Journal of Food Composition and Analysis
Volume 39, May 2015, Pages 62–68
Original Research Article

Polyphenols, vitamin C and antioxidant activity in wines from Rosa canina L. and Rosa rugosa Thunb.

doi:10.1016/j.jfca.2014.11.009

Highlights

•
Wild rose wines have high phenolic and ascorbic acid content.
•
High antioxidant activity of wines from R. rugosa and R. canina confirmed.
•
Wild rose wines reduce mutation intensity induced by MNNG in dose-dependent manner.

Abstract

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 2786–3456 and 3389–3990 mg/L GAE, respectively. The final 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. Significant differences were found between the tested wines terms of their reactivity against the ABTS and DMPD radicals. The wines inhibited in vitro N-methyl-N′-nitro-nitrosoguanidine (MNNG) and the number of induced His+ revertants increased in a dose-dependent manner by 16–48% in Salmonella Typhimurium TA98 and 12–52% in Salmonella Typhimurium TA100. Wines from dog rose (Rosa canina L.) showed a greater ability to reduce mutations.

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)

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

1. Introduction

The genus Rosa comprises over 100 species, found in Europe, Asia, the Middle East and North America ( Ercisli, 2007 and Nilsson, 1997), of which 23 occur in the wild in Poland. They grow both in the lowlands and in mountainous regions, where some are found above the tree line and among mountain pine thickets ( Grochowski, 1990). Generally hardy, these plants flourish in natural environments without needing chemical fertilizers or irrigation ( Ercisli, 2007).
Rose species have long been used for food and medicinal purposes in many cultures. Rose hips are used in many foodstuffs and drinks including teas, jellies, jams, and alcoholic beverages (Grochowski, 1990 and Zhang et al., 2008). As an herbal remedy, rose hips are used in skin care as well as for the treatment of various ailments including colds, flu, inflammations, chronic pain and ulcers (Zhang et al., 2008, Chrubasik et al., 2006 and Chrubasik et al., 2007).
In French folk medicine, the rose flower is used as a cure for scurvy and hemorrhoids, as an anthelmintic and fortifying agent. In Bulgaria, the rose flower is still used to cure diseases of the gastrointestinal tract, while in Russia it is recommended for the treatment of lung diseases and infections of the upper respiratory tract (Osińska, 2004). As Łuczaj and Szymański (2007) observe, fruits from Rosa sect. Caninae (folk species) are used to make preserves, medicines and, occasionally, children's snacks, in many regions of Poland. Until the turn of the 20th century, they were used as baby food, ground in a hand mill and cooked with milk. Wine made from wild rose species is a traditional beverage which has been made in Poland for centuries. In Poland, three native species (R. canina L., R. gallica L., R. rubiginosa L.) and three introduced species (R. centifolia L., Rosa rugosa Thunb., R. damascena L.) are of medical importance ( Góra and Lis, 1996).
Ethnobotanical works cited by Łuczaj and Szymański report the use of Rosa canina L., the most common of the species from this section found in Poland. Dried rose fruits are used for the treatment of all illnesses caused by vitamin C deficiency, including diarrhea, weakened activity of the gastrointestinal tract, as well as to treat diseases of the kidneys, liver and bladder. In Polish phytotherapy, Rosa canina L. fruits are further used as an anti-flu, diuretic and cardiotonic agent ( Pawlaczyk et al., 2009 and Strzelecka and Kowalski, 2000).
Dry powder from Rosa canina L. false fruits is a widely used herbal remedy against arthritis. A review published by Chrubasik et al. (2006) and a meta-analysis by Christensen et al. (2008) investigating clinical evidence for the medicinal use of rose hip powder, report that consuming powdered rose hips from Rosa canina L. can result in a moderate reduction of pain in patients suffering from osteoarthritis ( Saaby et al., 2011). Daily consumption of 40 g of rose hip powder for 6 weeks has also been shown to significantly reduce cardiovascular risk in obese persons, by lowering systolic blood pressure and plasma cholesterol levels ( Andersson et al., 2012).
Rosa rugosa Thunb. (common names: Japanese rose, rugosa rose) occurs naturally in Eastern Asia from Okhotsk and southern Kamchatka to Korea and the northern parts of Japan and China. In Poland, Rosa rugosa Thunb. was introduced in 1960 ( Tokarska-Guzik, 2003). The species is scattered throughout Poland, especially in the southwestern regions, and is still spreading ( Weidema, 2006). It is found in dry meadows and thickets as well as at the edges of forest ( Tokarska-Guzik, 2003).
Rosa rugosa Thunb. is used to make preserves, jellies, and wines. Extracts from the flowers or hips are used in herbal medicines and vitamin products ( Weidema, 2006). It is also used in traditional Chinese medicine as an effective vasodilator and to improve microcirculation ( Xie and Zhang, 2012).
Fu et al. (2006) have demonstrated that compounds found in Rosa rugosa Thunb. flowers have inhibitory activity against HIV reverse transcriptase. A study of Xie and Zhang (2012) has further shown that extracts from the flowers can inhibit the angiotensin-converting enzyme.
The physiological functions of Rosaceae fruits may be partly attributed to the fact that they contain an abundance of compounds including phenolics, β-carotene, lycopene, ascorbic acid, tocopherol, bioflavonoids, fruit acids, tannins, pectin, sugars, organic acids, amino acids and essential oils ( Demir and Ozcan, 2001, Uggla et al., 2003, Uggla et al., 2005 and Ercisli, 2007).
The vitamin C content of wild roses exceeds that in other raw materials, by a factor of up to several dozens, while polyphenols (especially flavonoids, their most active group) stabilize this vitamin in food products (Grochowski, 1990, Demir and Ozcan, 2001 and Jaroniewski, 1992). It has been shown that juice obtained from R. rugosa fruits contains significant quantities of catechins and proanthocyanidins ( Oszmiański and Chomin, 1993 and Wilska-Jeszka et al., 1991). The synergistic activity of l-ascorbic acid and flavonoids, or the so-called sparing effect, has been observed by Saucier and Waterhouse (1999) and by Vinson et al. (2001). Brand-Williams et al. (1995) have studied the reaction of various antioxidants toward DPPH and have shown that the effects of particular compounds on this radical depend on their structure.
The two main objectives of our research were, firstly, to determine the concentration of biologically active compounds (polyphenols and l-ascorbic acid) in wines form Rosa canina L. and Rosa rugosa Thunb. and secondly, to assess their antioxidant capacity and possible mutagenicity or antimutagenicity.

