ANTIOXIDANT, ANTIPROLIFERATIVE AND ANTIMICROBIAL …Raspberries (Rubus ideaus, cv. “Meeker”)...

APTEFF, 45, 1-283 (2014) UDC: 634.711+664.037.5:615.79 DOI: 10.2298/APT1445099V BIBLID: 1450-7188 (2014) 45, 99-116

Original scientific paper

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ANTIOXIDANT, ANTIPROLIFERATIVE AND ANTIMICROBIAL ACTIVITY OF FREEZE-DRIED RASPBERRY

Jelena J. Vulić1, Aleksandra S. Velićanski1, Dragana D. Četojević-Simin2, Vesna T. Tumbas Šaponjac1, Sonja M. Djilas1, Dragoljub D. Cvetković1, Siniša L. Markov1

1 University of Novi Sad, Faculty of Technology Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

2 Oncology Institute of Vojvodina, Dr Goldmana 4, 21204 Sremska Kamenica, Serbia

The main chemical composition, i.e. the total content of bioactive compounds (phe-nolics 2209.86 ± 70.32 mg GAE/100g FDR, flavonoids 831.87 ± 12.61 mg R/100g FDR and anthocyanins 144.55 ± 0.39 mg CGE/100g FDR), in freeze-dried raspberry (FDR) was evaluated spectrophotometrically. Vitamin C content was determined by HPLC ana-lysis (88.81 ± 4.38 mg vit C/100g FDR). Antioxidant activities of FDR extract were eva-luated spectrophotometrically on stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free ra-dicals and by electron spin resonance spectroscopy (ESR) method on hydroxyl radicals (•OH). EC50 values were evaluated. EC50

DPPH• was 0.127 ± 0.013 mg/ml, while EC50

•OH

was 1.366 ± 0.026 mg/ml. Antiproliferative activity of the FDR extract was evaluated in vitro in three human cell lines by colorimetric sulphorhodamine B (SRB) assay. The most pronounced effects were obtained in the breast adenocarcinoma cell line (MCF7). EC50 value was 395.07 ± 96.38 µg/ml. Antimicrobial activity was determined by disk diffusion method. The FDR extract produced a clear inhibition zone (without visible colonies) only toward Staphylococcus aureus. The minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations of FDR extract were evaluated. The values MIC were in the range of 4.7 - 100 mg/ml, and of MBC in the range of 6.3 - > 100 mg/ml.

KEY WORDS: raspberry, freeze-drying, DPPH• and ESR antioxidant methods, antipro-

liferative and antimicrobial activity

INTRODUCTION

Fruits are well-known sources of health-promoting compounds. These components comprise vitamins, minerals, and a range of different polyphenolic antioxidants, such as flavonoids and tannins (1). Numerous studies demonstrated that various phytochemical constituents of berry fruits exhibit a wide range of biological effects, including antioxi-dant, anticarcinogenic, vasodilatory and antimicrobial properties (2, 3). Due to their anti-proliferative and anticancer activity berry fruits are considered as a top class of healthy food (4, 5). Ellagic acid has been detected in berries of the genera Rubus and Fragaria, * Corresponding author: Jelena J. Vulić, University of Novi Sad, Faculty of Technology Novi Sad, Bulevar cara

Lazara 1, 21000 Novi Sad, Serbia, e-mail: [email protected]



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and can be present as ellagitannins, free ellagic acid and ellagic acid glycosides (6, 7, 8). The interest in ellagic acid has increased during the past decade due to its possible anti-mutagenic, anticarcinogenic and antioxidative effects (8, 9). Raspberries (Rubus ideaus) are high in phenolic phytochemicals, particularly flavo-noids such as anthocyanin pigments, which give raspberries their deep red color (10). Raspberries also contain a wide variety of quercetin and kaempferol-based flavonol co-njugates with the major components being quercetin-3-glucuronide and quercetin-3-glu-coside (11, 12, 13, 14). Eleven anthocyanins were reported in red raspberries, but cyani-din-3-sophoroside and cyanidin-3-(2-glucosylrutinoside) were the major ones (2). How-ever, significant differences in the amounts of individual anthocyanins were reported for various cultivars within the same species (1). Since the season during which fresh raspberries are available is short, only a small proportion of raspberries are consumed fresh. The most of the harvest is preserved by freezing or by processing to juices, jams, and jellies (8). Dehydration is one of the most effective methods of preserving delicate fruits, with high moisture content, and short har-vest time (15). It is widely known that freeze-drying is much more effective in preserving valuable food compounds than traditional methods of drying (16). In the case of freeze-drying, food is first frozen and then water is removed by sublimation. Due to the absence of liquid water and the low temperature required for the process, most of deterioration and microbiological reactions are stopped, which gives a final product of excellent qua-lity (17). The aim of this this study was to investigate the main chemical composition, total phenolics, flavonoids and anthocyanins, vitamin C content, as well as the antioxidant, antiproliferative and antimicrobial activities of freeze-dried raspberry (FDR).

EXPERIMENTAL

Chemicals

DPPH•, Folin-Ciocalteu reagent, 5,5-dimethyl-1-pyroline-N-oxide (DMPO) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). These chemicals were of analytical reagent grade. The other chemicals and solvents used were of the highest analytical grade and obtained from “Zorka” Šabac (Serbia).

Plant material

Raspberries (Rubus ideaus, cv. “Meeker”) were purchased from Alfa RS (Lipolist, Serbia) in July 2011. Fresh undamaged berries were frozen and stored at -20ºC until use. For the freeze-drying process, the raspberry samples were frozen at -40ºC for 2 h in a Martin Crist Alpha 2-4 (Osterode, Germany) lyophilizator. The main drying process was performed at p = 0.01 mbar and temperatures from -40ºC to 20ºC for 59.5 h. The final drying lasted 5.5 h at p = 0.005 mbar and temperatures from 20ºC to 30ºC. The dried material had a moisture content of 3.88 ± 0.17 %.



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Sample extraction Samples of FDR (20 g) were extracted at room temperature using a high performance homogenizer (Heidolph, Silent Crusher M, Kelheim, Germany). The extraction was per-formed on a laboratory shaker (200 rpm, Heidolph Unimax 1010, Kelheim, Germany), two times with different amounts of 80% methanol aqueous solution containing 0.05% acetic acid: 160 ml in 60 min and 80 ml for 30 min at room temperature. The obtained extracts were combined and evaporated to dryness under reduced pressure. The yield of FDR extract was 10.18 ± 0.48 g.

