Variability of evaluated morphometric traits of rugosa rose in collection of genotypes is high.

Antiradical activity of preserved and separated pulp of matured fruit achieved by heating with addition of honey is higher versus pulp of matured fruit.

The 4 genotypes of rugose rose originated from arboretum Mlyňany (Slovakia) in altitude 167 above sea level, with average year temperature 9.5 °C. Fruit were collected in physiological stage of maturity at the end of september. Genotypes were marked according to latin name Rosa rugosa (RR) from RR-01 up to RR-04.

The representative sample was 30 fruit. In the collection of fruit were measured the following morhometric traits: weight of fruit (g), weight of stalk (g), weight of fruit pulp with seeds (g), weight of calyx (g), weight of seeds (g), length of stalk (mm), length of calyx (mm), length of fruit (mm), width of fruit (mm), thickness of pulp (mm) and number of seeds in fruit. Separation of individual parts was provided by mechanical separation.

Weight was measured using analytical scales (Kern ADB-A01S05, Germany) with an accuracy of 0.01 mg, length and thickness were measured using by Metrica 10002 Digital calliper with an accuracy of 0.01 mm.

The pulp of fruit of mature rugose rose were separated by heating at 40 °C (variant I) and 80 °C (variant II). The seeds from overcooked fruit were separated mechanically. For the preparation of samples of pulp 25% proportion from each genotype were used. The pulp was mixed with black locust (Robinia pseudoacacia L.) honey (M1) and honeydew honey (M2) in variable proportion of fruit pulp and honey (Table 3).

The bevarages were prepared by mix of 15 g of samples 1 – 8 (Table 3) with 150 mL of water.

The antiradical activity of fresh fruit pulp of rugose rose was determined in methanolic (RRM) and water extract (RRW). The samples 1 g in 25 mL water/methyl alcohol were mixed for 12 hours and antiradical activity was determined after filtration of samples.

In the frame of antiradical activity (ability to eliminate the free radicals) was tested the capacity of rugosa rose to remove DPPH^• radicals (2.2-diphenyl-1-picrylhydrazyl) using methods of Brand-Williams, Cuvelier and Berset (1995) and of Sánchez-Moreno, Larrauri and Saura-Calixto (1998). Absorbance at 515 nm has been registered in regular time intervals until the reaction equilibrium was reached – using the GENESYS 20 Vis Spectrophotometer (Thermo Fisher Scientific Inc., USA). First was measured the DPPH^• (Sigma Aldrich, USA) absorbancy without antioxidant substance (control). The inhibition of DPPH^• radicals was calculated in percent of free DPPH^• radicals in the samples using the method of Von Gadow, Joubert and Hansmann (1997):

%   o f i n h i b i t i o n = [ ( A C 0 - A A t ) / A C 0 ]   x   100 ;

Where: A_C0 is absorbance of control in time t = 0 min (DPPH• solution), AAt is absorbance in the presence of antioxidant in time t min, the result is in % of DPPH• radicals inhibition.

The degree of variability was determined by values of variation coefficients (Stehlíková, 1998). Probability of differencies among genotypes was tested by Tukkey test in programme Statistica 13.1. Correlation analyses explored relation between fruit weight and assayed morphometric traits of fruit.

Investigations of morphology of roses are still relatively few numbers (Khapugin, 2015). Fruit weight represents the very important economic trait in respect of the practical utilisation of fruit. Fruit presents potential raw material for food industry. This is a reason for selection of cultivars with high proportion of pulp and lower amount of seeds and another part of fruit (Najda and Buczkowska, 2013). Fruit of rugose rose are presented on Figure 1.

The results of morphometric analyses showed that the average weight of fruit ranged from 5.14 g (RR-02) up to 5.46 g (RR-01), weight of pulp with seeds from 4.8 (RR-02) to 5.13 g (RR-01). Variation coefficients confirmed the average and high degree of variability in the both evaluated traits (Table 4). It means that on shrubs are fruit of different weight. These facts were verified within tested collection by analysis of variance. There has not been proved statistically significant differencies among genotypes (Table 4). Najda and Buczkowska (2013) evaluated the Polish genotypes of rugose rose and found out the average weight 1.58 ± 0.16 g that represent lower value in comparison with our assayed cultivars (Table 4). The average fruit weight is very variable and influence by many factors such as cultivar, conditions of cultivation etc.

