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Abstract

Background

Phenolic compounds are a large group of secondary metabolites that are widely distributed in plants and are known for their antioxidant, anti-inflammatory, and antimicrobial properties.

Objective

The aim of this research was to determine the content of bioactive compounds and antioxidant activity of different extracts and infusions of two samples, wild and commercial, of Rosa canina L.fruits.

Materials and Methods

The samples were extracted in a Soxhlet apparatus using three different solvents: ethanol, acetone and hexane.Water extracts were prepared as infusions for 5, 15 and 30 min. The contents of total phenolics (TPC), flavonoids (TFC), tannins (TTC) were analyzed. To assess the antioxidant potential, the DPPH and FRAP assay were employed.

Results

The highest TPC content was observed in the acetone extract of the commercial sample (177.63 mg GAE/g dw) and in the infusion of the wild sample extracted for 30 min. (26.66 mg GAE/g dw). The highest TFC content was found in the hexane extract of the wild sample (64.2 mg QE/g dw). The highest TTC content was recorded in the acetone extract of the wild sample (21.74 mg TAE/g dw) and the infusion of the commercial sample prepared for 30 min. (17.98 mg TAE/g dw). According to the DPPH assay, ethanolic extract of the commercial sample (IC₅₀ 115.98 µg/mL) and the infusion of the same sample prepared for 5 min (IC₅₀ 4.2 µg/mL) showed the highest antioxidant activity. According to the FRAP assay, the acetone extract of the wild sample (23.47 mmol Fe²⁺/g dw) and the infusion of the same sample prepared for 30 min. (51.30 mmol Fe²⁺/g dw) showed the highest antioxidant activity.

Conclusion

The results indicate that wild samples generally exhibited higher phenolic content and antioxidant activity compared to commercial ones, with infusions showing strong antioxidant potential. A positive correlation between total phenolic content and antioxidant activity was observed in some extracts, suggesting that phenolic compounds contribute significantly to the reducing capacity of Rosa canina L. preparations.

Keywords

antioxidant activity phenolics flavonoids tannins DPPH FRAP

Introduction

The growing interest in medicinal plants over recent decades is largely attributed to their therapeutic efficacy, low toxicity, and reduced side effects compared to synthetic drugs.¹ Their bioactivity is primarily linked to a wide range of secondary metabolites such as phenolic compounds, flavonoids, and tannins which contribute to the antioxidant potential of plant derived extracts.² These compounds not only play a protective role in human health but are also widely applied in various industries. Due to their nutritional and functional properties, plant extracts are used as natural flavoring agents in the food sector, and as valuable bioactive ingredients in the development of pharmaceutical and cosmetic products.^1,2

Phenols are a group of secondary metabolites that are distinguished by the level of biological capacity due to pronounced antioxidant, antimicrobial, antifungal and even anticancer activity.^3,4

The fruits of several species of the genus Rosa are important for their bioactive components: Rosa rugosa, R. canina, R. acicularis, R. cinnamemea, R. corymbifera.^5,6 These species contain high amounts of substances responsible for their antioxidant properties: vitamin C, phenolics, carotenoids, and fatty acids.^7,8

Rosa canina L. (dog rose, wild rose) is a perennial deciduous shrub native to Southeastern Europe, North Africa and Western Asia.^9,10 It can be found in light deciduous forests and on the forest edges, in meadows, forest glades and fire-affected areas. It is extremely resistant to unfavorable environmental conditions, such as rocky habitats with a steep slope, nutrient-poor soils and water scarcity. It is also grown for ornamental purposes, so it is common in urban areas.^11–13

Rosehip, the fruit of the Rosa canina L, is very important in the food and pharmaceutical industry. It is used in fresh and dry form. It contains numerous biologically and physiologically active ingredients, such as vitamin C, phenolic compounds, tannins, flavonoids, carotenes, organic acids, and carbohydrates.^14,15 Research shows that the fruit of the species Rosa canina L. contains the highest amount of vitamin C compared to almost all other examined species.⁵ It is used for the prevention and treatment of cold, gastric mucosa inflammation and stomach ulcers, as well as for gallstones. It is used as a laxative, for diseases of the kidneys and the lower urinary tract. It can be used as a diuretic for edema and as an astringent.¹⁶ Recently, many studies have focused on examining plant species used in traditional medicine. However, the content of bioactive substances and antioxidant properties is influenced not only by the plant species but also by various other factors, such as geographical origin, weather conditions, maturity stages, collection method, storage conditions, extraction methods and solvents. Consequently, research is increasingly based on the assessment of the influence of these factors on the quality of plant extracts.^7,15 Cunja et al¹⁷ employed HPLC/MS to examine the secondary metabolites of R. canina harvested at different stages of ripeness. They identified 45 unique phenolic compounds, which together accounted for 90% of the total flavanols and proanthocyanins, with no significant changes observed during the ripening process. However, the researchers noted that the concentrations of catechins, phloridzin, flavanones, and quercetin glycosides were highest at the early stages of harvest and significantly decreased after frost exposure.