2. Materials and methods

2.1. Chemicals and reagents

N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD), 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, DMSO and Folin–Ciocalteu reagent were purchased from Sigma–Aldrich (Poznan, Poland); MNNG from Fluka, St. Gallen, Switzerland; formic acid and HPLC grade methanol from J.T. Baker (Witko, Lodz, Poland); and gallic acid, syringic acid, chlorogenic acid, ferulic acid, p-coumaric acid, quercetin glucoside, quercetin rutinoside, and cyanidin-3-glucoside from Extrasynthese (Genay, France). HPLC grade water was obtained using an Aquinity E60 Lifescience TI system (membraPure GmbH, Bodenheim, Germany).

2.2. Plant material

Ripe fruits were collected in October from wild bushes growing in the Lodz region (central Poland, 18°36′E, 51°25′N, from 169 to 173 m) and transported to the laboratory. Five ≈1000 g samples of each rose species were enclosed in polyethylene bags and stored at −20 °C prior to use.

2.3. Winemaking procedure

Musts from Rosa canina L. and Rosa rugosa Thunb. were prepared as follows: having removed the stems (stalks), the fruits were thawed and crushed. Boiling water was added to the resulting pulp in a ratio of 1:1, and the temperature of the mixture was adjusted to 50–55 °C. The enzyme preparation Pektopol PT 400 (Jaslo, Poland) was added (2.6 g/kg of fruit pulp) and the temperature maintained in a range of between 50 and 55 °C for 2 h. After 2 h of enzymatic treatment, the must was pressed out and pitchings prepared. The must consumption in the pitchings reached 70%.
The pitchings were poured into flasks and then inoculated with the yeast Saccharomyces bayanus (0.4 g/L of pitching). Fermentation was performed at 25 °C and controlled using the gravimetric method by determining weight loss during the process. Vinification was performed in triplicate for each rose fruit sample.

2.4. Wine aging

After completing the fermentation process, the wine was decanted from the sediment and aged in bottles at 10 °C for a period of 3 months.

