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Journal of Food Composition and Analysis xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

1
2 Original Research Article

3 Polyphenols, vitamin C and antioxidant activity in wines from

4 Rosa canina L. and Rosa rugosa Thunb.
5 Q1 A. Czyzowska *, E. Klewicka, E. Pogorzelski, A. Nowak
6 Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Wolczanska 171/173, 90-924 Lodz, Poland

A R T I C L E I N F O A B S T R A C T

Article history:
Received 12 May 2013
Received in revised form 17 November 2014
Accepted 19 November 2014
Available online xxx
Chemical compounds studied in this article:
Vitamin C (PubChem CID: 54670067)
Gallic acid (PubChem CID 24721416)
Chlorogenic acid (PubChem CID 1794427)
Ferulic acid (PubChem CID: 445858)
Syringic acid (PubChem CID: 10742)
p-Coumaric acid (PubChem CID: 637542)
Quercetin rutinoside (PubChem CID:
5280805)
Quercetin glucoside (PubChem CID:
25203368)
The purpose of this study was to determine the concentration of biologically active compounds
(polyphenols and L-ascorbic acid) in Rosa canina L. and Rosa rugosa Thunb. wines. The antioxidant
capacity and antimutagenicity of the wines were also investigated. Aged and young wines contained
phenolics levels of 27863456 and 33893990 mg/L GAE, respectively. The nal concentrations of
ascorbic acid were 1200 for Rosa rugosa Thunb. and 600 mg/L for Rosa canina L. R. rugosa and R. canina
wines revealed high antioxidant activity in different assays (with ABTS, DPPH, and DMPD radicals).
Expressed in terms of Trolox equivalent antioxidant capacity (TEAC), the activity ranged from 8 to
13.5 mM. Signicant differences were found between the tested wines terms of their reactivity against
the ABTS and DMPD radicals. The wines inhibited in vitro N-methyl-N0 -nitro-nitrosoguanidine (MNNG)
and the number of induced His+ revertants increased in a dose-dependent manner by 1648% in
Salmonella Typhimurium TA98 and 1252% in Salmonella Typhimurium TA100. Wines from dog rose
(Rosa canina L.) showed a greater ability to reduce mutations.
2014 Published by Elsevier Inc.

Keywords:
Wild rose
Rosa canina L.
Rosa rugosa Thunb.
Food analysis
Food composition
Fruit wines
Fermentation process
Polyphenols
Vitamin C
Antioxidants
Antimutagenicity
Bioactive non-nutrients

7
8 1. Introduction natural environments without needing chemical fertilizers or 15
irrigation (Ercisli, 2007). 16
9 Q2 The genus Rosa comprises over 100 species, found in Europe, Rose species have long been used for food and medicinal 17
10 Asia, the Middle East and North America (Ercisli, 2007; Nilsson, purposes in many cultures. Rose hips are used in many foodstuffs 18
11 1997), of which 23 occur in the wild in Poland. They grow both in and drinks including teas, jellies, jams, and alcoholic beverages 19
12 the lowlands and in mountainous regions, where some are (Grochowski, 1990; Zhang et al., 2008). As an herbal remedy, rose 20
13 found above the tree line and among mountain pine thickets hips are used in skin care as well as for the treatment of various 21
14 (Grochowski, 1990). Generally hardy, these plants ourish in ailments including colds, u, inammations, chronic pain and 22
ulcers (Zhang et al., 2008; Chrubasik et al., 2006, 2007). 23
In French folk medicine, the rose ower is used as a cure for 24
scurvy and hemorrhoids, as an anthelmintic and fortifying agent. In 25
* Corresponding author. Tel.: +48 426313492; fax: +48 426365976. Bulgaria, the rose ower is still used to cure diseases of the 26
E-mail address: agata.czyzowska@p.lodz.pl (A. Czyzowska). gastrointestinal tract, while in Russia it is recommended for the 27

http://dx.doi.org/10.1016/j.jfca.2014.11.009
0889-1575/ 2014 Published by Elsevier Inc.