Although comparisons between wild and cultivated or commercial Rosa canina L.fruits have been reported, the available data remain limited and often inconsistent particularly for samples originating from the Western Balkans and for studies that include both extracts and infusions.

Therefore, to expand existing knowledge on the biological potential of rosehip, this study aimed to determine the content of bioactive compounds (phenols, flavonoids, and tannins) and the antioxidant activity of different extracts and infusions from two types of Rosa canina L. fruits: wild and commercial samples. Correlations between the content of different groups of polyphenols and antioxidant activity, assessed using Pearson’s correlation coefficient, may help clarify the role of these compounds in protecting against oxidative damage.

Materials and Methods

Chemicals and Instruments

All standards of phenolic compounds and other chemicals/reagents were sourced from the following suppliers: Cayman Chemical (Michigan, USA), Acros Organics (Antwerp, Belgium), Sigma-Aldrich Chem (Steinheim, Germany), VWR International (Pennsylvania, USA), MP Biomedicals (California, USA), Alfa Aesar (Massachusetts, USA), Carl Roth (Karlsruhe, Germany), and Centrohem (Stara Pazova, Serbia). All reagents utilized in this study were of analytical grade. The instruments employed for this research included a UV-Vis spectrophotometer (Cecil Aurius Series CE 2021, UK), a digital pH meter (Jenway 3505, UK), an incubator (LSW-33, Vims Elektrik, Serbia), and an analytical balance (Kern ABS 220-4, Philippines).

Plant Samples

The wild rosehip sample was collected at the site Moračke Planine (Tali, 42o 48′ 29″ N; 19 o 19′ 28″ E), Montenegro, in September 2022.during the full ripeness stage (late phenological phase), when the fruits had developed their characteristic red color and soft texture, indicating optimal maturity for harvest.The commercial sample was purchased in October 2022 from a local herbal market in Podgorica, Montenegro. The product was manufactured by “Bilje Borča” (Serbia), and the declared harvest year was 2022.2.3. Preparing samples for extraction

The collected wild rose fruits were dried at room temperature and stored in a dry, dark place in glass jars. The commercial sample was used as purchased, ie, as previously dried whole fruits, without any additional drying or cleaning. Before extraction, the whole dried fruits (including both the pericarp and seeds) were ground into a fine powder using a laboratory mill. No separation of fruit parts was performed.

Soxhlet Solid–Liquid Extraction

The samples were extracted in a Soxhlet apparatus using three different solvents: ethanol (96%), acetone (99.5%) and hexane(95%). For each extraction, 40 g of the mechanically ground sample was placed in a filter paper bag and inserted into the extractor of the Soxhlet apparatus connected to a reflux condenser. The extraction process, using 300 mL of the solvent heated to the boiling point, was carried out continuously for 6 h. Solvents were evaporated by air-drying at room temperature until a constant weight was achieved. The procedure was repeated for each of the three solvents, for both wild and commercial rosehip samples, yielding 6 different samples. A total of six final extracts were stored at 4 °C in an airtight container until further use. Stock samples were made by dissolving 0.01 g of each of the six obtained extracts in 10 mL of methanol. Their concentration was 1 mg/mL.

Infusion Preparation

Infusions were prepared using filter bags containing 2 g of mechanically ground rosehip material from both wild and commercial samples. Each filter bag was infused with 200 mL of boiling distilled water. For each sample type, three infusion durations were applied 5, 15, and 30 min resulting in a total of six different extracts. After the designated extraction time elapsed, the infusions were filtered once more to eliminate any residual particles that may have passed through the filter bags. The resulting extracts were transferred into glass bottles and stored at refrigeration temperature until further analysis. Infusions were treated as solutions containing 1 mg of starting plant material per mL for analytical purposes.^18,19

Determination of Total Phenolics

The total phenolic content of different fruit extracts was determined using the Folin-Ciocalteu colorimetric method.^20,21 The reaction mixture was prepared with 1 mL of the sample, 0.5 mL of Folin-Ciocalteu reagent and 2.5 mL of 7.5% Na₂CO₃. A blank was prepared in the same way, using distilled water instead of the sample. Four repetitions were done for each of the samples. The prepared mixtures were left in a dark place for 120 min, after which absorbance was measured at 740 nm. The content of total phenols was determined based on the gallic acid calibration curve and expressed as milligrams per gram of gallic acid equivalent (mg/g GAE).

Determination of Flavonoids

The total flavonoid content in different fruit extracts was determined using the colorimetric aluminum-chloride method.²² The reaction mixture was prepared with 1.2 mL of the sample and 1.2 mL of 2% AlCl3 solution. For each sample, four technical replicates were initially performed; however, occasionally one replicate was excluded due to outlier values. Therefore, the results are presented as the mean ± SD calculated from three replicates. The extraction solvent, methanol, was used as a blank, and distilled water was used for the infusions. The reaction mixture was incubated at room temperature for 60 min, after which the absorbance was measured at a wavelength of 420 nm. The flavonoid content was determined from a calibration curve expressed as quercetin equivalents and presented as milligrams per gram of quercetin equivalent (mg/g QuE).