Total phenolics content

The amount of total phenolics (TPh) in FDR was determined spectrophotometrically (UV-1800 Shimadzu spectrophotometer, Kyoto, Japan) according to the Folin-Ciocalteu method (18). The results were expressed as gallic acid equivalents (GAE) in mg per gram of dry extract (mg GAE/g DE) and per 100 g of FDR (mg GAE/100 g FDR).

Total flavonoid content The amount of total flavonoids (TF) in FDR was determined spectrophotometrically according to the Zhishen et al. (19). The results were expressed as mg of rutin equivalents (R) per g of dry extract (mg R/g DE) and per 100 g of FDR (mg R/100 g FDR).

Total anthocyanins content Total monomeric plus polymerized anthocyanins (TA) in the extracts were estimated spectrophotometrically using the pH single method (20). The extracts were diluted with two buffer solutions of pH 1 and 4.5. The absorbance of each dilution was measured at 510 nm (A510) and 700 nm (A700) against a distilled water control. The TA concentration was obtained from the equation:

Ctot(mg/l) = (Atot ×MW×DF×1000)/ε×L,

where Atot is calculated as Atot =A510–A700, ε is the molar absorbance coefficient of cyanidin-3-glucoside (26900 l mol-1cm-1); MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol); DF is the dilution factor, and L is the cell path length (1 cm). The results were expressed as mg of cyanidin-3-glucoside equivalents (CGE) per g of FDR dried extract (mg CGE/g DE) and per 100 g of FDR (mg CGE/100g FDR).

Vitamin C content

The HPLC analysis of the vitamin C content in FDR was performed according to Tumbas et al. (21) using a Shimadzu Prominence liquid chromatograph (Shimadzu, To-kyo, Japan) connected to a UV/Vis detector. The results were expressed as mg of vitamin



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C per g of FDR dried extract (mg vitC/100 g DE) and mg vitamin C per 100 g of FDR (mg vitC/100 g FDR).

Free radical assays

DPPH free radical scavenging assay. The DPPH radical scavenging activity (SADPPH•) of FDR was determined spectrophotometrically using the modified DPPH method of Yen and Chen (22). The hydrogen atom or electron donation abilities of the extract was measured based on the bleaching of a purple-colored methanol solution of the stable DPPH radical. Briefly, 1 ml of the solution (water solution of extract) was added to 2 ml of 90 μM DPPH solution (18 mg in 50 ml 95% methanol prepared daily). The mixture was vortexed thorough-ly for 1 min and left at room temperature for 30 min, then the absorbance was measured against a control at 517 nm (UV-1800 Shimadzu spectrophotometer, Kyoto, Japan). The ran-ge of the investigated concentrations was 0.008 - 0.5 mg/ml. The capability to scavenge the DPPH radicals was calculated using the following equation:

SADPPH• (%) = (AControl – ASample)/AControl × 100 where AControl is the absorbance of the blank and ASample is the absorbance in the presence of extract. The EC50, defined as the concentration of extract required for a 50% scavenging of DPPH radicals under experimental condition employed, was used to measure the free radical scavenging activity (23). Trolox was used as a control compound. Hydroxyl radical scavenging activity. Hydroxyl radicals were generated in the Fen-ton reaction system obtained by mixing 0.2 ml of 112 mM DMPO, 0.2 ml of H20, 0.2 ml of 2 mM H2O2, and 0.2 ml of 0.3 mM Fe2+ (control) (24). The influence of the extract on the formation and stabilization of hydroxyl radicals was investigated by adding the water solutions of FDR extract (0.25 - 4.0 mg/ml) into the Fenton reaction system. The ESR spectra were recorded with the following spectrometer settings: field modulation 100 kHz, modulation amplitude 0.226 G, receiver gain 5x105, time constant 80.72 ms, con-version time 327.68 ms, center field 3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23ºC. The SA•

OH value of the extract was defined as: SA•

OH (%) = 100 × (h0 – hx) / h0, where h0 and hx are the hight of the second peak in the ESR spectrum of DMPO-OH spin adduct of the control and the probe, respectively. Trolox was used as a control compound.

Antiproliferative activity Cell lines. Cell growth activity was evaluated in vitro in the human cell lines: HeLa (cervix epithelioid carcinoma, ECACC No. 93021013), MCF7 (breast adenocarcinoma, ECACC No. 86012803), HT-29 (colon adenocarcinoma, ECACC No 91072201) and MRC-5 (human fetal lung, ECACC 84101801). The cell lines were grown in a DMEM medium with 45 mg/ml glucose, supplemented with 10% heat-inactivated FCS, 100 IU/ml of penicillin and 100 µg/ml of streptomycin. The cells were cultured in 25 ml flasks (Corning, New York, USA) at 37ºC in the atmosphere of 5% CO2 and high hu-



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midity, and sub-cultured twice a week. A single cell suspension was obtained using 0.1% trypsin with 0.04% ethylenediamine tetraacetic acid (EDTA). Cell growth activity. The cell lines were harvested and plated into 96-well microtitre plates (Sarstedt, Newton, USA) at a seeding density of 3-5 x 103 cells/well, in a volume of 199 or 180 µl, and preincubated in the complete medium supplemented with 5% fetal calf serum (FCS), at 37ºC for 24 h. Serial twofold dilutions of the extracts and standards (1 µl) in dimethyl sulfoxide (DMSO) were added to 199 µl of the medium to achieve the required final concentrations. Serial twofold (Vitamin C, Aspirin®) or tenfold dilutions (Doxorubicin® and Gemzar®) (20 µl) were added to 180 µl of medium to achieve the required final concentrations. Equal volumes of the solvents were added in the control wells. The DMSO concentration in the cell culture was ≤ 5 µl/ml. After the addition of dilutions, the microplates were incubated at 37ºC for 48 h. The cell growth was evaluated by the colorimetric sulphorhodamine B (SRB) assay of Skehan et al. (25), modified by Cetojevic-Simin et al. (26). The color development was measured using a Multiscan Ascent (Labsystems; Helsinki, Finland) photometer at 540 nm against 620 nm as background. The effect on the cell growth was calculated as: 100 x (AT/AC) (%), where AT is the absorbance of the test sample and AC of the control. EC50 values, concentration that inhibits cell growth by 50%.

