The HPLC/UV–vis system was used to evaluate the concentration of ascorbic acid in frozen rose hips pulp, with L-ascorbic as standard for the calibration curve (R2 = 0.9949). The HPLC chromatograms of the standard L-ascorbic acid and L-ascorbic acid from frozen rose hips pulp of RC1 (var. transitoria f. ramosissima) are presented in Figure 1.
The amounts of ascorbic acid in samples (frozen rose hips pulp) are presented in Table 1 and Figure 2. Regarding the content of vitamin C, the results have large variability between the average amounts of vitamin C within the studied genotypes: 360.22 mg/100 g frozen pulp for RC1 (var. transitoria f. ramosissima Bistrita-Nasaud, Agiesel, altitude 1250 m) followed by RC2 (var. transitoria f. montivaga Bistrita-Nasaud, altitude 950 m), while the lowest concentration was obtained for RC5 (var. assiensis Cluj, altitude 440 m), respectively 112.20 mg/100 g frozen pulp.
These results are in agreement with the literature data. The concentration of ascorbic acid in different rose species was reported to be of 106–2712 mg/100 g fresh pulp in some studies conducted in different agro-climatic regions of Turkey [4, 9, 21–26]. Also, Novajan et al.  reported that the ascorbic acid amount found in rose hip varied between 211–417.5 mg/100 g. fresh pulp, which is in agreement with our results. Other studies reported higher values of ascorbic acid, for example, Jablanska-Rys et al.  (1252 mg/100 g fresh pulp), Rosu et al.  (643 mg/100g fresh pulp), or lower amounts of vitamin C between 40–47 mg/100 g fresh pulp [30, 31].
The vitamin C content is one of the most important features in the rose hip study areas. Differences in ascorbic acid contents could result from the variations in altitude, species, variety, ecological factors, and harvesting period . The decrease in vitamin C content in plants may also be the result of the environment oxygen level, the amount of light reaching the plants, variations in endogenous plant growth regulators and the temperature. The rose hips frozen fruits have a lower content of vitamin C than fresh fruit - approximately 30.1% when compared with literature data .
Our study revealed the increase in vitamin C content with altitude; the highest concentration of ascorbic acid being found in RC1 (360.22 mg/100 g) and RC2 (347.50 mg/100), situated in the same region of Transylvania (Tibles Mountain and Salva) at 1250 m, respectively 950 m (Table 1 and Figure 2). The lowest concentration in vitamin C determined for RC5 (112.20 mg/100), a variety that grows in Cluj-Napoca, could be attributed to the polluted air or soil of this city. Differences are also noticed in the morphological features of RC1, RC2 and RC5 regarding the colour, fruit shape, fruit taste, flesh content, presence or absence of thorns. Some authors  assert that the taxonomic assignment level lower than species (i.e. subspecies and variety) plays a great role in what concerns the level of vitamin C in rose hips.
The literature data shows important differences in the content of ascorbic acid in the hips of roses from the Caninae section . Rosa canina L. usually has low content of ascorbic acid (510 mg/100g) when compared to other studied species, such as Rosa drumalis, Rosa villosa.
Significant differences were found in the vitamin C content of all the rose hips’ varieties that were analysed (p<0.05).
Total polyphenol content (TPC)
The polyphenol compounds are important plant-constituents because of their free radical scavenging ability, facilitated by their hydroxyl groups. Total polyphenolic content (TPC) was estimated and expressed using the Folin–Ciocalteu method. Gallic acid was used as standard and the results (as gallic acid equivalent, mg/100 g frozen pulp) were expressed as means ± standard deviation of triplicate analysis.
Table 2 and Figure 3 depict the total polyphenols content of the extracts that were analysed. The total polyphenols content of the Rosa canina L. hips extracts registered values between: 575.0 mg GAE/100 g for RC1 (var. transitoria f. ramosissima from Bistrita-Nasaud, Agiesel) and 326.5 mg GAE/100 g for RC6 (var. lutetiana f. fallens from Satu-Mare, Petea). High concentrations of total phenolics were also found in the extracts of RC2 (var. transitoria f. montivaga (Bistrita-Nasaud, Salva) 548.0 mg GAE/100 g, followed by RC4 (var. andegavensis f. vinealis (Bistrita-Nasaud, Beclean) 534.5 mg GAE/100 g.