Determination of Tannins

The total tannin content in different fruit extracts was determined using the modified Price and Butler method (1977).²³ The reaction mixture was prepared by mixing 500 µL of the sample, 8 mL of distilled water, 0.5 mL of 0.1 M FeCl₃ and 0.5 mL of 0.008 M K₃Fe(CN)₆. For each of the samples, 4 repetitions were done. A blank sample was also prepared: 8 mL of distilled water, 0.5 mL of 0.1 M FeCl₃ and 0.5 mL of 0.008 M K₃Fe(CN)₆. After 10 min at room temperature, the absorbance was read at 720 nm. The result is expressed as the mean value of four measurements. From the calibration curve of the tannic acid standard, based on the measured absorbances, the tannin content was read, expressed as milligrams per gram of tannic acid equivalent (mg/g TAE).

Determination of Antioxidant Activity

DPPH Radical-Scavenging Activity

The DPPH test was used to assess the free radical scavenging activity of various fruit extracts.²⁴

For DPPH analysis, 7 dilutions of the extracts with methanol in the following concentrations were used: 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.015 mg/mL. The test with each dilution was performed in 4 repetitions, a total of 28 test tubes for each of the samples. In each test tube, 3 mL of the extract of the appropriate dilution and 1 mL of 0.1 mM DPPH solution were mixed. A control (by mixing 1 mL of DPPH solution and 3 mL of methanol) and a blank sample (3 mL of methanol) were prepared. All test tubes were left in a dark place at room temperature for 30 min, after which the absorbance was measured at 517 nm.

When it comes to infusions, dilutions of samples of the same concentrations were used, but they were made with distilled water, not with methanol, as in the case of extracts. The further procedure was the same. The control was prepared by mixing 1 mL of DPPH solution and 3 mL of distilled water, and distilled water was used for the blank test.

The percent DPPH inhibition (percentage of antioxidant activity - %AA) was calculated by the equation: %AA = (A₀ – A₁) / A₀×100, where A0 was the absorbance of the control, and A1 is the absorbance of the sample. Antioxidant activity was expressed as the IC₅₀ value, which represents the concentration of the extract or reference compound required to inhibit 50% of DPPH radicals. IC₅₀ values were determined by plotting the percentage of DPPH inhibition against the sample concentration, followed by interpolation using nonlinear regression.

FRAP Assay

The total antioxidant activity of the extracts was determined using the FRAP assay (according to Benzie & Devaki, 2017).²⁵ The reaction mixture was prepared with 3 mL of TPTZ (24,6-Tripyridyl-S-triazine) reagent, 1 mL of H₂O and 100 µL of the sample with a concentration of 5 mg/mL, given that at a concentration of extracts and infusions of 1 mg/mL, the absorbance values were too low, so it was not possible to read the value of antioxidant capacity. Four repetitions were done for each sample. A blank sample was prepared by mixing 3 mL of TPTZ reagent, 1 mL of distilled water and 100 µL of distilled water. The tubes were placed in a thermostat for 15 min at 37 °C. After that, the test tubes were left for another 5 min at room temperature, and then the absorbance was read at 595 nm. From the calibration curve of the standard (2000 µmol FeSO₄×7H₂O), the antioxidant capacity values were read, expressed as the equivalent of mmol Fe2 + in 1 mL of the sample.

Statistical Analysis

The obtained results were analyzed using the statistical software package Statistica 10.0. The correlation between the levels of total phenols, flavonoids, and tannins in the plant extracts and their antioxidant potential (DPPH, FRAP) was assessed using the Pearson correlation coefficient. The Pearson correlation coefficient was calculated (P < .05), and the correlation strength (r) was interpreted as follows:: -|r|< 0.3 (no or negligible correlation), 0.3 to 0.5 (low correlation), 0.5 to 0.7 (moderate correlation), 0.7 to 0.9 (strong correlation), and 0.9 to 1 (very strong correlation). One-way analysis of variance (ANOVA) was applied to assess differences among groups, followed by Tukey’s HSD post hoc test to determine significant differences between individual means. A P-value of <.05 was considered statistically significant. Principal component analysis (PCA) was performed to describe the content of bioactive substances of the tested extracts of the species Rosa canina L. in relation to the type of solvent used for extraction.

Results

Extraction Yields

The analysis results showed that for both wild and commercial rose hips, the highest amount of extract was obtained using ethanol (36% of the dry plant material) as the solvent while the lowest yield was obtained using hexane(3.75%). Overall, the yield of wild rose hip extracts was higher compared to the commercial ones.

Phenolic Compounds Content in Plant Extracts

The content of bioactive compounds in extracts and infusions of wild and commercial fruit of Rosa canina L. is presented in Tables 1 and 2.