2.5. Determination of total phenolics using Folin–Ciocalteu reagent

Total phenolic content (TPC) was determined using the Folin–Ciocalteu method (Waterhouse, 2001).
To prepare the calibration curve, solutions of gallic acid at concentrations of between 0 and 500 mg/L were used. The R2 coefficient of the standard curve was 0.9892.
20 μL of the calibration solution (sample or blank) 1.58 mL of distilled water and 100 μL of F–C reagent were added to 7 mL tubes and the mixture stirred. After 30 s 300 μL of 20% sodium carbonate was added. The solution was left at room temperature for 1 h. The absorbance of the solution at 765 nm was measured against blank (0 mL solution) using a Cecil CE2041 spectrophotometer (Cecil Instruments Limited, Cambridge, UK). The must and pitching samples were diluted 20 times, while the wine samples were diluted 10 times. The results were expressed as mg of gallic acid per liter.

2.6. Determination of total flavonoids

Total flavonoids were determined using the method developed by Di Stefano et al. (1989).
First, 0.5 mL of twofold-diluted must, pitching or wine was loaded onto a Sep-Pak C18 cartridge (Waters Assoc.), initially activated using 2 mL of methanol and 5 mL of distilled water. After the bed had been washed with 0.5 mL of water, flavonoids were eluted using 5 mL of methanol, and the content was collected in a volumetric flask with a capacity of 10 mL.
The sample in the flask was acidified by adding of 0.01 mL of concentrated HCl and supplemented with methanol. The absorbance spectrum was recorded in a range of between 230 nm and 700 nm.
The results were expressed as mg (+)-catechin equivalents (CE) per liter of wine. To prepare the calibration curve, solutions of catechin were used in concentrations of between 0 and 300 mg/L. The R2 coefficient of the standard curve was 0.9975.

2.7. Determination of ascorbic acid

Ascorbic acid was determined according to PN-90/A-75101/11. This method involves the oxidation of l-ascorbic acid in an acidic medium to form dehydroascorbic acid with 2,6-dichloroindophenol. An excess of 2,6-dichloroindophenol was extracted using xylene and determined spectrophotometrically at a wavelength of 500 nm. The content of ascorbic acid was calculated on the basis of a calibration curve with l-ascorbic acid, and the results expressed as mg of ascorbic acid per liter of beverage. The R2 coefficient of the standard curve was 0.9988.

2.8. HPLC polyphenol analysis

Samples were filtered through a 0.22 μm membrane prior to analysis and injected into the HPLC system. HPLC-DAD analyses were performed using a Finnigan Surveyor equipped with autosampler, diode array detector (Finnigan Surveyor-PDA Plus), and ChromQuest 5.0 chromatography software (Thermo Fisher Scientific Inc, Waltham, MA, USA). Separation was performed on a LiChrospher RP 18-5 (Hichrom, Berkshire, UK) (250 mm × 4.6 mm, 5 μm packing) protected with a guard column (LiChrospher guard cartridge (Hichrom, Berkshire, UK), 10 mm × 4.6 mm, 5 μm packing). The elution conditions were as follows: 0.9 mL/min flow rate; oven temperature 25 °C; solvent A, water/formic acid (95:5 v/v); solvent B, methanol. Elution began with linear gradients from 10% to 30% B for 2 min, the same for 6 min; from 30% to 35% B for 5 min; from 35% to 50% B for 7 min; from 50% to 70% B for 2 min; and from 70% to 80% B for 8 min. The column was then washed and re-equilibrated. The injection volume for all samples was 50 μL. The calibration curve was established using an HPLC grade gallic acid commercial standard (Extrasynthese, Z.I. Lyon Nord, France) to quantify polyphenols at 280, 320 and 360 nm. To prepare the calibration curve, solutions of gallic acid in concentrations from 0 to 500 mg/L were used. The R2 coefficient of the standard curve was 0.9917.