These data are in agreement with some literature data, such as: Nowak et al. , who obtained 990 mgGAE/100g dry plant, Fattahi et al.  with an average of 199 mg GAE/100g and Yoo et al. , who reported 818 mg GAE/100g fresh fruit. Duda-Chodak et al. , who studied the content of the main bioactive compounds in 15 herbal products used for their antioxidant activity, like oak bark, elder fruits, dog rose, reported for Rosa canina L. the lowest concentration in total polyphenols, an average of 110 mg/100g dry fruits. Same results were obtained by Yilmaz et al.  for rose hip 102 mg/100 g dry fruits, Coruh et al. , who reported 78.13 mg GAE/100 g dry weights, data which are lower than in our study. Ercisli  reported an average that is much higher than ours in what concerns the polyphenols in rose hip, about 9600 mg/100 g dry fruit.
According to these results, the total polyphenols of Rosa canina L. are higher than blueberry (2.7-3.5 mg/g), black currant (3–4 mg/g) or raspberry (2.7-3.0 mg/g), reported by Heinonen, Meyer and Frankel . The content of the phenolic shows that the rose hips could be considered a very good source of this compound compared to other fruit species.
The differences among the rose species regarding the phenolics compounds could be due to genetic derivation, because all plants were harvested in the same period (september-october), but in different ecological conditions. It was reported that the plant genotype, cultivation site and technique affect the total phenolic content in fruit .
The isolation of flavonoids from biological samples is important for the quantitative analysis. Because of the complexity of the biological matrix, other components could interfere in the analysis. The most common procedure used to evaluate the total flavonoid content is a spectrophotometric assay, based on the formation of a complex between the aluminium ion and the carbonyl and hydroxyl groups of the flavonoids. Some works have demonstrated that this procedure has different responses depending on the flavonoid structure . Our results are expressed as mg. quercitin/100g frozen pulp.
The results regarding the total flavonoid content are depicted in Table 3 and Figure 4; significant differences can be observed in the total flavonoids content for all the rose hips varieties that were analysed (p<0.05).
The results show variability in the content of flavonoids in the hips of roses from sect. Caninae and are in agreement with Montazeri et al. , who reported 104 mg QE/100 g fresh weight for methanolic extract. Lower concentration in flavonoids was reported by Fattahi et al.  with an average of 2 mg/100 mg rose hip, Adamczak et al.  and Ghazghazi et al.  about 41 mg QE/100 g dry fruit, respectivelly 0.11-0.41mg RE/ml extract. As for Rosa canina L., Ghazghazi also reported an inverse ratio between the content in ascorbic acid and flavonoid which is in agreement with our results; for RC5, with the lowest concentration of ascorbic acid (112 mg/100 g), the corresponding flavonoids content is the highest (163.2 mg QE/100 g), followed by RC6 with 147 mg/100 g vitamin C and 137.2 mg QE/100 g frozen pulp.
Yoo et al.  reported a higher content in flavonoids (400 mg QE/100 g fresh fruit) but in Rosa rubiginosa, data which is in concordance with Adamczak et al.  who obtained the highest concentration in flavonoids for Rosa rubiginosa among other 11 species of Rosa L.
Table 4 illustrates the results for total phenolic and total flavonoid content and the flavonoids/phenolics ratio in the studied rose hips.
These data outline the richest phenolic sources – RC1, RC2, RC4, but the total flavonoids have a smaller share of total phenolics in comparison with RC5, with a ratio of 0.49 and RC6 with a ratio of 0.41, which have the lowest contents in total phenols. It could be supposed that this is due to the rich abundance of anthocyanidines in combination with the other flavonoids. The variation of phenolic compounds content depends on fruits development; in red coloured varieties (like RC1, RC2, RC4) it increases during the ripening stage due to the maximal accumulation of anthocyanines and flavonols . The phenolic acids prevail in RC1, RC2, RC4 and this fact could explain the sour astringent taste.