Table 1.

Total Phenolic (TPC), Flavonoid (TFC), Tannin (TTC) and in Extracts of Wild (WF) and Commercial Fruit (CF) of Rosa canina L; Values Represent Mean + SD of Three Measurements (n = 3).

Sample/solvent	TPCmg GAE/g dw	TFCmg QE/g dw	TTCmg TAE/g dw
WF/ethanol	108.65 ± 0.31^a	n.d	10.84 ± 1.22^b
WF/acetone	50.41 ± 1.12^b	6.65 ± 0.89^d	21.74 ± 0.78^a
WF/hexane	1.78 ± 0.22^d	64.2 ± 0.63^a	6.49 ± 0.76^b
CF/ethanol	11.06 ± 0.31^e	42.19 ± 1.34^c	14.81 ± 1.82^b
CF/acetone	177.63 ± 0.88^a	58.39 ± 2.11^b	14.31 ± 1.32^b
CF/hexane	47.8 ± 1.06^c	54.14 ± 1.33^b	4.74 ± 0.97^c

Different superscript letters within each column indicate statistically significant differences (Tukey HSD test, P < .05).

Abbreviations: GAE, gallic acid equivalent; QE, quercetin equivalent; TAE, tannic acid equivalent; n.d., not detected with the standard concentration range.

Table 2.

Content of Bioactive Compounds in Infusions of Wild and Commercial Fruit of Rosa canina L; Values Represent Mean + SD of Three Measurements (n = 3).

Sample/preparation time of infusion	TPCmg GAE/g dw	TTCmg TAE/g dw
WF/_{5 min}	17.92 ± 1.34^b	8.76 ± 0.88^c
WF/_{15 min}	25.68 ± 0.51^a	11.53 ± 1.65^b
WF/_{30 min}	26.66 ± 0.31^a	13.64 ± 0.77^b
CF/_{5 min}	7.7 ± 0.46^c	7.48 ± 0.34^c
CF/_{15 min}	11.91 ± 1.26^c	10.21 ± 0.62^b
CF/_{30 min}	26 ± 0.24^a	17.98 ± 0.45^a

Different superscript letters within each column indicate statistically significant differences (Tukey HSD test, P < .05).

Abbreviations: GAE, gallic acid equivalent; TAE, tannic acid equivalent;

Flavonoids were not detected in the infusion samples (values below the quantification limit)

The analysis of the results revealed that the TPC in the tested extracts varied over a wide range, from 11.06 to 177.63 mg GA/g dw with greater variations observed in the commercial sample. The highest phenolic content was found in the acetone extract of the commercial rose hip, while the lowest was in the ethanol extract of the same sample. For wild rose hip fruits, ethanol was found to be the most efficient solvent for phenolic extraction, while for the commercial sample, acetone was the most effective. The total phenolic content in the infusions of wild and commercial rose hips, prepared over different time intervals of 5, 15, and 30 min, ranged from 7.7 to 26.66 mg GA/g dw. Comparison of the obtained values clearly indicates substantial variations in total phenolic content depending on the extraction solvent, with acetone generally yielding higher TPC values than ethanol or hexane, particularly in the commercial sample. In both samples, wild and commercial rose hips, the highest total phenolic content was observed in the 30-min infusions, while the lowest was in the 5-min infusions.

Observing the obtained TFC values in different extracts of wild rose fruit, significant differences can be noted, particularly for the wild sample. Hexane proved to be an effective solvent for isolating flavonoids from wild rose hips, yielding the extract with the highest flavonoid content measured in this study (64.2 mg QE/g dw). On the other hand, the acetone extract of wild rose hips contained a significantly lower level of flavonoids (6.65 mg QE/g dw). Based on the results from testing the commercial rose hips, it can be concluded that the variations in flavonoid content across different extracts were much smaller. In this case, acetone emerged as the most effective solvent.

The total tannin content (TTC) values in the extracts of this study ranged from 4.74 to 21.74 mg TAE/g dry weight. Hexane proved to be the weakest solvent for isolating tannins in both samples. For the wild sample, acetone was the most efficient solvent (21.74 mg TAE/g dw), while for the commercial sample, ethanol was the most effective (14.81 mg TAE/g dw). The highest tannin content in the infusions of wild and commercial rose hips was recorded in the 30-min infusion (17.98 mg TAE/g dw) while the lowest was observed in the 5-min infusion (7.48 mg TAE/g dw) of the commercial rose hip.

Significant differences were observed in TPC, TFC, and TTC between extracts obtained using different solvents and between wild (WF) and commercial (CF) Rosa canina L. fruits. The highest TPC was found in CF/acetone and WF/ethanol extracts, with no statistically significant difference between them (P > .05), while the lowest was in WF/hexane extracts. For TFC, WF/hexane extracts showed significantly higher values compared to others, while WF/ethanol extracts had undetectable flavonoid levels. Regarding TTC, WF/acetone extracts had the highest content, significantly different from CF/hexane extracts, which showed the lowest levels.

