABTS radical (ABTS+), converting it into a colourless product. The degree of decolourization reflects the amount of ABTS+ that has been scavenged and can be determined spectrophotometrically. The TEAC value is assigned by comparing the scavenging capacity of an antioxidant to that of Trolox (Badarinath et al., 2010). The TEAC values at 30 min were calculated, the Trolox calibration curve was linear (r2 0.996) and TEAC values of all extracts were calculated according to this curve. The TEAC values of the extracts ranged between 0.89 and 1.97 mmol Trolox equivalents/l. According to our results, the 70% acetone extract (1.96 mM) was as effective as ascorbic acid (1.97 mM) on ABTS+ radical scavenging. Cyanidin-3-glucoside at 1 mg/ml1 conc. had a lower activity than ascorbic acid at the same concentration, whereas it showed better activity than the infusion (1.23 mM), methanol (1.0 mM), acidified methanol (0.89 mM) and 70% ethanol (1.52 mM) extracts. The water extract (1.85 mM) was as active as cyanidin-3-glucoside (1.87 mM). Several berries are used as natural colourants in different industries and elderberries draw attention because of their anthocyaninrich content and high antioxidant activity. In elderberry fruits, cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3,5-diglucoside and cyanidin-3-sambubioside-5-glucoside are the main anthocyanins (Inami et al., 1996; Nakajima, Tanaka, Seo, Yamazaki, & Saito, 2004; Wu et al., 2004). The antioxidant and antiradical activities of the juice and extracts of elderberry have been reported. Espin et al. (2000) determined the antiradical activity on DPPH radical of commercial elderberry juice and other fruit extracts by a spectrophotometric method. According to their results, 3-monoglycoside (97%) and cyanidin 3,5-diglycoside (3%) identified in the juice had a lower DPPH radical scavenging activity when compared with vitamin E, BHT and BHA. Lugasi and Hovari (2003) reported that elderberry juice had a total polyphenol concentration of 5680 mg/l and this was significantly higher than that of high quality red wines. Hydrogen-donating ability and the reducing power found to be the highest in the juice of elderberry exhibited both antioxidant and DPPH scavenging properties strongly correlated with the total polyphenol content. In another study, the potent antiradical activity of some berry extracts or commercial juice were determined and their anthocyanin composition was analysed by LC/PDA/ESI–MS. Cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3-sambubioside-5-glucoside and cyanidin-3,5-diglucoside were identified in the commercial juice of elderberry. All extracts were treated by DPPH and compared with Trolox. According to the results, all the extracts showed lower DPPH radical scavenging activity than Trolox (Nakajima et al., 2004). Elderberry juice concentrate was found to have a rich phenolic content and high free-radical scavenging activity against both ABTS+ and DPPH radicals. Cyanidin-3-sambubioside was the most abundant anthocyanin in the elderberry juice concentrate (Bermudez-Soto & Thomas-Barberan, 2004). The alcoholic extracts (80% ethanol in water) from the leaves, berries and flowers of S. nigra act as antioxidants, neutralizing the activities of free

radicals and inhibiting the co-oxidation reactions of linoleic acid and b-carotene. Cyanidin-3-sambubioside, cyanidin-3-glucoside, rutin, isoquercitrin and astragalin were also identified in elderberry extracts (Dawidowicz, Wianowska, & Baraniak, 2006). According to our results, the highest total phenolic content was calculated in the water extract, which had a similar TEAC activity when compared to cyanidin-3-glucoside. Furthermore, the water extract was as effective as the 70% acetone extract on DPPH radical scavenging activity. The infusion of berries was found to possess good antiradical activity correlated with its cyanidin-3-glucoside, total phenol and anthocyanin contents. The methanol extract was found to have the lowest total phenolic, total anthocyanin and cyanidin-3-glucoside levels when compared with the other extracts. Because of this, the methanol extract showed the lowest antiradical and antioxidant activity in all the tested assays. 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In vitro antioxidant properties and anthocyanin compositions of elderberry extracts.

In this study, dried elderberry fruits growing wild in Turkey were macerated using different solvents and an infusion was prepared according to tradit...
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