2.9. ABTS•+, DMPD, DPPH TEAC assays

2.9.1. ABTS assay

The ABTS assay is based on the ability of antioxidants to scavenge the long-lived radical cation ABTS (Re et al., 1999).
ABTS•+ was generated by mixing an aqueous solution of ABTS with a solution of potassium persulfate to achieve a final concentration of 7.0 mM ABTS•+ and 2.45 mM sodium persulfate. This solution was kept in the dark at room temperature for 16 h before use. The ABTS•+ solution was then diluted with ethanol (approximately 1:90, v/v) to obtain an absorbance reading of 0.7 at 734 nm. Then 0.1 mL of appropriate dilutions (determined in preliminary experiments) of the wines were transferred to test tubes containing 3.9 mL of ABTS•+ solution. The solutions were mixed, and after 6 min absorbance was measured at 734 nm. The samples were tested in triplicate and the percentage of inhibition (% I) was calculated using the following formula:
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The results were also expressed as Trolox-equivalent antioxidant capacity (TEAC) values (mM Trolox/g extract), defined as the amount of Trolox (in mM) that exhibited the same antioxidant activity as 1 g of wine fractions.

2.9.2. Measurement of antioxidant ability by the DMPD method (Fogliano et al., 1999)

100 mM DMPD solution was prepared by dissolving 209 mg of DMPD in 10 mL of deionized water. 1 mL of this solution was added to 100 mL of 0.1 M acetate buffer, pH 5.25, and the colored radical cation (DMPD•+) was obtained by adding 0.2 mL of 0.05 M ferric chloride solution (to a final concentration of 0.1 mM). 2 mL of this solution was immediately placed in a cuvette and its absorbance measured at 505 nm. 0.1 mL of each diluted wine sample was added and absorbance measured at 505 nm after 10 min. The buffered solution was placed in a reference cuvette.
A dose–response curve was derived for Trolox by plotting absorbance at 505 nm as a percentage of the absorbance of an uninhibited radical cation solution (blank) according to the equation:
A505 inhibition (%)=(1−At/A0)×100,
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where A0 is the absorbance of the uninhibited radical cation and At is the absorbance measured 10 min after the addition of antioxidant samples.
The antioxidant activity of wine samples was expressed as TEAC values (mM Trolox/g extract), defined as the amount of Trolox (in mM) that exhibited the same antioxidant activity as 1 g of wine fractions.

2.9.3. DPPH assay (Sanchez-Moreno et al., 1998)

This method involves measuring changes in absorbance at a wavelength of λ = 515 nm at different time intervals, until the reaction reaches minimum absorbance. The violet color of DPPH in methanol (1,1-diphenyl-picrylhydrazyl) decreases as the degree of reduction increases. The percentage of reduced DPPH was calculated from the formula:
% red. DPPH⋅+=(1−At/A0)×100 [%]
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The number of millimoles of Trolox solution equivalent to the antioxidant activity of 1 dm3 of wine was calculated from the calibration curve.

2.10. Antioxidant capacity in linoleic acid emulsion

The ferric thiocyanate (FTC) method reported by Larrauri et al. (1996) was used with slight modifications.
A mixture of 0.5 mL wine, 0.5 mL linoleic acid emulsion, 0.1 mL sodium phosphate buffer (pH = 7) and 5 mL distilled water was placed in a tube with a screw cap, then shaken and placed in an oven at 40 °C in the dark. A control without an extract sample was used. To 0.1 mL of this solution were added 9.7 mL of ethanol and 0.1 mL of 300 g/L ammonium thiocyanate. Absorbance was measured against a reagent blank at 500 nm every 24 h (t) exactly 3 min after the addition of 0.1 mL of ferrous chloride to 35 g/L HCl of the reaction mixture, until the day after the absorbance of the control reached maximum. The oxidation index (OI) and antioxidant activity (AA) were calculated as
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2.11. Antimutagenic activity of wines from Rosa rugosa Thunb. and Rosa canina L.

