Changes in various secondary metabolites by crossing modern rose cultivars

Phenolic compounds are aromatic compounds of the benzene ring that make up a significant portion of plant secondary metabolites. They are extremely diverse and are classified according to the number of carbons. Structural diversity determines their functional properties and distribution in different plant species. In plants, they play a key role in physiological and mechanical activities. Plants produce them primarily for their growth, development and protection against pathogens, including fungi, viruses and bacteria, and as a defence mechanism against major abiotic stresses such as salinity, drought and UV radiation. Plants respond to these stressful abiotic and biotic conditions by accumulating phenolic compounds. Their accumulation depends on the type of stress and the response of the plant species. In addition to defence mechanisms, they play an important role in the interaction between the plant itself and microbes (Bhattacharya et al., 2010; Dai and Mumper, 2010; Kumar et al., 2020; Chowdhary et al., 2021; Pratyusha, 2022). The main group of natural pigments classified as phenolic compounds responsible for the colours of flowers, fruits and leaves is anthocyanins. Abiotic and biotic stress conditions where changes in the total anthocyanin content were observed were heat, light, water, salt, insect and fungal attack (Mannino et al., 2021).

The main bioactive compounds of roses are phenolic acids (protocatechuic acid, gallic acid, methyl gallate, vanillic acid, syringic acid, chlorogenic acid, cichoric acid), anthocyanins (cyanidin-3-O-glucoside), tannins, including proanthocyanidins (condensed tannins), flavonoids (flavones (apigenin), flavonols (rutin, catechin, quercetin, quercitrin)), flavanones (hesperidin, eriodictyol, taxifolin), dihydrochalcones (phloridzin), phytosterols (β-sitosterol), pentacyclic triterpenes (ursolic acid and oleanolic acid), organic acids (ascorbic acid, malic acid, citric acid), fatty oils (α-linolenic acid, linoleic acid, palmitic acid), pectins, carotenoids (lycopene, β-carotene), tocopherols and galactolipids (Deliorman Orhan et al., 2012; Dubtsova et al., 2012; Mihaylova et al., 2015; Nadpal et al., 2016). Roses are considered excellent antioxidants due to the antioxidant properties of the listed components, which inhibit or prevent the oxidation of oxidative substances by scavenging free radicals and reducing oxidative stress. Oxidative stress is an imbalanced state in which excessive amounts of reactive oxygen and/or nitrogen species overcome endogenous antioxidant capacity, resulting in the oxidation of various biomacromolecules, such as enzymes, proteins, DNA and lipids (Dai and Mumper, 2010). It is associated with many diseases such as cardiovascular disease, diabetes, obesity and cancer. Some studies could show that foods classified as antioxidants at least partially alleviate diseases associated with oxidative stress (Arts and Hollman, 2005). Cohen (2012) reported that roses have antioxidant, anti-inflammatory, antidiabetic, lipid-lowering and antiobesogenic effects. They have beneficial effects on osteoarthritis, rheumatoid arthritis and back pain. Because of these beneficial effects, foods rich in polyphenolics are increasingly valued and in demand, according to Dai and Mumper (2010). Plant foods are reported to have organoleptic properties (bitterness, astringency), and it is also reported that phenolic compounds are even better antioxidants than vitamins C and E and carotenoids (Dai and Mumper, 2010).

Kunc et al. (2022, 2023b) reported that various roses native to Slovenia, R. pendulina L. × R. glauca, R. corymbifera, R. gallica and R. subcanina, as well as the hybrid R. pendulina L. × R. spinosissima L., could be a rich source of various secondary metabolites. They found that the content of phenolic compounds was higher in the flesh with skin than in the seeds. The content of cyanidin-3-glucoside was higher in the studied hybrid. Demir et al. (2014) studied the phenolic compounds of five Turkish rose hips (R. canina, R. dumalis, R. gallica, R. dumalis subsp. boissieri and R. hirtissima). They found that R. dumalis subsp. boissieri contained the most diverse phenolic compounds. R. canina is an intensively studied species. Javanmard et al. (2018) found that the different ecotypes of R. canina are a rich source of phenolic compounds. Of all the ecotypes studied, the Aghcheh ecotype was the richest in phenolics. Cunja et al. (2015) added that the phenolic compound content was highest in R. canina rose hips harvested in late September. Kunc et al. (2023b) found that the content of phenolics in the flesh with skin was highest in R. gallica (as well as the content of cyanidin-3-glucoside) and lowest in the hips of R. corymbifera. In seeds, the highest content was in R. subcanina and the lowest in R. × R. glauca. The lowest content of cyanidin-3-glucoside was found in R. subcanina. Koczka et al. (2018) focussed on studying the phenolic composition and content of R. canina, R. gallica, R. rugosa and R. spinosissima, with R. spinosissima having the highest content.