Antimicrobial activity

Sample for the determination of antimicrobial activity. The FDR extract was dis-solved in sterile distilled water to a concentration of 50 mg/ml for disk diffusion method. For the determination of the minimal inhibitory concentration (MIC) and minimal bacte-ricidal concentrations (MBC) the FDR extract was dissolved in sterile distilled water to obtain the following concentrations: 200, 175,150, 125, 100, 75, 50, 25, 12.5, 9.4 and 6.3 mg/ml. Test microorganisms. The tested bacteria were the strains of Gram–negative bacte-ria: Salmonella typhimurium (ATCC 14028), Escherichia coli (ATCC 25922), Salmo-nella sp.w, Escherichia coliw and Pseudomonas aeruginosaw (w-wild strain) and Gram–positive bacteria: Staphylococcus aureus (ATCC 11632), Bacillus cereus (ATCC 10876), Listeria monocytogenes (ATCC 13932), Staphylococcus saprophyticusw and Bacillus sp.w. The tested yeasts were the strains of: Saccharomyces cerevisiae (112, Hefebank Weihenstephan) and Candida albicansw and moulds: Aspergillus niger (ATCC 16404) and Penicillium aurantiogriseumw. Wild strains were obtained from food and tap water. Wild bacterial strains were identified using Vitek®2 Compact System (bioMérieux, France) and Penicillium aurantiogriseum was identified according to Samson et al. (2004) (23). The strains were stored at -80oC in Nutrient broth (Himedia, Mumbai, In-dia), supplemented with 20% (v/v) glycerol in the Laboratory of Microbiology, Faculty of Technology, University of Novi Sad. Antimicrobial assay. Antimicrobial activity was determined by disk diffusion met-hod (27). The bacterial strains were grown on the Müeller–Hinton agar (MHA, Himedia, Mumbai, India) slants for 24 h at 37ºC, except for Bacillus cereus and Bacillus sp., which were grown at 30ºC. The yeasts were grown on the Sabouraud Maltose Agar (SMA, Himedia, Mumbai, India) slants 24 h, and the moulds on the Dichloran Rose



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Bengale Chloramphenicol Agar (DRBC, Himedia, Mumbai, India) slants during 7 days at 25ºC. The bacterial and yeast cells were then suspended in a sterile 0.9% NaCl solution. The moulds spores were suspended in a sterile 0.01% peptone salt solution with the addition of 0.05% Tween 80 and filtered through glass wool. The bacterial and yeasts suspensions for the inoculation were adjusted to a concentration of 1×106 cfu/ml, estimated using Densichek (Biomerieux, France), while the number of moulds spores was estimated using the Neubauer chamber. 1 ml of the suspensions for inoculation were homogenized with 9 ml of melted (45ºC) MHA, SMA or DRBC and poured into Petri dishes. Sterile 6 mm disks (Himedia, Mumbai, India) were placed on the inoculated agar plates and impregnated with 15 µl of the extract solution. The antibiotic (30 µg cefotaxime /10 µg clavulanic acid per disc, Bioanalyse®, An-kara, Turkey) and antifungal agent (cycloheximide solution, 0.03g/ml, Sigma-Aldrich, Co. St. Louis, USA) were used as controls. The antibiotic disks were placed on the ino-culated agar plates, while the sterile 6-mm disks (Himedia, Mumbai, India) after placing were impregnated with 15µl of the antifungal agent solution. Test plates were refrigerated at 8ºC for 1 h to allow the extract to diffuse into the medium, and then were incubated at 37ºC or 30ºC for 24 h (bacteria), at 25ºC for 48 h (yeasts) and at 25ºC for 72 h (moulds). After the incubation, the diameters of the inhi-bition zones were measured and recorded in millimeters (mm). The evaluation of antibac-terial activity was carried out in three repetitions. MIC and MBC were determined in order to quantify the antimicrobial activity of the FDR extract. The test was done by using microdilution method in sterile flat-bottom 96-well microtiter plates. The growing conditions and preparation of suspensions were the same as in the disk diffusion method. 1 ml of each prepared suspension was homogenised with 9 ml of Mueller–Hinton broth. In each well (n=3), 100 µl of the inoculated media were mixed with 100 µl of the extract dilutions to obtain final concentrations in the range of 100 - 3.2 mg/ml. All test plates contained an uninoculated control (blank) (100 µl of medium + 100 µl of extracts) and a positive growth control (inoculated media without extracts). The microtiter plates were incubated in the same conditions as in the disk diffusion method. After the incubation, a 100 µl aliquot from each well was poured into Petri dishes and homogenised with Nutrient agar (Himedia, Mumbai, India). The Petri dishes were incubated under the same conditions as the microtiter plates, and, after the incubation, the colonies were enumerated by viable count. MIC and MBC were defined as the lowest concentration of the extract resulting in a significant decrease in the inoculum viability (≥ 90%) and the concentration where ≥ 99.9% of the initial inoculum is killed (28), respectively, and calculated as: 100×(NC–NT)/NC (%), where NC and NT are the numbers of cells of the positive growth control and treatment, respectively.

Statistical analysis

All measurements for chemical composition, antioxidant and antimicrobial activity were carried out in triplicate (n=3), and were presented as the means ± SD. The EC50 va-lues were calculated using Microsoft Office Excel 2003. The measurements of the anti-proliferative activity were represented as the means ± SD of eight measurements (n = 8),



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while EC50 values were determined using OriginPro 8 SRO (Origin- Lab Corporation, Northampton, USA).

RESULTS AND DISCUSSION

The results of the spectrophotometric determination of the contents of total phenolics, flavonoids, anthocyanins and of the HPLC determination of the content of vitamin C in FDR are shown in Table 1.