TPC values increased with infusion time in both wild and commercial samples. The highest concentrations were observed in 30-min infusions (WF: 26.66 ± 0.31 mg GAE/g dw; CF: 26.00 ± 0.24 mg GAE/g dw) and 15-min infusions (WF: 25.68 ± 0.51 mg GAE/g dw), with no significant differences among these groups (P > .05). In contrast, shorter infusions (5 min and CF/15 min) had significantly lower TPC.Similarly, TTC showed an increasing trend with infusion duration. The highest TTC was recorded in the 30-min commercial infusion (17.98 ± 0.45 mg TAE/g dw), significantly higher than all other groups (P < .05). Wild fruit infusions at 30 and 15 min showed intermediate TTC values, while 5-min infusions exhibited the lowest tannin content. These findings suggest that a longer infusion time enhances the extraction of phenolic compounds and tannins from Rosa canina L. fruits, which may positively influence the antioxidant properties and potential health benefits of the infusions.

Antioxidant Capacity of Plant Extracts

The antioxidant potential of different extracts and infusions of Rosa canina L. fruits, determined by the DPPH test and expressed through IC₅₀ values, is presented in Tables 3 and 4.

Table 3.

Antioxidant Activity of Extracts of Wild and Commercial Fruit of Rosa canina L.; Values Represent Mean + SD of Three Measurements (n = 3).

Sample/solvent	DPPH IC₅₀(µg/mL)	FRAP(mmol Fe²⁺/g dw)
WF/ethanol	118.13 ± 1.41^a	11.91 ± 0.81^c
WF/acetone	119.25 ± 1.51^a	23.47 ± 1.13^a
WF/hexane	1154.22 ± 0.97^d	n.d. ¹
CF/ ethanol	115.88 ± 2.11^a	15.24 ± 1.21^b
CF/ acetone	201.94 ± 2.31^b	9.51 ± 0.94^d
CF/hexane	640.48 ± 1.33^c	n.d.

Different superscript letters within each column indicate statistically significant differences (Tukey HSD test, P < .05). For DPPH IC50, lower values indicate stronger antioxidant activity; for FRAP, higher values indicate stronger antioxidant activity.

Abbreviation: n.d., not detected with the standard concentration range.

Table 4.

Antioxidant Activity of Infusions of Wild and Commercial Fruit of Rosa canina L.; Values Represent Mean + SD of Three Measurements (n = 3).

Sample/preparationtime of infusion	DPPH IC₅₀(µg/mL)	FRAP(mmol Fe²⁺/g dw)
WF/5 min	9.73 ± 1.08^b	31.57 ± 1.34^b
WF/15 min	7.18 ± 2.51^bc	49.76 ± 1.28^a
WF/30 min	151.36 ± 1.56^a	51.30 ± 0.51^a
CF/5 min	4.2 ± 2.11^c	6.10 ± 1.67^d
CF/15 min	7.41 ± 1.56^bc	13.05 ± 0.91^c
CF/30 min	6.31 ± 0.97^bc	41.4 ± 1.03^ab

The IC₅₀ values for the extracts ranged from 118.13 µg/mL for the ethanol extract of the wild sample to 1154.22 µg/mL for the hexane extract of the same sample. Both ethanol and acetone extracts, from both wild and commercial samples, demonstrated similarly high antioxidant activity in the DPPH assay, with negligible differences in their IC₅₀ values. Significantly higher antioxidant activity was observed in the infusions, with IC₅₀ values ranging from 4.2 µg/mL for the 5-min infusion of the commercial sample to 151.36 µg/mL for the 30-min infusion of the wild sample.

The results of the FRAP test for the extracts and infusions of Rosa canina L. fruits are presented in Tables 3 and 4. The antioxidant capacity of the samples, determined by this test, is expressed as mmol Fe^2+/g dry weight. Reducing power of the tested extracts ranged from 9.51 for the of the acetone extract of the commercial sample to 23.47 mmol Fe²⁺/g dw for the acetone extract of wild fruit while the infusions ranged from 6.10 for the 30-min infusion of the commercial sample to 51.30 mmol Fe²⁺/g dw for the 30-min infusion of the wild sample. The FRAP test also confirmed a significantly higher antioxidant capacity in the infusions compared to the extracts.