Please cite this article in press as: Czyzowska, A., et al., Polyphenols, vitamin C and antioxidant activity in wines from Rosa canina L. and
Rosa rugosa Thunb.. J. Food Compos. Anal. (2014), http://dx.doi.org/10.1016/j.jfca.2014.11.009
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28 treatment of lung diseases and infections of the upper respiratory antioxidants toward DPPH and have shown that the effects of 94
29 tract (Osinska, 2004). As uczaj and Szymanski (2007) observe, particular compounds on this radical depend on their structure. 95
30 fruits from Rosa sect. Caninae (folk species) are used to make The two main objectives of our research were, rstly, to 96
31 preserves, medicines and, occasionally, childrens snacks, in many determine the concentration of biologically active compounds 97
32 regions of Poland. Until the turn of the 20th century, they were (polyphenols and L-ascorbic acid) in wines form Rosa canina L. and 98
33 used as baby food, ground in a hand mill and cooked with milk. Rosa rugosa Thunb. and secondly, to assess their antioxidant 99
34 Wine made from wild rose species is a traditional beverage which capacity and possible mutagenicity or antimutagenicity. 100
35 has been made in Poland for centuries. In Poland, three native
36 species (R. canina L., R. gallica L., R. rubiginosa L.) and three 2. Materials and methods 101
37 introduced species (R. centifolia L., Rosa rugosa Thunb.,
38 R. damascena L.) are of medical importance (Gora and Lis, 1996). 2.1. Chemicals and reagents 102
39 Ethnobotanical works cited by uczaj and Szymanski report the
40 use of Rosa canina L., the most common of the species from this N,N-dimethyl- p-phenylenediamine dihydrochloride (DMPD), 103
41 section found in Poland. Dried rose fruits are used for the treatment 2,20 -azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) dia- 104
42 of all illnesses caused by vitamin C deciency, including diarrhea, mmonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 105
43 weakened activity of the gastrointestinal tract, as well as to treat Trolox, DMSO and Folin Ciocalteu reagent were purchased from 106
44 diseases of the kidneys, liver and bladder. In Polish phytotherapy, Sigma Aldrich (Poznan, Poland); MNNG from Fluka, St. Gallen, 107
45 Rosa canina L. fruits are further used as an anti-u, diuretic and Switzerland; formic acid and HPLC grade methanol from J.T. Baker 108
46 cardiotonic agent (Pawlaczyk et al., 2009; Strzelecka and Kowalski, (Witko, Lodz, Poland); and gallic acid, syringic acid, chlorogenic 109
47 2000). acid, ferulic acid, p-coumaric acid, quercetin glucoside, quercetin 110
48 Dry powder from Rosa canina L. false fruits is a widely used rutinoside, and cyanidin-3-glucoside from Extrasynthese (Genay, 111
49 herbal remedy against arthritis. A review published by Chrubasik France). HPLC grade water was obtained using an Aquinity 112
50 et al. (2006) and a meta-analysis by Christensen et al. (2008) E60 Lifescience TI system (membraPure GmbH, Bodenheim, 113
51 investigating clinical evidence for the medicinal use of rose hip Germany). 114
52 powder, report that consuming powdered rose hips from Rosa
53 canina L. can result in a moderate reduction of pain in patients 2.2. Plant material 115
54 suffering from osteoarthritis (Saaby et al., 2011). Daily consump-
55 tion of 40 g of rose hip powder for 6 weeks has also been shown to Ripe fruits were collected in October from wild bushes growing 116
56 signicantly reduce cardiovascular risk in obese persons, by in the Lodz region (central Poland, 188360 E, 518250 N, from 169 to 117
57 lowering systolic blood pressure and plasma cholesterol levels 173 m) and transported to the laboratory. Five 1000 g samples of 118
58 (Andersson et al., 2012). each rose species were enclosed in polyethylene bags and stored at 119
