Available Online: http://www.ejpau.media.pl/volume17/issue4/art-08.html
CHANGES IN POLYPHENOL AND ANTHOCYANIN CONTENT AND ANTIOXIDANT ACTIVITY IN METHANOL AND WATER EXTRACTS PREPARED FROM STORED DRIED WRINKLED ROSE (ROSA RUGOSA THUNB.) PETALS
Agnieszka Zawi¶lak, Magdalena Michalczyk
Department of Refrigeration and Food Concentrates, University of Agriculture in Kraków, Poland
The aim of the present study was to follow changes in the content of total
polyphenols and anthocyanins as well as antioxidant activity in methanol and
water extracts prepared from dried wrinkled rose (Rosa rugosa) petals
stored for a year at 21 ± 1ºC. Polyphenols were evaluated
by Folin-Ciocalteau assay and anthocyanins were determined by the pH-differential
method. Antioxidant properties were assayed by DPPH radical scavenging activity
and by measuring the reducing power of both extracts. The extracts were a rich
and stable source of polyphenols (800–1300 mg/100 g) and anthocyanins (85–150
mg/100 g). Extract antioxidant activity expressed as EC50 value did
not change significantly during storage of dry material and was quite
g/g DPPH). Reducing power was strongly correlated with EC50 (r = -0.86,
p < 0.01). The
antioxidant power of both extracts correlated more closely with polyphenol content
than with anthocyanins. Extracts of methanol showed better antioxidant properties
than those of water, probably due to higher polyphenol and anthocyanin content.
The aim of the present study was to follow changes in the content of total polyphenols and anthocyanins as well as antioxidant activity in methanol and water extracts prepared from dried wrinkled rose (Rosa rugosa) petals stored for a year at 21 ± 1ºC. Polyphenols were evaluated by Folin-Ciocalteau assay and anthocyanins were determined by the pH-differential method. Antioxidant properties were assayed by DPPH radical scavenging activity and by measuring the reducing power of both extracts. The extracts were a rich and stable source of polyphenols (800–1300 mg/100 g) and anthocyanins (85–150 mg/100 g). Extract antioxidant activity expressed as EC50 value did not change significantly during storage of dry material and was quite high (0.9–1.5 g/g DPPH). Reducing power was strongly correlated with EC50 (r = -0.86, p < 0.01). The antioxidant power of both extracts correlated more closely with polyphenol content than with anthocyanins. Extracts of methanol showed better antioxidant properties than those of water, probably due to higher polyphenol and anthocyanin content.
Key words: Rosa rugosa, dried petals, polyphenols, anthocyanins, antioxidant activity.
High levels of fruit and vegetable consumption lower the risk of morbidity caused by cancer and cardiovascular diseases . This protective health effect had for some time been attributed to vitamins C and E as well as to β-carotene, which were considered the main antioxidants present in these food groups. However, clinical research, particularly controlled attempts of supplementing diets with antioxidant vitamins, failed to demonstrate beneficial effects on health [19, 33]. It may therefore be assumed that other components in foods of plant origin must be responsible for the antioxidant activity of such foods. Among the mass of phytonutrients supplied to the body by plant-based foods, polyphenols appear to be the most important group of health-promoting constituents . Phenolic compounds form a very diverse group of secondary metabolites whose activity is dependent on their chemical structure. There are scientific studies confirming the antioxidant, anticarcinogenic, antimutagenic, antibacterial and antiallergic properties of this group of compounds [16, 24, 36].
Plants of the Rosaceae family have enormous possibility to become a source of health-promoting components. One species belonging to this group, Rosa rugosa,contains a number of bioactive constituents. Wrinkled rose (Rosa rugosa) was first described by Thunberg in 1784 in a work entitled Flora Japonica sisteus plantas insolarum japonicarum. This plant is native to eastern Asia and was introduced into European and North American gardens in the middle of the nineteenth century . Apart from being used in perfume production, wrinkled rose was cultivated in China for use in traditional medicine, no doubt due to the biological activity of components found in the petals. When dried, the petals were used as an antidiarrhoeal and haemostatic agent . Every part of the Rosa rugosa plant contains large amounts of phytochemicals, especially phenolic constituents [6, 10, 17]. The petals are rich in hydrolysable tannins, from which their medicinal properties are believed to derive. Two ellagitannins present in wrinkled rose petals, tellimagrandin I and pedunculagin, have been demonstrated to inhibit human immunodeficiency virus type-1 reverse transcriptase (HIV-1RT) in vitro [2, 14, 17]. Polyphenols isolated from R. rugosa petals have also been shown to have antioxidant properties [28, 29]. Cho et al.  demonstrated that certain phenolic compounds from wrinkled rose petals inhibited lipid peroxidation. Water extracts of dried flowers increased the activity of antioxidant enzymes and their gene expression, while at the same time reducing lipid peroxidation.