The antimutagenic effect of wines from Rosa rugosa Thunb. and Rosa canina L. was determined using the method described by Maron and Ames (1983). The Ames test employs two cultures of Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium), designated as TA98 and TA100 ( McCann et al., 1975). In our study, MNNG (Fluka, St. Gallen, Switzerland) was used as a direct-acting mutagen. MNNG does not require any metabolic activation with liver fraction S9 to induce a mutagenic effect ( Lankaputhra and Shah, 1998). The concentration of MNNG solution in the DMSO (Sigma, St. Louis, MO, USA) was 10 μg/test for experiments with Salmonella Typhimurium TA98 and 0.1 μg/test for experiments with Salmonella Typhimurium TA100. These doses of the mutagen were determined on the basis of experimental curves presenting the relationship between the quantity of His+ revertants and mutagen concentration.
For Salmonella Typhimurium TA98 and TA100 respectively, the applied mutagen doses caused the appearance of 200 and 2000 colonies of His+ revertants. Wine samples were filtered through 0.2 μm membranes (Millipore, Cork, Ireland). Wine doses of 10.0, 20.0, 50.0, and 100.0 μL/plate were mixed with 0.1 mL samples of Salmonella Typhimurium overnight cultures (cell density of 4.5 · 109 CFU/mL) and aliquots of MNNG (10 μg/plate for TA98 and 0.1 μg/plate for TA100) and incubated at 37 °C for 20 min before adding 2 mL of top agar. The mixtures were then spread on minimal essential agar plates and incubated for 48 h at 37 °C in darkness, after which the Salmonella His+ colonies were counted. All samples were prepared in triplicate. The decrease in the number of mutations was calculated using the equation:
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where N0 is the number of Salmonella His+ colonies (corrected for the number of spontaneous revertants) visible on plates containing samples with MNNG without rose hip wine, and N1 is the number of Salmonella His+ colonies (corrected for the number of spontaneous revertants) visible on plates containing samples supplemented with MNNG and rose hip wine. The results were corrected for spontaneous reversion. The number of spontaneous His+ revertants was 33 ± 7 in Salmonella TA98 and 198 ± 32 in Salmonella TA100. The MNNG concentration was 10.0 and 0.1 μg/plate in Salmonella Typhimurium TA98 and TA100, respectively.

2.12. Statistical analysis

All measurements were performed in triplicate and the results presented as mean values ± standard deviations (SD). Using STATISTICA 10 PL software (StatSoft, Cracow, Poland), the standard deviation was determined and the ANOVA test conducted, assuming a significance level of 0.05.