To date, very little is known about the effects of hybridisation on the secondary metabolite content. We know that there are approx. 100 cultivars derived from R. centifolia, 500 from R. spinosissima and >200 from R. gallica. The current study will enable a clearer overview of how hybridisation affects the composition and the content of phenolic compounds, which has been very poorly studied until now. The study was based on different original species and several older and modern varieties originating from these species. All original species are also very common in Slovenia. We were interested in how the content of phenolic compounds changes during crossing. We checked whether related varieties derived from the same original species can be assigned to the same chemotype based on the phenolic compound content. Due to the increasing importance of foods with a high content of phenolic compounds, we will additionally try to determine whether the examined rose hips are a rich source of phenolic compounds.

As part of research work, we determined the colour parameters and the content of bioactive compounds in the rose hips of three native Slovenian roses and cultivars derived from them, growing in Arboretum Volčji Potok (Slovenia).

Lancaster et al. (1997) reported that fruit skin colour results from several parameters, among which pigments, chlorophyll and carotenoids in the chloroplast and chromoplast, and phenolic pigments (anthocyanins, flavonols and proanthocyanidins) in the vacuole are of great importance. The expression of pigment colour also depends on physical factors such as the presence of cuticular growth, epidermal hairs, the shape and orientation of cells in the epidermis and subepidermis. They concluded that pigments and surface topography selectively absorb and refract incident visible light to produce a reflectance spectrum characteristic of the particular fruit skin. Comparing the Hunter Lab colour content determined in the current study (Sahingil and Hayaloglu, 2022) with the results of the study by Cunja et al. (2015), who measured the colour parameters of R. canina in six different periods, it is noticeable that the value of the parameter L* was quite similar to the values measured in the current study (20.28 to 43.38). Cunja et al. (2015) reported that in the value was 43.1 the initial stage of harvesting, followed by 35.0, 32.6, 29.4 and 28.9, and the value was 28.2 in the last stage. It was reported that the analysis of the parameter L* showed significant differences between the ripening of the rose hips and the increase in the darkness of the fruits in the last sampling. The values were highest (43.1) at the first sampling in September and lowest after fruit freezing. The decrease in the a* value was measured at the last sampling after freezing (Cunja et al., 2015). The parameter b* also decreased as the rose hip gradually lost its yellow colouration. Similar values and a significant decrease in the above parameters are also reported by Uggla et al. (2005), who observed the ripening of R. dumalis and R. rubiginosa. Ercişli (2007) found similar results but also reported a decrease in the parameter h° during maturation, which Cunja et al. (2015) could not confirm. In their case, h° initially decreased until the fourth sampling date and increased again in the last two sampling dates. The values of the mentioned parameter ranged from 19.5 to 34.2 in the results of Cunja et al. (2015). In our samples, the parameter h° was slightly higher and ranged from 22.14 (R. spinosissima) to 59.18 (‘Single Cherry’), which means the hips of R. spinosissima were more red than ‘Single Cherry’ hips, which were more red and yellow. Goztepe et al. (2022) measured the colour of fresh fruit of R. canina and reported that the values of the parameters L*, a* and b* were 42.5, 24.7 and 13.3, respectively. Their results are comparable to ours. The parameter L* corresponds most closely to the value we determined for R. gallica and a* and b* for the cultivar ‘Frühlingsmorgen’.

The total analysed phenolic content (excluding anthocyanins) in the flesh with skin was lowest in the modern cultivar of R. pendulina, ‘Mount Everest’, 3603.57 mg · kg⁻¹ f.w. The highest total contentin the flesh with skin was found in the cultivar ‘Splendens’, which is derived from R. gallica, with 68789.39 mg · kg⁻¹ f.w.. From the dendrograms, it can be concluded that the cultivars ‘Single Cherry’ and ‘Splendens’ are similar and also characterised by a high total phenolic compound content. Based on the total phenolic compound content in the hips, ‘Frühlingsduft’ and ‘Violacea’, ‘Frühlingsmorgen’ and R. gallica, R. pendulina and ‘Poppius’, ‘Bourgogne’ and ‘Mount Everest’, and R. spinosissima are also similar. The only cultivar that differed from all others was ‘Harstad’.