Table 1. Contents of the analyzed parameters in FDR

Chemical compound Content

Total phenolics 43.43 ± 1.38 mg GAE/g DE

2209.86 ± 70.32 mg GAE/100g FDR

Total flavonoids 16.35 ± 0.25 mg R/g DE

831.87 ± 12.61 mg R/100g FDR

Total anthocyanins 2.84 ± 0.01 mg CGE/g DE

144.55 ± 0.39 mg CGE/100g FDR

Vitamin C 1.75 ± 0.06 mg vit C/g DE

88.81 ± 4.38 mg vit C/100g FDR

The phenolics content in FDR was 2209.86 ± 70.32 mg GAE/100g FDR. Weber et al. (10) reported that the phenolic content that, depending on the raspberry variety (Heritage, Kiwigold, Goldie and Anne), varied from 359.19 to 512.70 mg GAE/100g fruit. Sava-tović et al. (29) reported 265.38 ± 10.75 mg GAE/100g fresh weight in Meeker raspberry pomace. Also, comparing the results in Table 1 and those reported by Sariburun et al. (30), the contents of phenolic compounds (1040.90 to 2062.30 mg GAE/100g fresh weight) in the raspberry cultivars (Aksu Kirimizisi, Newburgh, Rubin, Heritage and Hollanda Boduru), extracted with methanol or water, were higher in our study. Novako-vic et al. (31) suggested that that concentration of phytochemicals rises in the same di-rection as dehydration, which means that the freeze-drying process did not degrade phe-nolics content in FDR. Also, these differences could be due to the environmental cha-racteristics, period of harvesting, cultivar variability, fruit maturity, and the extraction solvent and procedure (30). The total flavonoid content reported by Sariburun et al. (30) varied greatly among the raspberry cultivars, ranging from 15.41 ± 0.12 mg (Heritage) to 41.08 ± 0.92 mg CTE/ 100 g fresh weight (Hollanda Boduru), while the results in Table 1 show a much higher content of flavonoids (831.87 ± 12.61 mg R/100g FDR). According to de Torres et al. (32), the lyophilized skins preserve their volatile and phenolic composition in comparison with the original skins. Also, Wojdy1o et al. (33), who analyzed phenolics (anthocyanins, flavanols, hydroxycinnamic acids and flavonols) in strawberry dehydrated by different methods, reported that the highest concentration of phenolic compounds was found in the samples that were freeze-dried and vacuum-microwave dried. The climate conditions, the



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soil composition and the management of berries are the main factors by which are possible to explain the difference in bioactive compounds (34). Sariburun et al. (30) reported the anthocyanin content in red raspberry which varied from 12.4 to 69.5 mg CGE/100g fresh weight. De Souza et al. (34) reported 14.69 ± 2.03 mg CGE/100 g fresh weight, while the FDR content was 144.55 ± 0.39 mg CGE/100g FDR (Table 1). The content of anthocyanin in our study is much higher than in the other reports. Zabetakis et al. (35) showed that anthocyanins are very sensitive to temperature denaturation, and a combined time/temperature process can greatly reduce the level of pigments in the final product. In most species, the concentrations of fruit anthocyanin in-crease with ripening, as their biosynthesis proceeds faster than fruit expansion (32). Also, the different conditions (year, location, climate) appear to influence the anthocyanin accumulation. In FDR, the vitamin C was present in the amount of 88.81 ± 4.38 mg mg/100g FDR (Table 1), while De Souza et al. (34) reported a content of 92.17 ± 10.11 mg/100 g fresh weight in red raspberry, which is in accordance with our results. De Ancos et al. (36) reported the vitamin C contents of Rubi (310.89 ± 16.3 mg/kg), Zeva (296.59 ± 3.8 mg/kg), and Autumn Bliss (301.89 ± 8.00 mg/kg) raspberry cultivars, which showed very similar initial vitamin C contents, while the lowest level was found in the Heritage rasp-berry (220.67 ± 10.8 mg/kg). Significant changes in the vitamin C content due to long-term frozen storage were observed in the four cultivars studied. Although the vitamin C contents in the four cultivars studied were similar after 365 days, the percentage of the loss depended on the cultivar: The Rubi and Zeva cultivars suffered vitamin C losses of 49 and 47%, respectively, whereas the losses for Heritage and Autumn Bliss were 34 and 56%, respectively, based on the raw fruit values. Compared with other major fruit and ve-getable antioxidants, ascorbic acid is more susceptible to significant loss during the post-harvest handling and storage. Also, ascorbic acid requires an acidic environment for sta-bility (37). The changes in the vitamin C content could be a good indicator for enzymatic or nonenzymatic degradative reactions taking place during the processing or storage of the fruit (38).

Antiradical assays

The free radical scavenging activities of the FDR extract and Trolox were determined by the DPPH radical assay and ESR spectroscopy (Table 2).

Table 2. EC50 values of the FDR extract/FDR and control

FDR extract/FDR and control

EC50DPPH•

(mg FDR extract or Trolox/ml)

EC50DPPH•

(mg FDR/ml)

EC50•OH

(mg FDR extract or Trolox/ml)

EC50•OH

(mg FDR/ml)

FDR 0.127 ± 0.013 0.250 ± 0.011 1.366 ± 0.026 2.685 ± 0.131 Trolox 0.005 ± 0.001 - 0.424 ± 0.020 -

The antioxidant molecules can quench DPPH free radicals (i.e. by providing hydrogen atoms or by electron donation, conceivably via a free-radical attack on the DPPH mole-



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cule) and convert them to a colorless/bleached product (i.e. 2,2-diphenyl-1-hydrazyne, or a substituted analogous hydrazine), resulting in a decrease in the absorbance at 517 nm (39). Figure 1 shows the dose response for the DPPH radical scavenging activity of the FDR extract. The DPPH free radical scavenging activity of the FDR extract increased with the increasing concentration. The EC50 values of FDR extract and Trolox are shown in Table 2.

Figure 1. DPPH radical scavenging activity (SADPPH•) of the FDR extract. Values

represent means ± SD of three independent measurements (n=3)

Weber et al. (10) reported that the EC50 values for Heritage, Kiwigold, Goldie and Anne raspberry extracts determined by DPPH• assay varied from 0.312 – 0.802 mg/ml. Our results reported in Table 2 are in accordance with the reported results. Stajčić et al. reported the EC50

DPPH• value for Meeker raspberry pomace extract (0.1996 ± 0.0054 mg/ml) (29). The FDR extract exhibited a slightly better scavenging activity comparing with the reported results. Trolox showed better antioxidant activity than FDR extract (Table 2). As hydroxyl free radicals are highly reactive, with relatively short half-lives, the con-centrations found in natural systems are usually inadequate for direct detection by ESR spectroscopy. Hydroxyl radicals are identified thanks to their ability to form nitroxide adducts (stable free radicals form) from the commonly used DMPO as the spin trap (40). Figure 2 shows the scavenging activity (SA•OH) of different concentrations of FDR extract on the DMPO-OH spin adduct during the Fenton reaction.

0

20

40

60

80

100

0.008 0.05 0.1 0.15 0.25

SA

DP

PH

•(%

)

Concentration (mg/ml)



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Figure 2. Scavenging activity (SA•OH) of different concentrations of FDR extract on the DMPO-OH spin adduct during the Fenton reaction. Data are given as inhibition percen-tages of DMPO-OH spin adduct peak height measured by ESR and are the means SD

of three independent experiments (n=3) The intensity of the ESR signal, corresponding to the concentration of free radicals formed decreased in the presence of different amounts of the FDR extract (Fig. 2). A complete elimination of hydroxyl radicals (SA•OH = 100%) was achieved at the concen-tration of 4.0 mg/ml of the FDR extract. Trolox expressed a stronger antioxidant activity comparing with the FDR extract. According to our unpublished data, the EC50

•OH of Meeker raspberry pomace (MRP) extract was 3.73 mg/ml (41). FDR showed a higher activity on •OH than the MRP extract, which shows that freeze-drying is a good way to preserve antioxidant compounds in raspberry fruits.