Discussion

Phenolic Compounds Content

The content of bioactive compounds differed depending on the analyzed sample and solvent in the case of extracts, that is, the time of extraction of the prepared infusions. The total phenolic content ranged from 11.06 mg GAE/g dw to 177.63 mg GA/g dw. These values are lower than 197.24 mg/100 g dried rose hips for EtOAc extract (Daels-Rakotoarison et al, 2002)²⁶ and 255.9 to 766 mg GAE/g dw, which Koczka et al (2018)²⁷ obtained in their ethanolic extracts. Li et al (2023)²⁸ demonstrated that mid-polar solvents such as ethanol and acetone yield higher levels of phenolics from various medicinal plants, including Rosa species, in comparison to non-polar solvents such as hexane, which is consistent with the results of our study. In infusions, the phenolic content ranged from 7.7 mg GAE/g dw to 26.66 mg GAE/g dw, which is significantly lower than in extracts prepared using a Soxhlet extractor. The results presented in this research are consistent with those obtained by Mihaylova et al (2015)²⁹ since their water extract prepared for 30 min had a higher content of phenolics than the one prepared for 5 min. Organic solvents have been shown to be more suitable and effective than water in isolating phenolics. The significant differences observed in the content of phenolic compounds are likely attributable to several factors, including the variation in extraction methods, genetic differences, environmental influences in the cultivation regions, as well as the maturity of the fruits utilized in the aforementioned studies. These factors collectively impact the levels of bioactive substances present.^30–32 The literature provides a detailed array of both quantitative and qualitative assessments of the phenolic profile of Rosa L. species.^7,33 Some autors noted that the variation in phenolic compounds in rose hips is of considerable importance for their chemotaxonomic differentiation.³⁴ As observed in other fruits, the health-promoting effects of Rosa L. fruits are not attributed to a single bioactive compound. Instead, these benefits are primarily associated with their antioxidant properties, particularly due to their vitamin C content, and the presence of phenolic compounds.⁷ Fascella et al (2019)³⁵ investigated four Sicilian rose hip species: R. canina, R. corymbifera, R. micrantha, and R. sempervirens. Their analysis demonstrated considerable variability among the species. R. canina and R. sempervirens exhibited the highest concentrations of phenolic compounds, with galloyl derivatives, catechin derivatives, ellagic acid, and quercitrin identified as the primary phenolic components.

Relevant literature sources indicate that flavonoids in rose hips play a significant role in enhancing the stability and bioavailability of vitamin C by inhibiting its oxidation.³⁶ Among the various flavonoids found in plants, quercetin and kaempferol are among the most prevalent and biologically relevant. These compounds typically exist naturally as glycosides.In rose hips, the primary glycoside derivatives of quercetin include quercitrin (quercetin-3-O-rhamnoside), isoquercitrin (quercetin-3-O-glucoside), and hyperoside (quercetin-3-O-galactoside).³⁷

The analysis of the results of our research showed that the flavonoid content ranged from 6.65 mg QE/g dw to 64.2 mg QE/g dw, which is significantly lower than in aqueous and methanolic extracts in the study by Nadjpal et al (2016)^.³⁸ Beyhan et al (2017)³⁹ also observed higher values in their research. In infusions and ethanolic extract of the wild sample, the flavonoid content was extremely low, so it was not possible to read it using a spectrophotometer. This indicates that water, as a solvent, does not extract significant amounts of this group of bioactive compounds.⁴⁰ In line with our results, Adamczak et al (2012)³⁴ based on the comparison of flavonoid content in 11 different species of the Rosa L., detected a low average value of flavonoids for R. canina, the most common species, while flavonoids were the highest in R. rubiginosa.

Differences in the total tannins content between extracts and infusions were insignificant, which was not the case with the content of phenols and flavonoids in these two types of samples. The highest amount of TTC was identified in the acetone extracts of wild sample (21.74 mg TAE/g dw) representing 43.12% of TPC. Turker et al (2012)⁴¹ and Ousaaide et al (2020)⁴² examined the content of tannins in ethanolic and acetone extracts of wild rose fruit and obtained similar values for those two samples. That is consistent with the results of our research obtained for the commercial but not for the wild sample.

Antioxidant Activity

Plenty of medical studies suggested the importance of antioxidants in the prevention or reduction of oxidative stress caused by free radicals. In this regard, many in vitro antioxidant test methods are used to determine the total antioxidant activity.

DPPH assay is based on the following principle: when DPPH receives a hydrogen atom from an antioxidant molecule, it is reduced to 1,1-diphenyl-2-(24,6-trinitrophenyl)-hydrazine, whereby the purple color of the reaction mixture changes to yellow, with a simultaneous decrease in absorbance at 517 nm. This color change can be monitored spectrophotometrically and is used to determine antioxidant activity.³⁰ Hexane extracts showed the lowest antioxidant potential, which is consistent with results for bioactive compounds content. Montazeri et al (2011)⁴³ determined the IC₅₀ values of wild rose fruit extracts and obtained the following results: methanolic extract 11.58 μg/mL, aqueous extract 15.14 μg/mL, while the IC₅₀ value for the hexane extract was higher: 224.6 μg/mL. Those values were notably lower than those observed in extracts in our research, indicating a higher antioxidant potential. However, in their research, the hexane extract also showed the lowest antioxidant potential, which confirms that this solvent does not extract significant amounts of components that contribute to the antioxidant potential. The higher antioxidant potential observed in Montazeri et al (2011)⁴³ may be attributed to geographical and ecological differences in the plant material, as well as slight variations in extraction methodology. Although both studies used polar solvents (methanol vs ethanol), which extract similar classes of compounds, the phenolic composition of wild rose can vary significantly depending on environmental conditions.Infusions showed significantly better results than extracts.The higher DPPH radical scavenging activity in commercial fruit infusions, particularly after 5 min (CF/5 min), may suggest the presence of readily extractable low-molecular-weight antioxidants, such as ascorbic acid, which can dominate early extraction phases. However, as the infusion time increases, the contribution of polyphenolic compounds becomes more evident, as reflected in the increasing FRAP values over time (especially CF/30 min). This implies that prolonged extraction enhances the release of polyphenols, which act more effectively as reducing agents rather than as radical scavengers.⁴⁴