DPPH-scavenging activity assay
The radical scavenging activity of the methanolic Rosa canina L. extracts was determined from its DPPH radical quenching ability. Some studies showed that antioxidant activity of plants extracts is correlated with total phenolics rather than with individual phenolic compound , so the total phenol content was investigated in this study. It is important to state that different phenols develop different activities, depending on their chemical structure (phenolic acids, flavonols, antocyanidins, stilbens) and the capacity for scavenging free radicals from these classes of compounds differs. The antioxidant properties of a single compound within a group can be different, that’s why the same levels of phenolic compound do not necessarily correspond to the same antioxidant responses .
The results are presented in Table 5 and significant differences between Trolox concentration in all samples analysed (Figure 5) can be observed.
Antioxidant activity of Rosa canina L. fruits ranged from 63.35 μM Trolox/100 g sample for RC5 (var. assiensis, Cluj, Manastur) to 127.8 μM Trolox/100 g sample for RC1 (var. transitoria f. ramosissima, Bistrita-Nasaud, Agiesel).
Our results, expressed in μg Trolox/ml, varied between 18.12 μg Trolox/ml for RC5 to 36.6 μg Trolox/ml for RC1, which are in accordance with Wenzig et al.  (13.7 μg Trolox/ml – 25 μg Trolox/ml) and Ghazghazi et al.  (12.5 μg Trolox/ml - 22.6 μg Trolox/ml).
Phenolic compounds are considered to be a major group of compounds that contribute to the antioxidant activities of botanical materials because of their scavenging ability, made possible by their hydroxyl groups. The antioxidant capacity of phenolic compounds is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators. Phenolics are believed to be the major phytochemicals responsible for the antioxidant activity of plant materials .
The relation between the polyphenols content and the antioxidant capacity was determined by using linear correlations. There was a good linear correlation (R2 = 0.713, p < 0.05) between the total phenols content and the scavenging radical of the rose hip extracts (Figure 6). This result indicated that the radical scavenging capacity of each extract could be related to their concentration of phenolic hydroxyl groups. The antiradical activity of phenolic compounds depends on their molecular structure, on the availability of phenolic hydrogen, on the stabilization possibility of the resulting phenoxyl radicals formed by hydrogen donation, in a variety of extraction solvent polarity and thus different extractability .
In previous studies dealing with correlations of the phenolics compounds to DPPH, the scavenging activity showed that the polyphenols were involved with a very different coefficient of determination R2 = 0.723 ; R2 = 0.411 ; R2 = 0.086 .
For other wild fruits, the positive correlations between the total phenols and the antioxidant activity, were recorded by Egea et al. , Kilicgun et al. , Ghazghazi et al. . There are also reports concerning the lack of correlations between the total phenolic compounds and the radical scavenging . The high free radical scavenging capacity of wild fruits could also be attributed to the presence of other bioactive components, such as vitamin C, tocopherols, pigments, as well as the interaction of these compounds .
In our study, flavonoids are negatively correlated with scavenging radical of the extracts as it can be observed in Figure 7. This lack of a relationship is in agreement with other reports  that also obtained negative correlation. In other studies, a low correlation was obtained [45, 51, 52], pointing out that flavonoids did not contribute to the antioxidant activity of some fruits.
Regarding the ascorbic acid and the radical scavenging capacity, our study reveals a good correlation (R2 = 0.848) which is presented in Figure 8, being in accordance with Samee et al.  (R2 = 0.7725).
The correlation between the ascorbic acid and the altitude (Figure 9) with R2 = 0.802, suggests that the content in vitamin C increases with altitude. Due to the decrease in the oxygen content at the high altitude, plants get rid of oxidative stress and the vitamin C contents increases. The respiratory rate of fruit is also reduced at low temperature and at high altitude. As a result, the decrease in oxygen concentration in fruit prevents the degradation of the ascorbic acid in plants . Therefore, the decrease in the oxygen concentration prevents the degradation of vitamin C in plants. Biosynthesis and the accumulation of vitamin C in fruits of rosehip are significantly influenced by altitude.