59 Rosa rugosa Thunb. (common names: Japanese rose, rugosa 20 8C prior to use. 120
60 rose) occurs naturally in Eastern Asia from Okhotsk and southern
61 Kamchatka to Korea and the northern parts of Japan and China. In 2.3. Winemaking procedure 121
62 Poland, Rosa rugosa Thunb. was introduced in 1960 (Tokarska-
63 Guzik, 2003). The species is scattered throughout Poland, Musts from Rosa canina L. and Rosa rugosa Thunb. were 122
64 especially in the southwestern regions, and is still spreading prepared as follows: having removed the stems (stalks), the fruits 123
65 (Weidema, 2006). It is found in dry meadows and thickets as well were thawed and crushed. Boiling water was added to the resulting 124
66 as at the edges of forest (Tokarska-Guzik, 2003). pulp in a ratio of 1:1, and the temperature of the mixture was 125
67 Rosa rugosa Thunb. is used to make preserves, jellies, and wines. adjusted to 5055 8C. The enzyme preparation Pektopol PT 400 126
68 Extracts from the owers or hips are used in herbal medicines and (Jaslo, Poland) was added (2.6 g/kg of fruit pulp) and the 127
69 vitamin products (Weidema, 2006). It is also used in traditional temperature maintained in a range of between 50 and 55 8C for 128
70 Chinese medicine as an effective vasodilator and to improve 2 h. After 2 h of enzymatic treatment, the must was pressed out 129
71 microcirculation (Xie and Zhang, 2012). and pitchings prepared. The must consumption in the pitchings 130
72 Fu et al. (2006) have demonstrated that compounds found in reached 70%. 131
73 Rosa rugosa Thunb. owers have inhibitory activity against HIV The pitchings were poured into asks and then inoculated with 132
74 reverse transcriptase. A study of Xie and Zhang (2012) has further the yeast Saccharomyces bayanus (0.4 g/L of pitching). Fermenta- 133
75 shown that extracts from the owers can inhibit the angiotensin- tion was performed at 25 8C and controlled using the gravimetric 134
76 converting enzyme. method by determining weight loss during the process. Vinica- 135
77 The physiological functions of Rosaceae fruits may be partly tion was performed in triplicate for each rose fruit sample. 136
78 attributed to the fact that they contain an abundance of
79 compounds including phenolics, b-carotene, lycopene, ascorbic 2.4. Wine aging 137
80 acid, tocopherol, bioavonoids, fruit acids, tannins, pectin, sugars,
81 organic acids, amino acids and essential oils (Demir and Ozcan, After completing the fermentation process, the wine was 138
82 2001; Uggla et al., 2003, 2005; Ercisli, 2007). decanted from the sediment and aged in bottles at 10 8C for a 139
83 The vitamin C content of wild roses exceeds that in other raw period of 3 months. 140
84 materials, by a factor of up to several dozens, while polyphenols
85 (especially avonoids, their most active group) stabilize this 2.5. Determination of total phenolics using FolinCiocalteu reagent 141
86 vitamin in food products (Grochowski, 1990; Demir and Ozcan,
87 2001; Jaroniewski, 1992). It has been shown that juice obtained Total phenolic content (TPC) was determined using the Folin 142
88 from R. rugosa fruits contains signicant quantities of catechins Ciocalteu method (Waterhouse, 2001). 143
89 and proanthocyanidins (Oszmianski and Chomin, 1993; Wilska- To prepare the calibration curve, solutions of gallic acid at 144
90 Jeszka et al., 1991). The synergistic activity of L-ascorbic acid and concentrations of between 0 and 500 mg/L were used. The R2 145
91 avonoids, or the so-called sparing effect, has been observed by coefcient of the standard curve was 0.9892. 146
92 Saucier and Waterhouse (1999) and by Vinson et al. (2001). Brand- 20 mL of the calibration solution (sample or blank) 1.58 mL of 147
93 Williams et al. (1995) have studied the reaction of various distilled water and 100 mL of FC reagent were added to 7 mL 148