The FRAP assay results ranged from 6.10 to 51.30 mmol Fe²⁺/g dw. Hexane extracts exhibited very low antioxidant potential, which is consistent with the findings of the DPPH test. Overall, ethanol proved to be the most effective solvent for extracting compounds with high antioxidant activity in our study. A strong correlation was observed between total phenolic content and antioxidant capacity, particularly in the FRAP assay, supporting the widely accepted role of phenolic compounds as major contributors to antioxidant potential. These findings are consistent with previous studies on Rosa canina and related species, which emphasize the efficiency of polar solvents in extracting bioactive phenolics⁴⁵ The FRAP test for the infusions yielded results showing an extremely high antioxidant potential, which is also in line with the results of the DPPH test. Nadjpal et al (2016)³⁸ obtained results consistent with those presented here, because in their research, based on the results of the FRAP test, the water extract had a higher potential than the extract with the organic solvent. Bratu et al (2018)⁴⁶ examined the antioxidant potential of water extract from fruits from different species of the genus Rosa and obtained a result of 19.45 ± 0.78 mmol, which is a much lower potential than that obtained in this study when it comes to infusions. The results of the research conducted by Mihaylova et al (2015)²⁹ are in line with what is described in this research, given that 30-min infusions had a higher antioxidant potential than 5-min infusions, when analyzed by FRAP.

Interestingly, wild fruit infusions at 30 min (WF/30 min) showed the highest FRAP value but the lowest DPPH activity, suggesting that some compounds with strong reducing power might not effectively neutralize free radicals in the DPPH assay. Alternatively, thermal degradation of radical-scavenging components during longer infusion may reduce their efficacy in DPPH reaction.⁴⁷ Similar patterns have been reported in previous studies, such as by Nowak et al (2012)⁴⁸ and Koczka et al (2018)²⁷ where processing and extraction time were shown to significantly influence both the yield and the mode of antioxidant action.

The extracts exhibited a higher content of total phenols and flavonoids; however, the infusions demonstrated a superior antioxidant potential. This discrepancy can be attributed to several factors, including the dependence of antioxidant activity on the type of solvent used, the presence of antioxidants with varying chemical properties, and the polarity of the compounds in the extraction solvent.⁴⁹ Górnaś et al (2023)⁵⁰ reported that both drying method and extraction time significantly affect the recovery of bioactive compounds in rosehip products. These findings are consistent with our observation that extended infusion times (15 and 30 min) led to higher concentrations of phenolic compounds, although the rate of increase plateaued after a certain point.

Furthermore, Vasić et al (2024)⁵¹ found that combining rosehip with other botanicals such as hibiscus and saffron in herbal teas significantly improved total phenolic content (TPC) and antioxidant potential. This supports the idea that both the intrinsic composition of the plant material and its formulation can substantially influence the final bioactivity of the infusion. Recent studies by Hellström et al (2024)⁵² and Zhang et al (2025)⁵³ also emphasized the strong impact of geographical origin and post-harvest processing on the nutritional and functional properties of rosehip-based products. These factors likely contribute to the differences observed between wild and commercial samples in our study.

Correlation of Phenolic, Flavonoid and Tannin Content and Antioxidant Activity of Different Extracts of Rosa canina L. Fruit

A very high positive and significant correlation was recorded between tannin content and antioxidant activity (r = 0.99, P < .05) measured by the FRAP test in the hexane extract of the wild sample.In the commercial sample, the results also indicate the existence of a very high positive and significant correlation (r = 0.99, P ˂ .05) between total phenolic content in the acetone extract and the antioxidant activity determined by the DPPH test, as well as a high significant positive correlation (r = 0.86, P < .05) between the total phenolic content in the acetone extract and antioxidant activity determined by the FRAP test (Figure 1). In the research conducted by Roman et al (2013),⁵⁴ a positive correlation was found between total phenolic content and the antioxidant activity of fruit extracts of different varieties of Rosa canina L, as determined by the DPPH method. Turker et al (2012)⁴¹ obtained a positive correlation between the content of tannins and the antioxidant activity determined by the DPPH method (r = 0.85, P < .05), while the correlations between the content of total phenols and flavonoids and the antioxidant activity were negative.