Cell growth activity The effects of the FDR on the cell growth depended on the concentration and cell line. The most pronounced effects, i.e. the lowest EC50 value, were obtained in the breast adenocarcinoma cell line (MCF7), reaching 395.07 ± 96.38 µg/ml (Table 3). The least sensitive cell line was healthy tissue (MRC-5), with the EC50 value higher than 1000 µg/ml (Table 3).



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Table 3. EC50 (µg/ml) values of the FDR extract, standards and drugs obtained after 48-h exposure

Values represent means ± SD of eight measurements (n = 8).

The activity of the FDR extract was compared to the activity of standard solutions and well-known drugs with proven high (Doxorubicin and Gemzar®) and mild (Aspirin®) cytotoxic activities. All standard solutions exhibited enviable antiproliferative activity towards tumor cells but also, apart from vitamin C, toward the cell line derived from the healthy tissue, with the majority of the EC50 values below 20 µg/ml (Table 3). The FDR extracts exhibited a substantial cytotoxicity towards the most sensitive tu-mor cell lines (breast and cervix), and they were from 5 to 20-fold more toxic (with lower EC50 values) than was Aspirin®. On the other hand, their activity was 100-fold lower compared to the most potent cytotoxic drugs, where the most EC50 values were below 1 µg/ml (Table 3). Cetojević-Simin et al. (42) reported that the most pronounced cell growth inhibition effect was obtained in the MCF7 cell line, reaching EC50 values of 34.8 and 60.3 µg/ml for Willamette and Meeker raspberry pomace extracts, respectively. Also, the FDR ex-tract was the most efficient on this cell line, while EC50 values were higher than the re-ported results. Based on the selectivity towards cell lines, it can be assumed that the antiproliferative activity of the investigated FDR extract can be attributed to the presence of phenolic compounds and vitamin C and to their synergistic actions in extract mixtures (Table 1 and Table 3).

Antimicrobial activity

The results of the antimicrobial activity of the FDR extract against both Gram-positi-ve and Gram-negative bacteria, yeasts and moulds are presented in Table 4.

Extract, standards and

drugs

Cell line HeLa MCF7 HT-29 MRC-5

FDR extract 602.45 ± 81.15 395.07 ± 96.38 721.54 ± 91.45 >1000 Standard

Gallic acid 2.33 ± 0.29 6.59 ± 0.27 170.00 ± 13.00 19.2 ± 6.12 Vitamin C 26.70 ± 5.07 32.90 ± 1.84 98.40 ± 35.60 399 ± 25.0

Ellagic acid 2.47 ± 0.40 11.1 ± 0.73 >25 2.89 ± 0.91 Quercetin 12.80 ± 1.23 12.2 ± 0.60 38.90 ± 5.89 11.7 ± 2.98

Drug Doxorubicin® 0.25 ± 0.09 0.26 ± 0.02 0.38 ± 0.04 0.40 ± 0.03 Gembitacin® 0.13 ± 0.10 0.04 ± 0.00 31.30 ± 0.00 0.08 ± 0.03

Aspirin® > 1000 >1000 >1000 >1000



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Table 4. Antimicrobial activity of the FDR extract

Group Tested strain Disk

diffusion methoda

MIC/MBCb (mg/ml)

Controls (antibiotic/

antifungal agent)

G (–) bacteria

Escherichia coli 6.5*

(1.91) 50/62.5

30 (1.0)

Escherichia coliw nd >50/>50 25.33 (0.58)

Salmonella typhimurium

8.5* (0.58)

50/62.5 33.6

(0.85)

Salmonella sp.w 9.5* (1)

50/62.5 27.83 (0.76)

Pseudomonas aeruginosaw nd 25/37.5

15.33 (0.58)

G (+) bacteria

Staphylococcus aureus

9.25 (0.96) 4.7/6.3 34.3

(0.75) Staphylococcus saprophyticusw nd 12.5/25

24.0 (0.5)

Bacillus cereus 8*

(0.82) 100/>100

34.5 (1.5)

Bacillus sp.w 9.75*

(0) 50/>100

39.33 (0.58)

Listeria monocytogenes

nd 4.7/6.3 14.25 (0.55)

Yeast

Saccharomyces cerevisiae

nd nt >38

Candida albicans

nd nt 15.33* (0.58)

Moulds Aspergillus niger nd nt

27.67 (0.58)

Penicillium aurantiogriseum

nd nt 31.33 (0.58)

a - mean of diameter of the inhibition zone (mm) including disk (6 mm) with the standard deviation in the paren-theses; b - results of MIC/MBC(MFC) are presented without standard deviations because all three repetitions gave the same concentration values; * - zone of reduced growth; nd - non detected inhibition zone; nt – not tested

The FDR extract produced clear inhibition zone (without visible colonies) only tow-ard Staphylococcus aureus (Table 4). Toward Escherichia coli, Salmonella and Bacillus strains only the zones of reduced growth appeared. The other tested bacteria were the least susceptible to the effect of the FDR extract and were able to grow in its presence. The control sample (antibiotic combination cefotaxime/clavulanic acid) showed bac-tericidal activity against all bacteria. The most expressive activity was against Bacillus strains, Salmonella typhimurium and Staphylococcus aureus (the zones higher than 30