Please cite this article in press as: Czyzowska, A., et al., Polyphenols, vitamin C and antioxidant activity in wines from Rosa canina L. and
Rosa rugosa Thunb.. J. Food Compos. Anal. (2014), http://dx.doi.org/10.1016/j.jfca.2014.11.009
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149 tubes and the mixture stirred. After 30 s 300 mL of 20% sodium 2.9. ABTS+, DMPD, DPPH TEAC assays 209
150 carbonate was added. The solution was left at room temperature
151 for 1 h. The absorbance of the solution at 765 nm was measured 2.9.1. ABTS assay 210
152 against blank (0 mL solution) using a Cecil CE2041 spectropho- The ABTS assay is based on the ability of antioxidants to 211
153 tometer (Cecil Instruments Limited, Cambridge, UK). The must and scavenge the long-lived radical cation ABTS (Re et al., 1999). 212
154 pitching samples were diluted 20 times, while the wine samples ABTS+ was generated by mixing an aqueous solution of ABTS 213
155 were diluted 10 times. The results were expressed as mg of gallic with a solution of potassium persulfate to achieve a nal 214
156 acid per liter. concentration of 7.0 mM ABTS+ and 2.45 mM sodium persulfate. 215
This solution was kept in the dark at room temperature for 16 h 216
157 2.6. Determination of total avonoids before use. The ABTS+ solution was then diluted with ethanol 217
(approximately 1:90, v/v) to obtain an absorbance reading of 0.7 at 218
158 Total avonoids were determined using the method developed 734 nm. Then 0.1 mL of appropriate dilutions (determined in 219
159 by Di Stefano et al. (1989). preliminary experiments) of the wines were transferred to test 220
160 First, 0.5 mL of twofold-diluted must, pitching or wine was tubes containing 3.9 mL of ABTS+ solution. The solutions were 221
161 loaded onto a Sep-Pak C18 cartridge (Waters Assoc.), initially mixed, and after 6 min absorbance was measured at 734 nm. The 222
162 activated using 2 mL of methanol and 5 mL of distilled water. After samples were tested in triplicate and the percentage of inhibition 223
163 the bed had been washed with 0.5 mL of water, avonoids were (% I) was calculated using the following formula: 224
164 eluted using 5 mL of methanol, and the content was collected in a 225
165 volumetric ask with a capacity of 10 mL. ABTS free radical scavenging activity %
166 The sample in the ask was acidied by adding of 0.01 mL of  
1  sample absorbance
167 concentrated HCl and supplemented with methanol. The absor-  100
control absorbance
168 bance spectrum was recorded in a range of between 230 nm and
169 700 nm. The results were also expressed as Trolox-equivalent antioxi- 228
227
226
170 The results were expressed as mg (+)-catechin equivalents (CE) dant capacity (TEAC) values (mM Trolox/g extract), dened as the 229
171 per liter of wine. To prepare the calibration curve, solutions of amount of Trolox (in mM) that exhibited the same antioxidant 230
172 catechin were used in concentrations of between 0 and 300 mg/L. activity as 1 g of wine fractions. 231
173 The R2 coefcient of the standard curve was 0.9975.
2.9.2. Measurement of antioxidant ability by the DMPD method 232
174 2.7. Determination of ascorbic acid (Fogliano et al., 1999) 233
100 mM DMPD solution was prepared by dissolving 209 mg of 234
175 Ascorbic acid was determined according to PN-90/A-75101/ DMPD in 10 mL of deionized water. 1 mL of this solution was added 235
176 11. This method involves the oxidation of L-ascorbic acid in to 100 mL of 0.1 M acetate buffer, pH 5.25, and the colored radical 236
177 an acidic medium to form dehydroascorbic acid with 2,6- cation (DMPD+) was obtained by adding 0.2 mL of 0.05 M ferric 237
178 dichloroindophenol. An excess of 2,6-dichloroindophenol was chloride solution (to a nal concentration of 0.1 mM). 2 mL of this 238
179 extracted using xylene and determined spectrophotometrically solution was immediately placed in a cuvette and its absorbance 239
180 at a wavelength of 500 nm. The content of ascorbic acid was measured at 505 nm. 0.1 mL of each diluted wine sample was 240
181 calculated on the basis of a calibration curve with L-ascorbic added and absorbance measured at 505 nm after 10 min. The 241
182 acid, and the results expressed as mg of ascorbic acid per buffered solution was placed in a reference cuvette. 242
183 liter of beverage. The R2 coefcient of the standard curve was A doseresponse curve was derived for Trolox by plotting 243
184 0.9988. absorbance at 505 nm as a percentage of the absorbance of an 244
uninhibited radical cation solution (blank) according to the 245
185 2.8. HPLC polyphenol analysis equation: 246
247
186 Samples were ltered through a 0.22 mm membrane prior to A505 inhibition % 1  At =A0  100;
187 analysis and injected into the HPLC system. HPLC-DAD analyses
188 were performed using a Finnigan Surveyor equipped with where A0 is the absorbance of the uninhibited radical cation and At 248
249
189 autosampler, diode array detector (Finnigan Surveyor-PDA Plus), is the absorbance measured 10 min after the addition of 250
190 and ChromQuest 5.0 chromatography software (Thermo Fisher antioxidant samples. 251
191 Scientic Inc, Waltham, MA, USA). Separation was performed on The antioxidant activity of wine samples was expressed as TEAC 252
192 a LiChrospher RP 18-5 (Hichrom, Berkshire, UK) (250 mm  values (mM Trolox/g extract), dened as the amount of Trolox (in 253
193 4.6 mm, 5 mm packing) protected with a guard column (LiChro- mM) that exhibited the same antioxidant activity as 1 g of wine 254
194 spher guard cartridge (Hichrom, Berkshire, UK), 10 mm  fractions. 255
195 4.6 mm, 5 mm packing). The elution conditions were as follows:
196 0.9 mL/min ow rate; oven temperature 25 8C; solvent A, 2.9.3. DPPH assay (Sanchez-Moreno et al., 1998) 256
197 water/formic acid (95:5 v/v); solvent B, methanol. Elution This method involves measuring changes in absorbance at a 257
198 began with linear gradients from 10% to 30% B for 2 min, the wavelength of l = 515 nm at different time intervals, until the 258
199 same for 6 min; from 30% to 35% B for 5 min; from 35% to 50% B reaction reaches minimum absorbance. The violet color of DPPH in 259
200 for 7 min; from 50% to 70% B for 2 min; and from 70% to 80% B methanol (1,1-diphenyl-picrylhydrazyl) decreases as the degree of 260
201 for 8 min. The column was then washed and re-equilibrated. The reduction increases. The percentage of reduced DPPH was 261
202 injection volume for all samples was 50 mL. The calibration calculated from the formula: 262
203 curve was established using an HPLC grade gallic acid 263