The average weight of seed of rugose rose were determined in range from 0.55 g (RR-02) up to 0.72 g (RR-03) as we can see at Table 6. Variation coefficients pointed to high degree of variability (27.47 – 50.24%). Analyse of variance confirmed statistically significant differences among genotypes with the highest value in RR-03 genotype. This genotype seems to be suitable in respect of extraction of high quality oil. Najda and Buczkowska (2013) found out lower values of average weigh of seed 0.19 ±0.1 g among Polish genotypes. Table 5

In the population of rugose rose has been noticed the variability in fruit shapes. Shape of fruit can be characterised by shape index, it means the ratio of length and width of fruit. The larger value of the parameter, the fruit is more elongated. The shape index of assayed genotypes varied from 0.73 – 0.84 (Table 5) that represent lower value in comparison with Polish genotypes 1.66 ±0.11% (Najda and Buczkowska, 2013). The most important shapes (predominantly spherical and oval shape) are presented on Figure 1.

Secondly, variability of another morphometric traits (weight of fruit stalk, weight of calyx, weight of seeds, thickness of fruit pulp, average number of seeds in fruit) were determined (Table 6). Results of morphometric traits showed the high variability, so the scientific hypothesis have been proved.

The correlation coefficients were calculated among evaluated morphometric traits of rugose rose (Rosa rugosa) (Table 7). It has been proved positive correlation between weight of fruit and weight of pulp with seeds that is crucial for selection of fruit on maximum weight of fruit with the highest proportion of pulp with seeds (r = 0.976). It is also significant correlation between weight of fruit and weight of seeds (r = 0.613) that make possible the production of seeds with the content high quality oil. Negative correlation was evaluated between fruit weight, length of fruit and thickness of pulp. The increased size of fruit lead to the decreased thickness of pulp (r = -0.564) that was presented in Table 4 (genotype RR-03).

Extracts of fruit pulp were prepared in two versions: water and methanolic. Previous studies (Dudra et al., 2015; Olech et al., 2017a; Olech et al., 2017b) confirmed that extracts of Rosa rugosa displayed strong antioxidant and antiradical activity (up to EC50 0.85 mg.mg^-1 DPPH) indicating widespread utilisation of rugose rose as natural antioxidant in food and pharmaceutical industry. In same way our results pointed to strong antiradical activity of aqueous 90.22 – 90.84% and methanolic extract as well 92.87 – 94.59% (Table 8). The notable antioxidant activity was also confirmed Rosa dumalis Bechst., R. dumetorum Thuill. and R. sempervirens LM (Nadpal et al., 2018).

There has not been proved statistically significance between assayed extracts. Dudra et al. (2015) compared antioxidant activity of Rosa canina fruit in relation to type of solvent and found out that ethylacetate and water extract showed lower values of antioxidant activity than methanolic and diethyleter extracts. Similarly, Olech and Nowak (2012) noticed the significant influence of extraction technique on antiradical activity of rugose rose petals. According to results of experiment they reccomended polar organic solvents. High efficacy of 15% methanolic extract of rugose fruit was proved in respect of antiradical activity in studies of Lee et al. (2008).