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mm), and the least against Listeria monocytogenes and Pseudomonas aeruginosa (the zones less than 15 mm). Microorganisms with eukaryotic type of cells (yeasts and moulds) were resistant to the applied amounts of FDR extract. Also, for yeasts and moulds, agar-well diffusion method was done (by applying 50 µl and 100 µl of the extract) and inhibition zones were absent (data not shown). So, further analyses (determination of MIC and MBC) for these organisms were not done. Candida albicans is probably the most resistant tested euca-ryotic strain, because the control antifungal agent (cycloheximide solution) showed only a zone of reduced growth, while for the other eucariotic strains halo zones of 27 mm or more were obtained. The results MIC and MBC showed that the FDR extract inhibited growth of almost all tested bacteria. The values of MIC were in the range of 4.7 - 100 mg/ml and of MBC in the range of 6.3 - > 100 mg/ml. Gram-positive bacteria (Staphylococcus aureus, Staphy-lococcus saprophyticus and Listeria monocytogenes) were more susceptible than Gram-negative bacteria, with lower MIC/MBC values obtained. Exceptions among the Gram-positive bacteria were Bacillus strains, for which the MBC values were not found in the tested concentration range (they were higher than 100 mg/ml). The most resistant Gram-negative bacteria was Escherichia coliw, whose growth was not inhibited by 50 mg/ml of FDR extract, and the most susceptible was Pseudomonas aeruginosaw. However, the absence of antifungal activity or the presence of a slight activity in the case of some bacteria (Bacillus strains and Escherichia coli), do not indicate the absence of bioactive constituents in the FDR extract. Namely, active components may be present in insufficient quantities to inhibit the cells growth. The lack of activity can thus only be proven by using large doses. On the other hand, by applying higher extract quantities, some other constituents may be introduced which exert antagonistic effects or negate the positive effects of the bioactive agents (43). In the literature, several studies refer to the antimicrobial potential of raspberry fruit, juices and pomace extracts. The previous results on antibacterial activity (tested by disk diffusion method) of raspberry extracts from cultivars grown in Serbia showed that both Meeker and Willamette cultivar possess antibacterial potential against all tested Gram-positive and Gram-negative bacteria (44). It is confirmed in the study of Četojević Simin et al. (42) who determined low MIC (0.29-0.59 mg/ml) and MBC (0.39-0.78 mg/ml) values of raspberry pomace extract against eleven bacterial strains. In a study of Ördögh et al. (45) raspberry juice showed antibacterial activity only against Staphylococcus epi-dermidis (MIC at pH 7 was 12.35 mg/ml, and 9.18 mg/ml at the pH 5.5), while pomace extracts did not show any antibacterial activity. By testing antimicrobial activity of rasp-berry extract (concentration 1 mg/ml in growth media) in liquid culture, Nohynek et al. (46) obtained strong activity of raspberry extract toward Helicobacter pylori, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Campylobacter jejuni and Clostridium perfringens. Using the same method, Pupponen-Pimiä et al. (47) obtained strong inhibition of FDR extract (concentration 10 mg/ml in growth media) against Salmonella enterica sv. typhimurium and Staphylococcus aureus, and no effects on the growth of Listeria monocytogenes. The observed differences obtained in different studies are probably caused by the dif-ferent methods, extract solution concentrations, and different susceptibility of tested natu-



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ral isolates. Also, the composition and amount of active components extracted from tes-ted materials originated from different geographic areas, growth conditions of plant ma-terial, climatic factors, ripening stage, harvesting time and storage conditions affected the results (48, 49).

CONCLUSION

The contents of total phenolics (2209.86 ± 70.32 mg GAE/100g FDR), flavonoids (831.87 ± 12.61 mg R/100g FDR), anthocyanins (144.55 ± 0.39 mg CGE/100g FDR) and vitamin C (88.81 ± 4.38 mg vit C/100g FDR) were determined in FDR. The FDR extract showed very good free radical scavenging activity towards stable DPPH radicals. EC50

DPPH• value in DPPH• assay was 0.127 ± 0.013 mg/ml. Also, good antiradical activity of FDR extract against hydroxyl radicals was obtained (EC50

•OH = 1.366 ± 0.026 mg/ml).

The antiproliferative activity was evaluated in vitro in three human cell lines. The lowest EC50 value of FDR extract was obtained for the MCF7 cell line (395.07 ± 96.38 µg/ml). The FDR extract produced clear inhibition zone only toward Staphylococcus aureus. The MIC values for the test organisms were in the range of 4.7 - 100 mg/ml, and MBC in the range of 6.3 - > 100 mg/ml. The presented results indicate that bioactive compounds present in FDR have good influence on the antioxidant, antiproliferative and antimicro-bial activity. Based on the reported study and increasing concern about food quality, the freeze-drying process could be cosidered as a valuable alternative to preserve food and good choice for the food processing industry.

Acknowledgement This research is part of the Project TR 31044 which is financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia.

REFERENCES

1. Beekwilder, J., Jonker, H., Meesters, P., Hall, R. D., van der Meer, I. M. and Ric de Vos, C. H.: Antioxidants in raspberry: On-line analysis links antioxidant activity to a diversity of individual metabolites. J. Agric. Food Chem. 53 (2005) 3313-3320.

2. Mullen, W., McGinn, J., Lean, M. E. J., MacLean, M. R., Gardner, P., Duthie, G. G., Zokota, T. and Croyier, A.: Ellagitannins, flavonoids, and other phenolics in red rasp-berries and their contribution to antioxidant capacity and vasorelaxation properties. J. Agric. Food Chem. 50 (2002) 5191-5196.

3. Paredes-Lopez, O., Cervantes-Ceja, M. L., Vigna-Perez, M. and Hernandez-Perez, T.: Berries: Improving human health and healthy aging, and promoting quality life – A review. Plant Foods Hum. Nutr. 65 (2010) 299-308.

4. Cates, E.M., Popa, G., Gill, C.I., McCann, M.J., Mc Dougall, G.J., Stewart, D. and Rowland, I.: Colonavailable raspberry polyphenols exhibit anticancer effects on in vitro models of colon cancer. J. Carcinog. 6 (2007) 4-9.



113

5. Seeram, N.P.: Berry fruits: compositional elements, biochemical activities, and the impact of their intake on human health, performance, and disease. J. Agric. Food Chem. 56 (2008) 627-629.

6. Koponen, J.M., Happonen, A.M., Mattila, P.H. and Törrönen, A.R.: Contents of anthocyanins and ellagitannins in selected foods consumed in Finland. J. Agric. Food Chem. 55 (2007) 1612-1619.

7. Kahkonen, M.P., Hopia, A.I. and Heinonen, M.: Berry phenolics and their antioxidant activity. J. Agric. Food Chem. 49 (2001) 4076-4082.

8. Häkkinen, S., Kärenlampi, S., Mykkänen, H., Heinonen, M. and Törrönen, R.: Ellagic acid content in berries: Influence of domestic processing and storage. Eur. Food Res. Technol. 212 (2000) 75-80.

9. Juranić, Z., Žižak, Ž., Tasic, S., Petrović, S., Nidzović, S., Leposavić, A. and Stanoj-ković, T.: Antiproliferative action of water extracts of seeds or pulp of five different raspberry cultivars. Food Chem. 93 (2005) 39-45.

10. Weber, C., Liu, M., Qi Li, X. and Hai Liu, R.: Antioxidant Capacity and Anticancer Properties of Red Raspberry. New York Fruit Quarterly 9, 3 (2001) 13-15.