Since wrinkled rose not only contains large amounts of phytonutrients but also exhibits attractive sensory qualities, it could be used as homogenized pulp in food products, preserved with sucrose, or in dried form as a basis for extracts. Therefore the content and stability of specific components during storage is of the utmost importance.
The aim of this study was to determine the levels of polyphenols, anthocyanins and antioxidant activity in methanol and water extracts of dried wrinkled rose petals and to evaluate the stability of these values in mentioned extracts prepared from dried products stored for one year at ambient temperature. Up till now, there has been no data available concerning properties of extracts from long-time stored dried Rosa rugosa petals.
MATERIALS AND METHODS
Wrinkled rose (Rosa rugosa) petals were obtained from a plantation located in the north-west suburbs of Kraków (Poland). Petals were harvested, sorted and dried in a convection dryer at 50ºC for 36 hours (final moisture level: 4%). They were then sealed in glass jars and stored for a year without light at ambient temperature (21 ± 1ºC).
Methanol extract. 100 ml of methanol/HCl 0.5% (80:20 v/v) was added to 1.608 g of dried petals (equivalent to 10 grams of fresh petals based on dry matter), homogenized (Heidolph Diax 900) and left for 15 minutes. The extract was then centrifuged (3000 rpm, 15 min). The supernatant solution was filtered into a 200 mL volumetric flask and the residue was extracted twice more, using 50 mL of the above-mentioned acidified methanol each time. The filtration procedure was repeated until the volumetric flask was filled.
Water extract. 200 mL of boiling distilled water was added to 1.608 g of dried petals and the mixture stirred with a glass rod for 5 minutes. The extract was then filtered and cooled to 20ºC.
Total phenolic content was determined by means of the Singleton et al.  method with Folin-Ciocalteau’s reagent. Absorbance was measured (Cecil CE 9500) at 750 nm and results were expressed as mg of gallic acid per 100 g of fresh weight.
Total anthocyanin content was determined using the pH-differential method described by Lee et al. . Absorbance was measured at 510 and 700 nm and total anthocyanin content was calculated as mg of cyanidin-3-glucoside per 100 g of fresh weight.
The DPPH radical scavening activity of rose petal extract was measured using the method described by Brand-Williams et al. . The percentage of DPPH remaining in the reaction solution at steady state was calculated and EC50 values were counted on the basis of these results.
The reducing power was determined according to Yen & Chen . Absorbance was measured at 700 nm; the greater the increase in absorbance, the stronger the reducing power of the reaction mixture.
Polyphenol oxidase activity was determined according to Cano et al.  with use of catechol (in fresh material, immediately after drying and the last day of experiment). Enzyme activity was calculated from the changes in slope of the linear part of the absorbance (measured at 420 nm) curve during the first 3 minutes of the reaction. The results were expressed as ΔA420min-1g-1 of fresh weight.
Microbiological analysis was conducted according to Burbianka et al. . Total bacterial count was determined on plate count agar (PCA) incubated at 30ºC for 72 hours. Yeasts and moulds were determined applying maltose agar (Oxoid) with 4-day incubation at 25 ± 1ºC (pH 3.5).
The measurements were carried out every 42 days and taken in triplicate. Every time the fresh extracts from stored wrinkled rose petals were prepared and analysed. The Pearson correlation coefficient (r) was used as the basic magnitude to measure the degree of correlations between the values of experimental variables. The statistical significance of the difference between mean values was determined on the basis of Student’s t-test.