3. Results and discussion

3.1. Total polyphenols and flavonoids and ascorbic acid content

The measured concentrations of polyphenols in musts from R. canina and R. rugosa fruits respectively were 9007 ± 345 and 7400 ± 520 mg/L GAE ( Table 1). This compares with a study by Gao et al. (2000), which found a TPC of 59–122 mg GAE/g DW in 8 rose species (Rosa moschata had the lowest content, R. villosa hybrid the highest). R. canina from Chile was found to have a TPC from 60 to almost 63 mg GAE/g DW. A study by Ercisli (2007) found the total phenolics in six Rosa species to be between 73 mg GAE/g DW (Rosa villosa) and 96 mg GAE/g DW (Rosa canina L.). The TPC of Rosa rugosa Thunb. fruits from Poland, as studied by Hallmann et al. (2011), was found to be 176 mg/100 g FW. A study by Oszmiański and Chomin (1993) also revealed high flavonoid content in Rosa rugosa Thunb.
Table 1. Total polyphenol content in musts, pitchings, and wines [mg/L GAE].
SampleRosa rugosaRosa canina
Must7400 ± 5209007 ± 345*
Pitching4200 ± 2005670 ± 139*
Wine after fermentation3389 ± 2453990 ± 256*
Wine after aging2786 ± 1563456 ± 134*
All values are presented as means ± SD (n = 3).
*
Statistically significant difference between Rosa species, ANOVA test (p < 0.05).
Table options
The results presented in this study clearly demonstrate that polyphenol levels depend not only on the species but also on climatic conditions. This is in agreement with Scalzo et al. (2005), who highlight the importance of the plant genotype (species and variety within species) for in determining the phenolic compound content. The results of a study by Meng et al. (2012), similarly show that wines made from Cherokee rose (Rosa laevigata Michx.) have higher levels of total phenolics, total flavonoids and oligomeric proanthocyanidins than spine grape wines and Cabernet Sauvignon wines, while their TPC was measured at 2529 ± 16.9 mg/L GAE. The total phenolic content of the wines in our study was found to be in a range of 2786–3456 and 3389–3990 mg/L GAE for aged and young wines, respectively ( Table 1). Total flavonoid content in the final wines was 2666 mg/L for Rosa rugosa Thunb. and 3008 mg/L for Rosa canina L. ( Table 2).
Table 2. Total flavonoid content in musts, pitchings and wines [mg/L].
SampleRosa rugosaRosa canina
Must7000 ± 4208700 ± 345*
Pitching3890 ± 1205070 ± 339*
Wine after fermentation3089 ± 2453740 ± 253*
Wine after aging2666 ± 1563008 ± 134*
Number of analyzed samples n = 3. All values are presented as means ± SD (n = 3).
*
Statistically significant difference between Rosa species, ANOVA test (p < 0.05).
Table options
Rosa rugosa and Rosa canina wines thus exhibit higher TPC than wines made from Cherokee rose (Rosa laevigata Michx.).
Rose hips are well known for their high ascorbic acid content (Barros et al., 2011, Demir and Ozcan, 2001, Ercisli, 2007, Gao et al., 2000, Hallmann et al., 2011, Kazaz et al., 2009 and Rosu et al., 2011). For instance, the ascorbic acid content of Rosa canina L. collected in two different locations in Turkey varied from 2365 to 2712 mg/100 g ( Demir and Ozcan, 2001). In a study by Kazaz et al. (2009), the ascorbic acid contents of Rosa canina L. fruit and fruit flesh from Turkey were 411 and 2200 mg/100 g, respectively, and were 1.23 and 4.03 times higher than in the corresponding parts of Rosa damascena Mill. Our research indicates that the musts from Rosa rugosa and Rosa canina are also rich in l-ascorbic acid ( Table 3). l-ascorbic acid content in Rosa canina was found to be between 1571 ± 145 and 3182 ± 420 mg/L, and 2-fold higher in musts from Rosa rugosa Thunb.
Table 3. l-ascorbic acid content in musts, pitchings and wines [mg/L].
SampleRosa rugosaRosa canina
Must3182 ± 4201571 ± 145*
Pitching1546 ± 120700 ± 39*
Wine after fermentation1348 ± 245653 ± 25*
Wine after aging1200 ± 156600 ± 34*
Number of analyzed samples n = 3. All values are presented as means ± SD (n = 3).
*
Statistically significant difference between Rosa species, ANOVA test (p < 0.05).
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The final concentration of ascorbic acid in the investigated wines was 1200 and 600 mg/L for Rosa rugosa Thunb. and Rosa canina L., respectively. This indicates that in finished wines obtained from wild rose initial vitamin C content remains 50–70%.
The results for total polyphenols and flavonoids and ascorbic acid content clearly show that the investigated wild rose wines are some of the richest sources of antioxidants. The level of vitamin C in 100 mL of wine completely covers the daily dietary requirement.