11. Rommel, A. and Wrolstad, R.: Influence of acid and base hydrolysis on the phenolic composition of red raspberry juice. J. Agric. Food Chem. 41 (1993) 1237-1241.

12. Corthout, J., Peiters, L.A., Claeys, M., Vanden Berghe, D.A. and Vleitinck, A.J.: Antiviral ellagitannins from Spondia mombin. Phytochem. 30 (1991) 1129-1130.

13. Rao, C.V., Tokumo, K., Rigoty, J., Zang, E., Kelloff, G. and Reddy, B.S.: Chemopre-vention of colon carcinogenesis by dietary administration of piroxicam, difluoro-methlornithine, 16â-fluor-5-androsten-17-one and ellagic acid individually and in combination. Cancer Res. 51 (1991) 4528-4534.

14. Stoner, G. D. and Morse, M. A.: Isocyanates and plant polyphenols as inhibitors of lung and esophageal cancer. Cancer Lett. 114 (1997) 113-119.

15. Bórquez, R.M., Canales, E.R. and Redon, J.P.: Osmotic dehydration of raspberries with vacuum pretreatment followed by microwave-vacuum drying. J. Food Eng. 99 (2010) 121-127.

16. Michalczykk M., Macura, R. and Matuszak, I.: The effect of air-drying, freeze-drying and storage on the quality and antioxidant activity of some selected berries. J. Food Process. Preserv. 33 (2009) 11-21.

17. Ratti, C.: Hot air and freeze-drying of high-value foods: A review. J. Food Engin. 49 (2001) 311-319.

18. Singleton, V.L., Orthofer, R. and Lamuela-Raventos, R.M.: Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Meth. Enzymol., Oxidant and Antioxidants (Part A). Academic Press, San Diego (1999) p. 152-178.

19. Zhishen, J., Mengcheng, T. and Jianming, W.: The determination of flavonoid con-tents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64 (1999) 555-559.

20. Cheng, G. W. and Breen, P. J.: Activity of Phenylalanine Ammonialyase (PAL) and concentrations of anthocyanins and phenolics in developing strawberry fruits, J. Americ. Soc. Hort. Sci. 116 (1991) 865-869.



114

21. Tumbas, V.T., Canadanovic-Brunet, J.M., Cetojevic-Simin, D.D., Cetkovic, G.S., Ðilas, S.M., and Gille, L.: Effect of rosehip (Rosa canina L.) phytochemicals on stable free radicals and human cancer cells. J. Sci. Food Agric. 92, 6 (2012) 1273-1281.

22. Yen, G. C. and Chen, H. Y.: Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43 (1995) 27-32.

23. Samson, R.A., Hoekstra, E.S. and Frisvad, J.C.: Introduction to Food- and Airborne Fungi. Centraalbureau voor schimmelcultures – Utrecht, The Netherlands (2004), pp.174-243.

24. Čanadanović-Brunet, J.M., Ćetković, G.S., Djilas, S.M., Tumbas, V.T., Savatović, S.S., Mandić, A.I., Markov S.L. and Cvetković, D.D.: Radical scavenging and anti-microbial activity of horsetail (Equisetum arvense L.) extracts. Int. J. Food Sci. Tech. 44 (2009) 269-278.

25. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R.: New colorimetric cytotoxicity assay for anticancer-drug screening. J. National Cancer Inst. 82 (1990) 1107-1112.

26. Četojevic-Simin, D., Svirčev, Z. and Baltić, V.: In vitro cytotoxicity ofcyanobacteria from water systems of Serbia. J. Balkan Union Oncol. 14, 2 (2009) 289-294.

27. Mayo, W. J.: Chemical methods of control: Antimicrobial drugs, in Laboratory expe-riments in microbiology. Eds. Johnson, T. R. and Case, C. L., The Benjamin/Cum-mings Publishing Company, San Francisco (1998) pp. 179-181.

28. Burt, S.: Essential oils: Their antibacterial properties and potential applications in foods-a review. Int. J. Food Microbiol. 94 (2004) 223-253.

29. Stajčić, S., Tepić, A., Djilas, S., Šumić, Z., Čanadanović-Brunet, J., Ćetković, G., Vu-lić, J. and Tumbas, V.: Chemical composition and antioxidant activity of berry fruits. APTEF, 43 (2012) 93-105.

30. Sariburun, E., Şahin, S., Demir, C., Tűrkben, C. and Uylaşer, V.: Phenolic Content and Antioxidant Activity of Raspberry and Blackberry Cultivars. J. Food Sci. 75, 4 (2010) C328-C335.

31. Novakovic, M. M., Stevanovic, S. M., Gorjanovic, S. Z., Jovanovic, P. M., Tesevic, V. V., Jankovic, M. A. and Suznjevic, D. Z.: Changes of Hydrogen Peroxide and Ra-dical-Scavenging Activity of Raspberry during Osmotic, Convective, and Freeze-Dry-ing. J. Food Sci. 76, 4 (2011) C663-8.

32. De Torres, C., Diaz-Maroto, M.C., Hermosin-Gutierrez., I. and Perez-Coello, M.S.: Effect of freeze-drying and oven-drying on volatiles and phenolics composition of grape skin. Anal. Chim. Acta 660 (2010) 177-182.

33. Wojdy1o, A., Figiel, A., and Oszmianski, J.: Effect of drying methods with the appli-cation of vacuum-microwave on the phenolic compounds, color and antioxidant acti-vity in strawberry fruits. J. Agric. Food Chem. 57 (2009) 1337-1343.

34. De Souza, V.R., Pimenta Pereira, P.A., Teodoro da Silva, T. L., de Oliveira Lima, L.C., Rafael Pio, R. and Queiroz, F.: Determination of the bioactive compounds, anti-oxidant activity and chemical composition of Brazilian blackberry, red raspberry, strawberry, blueberry and sweet cherry fruits. Food Chem. 156 (2014) 362-368.

35. Zabetakis, I., Leclerc D., and Kajda, P.: The effect of high hydrostatic pressure on the strawberry anthocyanins. J. Agric. Food Chem. 48 (2000) 2749-2754.



115

36. De Ancos, B., Gonzales, E.M. and Cano, M.P.: Ellagic acid, vitamin C, and total phe-nolic contents and radical scavenging capacity affected by freezing and frozen storage in raspberry fruit. J. Agric.Food Chem. 48 (2000) 4565-4570.

37. Kalt, W.: Effects of Production and Processing Factors on Major Fruit and Vegetable Antioxidants. J. of Food Sci. 70, 1 (2005) R11-R19.