RESULTS AND DISCUSSION
Drying is one of the oldest methods of food preservation and generally applied to extend the shelf life of plants used for pharmaceutical purposes. Drying method and parameters (temperature, duration, etc.) should be carefully selected for specific raw materials in order to retain valuable components and limit energy costs. The water content in biological material is reduced during drying, concentrating chemical compounds and causing changes in the reaction environment. This can give rise to various kinds of chemical or enzymatic conversions as isolating water layers are replaced by air, leading to oxidative reactions and a deterioration in the quality of the raw material. At the same time, lower water activity provides dried products with microbiological stability. However, such products must be properly packed and stored in suitable conditions [4, 23, 34].
In the present study the plant material was dried at 50ºC to shorten the time of the process and quickly lower water activity as well as to limit the growth of microorganisms and inhibit polyphenol oxidase. Drying temperature between 50 and 60ºC on the basis of several studies was recognized as the most practicable for preservation of many medical plants .
The present investigation found that storage time of dried wrinkled rose petals had no effect on polyphenol and anthocyanin content, as well as on antioxidant activity, any data fluctuations observed probably being random (Fig. 1).
Figure 1. Effect of storage time on polyphenol (a) and anthocyanin (b) content as well as EC50 (c) and reducing power (d) in extracts prepared from dried and stored wrinkled rose (Rosa rugosa) petals
Most of the rose petals, which were investigated by other Authors, including Rosa rugosa petals, were dried in ambient temperature [1, 15, 30, 31, 41]. Nevertheless, the temperature implemented for drying in this study (50ºC) did not have an adverse effect neither on total polyphenol content nor anthocyanin amount both in the water and methanol extracts. Olech and Nowak  extracted antiradical ingredients from dried wrinkled rose petals with the use of different extraction procedures and diverse solvents. Total polyphenol content in hot water extracts (100ºC) and in alcoholic ones (ambient temperature) were similar to those presented in this study. In the next work  concerning biological activity of teas and tinctures prepared from wrinkled rose petals the results of polyphenol contents in both extracts’ types were comparable to those found earlier. However, the EC50 values obtained by Olech et al.  were higher than acquired in current study. It can be caused by changes in the structure of polyphenols as the result of elevated temperature application for drying procedure in the present study, and thus forming new compounds with higher antioxidant activities. Nonetheless, there is an agreement between results of Olech et al.  and ours in tendency that EC50 characterizing water extracts had higher values than those of alcohol ones. In turn, polyphenol content determined in methanol extracts prepared from dried Rosa damascena petals  coming from different plantations were influenced by the year of growing season. Data obtained for polyphenols in 2010 were similar to those determined in this work. However, the anthocyanin content found in current study was higher than those determined in extracts from dried Rosa damascena petals.
Vinokur et al.  measured polyphenol content in hot water infusions of twelve rose cultivars and its mean value was in accordance with our results. Anthocyanin concentrations were mostly lower than fount in the present study.
Tsai et al.  examined changes in antioxidant activity and followed fluctuations in polyphenol and anthocyanin content in dried hibiscus petals (Hibiscus Sabdariffa L.) processed and stored at different temperatures. Three drying temperatures (25, 50 and 75ºC) and two storage temperatures (20 and 40ºC) were used in their experiment. Drying at 25ºC combined with storage at 20ºC resulted in the smallest changes in anthocyanin content. However, water extracts obtained from material dried at 50ºC and stored at 20ºC (the same conditions as used in the present work), led to polyphenol loss of approximately 13% (determined as delfinidyno-3-sambubiozyd) after 15 weeks of storage, although this did not cause serious changes in antioxidant activity in the examined extracts, probably due to the transformation of monomeric anthocyanins into polymeric polyphenol compounds.
Garden sage (Salvia officinalis) dried at temperatures not exceeding 50ºC retained ethereal oils and did not change colour . Larrauri et al  examined the effect of three drying temperatures (60, 100 and 140ºC) on the polyphenol content and antioxidant activity of grape skin pomace. Only when the lowest of these temperatures was applied were there no significant changes in the analysed material. However, high drying temperatures did not always cause losses of valuable components or deterioration of the material. The kind of biological material to be dried is also of great importance.