3.2. Polyphenolic profile of wild rose wines

There is no information in the literature concerning the phenolic profile of rose hip wines. The concentrations of individual polyphenolic antioxidants were determined using HPLC. Some of the flavonols identified (e.g. quercetin glucoside) occurred in wines made from both rose species, while others (e.g. quercetin rutinoside) were present only in wines from Rosa rugosa Thunb.
The content of phenolic acids was also measured, the most abundant being gallic acid (Table 4). Cyanidin-3-glucoside was not found in any of the wines tested. As shown by previous studies by the authors, this compound is unstable during the process of fermentation (unpublished results). Moreover, polymerization of anthocyanins is known to occur during the aging process.
Table 4. Polyphenols in rose hip wines [mg/L].
CompoundRosa caninaRosa rugosa
Gallic acidnd144.02 ± 3.92*
Chlorogenic acid40.91 ± 1.354.45 ± 0.13*
Ferulic acidTrace0.01 ± 0.00*
Syringic acid1.25 ± 0.040.02 ± 0.00*
p-Coumaric acidTrace0.02 ± 0.00*
Unidentified compound Rt = 8.1a83.62 ± 0.9832.94 ± 1.01*
Unidentified compound Rt = 8.55b
(catechin derivative)		nd67.00 ± 2.25*
Unidentified compound Rt = 9.61cnd0.55 ± 0.01*
Quercetin rutinosidend14.55 ± 0.47*
Quercetin glucoside66.65 ± 2.6734.04 ± 1.24*
Unidentified compound Rt = 24.6d46.18 ± 1.3942.03 ± 1.10*
All values are presented as means ± SD (n = 3); unidentified compounds: a Uvmax = 264; b Uvmax = 280; c Uvmax = 286; d Uvmax = 277; nd–not detected (LOD 0.004 mg/L)
*
Statistically significant difference between Rosa species, ANOVA test (p < 0.05).
Table options
In studies by other authors, wild rose hips were found to contain all polyphenols identified in the present study of wines. Nowak (2007) found quercitrin and kaempferol 3-O-rhamnoside in rose fruits. Mikulic-Petkovsek et al. (2012) using HPLC-MSn identification of flavonol glycosides found quercetin galactoside, glucuronide, and arabinofuranoside, as well as kaempferol rutinoside and coumaroylglucoside in dog rose (R. canina) fruits. Other phenols found in Rosa canina L. fruits include: cyanidin-3-glucoside, taxifolin glycosides, eriodictyol, phloridzin, myricetin, gallic acid derivatives, ellagic and p-hydroxybenzoic acids ( Hvattum, 2002, Tumbas et al., 2012 and Zocca et al., 2011).
Zocca et al. (2011) found in extracts of dog rose (Rosa canina L.) derivatives of catechin and epicatechin, including epigallocatechin at almost 2500 mg/L, epicatechin gallate at 37.69 mg/L and ellagic acid at over 170 mg/L. However, as catechins are not very stable during the fermentation process, significantly lower contents of this group of compounds were found in our study of wines. Hallman et al. (2011) report that Rosa rugosa Thunb. fruits from Poland contain quercetin glucoside, kaempferol glucoside, and myricetin.
Losses and changes in antioxidant compounds occur at each stage of wine production.
The most substantial losses of vitamin C and polyphenolic compounds are incurred while the musts are being obtained, for instance during enzymatic and thermal processing of raw materials and technological procedures such as filtration, pasteurization, physicochemical stabilization and bottling.

3.3. Antioxidant capacity of rose hip wines

One method is usually insufficient to evaluation of the antioxidant activity of a complex substance.
In our study, three different assays (with ABTS, DPPH, and DMPD radicals) were used to evaluate the antioxidant properties of wines from R. rugosa and R. canina. High antioxidant activity was observed in all assays, ranging from 8 to 13 mM in terms of Trolox equivalent antioxidant capacity (TEAC) ( Table 5). Some substantial and significant differences between ABTS and DMPD radicals were found in the tested wines.
Table 5. Antioxidant capacity of wines from Rosa rugosa and Rosa canina.
Method of determinationRosa rugosaRosa canina
TEAC/ABTS10.4 ± 0.913.5 ± 0.6*
TEAC/DMPD13.4 ± 0.610.5 ± 0.8*
TEAC/DPPH8.9 ± 0.88.6 ± 0.5
LDL %20.1 ± 0.321.4 ± 0.2*
All values are presented as means ± SD (n = 3).
*
Statistically significant difference between Rosa species, ANOVA test (p < 0.05).
Table options
The next step was to evaluate the antioxidant activity of rose hip wines using the ferric thiocyanate method based on the inhibition of linoleic acid oxidation. Linoleic acid is a polyunsaturated fatty acid, which, similarly to LDL, undergoes oxidation in the human body and is used as a model in in vitro studies of fatty acids to determine the mechanisms of lipid oxidation which lead to the formation of free radicals.
Based on the absorbance measured, oxidation indices were measured and antioxidant activity calculated, expressed as a percentage of the autoxidation delay of linoleic acid. The oxidization indices of rose hip wines were observed over 72 h and found to be 20% and 21% for Rosa rugosa Thunb. and Rosa canina L., respectively ( Table 5).
A study by Gao et al. (2000) investigating the contribution of three different antioxidant fractions using an ABTS assay, showed the total antioxidant capacity of phenolics, ascorbic acid and lipophilic compounds to be slightly lower than those of crude extracts. The phenolic fraction made a major contribution to total activity (about 75%), followed by ascorbic acid (around 17%). This is consistent with our results for TEAC/ABTS. Wines from Rosa canina L. were found to have a significantly higher TEAC/ABTS value (13.5) and to contain higher levels of total polyphenols and flavonoids than wines from Rosa rugosa Thunb. However, they had only slightly more than half of the vitamin C content of the latter.
The results of a study by Brand-Williams et al. (1995) indicate that ascorbic acid is one of the fastest reacting antioxidants. This is also confirmed by our findings. The values for DPPH in Rosa rugosa Thunb. wines were slightly higher than those of R. canina. The level of ascorbic acid in wines made from R. rugosa was also found to be higher.