204 commercial standard (Extrasynthese, Z.I. Lyon Nord, France) % red: DPPH 1  At =A0  100 %
205 to quantify polyphenols at 280, 320 and 360 nm. To prepare the
206 calibration curve, solutions of gallic acid in concentrations from The number of millimoles of Trolox solution equivalent to the 266
265
264
207 0 to 500 mg/L were used. The R2 coefcient of the standard antioxidant activity of 1 dm3 of wine was calculated from the 267
208 curve was 0.9917. calibration curve. 268

Please cite this article in press as: Czyzowska, A., et al., Polyphenols, vitamin C and antioxidant activity in wines from Rosa canina L. and
Rosa rugosa Thunb.. J. Food Compos. Anal. (2014), http://dx.doi.org/10.1016/j.jfca.2014.11.009
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269 2.10. Antioxidant capacity in linoleic acid emulsion Salmonella TA100. The MNNG concentration was 10.0 and 0.1 mg/ 329
plate in Salmonella Typhimurium TA98 and TA100, respectively. 330
270 The ferric thiocyanate (FTC) method reported by Larrauri et al.
271 (1996) was used with slight modications. 2.12. Statistical analysis 331
272 A mixture of 0.5 mL wine, 0.5 mL linoleic acid emulsion, 0.1 mL
273 sodium phosphate buffer (pH = 7) and 5 mL distilled water was All measurements were performed in triplicate and the results 332
274 placed in a tube with a screw cap, then shaken and placed in an presented as mean values  standard deviations (SD). Using 333
275 oven at 40 8C in the dark. A control without an extract sample was STATISTICA 10 PL software (StatSoft, Cracow, Poland), the standard 334
276 used. To 0.1 mL of this solution were added 9.7 mL of ethanol and deviation was determined and the ANOVA test conducted, assuming a 335
277 0.1 mL of 300 g/L ammonium thiocyanate. Absorbance was signicance level of 0.05. 336
278 measured against a reagent blank at 500 nm every 24 h (t) exactly
279 3 min after the addition of 0.1 mL of ferrous chloride to 35 g/L HCl 3. Results and discussion 337
280 of the reaction mixture, until the day after the absorbance of the
281 control reached maximum. The oxidation index (OI) and antioxi- 3.1. Total polyphenols and avonoids and ascorbic acid content 338
282 dant activity (AA) were calculated as
283   The measured concentrations of polyphenols in musts from 339
OI  t  sample
AA 100   100 R. canina and R. rugosa fruits respectively were 9007  345 and 340
OI  t  controlAbs: max
7400  520 mg/L GAE (Table 1). This compares with a study by 341
285
284 Gao et al. (2000), which found a TPC of 59122 mg GAE/g DW in 8 rose 342
species (Rosa moschata had the lowest content, R. villosa hybrid the 343
286
Abs  t highest). R. canina from Chile was found to have a TPC from 60 to 344
OI 0
Abs  t almost 63 mg GAE/g DW. A study by Ercisli (2007) found the total 345
phenolics in six Rosa species to be between 73 mg GAE/g DW (Rosa 346
287
288
villosa) and 96 mg GAE/g DW (Rosa canina L.). The TPC of Rosa rugosa 347
289 2.11. Antimutagenic activity of wines from Rosa rugosa Thunb. and
Thunb. fruits from Poland, as studied by Hallmann et al. (2011), was 348
290 Rosa canina L.
found to be 176 mg/100 g FW. A study by Oszmianski and Chomin 349
(1993) also revealed high avonoid content in Rosa rugosa Thunb. 350
291 The antimutagenic effect of wines from Rosa rugosa Thunb. and
The results presented in this study clearly demonstrate that 351
292 Rosa canina L. was determined using the method described by
polyphenol levels depend not only on the species but also on 352
293 Maron and Ames (1983). The Ames test employs two cultures of
climatic conditions. This is in agreement with Scalzo et al. (2005), 353
294 Salmonella enterica subsp. enterica serovar Typhimurium (Salmo-
who highlight the importance of the plant genotype (species and 354
295 nella Typhimurium), designated as TA98 and TA100 (McCann et al.,
variety within species) for in determining the phenolic compound 355
296 1975). In our study, MNNG (Fluka, St. Gallen, Switzerland) was
content. The results of a study by Meng et al. (2012), similarly show 356
297 used as a direct-acting mutagen. MNNG does not require any
that wines made from Cherokee rose (Rosa laevigata Michx.) have 357
298 metabolic activation with liver fraction S9 to induce a mutagenic
higher levels of total phenolics, total avonoids and oligomeric 358
299 effect (Lankaputhra and Shah, 1998). The concentration of MNNG
proanthocyanidins than spine grape wines and Cabernet Sau- 359
300 solution in the DMSO (Sigma, St. Louis, MO, USA) was 10 mg/test
vignon wines, while their TPC was measured at 2529  16.9 mg/L 360
301 for experiments with Salmonella Typhimurium TA98 and 0.1 mg/
GAE. The total phenolic content of the wines in our study was found to 361
302 test for experiments with Salmonella Typhimurium TA100. These
be in a range of 27863456 and 33893990 mg/L GAE for aged and 362
303 doses of the mutagen were determined on the basis of
young wines, respectively (Table 1). Total avonoid content in the 363
304 experimental curves presenting the relationship between the
nal wines was 2666 mg/L for Rosa rugosa Thunb. and 3008 mg/L for 364
305 quantity of His+ revertants and mutagen concentration.
Rosa canina L. (Table 2). 365
306 For Salmonella Typhimurium TA98 and TA100 respectively, the
Rosa rugosa and Rosa canina wines thus exhibit higher TPC than 366
307 applied mutagen doses caused the appearance of 200 and
wines made from Cherokee rose (Rosa laevigata Michx.). 367
308 2000 colonies of His+ revertants. Wine samples were ltered
Rose hips are well known for their high ascorbic acid content 368
309 through 0.2 mm membranes (Millipore, Cork, Ireland). Wine doses
(Barros et al., 2011; Demir and Ozcan, 2001; Ercisli, 2007; Gao 369
310 of 10.0, 20.0, 50.0, and 100.0 mL/plate were mixed with 0.1 mL
et al., 2000; Hallmann et al., 2011; Kazaz et al., 2009; Rosu et al., 370
311 samples of Salmonella Typhimurium overnight cultures (cell
2011). For instance, the ascorbic acid content of Rosa canina 371
312 density of 4.5  109 CFU/mL) and aliquots of MNNG (10 mg/plate
L. collected in two different locations in Turkey varied from 2365 to 372
313 for TA98 and 0.1 mg/plate for TA100) and incubated at 37 8C for
2712 mg/100 g (Demir and Ozcan, 2001). In a study by Kazaz et al. 373
314 20 min before adding 2 mL of top agar. The mixtures were then
(2009), the ascorbic acid contents of Rosa canina L. fruit and fruit 374
315 spread on minimal essential agar plates and incubated for 48 h at
esh from Turkey were 411 and 2200 mg/100 g, respectively, and 375
316 37 8C in darkness, after which the Salmonella His+ colonies were
were 1.23 and 4.03 times higher than in the corresponding parts of 376
317 counted. All samples were prepared in triplicate. The decrease in
Rosa damascena Mill. Our research indicates that the musts from 377
318 the number of mutations was calculated using the equation:
  Rosa rugosa and Rosa canina are also rich in L-ascorbic acid 378
319 N1  100
Mutation reduction 100  %
N0
Table 1
320
321 where N0 is the number of Salmonella His+ colonies (corrected for Total polyphenol content in musts, pitchings, and wines [mg/L GAE].