In study of Al-Yafeai, Bellstedt and Böhm (2018) different degrees of ripeness affected the bioactive compounds as well as the antioxidant capacity in the fruit of R. rugosa (hips). The maximum concentration of carotenoids was observed at late harvesting. The maximum concentration of both vitamin E and vitamin C was obtained in the orange hips and total phenolic contents were determined in the mature hips (red colour) with significant difference. The highest hydrophilic and lipophilic Trolox equivalent antioxidant capacity (TEAC) values were determined in the mature red hips, whereas oxygen radical absorbance capacity (ORAC) showed significantly lower activity in the mature hips. Andersson et al. (2012) found that amounts of total tocopherols and vitamin E activity being decreased in rose hips during ripening. According to Uggla, Gao and Werlemark (2003) the contents of vitamin C in rosehips ranged from 200 to 2800 mg.100g^-1, rosehips are considered the most abundant source of natural vitamin C. Ercisli (2007) found that contents of ascorbic acid (vitamin C) in the fresh fruits of rose species were between 706 and 974 mg.100g^-1. According to Al-Yafeai, Bellstedt and Böhm (2018) the contents of ascorbic acid in R. rugosa hips at different ripening degrees ranged between 798 and 1090 mg.100g^-1. The medicinal value of rosehips depends largely on the vitamin C contents.

Black locust honey and honeydew honey were used for conservation of fruit pulp of rugose rose. Antiradical activity of black locust honey and honeydew honey represented 6.35 – 8.35% and 6.11 – 7.28% respectively. There have not been statistically significant differences between assayed honey types (Table 9).

The average values of antiradical activity of fruit pulp preserved in honey ranged from 33.55% (sample 8) up to 77.58% (sample 1) as it has been showed at Table 10. Addition of honey caused decrease antiradical activity of fruit pulp. On the other hand, honey displays unique antimicrobial properties (Israili, 2014; Faustino and Pinheiro, 2015). The addition of honey to samples leads to stabilization of final products and play an important role as natural sweetener. The high temperature 80 °C during separation and mix of fruit pulp cause the degradation of thermolabile compounds that generally lead to decrease of antiradical activity after addition of preservants by 36% (black locust honey) and by 41% (honeydew honey). The analyse of variance confirmed the statistically significant differences among assayed samples, variation coefficient ranged from 2.34 – 13.30% pointed to low up to high degree of variability.

The antimicrobial activity of bee honey is one of its most studied biological properties. The specificity of this activity, as well as the others honey's bioactivities such as antitumor, anti-inflammatory, antioxidant and antiviral properties, depends on honey's components, which vary according to its floral, geographical and entomological origin (Kačániová and Almeida-Aguiar, 2016). Kačániová et al. (2009) study the antimicrobial activity of honey samples against Candida species. The antimicrobial activity was determined as an equivalent of the inhibition zones diameters (in millimetres) after incubation of the cultures for 48 hours. There were not seen an inhibition zones against the yeasts investigated in the 25 and 50% concentration of honey samples. In another study Kačániová et al. (2011) the antifungal activities of honey samples were tested by 10, 25 and 50% (by mass per volume) concentration against fungi Penicillium crustosum, P. expansum, P. griseofulvum, P. raistrickii and P. verrucosum and by the agar well diffusion method. The solutions containing 10% (by mass per volume) of honey did not have any effect on the growth of fungi. The strongest antifungal effect was shown by 50% honey concentration against P. raistrickii.

Beverages were prepared by mixing of 15 g separated fruit pulp preserved in honey with 150 mL of water. The antiradical activity of beverages reached up 75.57% (sample 7) up to 83.63% (sample 2). The samples 7 and 8 displayed statistically significantly lower variability in antiradical activity in comparison with the rest of samples. Variation coefficients (Table 10) pointed to low or medium degree of variability (2.05 – 12.32%). Samples with addition of honeydew honey displayed the antiradical activity about 80%. Antiradical activity of beverages with pulp preserved in honey displayed higher values of antiradical activity in comparison with maturated fruit. The second hypothesis has been verified.

Total water-soluble antioxidants were also significantly varied among Rosa species. This value was significantly higher (up to 2 times) for the fruit of Rosa rugosa (p <0.05) (Czyzowska et al., 2015).

Horčinová Sedláčková, Grygorieva and Brindza (2018) determined antioxidant activity of activated beverages of dilute/aqueous honey prepared from fresh inflorescences elderberry (Sambucus nigra L.) in the saccharide extract. It was determined antioxidant activity in the range from 16.81 to 24.16%. The enlarging of activation positively correlated with an increasing in antioxidant activity.