38. Skrede, G.: Fruits. In Freezing Effects on Food Quality; Jeremiah, L. E., Ed.; Dekker: New York, 1996.

39. Ribeiro, B., Rangel, J., Valentao, P., Baptista, P., Seabra, R.M. and Andrade, P.B.: Contents of carboxylic acids and two phenolics and antioxidant activity of dried Por-tugese wild edible mushrooms. J. Agric. Food Chem. 54 (2006) 8530-8537.

40. Čanadanović-Brunet, M.J., Djilas, SM, Ćetković, G.S. and Tumbas, V.T.: Free-radi-cal scavenging activity of wormwood (Artemisia absinthium L.) extracts. J. Sci. Food Agric. 85 (2005) 265-273.

41. Čanadanović-Brunet, M., Vulić J., Ćebović, Т., Ćetković, G., Djilas, S., Tumbas Ša-ponjac, V. and Stajčić, S.: Antioxidant properties and cytotoxic effects of the raspber-ries (Rubus idaeus L.) pomace, unpublished data.

42. Četojević-Simin, D.D., Velićanski, A.S., Cvetković, D.D., Markov, S.L., Ćetković, G.S., Tumbas Šaponjac, V.T., Vulić, J.J., Čanadanović-Brunet, J.M. and Đilas, S.M.: Bioactivity of Meeker and Willamette raspberry (Rubus idaeus L.) pomace extracts. Food Chem. 166 (2015) 407-413.

43. Parekh, J. and Chanda, V.: Antibacterial Activity of Aqueous and Alkoholic Extracts of 34 Indian Medicinal Plants against Some Staphylococcus Species. Turk. J. Biol. 32 (2008) 63-71.

44. Velićanski, A.S.., Cvetković, D.D. and Markov, S.L.: Screaning of antibacterial acti-vity of raspberry (Rubus idaeus L.) fruit and pomace extracts. Acta Periodica Techno-logica. 43 (2012) 305-313.

45. Ördögh, L., Galgóczy, L., Krisch, J., Papp, T. and Vágvölgyi C.: Antioxidant and antimicrobial activities of fruit juices and pomace extracts against acne-inducing bac-teria. Acta Biologica Szegediensis 54, 1 (2010) 45-49.

46. Nohynek, L.J., Alakomi, H.-L., Kähkönen, M.P., Heinonen, M., Helander, I.M., Oks-man-Caldentey, K.-M. and Puupponen-Pimiä, R.H.: Berry Phenolics: Antimicrobial Properties and Mechanisms of Action Against Severe Human Pathogens. Nutr. Can-cer 54, 1 (2006) 18-32.

47. Puupponen-Pimiä, R., Nohynek, L., Hartmann-Schmidlin, S., Kähkönen, M., Heino-nen, M., Määttä-Riihinen, K. and Oksman-Caldentey, K.-M.: Berry phenolics selecti-vely inhibit the growth of intestinal pathogens. J. Appl. Microbiol. 98 (2005) 991-1000.

48. Ryan, T., Wilkinson, J.M. and Cavanagh, H.M.A.: Antibacterial activity of raspberry cordial in vitro. Res. Vet. Sci. 71 (2001) 155-159.

49. Jimenez Garcia, S.N., Guevara-Gonzalez, R.G., Miranda-Lopez, R., Feregrino-Perez, A.A., Torres-Pacheco, I. and Vazquez-Cruz, M.A.: Functional properties and quality characteristics of bioactive compounds in berries: Biochemistry, biotechnology, and genomics. Food Res. Int. 54, 1 (2013) 1995-1207.



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АНТИОКСИДАТИВНA, AНТИМИКРОБНА И АНТИПРОЛИФЕРАТИВНА АКТИВНОСТ ЛИОФИЛИЗИРАНЕ МАЛИНЕ

Јелена Ј. Вулић1, Александра С. Велићански1, Драгана Д. Четојевић-Симин2, Весна Т. Тумбас-Шапоњац1, Соња М. Ђилас1, Драгољуб Д. Цветковић1,

Синиша Л. Марков1

1 Универзитет у Новом Саду, Технолошки факултет Нови Сад, Булевар Цара Лазара 1, 21000 Нови Сад,

Србија 2 Институт за Онкологију Војводине, Др Голдмана 4, 21204 Сремска Каменица

Садржај укупних фенола, флавоноида и антоцијана у лиофилизираној малини испитан је спектрофотометријском методом, док је садржај витамина Ц испитан HPLC методом. Одређена количина биоактивних једињења у лиофилизираној ма-лини је: феноли 2209,86 ± 70,32 mg GAE/100g, флавоноиди 831,87 ± 1,61 mg R/100g и антоцијани 144,55 ± 0,39 mg CGE/100g. У лиофилизираној малини витамин Ц је био присутан у количини од 88,81 ± 4,38 mg витамина Ц/100g. Антирадикалска ак-тивност екстракта лиофилизиране малине утврђена је спектрофотометријским тес-том на DPPH радикале и ЕСР спектроскопијом на хидроксил радикале. ЕC50

DPPH• вредност износилa je 0,127 ± 0,013 mg/ml, а ЕC50

•OH 1,366 ± 0,026 mg/ml. Антипро-лиферативна активност екстракта лиофилизиране малине одређена је in vitro на три ћелијске линије, колориметријским сулфородамин Б (SRB) тестом. Најнижа ЕC50 вредност, а самим тим и најбоља антипролиферативна активност, постигнута је код MCF7 ћелијске линије (395,07 ± 96,38 µg/ml). Антимикробна активност екстракта лиофилизиране малине одређена је диск дифузионом методом. Екстракт лиофили-зиране малине дао је зону без раста (показатељ бактериостатског деловања) само за сојеве Staphylococcus aureus. Минималне инхибиторне концентрације су биле у оп-сегу од 4,7 до 100 mg/ml, а минималне бактерицидне концентрације у опсегу од 6,3 до > 100 mg/ml. Резултати овог истраживања указују да биоактивне компоненте присутне у екстракту лиофилизиране малине значајно утичу на антиоксидативну, антипролиферативну и антимикробну активност. Такође, на основу приказаних ре-зултата и све веће потребе потрошача за квалитетном храном, поступак конзерви-сања лиофилизацијом, могао би да буде одличан избор у прехрамбеној индустрији. Кључне речи: малина, лиофилизација, DPPH• тест и ЕSR антиоксидативна метода,

антипролиферативна и антимикробна активност

Received: 2 September 2014. Accepted: 23 October 2014.

ANTIOXIDANT, ANTIPROLIFERATIVE AND ANTIMICROBIAL …Raspberries (Rubus ideaus, cv. “Meeker”)...

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