322 the number of spontaneous revertants) visible on plates containing Sample Rosa rugosa Rosa canina
323 samples with MNNG without rose hip wine, and N1 is the number
Must 7400  520 9007  345*
324 of Salmonella His+ colonies (corrected for the number of Pitching 4200  200 5670  139*
325 spontaneous revertants) visible on plates containing samples Wine after fermentation 3389  245 3990  256*
326 supplemented with MNNG and rose hip wine. The results were Wine after aging 2786  156 3456  134*
327 corrected for spontaneous reversion. The number of spontaneous All values are presented as means  SD (n = 3).
328 His+ revertants was 33  7 in Salmonella TA98 and 198  32 in *
Statistically signicant difference between Rosa species, ANOVA test (p < 0.05).

Please cite this article in press as: Czyzowska, A., et al., Polyphenols, vitamin C and antioxidant activity in wines from Rosa canina L. and
Rosa rugosa Thunb.. J. Food Compos. Anal. (2014), http://dx.doi.org/10.1016/j.jfca.2014.11.009
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Table 2 Table 4
Total avonoid content in musts, pitchings and wines [mg/L]. Polyphenols in rose hip wines [mg/L].

Sample Rosa rugosa Rosa canina Compound Rosa canina Rosa rugosa
*
Must 7000  420 8700  345 Gallic acid nd 144.02  3.92*
Pitching 3890  120 5070  339* Chlorogenic acid 40.91  1.35 4.45  0.13*
Wine after fermentation 3089  245 3740  253* Ferulic acid Trace 0.01  0.00*
Wine after aging 2666  156 3008  134* Syringic acid 1.25  0.04 0.02  0.00*
p-Coumaric acid Trace 0.02  0.00*
Number of analyzed samples n = 3. All values are presented as means  SD (n = 3).
* Unidentied compound Rt = 8.1a 83.62  0.98 32.94  1.01*
Statistically signicant difference between Rosa species, ANOVA test (p < 0.05).
Unidentied compound Rt = 8.55b nd 67.00  2.25*
(catechin derivative)
Unidentied compound Rt = 9.61c nd 0.55  0.01*
Quercetin rutinoside nd 14.55  0.47*
379 (Table 3). L-ascorbic acid content in Rosa canina was found to be
Quercetin glucoside 66.65  2.67 34.04  1.24*
380 between 1571  145 and 3182  420 mg/L, and 2-fold higher in Unidentied compound Rt = 24.6d 46.18  1.39 42.03  1.10*
381 musts from Rosa rugosa Thunb.
All values are presented as means  SD (n = 3); unidentied compounds: a
382 The nal concentration of ascorbic acid in the investigated
Uvmax = 264; b Uvmax = 280; c Uvmax = 286; d Uvmax = 277; ndnot detected (LOD
383 wines was 1200 and 600 mg/L for Rosa rugosa Thunb. and Rosa 0.004 mg/L)
384 canina L., respectively. This indicates that in nished wines *
Statistically signicant difference between Rosa species, ANOVA test (p < 0.05).
385 obtained from wild rose initial vitamin C content remains 5070%.
386 The results for total polyphenols and avonoids and ascorbic
387 acid content clearly show that the investigated wild rose wines are Poland contain quercetin glucoside, kaempferol glucoside, and 421
388 some of the richest sources of antioxidants. The level of vitamin C myricetin. 422
389 in 100 mL of wine completely covers the daily dietary requirement. Losses and changes in antioxidant compounds occur at each 423
stage of wine production. 424
390 3.2. Polyphenolic prole of wild rose wines The most substantial losses of vitamin C and polyphenolic 425
compounds are incurred while the musts are being obtained, for 426
391 There is no information in the literature concerning the instance during enzymatic and thermal processing of raw 427
392 phenolic prole of rose hip wines. The concentrations of individual materials and technological procedures such as ltration, pasteur- 428
393 polyphenolic antioxidants were determined using HPLC. Some of ization, physicochemical stabilization and bottling. 429
394 the avonols identied (e.g. quercetin glucoside) occurred in wines
395 made from both rose species, while others (e.g. quercetin rutino- 3.3. Antioxidant capacity of rose hip wines 430