When the compounds present were examined, it was found that 45 phenolic compounds were determined in the flesh with the skin of R. pendulina and its cultivars, 76 in R. spinosissima and 67 in R. gallica. R. pendulina and its cultivars differ from the others by their extremely low composition of flavanols and flavonols, quite the opposite of R. gallica, where quite a lot of compounds belonging to the mentioned group of phenolics were present. In R. pendulina, on the other hand, compounds such as quercetin-3-arabinopyranoside, quercetin-3-rhamnoside, quercetin-3-furanoside, isorhamnetin-pentoside 1–2, quercetin-galloyl-pentoside 1–3 were absent. The same was true for the content in the seeds, with the difference that the contents of compounds and total content in seeds were lower. They were also the lowest in ‘Mount Everest’, 757.02 mg · kg⁻¹ f.w. and the highest in ‘Single Cherry’, 6823.21 mg · kg⁻¹ f.w. When comparing our results with the literature, it can be seen that Kunc et al. (2023c) reported that the total phenolic content in the flesh with skin of R. gallica was 15800 mg · kg⁻¹ f.w. and 5310 mg · kg⁻¹ f.w. in R. subcanina, which is also comparable with our results. Najda and Buczkowska (2013) reported the total content in the hips of R. villosa, R. californica, R. spinosissima, R. rugosa and R. × damascena, where the lowest content was determined in R. × damascena (1096.7 mg · kg⁻¹ f.w.) and the highest in R. rugosa (2151.4 mg · kg⁻¹ f.w.), which was, on average, lower than that determined in the current study. As reported by Roman et al. (2013), there are differences in the phenolic composition of rose hips due to genetic variations, growth location, cultivation techniques and altitude. Olsson et al. (2004) found that the predominant phenolic compounds were quercetin and catechin. The phenolic acids identified by Demir et al. (2014) and Elmastas et al. (2017) were gallic acid, 4-hydroxybenzoic acid, caftaric acid, 2,5-dihydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid and ferulic acid. Nadpal et al. (2016) listed methyl gallate, catechin, epicatechin, rutin, eriocitrin, quercetin, apigenin-7-O-glucoside, kaempferol, quercitrin and quinic acid as the major flavonoids. In our samples of R. pendulina, the predominant HBA was galloylquinic acid, from the HCAs, 5-caffeoyolquinic acid 2 was predominant, and among the gallotannins, digalloylquinic acid 1 and HHDP digalloylhexoside isomer 1 (ellagitannins) were predominant. The predominant flavanols were procyanidins. The high content ofprocyanidins, catechin and PA dimer diglycoside in R. spinosissima should be emphasised. R. gallica was very rich inp-coumaric acid hexo side 1. From the flavanols, procyanidins andPA dimer monoglycosides should be mentioned. The total content of phenolic compounds in the seeds of the studied rose hips ranged from 757.02 mg · kg⁻¹ f.w. (‘Mount Everest’) to 1714.72 mg · kg⁻¹ f.w. (‘Harstad’) for R. pendulina and cultivars and from 1684.59 mg · kg⁻¹ f.w. for R. spinosissima (‘Frühlingsmorgen’) to 6823.21 mg · kg⁻¹ f.w. (‘Single Cherry’). In contrast, for R. gallica, these levels ranged from 1347.75 mg · kg⁻¹ f.w. (‘Violacea’) to 2155.44 mg · kg⁻¹ f.w. (‘Splendens’). Kunc et al. (2023a) found that the total phenolic content in seeds of selected native-grown Slovenian roses ranged from 1263.08 mg · kg⁻¹ f.w. to 3247.89 mg · kg⁻¹ f.w. Kunc et al. (2022) reported that the content of flavanols in the seeds of R. pendulina was 391.4 mg · kg⁻¹ f.w. and that of flavonols was 39.4 mg · kg⁻¹ f.w. They listed phloridzin as the dominant compound. In the seeds of ‘Harstad’ and R. pendulina, HBA dominated (935.52 mg · kg⁻¹ f.w. and 976.50 mg · kg⁻¹ f.w.), while in ‘Bourgogne’ and ‘Mount Everest’, flavanols were present (384.61 mg · kg⁻¹ f.w. and 341.85 mg · kg⁻¹ f.w.). In the samples of R. spinosissima and its cultivars, the content of ellagitannins dominated, ranging from 880.66 mg · kg⁻¹ f.w. (R. spinosissima) to 2753.88 mg · kg⁻¹ f.w. (‘Single Cherry’). In R. gallica and its cultivars, the content of flavanols dominated, ranging from 751.96 mg · kg⁻¹ f.w. (R. gallica) to 1432.40 mg · kg⁻¹ f.w. (‘Splendens’).