3.4. Antimutagenic activity of rose hip wines

The antimutagenic activity of rose hip wines was determined in Salmonella Typhimurium TA98 and Salmonella Typhimurium TA100 using the Ames test. Wines from Rosa rugosa Thunb. reduced the intensity of mutations induced by MNNG in a dose-dependent manner by 16–48% in Salmonella Typhimurium TA98 and by 12–52% in Salmonella Typhimurium TA100 ( Table 6).
Table 6. Antimutagenic activity of Rosa rugosa wine in Salmonella Typhimurium TA98 and Salmonella Typhimurium TA100 as determined by the Ames test.
V (wine) [μL/plate]TA98
TA100

His+ mutants/plate ± SDMutation reduction [%]His+ mutants/plate ± SDMutation reduction [%]
0.0137 ± 1601783 ± 1640
10.0135 ± 3201572 ± 18212
20.0114 ± 12161343 ± 16124
50.090 ± 9*341234 ± 210*31
100.071 ± 7*48854 ± 86*52
Number of analyzed plates n = 3.
*
Statistically significant difference from control (sample without wine), ANOVA test (p < 0.05).
Table options
Wines from the dog rose (Rosa canina L.) showed a greater ability to reduce mutations ( Table 7). Statistically significant differences (p = 0.05) in relation to the control sample were observed with even the lowest dose of wine (10 μL/plate).
Table 7. Antimutagenic activity of Rosa canina wine in Salmonella Typhimurium TA98 and Salmonella Typhimurium TA100 as determined by the Ames test.
V (wine) [μL/plate]TA98
TA100

His+ mutants/plate ± SDMutation reduction [%]His+ mutants/plate ± SDMutation reduction [%]
0.0137 ± 1601783 ± 1640
10.060 ± 2*56767 ± 10*56
20.037 ± 6*72542 ± 22*69
50.040 ± 9*69403 ± 73*77
Number of analyzed plates n = 3.
*
Statistically significant difference from control (sample without wine), ANOVA test (p < 0.05).
Table options
In contrast, the addition of 10 μL of wine from Rosa rugosa Thunb. did not have a biocidal effect on the TA98 strain. Here, statistically significant differences were observed only after the addition of 50 μL of wine, and its ability to reduce mutations at this concentration was approximately 2-fold lower than that of wines from Rosa canina L.
A study by Karakaya and Kavas (1999) concerning, the antimutagenic activity of Rosa canina L., among other plant juices, leaves and seeds, showed that rose hips boiled at 100 °C for 10 min and stewed for a further 10 min were not mutagenic in Salmonella Typhimurium TA100. Rose hips decreased sodium azide mutagenicity by 44%.
Shinohara et al. (1988) observed a positive correlation between the antimutagenic activity and polyphenol content of vegetables and fruits, which is consistent with our results. Phenolics can create complexes or interact non-enzymatically with mutagens, thus reducing their bioavailability (Mejia et al., 1999).
In our study, wines from Rosa canina L. were found to contain higher levels of total polyphenols and flavonoids, but only about half the level of vitamin C. These results may indicate a lack of correlation between antimutagenic activity and the vitamin C content in these rose hip wines.

4. Conclusions

The results of our study show that wild rose wines are a rich source of antioxidants. Both, wines from Rosa rugosa Thunb. and Rosa canina L. contain high levels of polyphenols and l-ascorbic acid. According to Regulation (EU) no. 1160/2011 the recommended daily dosage of vitamin C for the adult male is 80 mg: 70 mL of wine from Rosa rugosa Thunb. or 140 mL of wine from Rosa canina L. would provide this level of vitamin C. However, due to its ethanol content, wild rose wine should not be treated as the sole source of this vitamin.
The high antioxidant activity of wild rose wines has been confirmed by different assays (using ABTS, DPPH, and DMPD radicals). Moreover, the wines have been found to reduce the intensity of mutations induced by MNNG in a dose-dependent manner.

Acknowledgments

This research was financially supported by Polish Grant2P06T 07627. The authors would like to thank J. Laskowska, Ph.D. for analysis of antioxidant activity.

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