Insignificant changes in polyphenol and anthocyanin content observed in this work during storage of dried wrinkled rose petals could have been caused by very low polyphenol oxidase activity assessed immediately after drying and the last day of storage; 0.029 ± 0.001 ΔA420min-1g-1 and 0.0266 ± 0.001 ΔA420min-1g-1, respectively. For this reason, the transformations in polyphenol structures could have been minimized. The drying temperature applied in this study (50ºC) probably reduced initial polyphenol oxidase activity (0.20 ± 0.002 ΔA420 min-1g-1) determined in fresh petals. The effect of different drying temperatures on polyphenol oxidase activity in various morphological parts of oregano (Origanum vulgaris spp plant. hirtum) was examined by Doğan et al. , who reported a reduction in such activity at temperatures higher than 30ºC resulting from thermal denaturation of the enzyme protein. Other studies also showed similar tendency [12, 26]. In addition, high stability of analyzed in this work dried wrinkled rose petals may also resulted from the low microbiological contamination of this product (TBC 1.9 × 103 CFU/g, yeasts and moulds 3.2 × 103 CFU/g), which values did not change during storage. It means that storage conditions were properly designed.
Results reported in Table 1 show that polyphenol and anthocyanin content were higher in methanol than in water extracts, and using the Student’s t-test to analyse mean values indicated significantly higher antioxidant activity in the methanol extract. The same trend was observed by several Authors [1, 18, 30, 31].
Table 1. Results of Student’s t-test comparing mean values of determinants characteristic for extracts prepared from dried wrinkled rose petals
X ± ΔX
High selectivity is one of the most important features of a good solvent. Selecting an appropriate solvent for polyphenols may be difficult because of the structural diversity of this group of compounds and their possible interactions with other components in the analysed material. There are several solvents which are commonly used to extract polyphenols and anthocyanins from the biological matrix but the most common solvent is an acidified solution of methanol [3, 13, 32], which was used in the present work, yielding extracts rich in polyphenols and anthocyanins with high antioxidant activity. Water extracts were only slightly inferior with regard to analysed values; however, the use of methanol is more in keeping with the principle: “similica similibus solvuntur”.
The correlation coefficient between EC50 and reducing power counted together for both extracts was r = 0.86 (p < 0.01). When determined separately, the correlation coefficients for water and methanol extracts were: r = 0.78 and r = 0.77 (p < 0.01), respectively. It can therefore be assumed that the results for reducing power indirectly confirm antioxidant activity measured as EC50. Table 2 presents the correlation coefficients between EC50, reducing power and polyphenol and anthocyanin content. On the basis of these data, polyphenol content appears to have had a greater effect on EC50 than anthocyanin concentration. Similar results were obtained by Vinokur et al. , Ginova , Olech et al.  Benvenuti et al. .
Table 2. Correlation coefficients between antioxidant activity (EC50 and reducing power) and polyphenol and anthocyanin content in assayed extracts
Reducing power as determined in this study also depended on anthocyanin and polyphenol content. Similar tendencies in the effect of polyphenol content on antioxidant activity were noticed by Moyer et al. , Lohachoompol et al.  and Orak . However, a number of studies found that anthocyanin content had a substantial effect on the antioxidant properties of the material investigated [20, 42, 43]. This variation in results may be due to differences in the chemical composition of products analyzed as antioxidant activity is strongly structure-related. Compounds can interact with different chemicals, thus altering their antioxidant properties.
The results of the present study provide justification for further research into the health-promoting properties of wrinkled rose since both extracts exhibited high levels of antioxidant activity, stable over time and attributable to the properties of dried Rosa rugosa petals.
- Different storage time of dried wrinkled rose petals had no significant effect on polyphenol and anthocyanin content as well as on antioxidant activity measured in water and methanol extracts prepared from this dry material.
- A stronger correlation was observed between antioxidant activity and polyphenol content (r = -0.72; p = 0.02 for EC50 and r = 0.67; p = 0.03 for reducing power) than for anthocyanins, although reducing power also depended on the latter group of compounds (r = 0.70; p = 0.02).
- Methanol extracts displayed stronger antioxidant activity (measured as reducing power and EC50) than water extracts.
Authors would like to thank Professor Mirosław Fik of the Department of Refrigeration and Food Concentrates, University of Agriculture in Kraków, for his support in this investigation.
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Accepted for print: 3.12.2014
Department of Refrigeration and Food Concentrates, University of Agriculture in Kraków, Poland
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ul. Balicka 122
Department of Refrigeration and Food Concentrates, University of Agriculture in Kraków, Poland
ul. Balicka 122
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