In the study, we included roses that were grown in the park, which means that they were regularly maintained (irrigation, fertilisation, treatments against pests and diseases), unlike some other, similar studies (Kunc et al., 2022), where the plants were grown in a natural environment and not maintained and were also highly exposed to stress factors. Despite all this, from the results obtained, it appears that the content of the compounds analysed is extremely higher. This also indicates a great influence of the genotype to the phenolic picture of the hips.

The content of the anthocyanin cyanidin-3-glucoside was determined in rose hips. The cultivar ‘Single Cherry’ derived from R. spinosissima had the highest content (3090.36 mg · kg⁻¹ f.w.). The extremely high content of the mentioned anthocyanin is the result of the pronounced dark purple colour. In all the samples studied, it is noted that R. spinosissima and its cultivar ‘Poppius’ have identical anthocyanin content (20.97 mg · kg⁻¹ f.w.). However, in the other samples, there is no obvious link between the original rose hip and its cultivars. Kunc et al. (2022) reported that the content of cyanidin-3-glucoside in the hips of wild R. pendulina was 19.3 mg · kg⁻¹ f.w. In comparison to R. pendulina grown in Arboretum Volčji Potok, the content is significantly lower (5.09 mg · kg⁻¹ f.w.). All varieties derived from R. pendulina had a lower content of cyanidin-3-glucoside, except for ‘Mount Everest’, whose content was even 56.61 mg · kg⁻¹ f.w. The content of cyanidin-3-glucoside in the cultivar ‘Splendens’ was only 0.85 mg · kg⁻¹ f.w., and its content in the cultivar ‘Violacea’ was higher than that in R. gallica, with 11.51 mg · kg⁻¹ f.w. The dendrogram of the classification of the samples according to the content of cyanidin-3-glucoside shows that the samples are quite similar to each other. There are three groups of species and cultivars. Cultivars ‘Splendens’, ‘Harstad’ and ‘Bourgogne’ and species R. gallica and R. pendulina form the first group; cultivars ‘Violacea’ and ‘Poppius’ and species R. spinosissima form the second group; and the cultivars ‘Mount Everest’ and ‘Frühlingsduft’ form the third group. Only ‘Frühlingsmorgen’ stands out, not fitting into any of the listed groups. It can be seen that the content of constituents in the cultivars does not often correspond to the content in the species from which they are derived. This indicates that modern breeding has other objectives than increasing the content of polyphenolic compounds in hips of new cultivars.

The phenolic compounds of rose hips from 12 different roses (R. pendulina, R. spinosissima, R. gallica, ‘Violacea’, ‘Splendens’, ‘Poppius’, ‘Frühlingsmorgen’, ‘Frühlingsduft’, ‘Single Cherry’, ‘Harstad’, ‘Bourgogne’ and ‘Mount Everest’) from Slovenia were studied. Research findings showed that the total phenolic content was higher in the flesh with skin than in the seeds. Due to the dark purple colour of rose hips, the variety ‘Single Cherry’ stood out, in which we found the highest content of cyanidin-3-glucoside. We can conclude that most of the samples studied are a rich source of phenolic compounds. Based on the composition of the phenolic compounds, the original species can be clearly distinguished from each other. However, we cannot say that a particular cultivar is derived from it solely based on its content, without knowing the original cultivar from which it is derived. The contents of studied phenolic compounds are very different, and we have found that some cultivars derived from different parent species are even more similar than those derived from the same species. We attribute this to the fact that only one known species of the origin (parent species) was considered in the ingredient and composition analysis, that the plants are subject to mutations and that the breeding houses have different breeding objectives, which is then reflected in the composition of phenolic compounds and their content. We suggest that further research studies should focus on investigating different new hybrids and modern cultivars to find out how different locations affect the content and composition of bioactive compounds in these genotypes. With this information, we would obtain information on which area is best suited for the growth of the varieties under study to obtain the highest content of bioactive substances useful for humans.