396 side) were present only in wines from Rosa rugosa Thunb.
397 The content of phenolic acids was also measured, the most One method is usually insufcient to evaluation of the 431
398 abundant being gallic acid (Table 4). Cyanidin-3-glucoside was not antioxidant activity of a complex substance. 432
399 found in any of the wines tested. As shown by previous studies by In our study, three different assays (with ABTS, DPPH, and 433
400 the authors, this compound is unstable during the process of DMPD radicals) were used to evaluate the antioxidant properties of 434
401 fermentation (unpublished results). Moreover, polymerization of wines from R. rugosa and R. canina. High antioxidant activity was 435
402 anthocyanins is known to occur during the aging process. observed in all assays, ranging from 8 to 13 mM in terms of Trolox 436
403 In studies by other authors, wild rose hips were found to equivalent antioxidant capacity (TEAC) (Table 5). Some substantial 437
404 contain all polyphenols identied in the present study of wines. and signicant differences between ABTS and DMPD radicals were 438
405 Nowak (2007) found quercitrin and kaempferol 3-O-rhamnoside in found in the tested wines. 439
406 rose fruits. Mikulic-Petkovsek et al. (2012) using HPLC-MSn The next step was to evaluate the antioxidant activity of rose 440
407 identication of avonol glycosides found quercetin galactoside, hip wines using the ferric thiocyanate method based on the 441
408 glucuronide, and arabinofuranoside, as well as kaempferol rutino- inhibition of linoleic acid oxidation. Linoleic acid is a polyunsatu- 442
409 side and coumaroylglucoside in dog rose (R. canina) fruits. Other rated fatty acid, which, similarly to LDL, undergoes oxidation in the 443
410 phenols found in Rosa canina L. fruits include: cyanidin-3- human body and is used as a model in in vitro studies of fatty acids 444
411 glucoside, taxifolin glycosides, eriodictyol, phloridzin, myricetin, to determine the mechanisms of lipid oxidation which lead to the 445
412 gallic acid derivatives, ellagic and p-hydroxybenzoic acids formation of free radicals. 446
413 (Hvattum, 2002; Tumbas et al., 2012; Zocca et al., 2011). Based on the absorbance measured, oxidation indices were 447
414 Zocca et al. (2011) found in extracts of dog rose (Rosa canina L.) measured and antioxidant activity calculated, expressed as a 448
415 derivatives of catechin and epicatechin, including epigallocatechin percentage of the autoxidation delay of linoleic acid. The 449
416 at almost 2500 mg/L, epicatechin gallate at 37.69 mg/L and ellagic oxidization indices of rose hip wines were observed over 72 h 450
417 acid at over 170 mg/L. However, as catechins are not very stable and found to be 20% and 21% for Rosa rugosa Thunb. and Rosa 451
418 during the fermentation process, signicantly lower contents of canina L., respectively (Table 5). 452
419 this group of compounds were found in our study of wines. A study by Gao et al. (2000) investigating the contribution of 453
420 Hallman et al. (2011) report that Rosa rugosa Thunb. fruits from three different antioxidant fractions using an ABTS assay, showed 454

Table 3 Table 5
L-ascorbic acid content in musts, pitchings and wines [mg/L]. Antioxidant capacity of wines from Rosa rugosa and Rosa canina.

Sample Rosa rugosa Rosa canina Method of determination Rosa rugosa Rosa canina

Must 3182  420 1571  145* TEAC/ABTS 10.4  0.9 13.5  0.6*
Pitching 1546  120 700  39* TEAC/DMPD 13.4  0.6 10.5  0.8*
Wine after fermentation 1348  245 653  25* TEAC/DPPH 8.9  0.8 8.6  0.5
Wine after aging 1200  156 600  34* LDL % 20.1  0.3 21.4  0.2*

Number of analyzed samples n = 3. All values are presented as means  SD (n = 3). All values are presented as means  SD (n = 3).
* *
Statistically signicant difference between Rosa species, ANOVA test (p < 0.05). Statistically signicant difference between Rosa species, ANOVA test (p < 0.05).