Figure 1.

Correlation of (a) Phenolic Content and Antioxidant Activity Determined by the DPPH Test and (b) phenolic Content and Antioxidant Activity Determined by the FRAP Test of the Acetone Extract of a Commercial Sample of Rosa canina L. Fruit.

On the other hand, numerous literature data indicate that no correlation exists between total phenolic content and antioxidant activity. The causes of this phenomenon can vary. The antioxidant activity of phenolic compounds depends on their structure, and the antiradical activity of an extract may also result from the presence of numerous other compounds, such as vitamin C, pigments and tocopherols.⁵⁵

Principal Component Analysis (PCA)

By applying Principal component analysis (PCA) to the set of obtained data, the content of bioactive substances in plant extracts of Rosa canina L. species is described in relation to the type of selected solvent. The graph was processed with a 95% confidence interval. Based on the data shown in Figure 2, it can be noted that the first two principal components describe more than 90% of the variability of the samples (PC1: 57.76%, PC2: 32.24%).The PCA results indicate that hexane extracts form a distinct group, suggesting that their composition particularly regarding phenolics, flavonoids, and tannins is markedly different from that of ethanol and acetone extracts.

Figure 2.

Principal Component Analysis (PCA) of the Total Content of Phenols, Flavonoids, and Tannins According to Solvent Type in Both Rosa canina L. Samples. Projection of the Samples on the First Two Principal Components (PC1: 57.76%, PC2: 32.24%).

Limitations of the Study

The extraction process employed in this study is time-consuming, which represents a major methodological limitation. Furthermore, the assessment of antioxidant activity is most reliable when multiple complementary assays are applied. The primary objective was to evaluate and compare the antioxidant potential of extracts and infusions derived from wild and commercial rosehip fruits, taking into account that Rosa canina L. is most commonly consumed in the form of herbal tea in everyday use. A significant limitation of the current study is the absence of detailed phytochemical profiling of individual bioactive constituents. Such profiling would provide a deeper understanding of the chemical composition and contribute to the interpretation of the observed bioactivities. Future research will address this gap by incorporating advanced analytical techniques such as LC-MS/MS and GC-MS for comprehensive chemical characterization of rosehip samples.

Conclusion

The tests conducted as part of this research revealed that both Rosa canina L. fruit samples exhibit significant antioxidant potential, with the commercial sample showing slightly better results. These samples can be utilized as safe and effective sources of antioxidants. Based on the obtained results, it is evident that the infusions demonstrate a significantly higher antioxidant capacity compared to the extracts in both tested samples.Our research also revealed a statistically significant positive correlation (r = 0.99, P < .05; r = 0.86, P < .05) between the DPPH/FRAP values and total phenolic content (TPC) in the acetone extract of the commercial sample, suggesting that the concentration of phenolic compounds may serve as a biochemical indicator of reducing capacity in the extracts of Rosa canina L. fruit. This study has also contributed significantly to the understanding of the biological potential of Rosa canina L. fruit and its applications in the medicinal and pharmacological fields. Further research should focus on identifying the active compounds and analyzing environmental factors (eg, light, temperature, soil nutrients) that may affect the metabolism and conversion of phenolics. These factors undoubtedly influence the content of bioactive substances as well as the antioxidant activity of the tested samples.

Footnotes

Acknowledgments

The authors are thankful to Mrs. Danka Caković and Mr Vukoica Despotović for their help with field work.

ORCID iDs

Dragana Petrović

Mijat Božović

Author Contributions

Conceptualization, T.Dž. and D.P.; data collection, T.Dž.; data analysis and interpretation, T.Dž., D.P. and M.B.; writing—original draft preparation, T.Dž., D.P. and M.B. .; writing—review and editing, T.Dž., D.P. and M.B. All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

The authors confirm that the data supporting of this study are available within the article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

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Antioxidant Activity and Total Phenolic Content of Different Extracts from Rosa canina L Fruits

Abstract

Background

Objective

Materials and Methods

Results

Conclusion

Keywords

Introduction

Materials and Methods

Chemicals and Instruments

Plant Samples

Soxhlet Solid–Liquid Extraction

Infusion Preparation

Determination of Total Phenolics

Determination of Flavonoids

Determination of Tannins

Determination of Antioxidant Activity

DPPH Radical-Scavenging Activity

FRAP Assay

Statistical Analysis

Results

Extraction Yields

Phenolic Compounds Content in Plant Extracts

Antioxidant Capacity of Plant Extracts

Discussion

Phenolic Compounds Content

Antioxidant Activity

Correlation of Phenolic, Flavonoid and Tannin Content and Antioxidant Activity of Different Extracts of Rosa canina L. Fruit

Principal Component Analysis (PCA)

Limitations of the Study

Conclusion

Footnotes

Acknowledgments

ORCID iDs

Author Contributions

Funding

Declaration of Conflicting Interests

Data Availability

Statement of Human and Animal Rights

Statement of Informed Consent

References