Please cite this article in press as: Czyzowska, A., et al., Polyphenols, vitamin C and antioxidant activity in wines from Rosa canina L. and
Rosa rugosa Thunb.. J. Food Compos. Anal. (2014), http://dx.doi.org/10.1016/j.jfca.2014.11.009
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455 the total antioxidant capacity of phenolics, ascorbic acid and A study by Karakaya and Kavas (1999) concerning, the 487
456 lipophilic compounds to be slightly lower than those of crude antimutagenic activity of Rosa canina L., among other plant juices, 488
457 extracts. The phenolic fraction made a major contribution to total leaves and seeds, showed that rose hips boiled at 100 8C for 10 min 489
458 activity (about 75%), followed by ascorbic acid (around 17%). This is and stewed for a further 10 min were not mutagenic in Salmonella 490
459 consistent with our results for TEAC/ABTS. Wines from Rosa canina Typhimurium TA100. Rose hips decreased sodium azide mutage- 491
460 L. were found to have a signicantly higher TEAC/ABTS value (13.5) nicity by 44%. 492
461 and to contain higher levels of total polyphenols and avonoids Shinohara et al. (1988) observed a positive correlation between 493
462 than wines from Rosa rugosa Thunb. However, they had only the antimutagenic activity and polyphenol content of vegetables 494
463 slightly more than half of the vitamin C content of the latter. and fruits, which is consistent with our results. Phenolics can 495
464 The results of a study by Brand-Williams et al. (1995) indicate create complexes or interact non-enzymatically with mutagens, 496
465 that ascorbic acid is one of the fastest reacting antioxidants. This is thus reducing their bioavailability (Mejia et al., 1999). 497
466 also conrmed by our ndings. The values for DPPH in Rosa rugosa In our study, wines from Rosa canina L. were found to contain 498
467 Thunb. wines were slightly higher than those of R. canina. The level higher levels of total polyphenols and avonoids, but only about 499
468 of ascorbic acid in wines made from R. rugosa was also found to be half the level of vitamin C. These results may indicate a lack of 500
469 higher. correlation between antimutagenic activity and the vitamin C 501
content in these rose hip wines. 502
470 3.4. Antimutagenic activity of rose hip wines

471 The antimutagenic activity of rose hip wines was determined in 4. Conclusions 503
472 Salmonella Typhimurium TA98 and Salmonella Typhimurium
473 TA100 using the Ames test. Wines from Rosa rugosa Thunb. The results of our study show that wild rose wines are a rich 504
474 reduced the intensity of mutations induced by MNNG in a dose- source of antioxidants. Both, wines from Rosa rugosa Thunb. and 505
475 dependent manner by 1648% in Salmonella Typhimurium TA98 Rosa canina L. contain high levels of polyphenols and L-ascorbic 506
476 and by 1252% in Salmonella Typhimurium TA100 (Table 6). acid. According to Regulation (EU) no. 1160/2011 the recom- 507
477 Wines from the dog rose (Rosa canina L.) showed a greater mended daily dosage of vitamin C for the adult male is 80 mg: 508
478 ability to reduce mutations (Table 7). Statistically signicant 70 mL of wine from Rosa rugosa Thunb. or 140 mL of wine from 509
479 differences (p = 0.05) in relation to the control sample were Rosa canina L. would provide this level of vitamin C. However, due 510
480 observed with even the lowest dose of wine (10 mL/plate). to its ethanol content, wild rose wine should not be treated as the 511
481 In contrast, the addition of 10 mL of wine from Rosa rugosa sole source of this vitamin. 512
482 Thunb. did not have a biocidal effect on the TA98 strain. Here, The high antioxidant activity of wild rose wines has been 513
483 statistically signicant differences were observed only after the conrmed by different assays (using ABTS, DPPH, and DMPD 514
484 addition of 50 mL of wine, and its ability to reduce mutations at this radicals). Moreover, the wines have been found to reduce the 515
485 concentration was approximately 2-fold lower than that of wines intensity of mutations induced by MNNG in a dose-dependent 516
486 from Rosa canina L. manner. 517

Acknowledgments 518

Table 6
Antimutagenic activity of Rosa rugosa wine in Salmonella Typhimurium TA98 and
This research was nancially supported by Polish Grant 2P06T Q3 519
Salmonella Typhimurium TA100 as determined by the Ames test. 07627. The authors would like to thank J. Laskowska, Ph.D. for 520
analysis of antioxidant activity. 521
V (wine) TA98 TA100
[mL/plate]
His+ mutants/ Mutation His+ mutants/ Mutation
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Rosa rugosa Thunb.. J. Food Compos. Anal. (2014), http://dx.doi.org/10.1016/j.jfca.2014.11.009