Next Article in Journal
Antifungal Activity of Genistein Against Phytopathogenic Fungi Valsa mali Through ROS-Mediated Lipid Peroxidation
Next Article in Special Issue
Artemisia herba-alba Essential Oil: Chemical Composition, Phytotoxic Activity and Environmental Safety
Previous Article in Journal
Metal Biomonitoring Through Arboreal Species in Riparian Ecosystems: Pithecellobium dulce as a Bioindicator Species
Previous Article in Special Issue
Chemodiversity and Antinociceptive Activity of Amorpha fruticosa L. Essential Oil
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 14
Issue 1
10.3390/plants14010119
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Phytochemical Characterisation of Sorbus Species: Unveiling Flavonoid Profiles Related to Ploidy and Hybrid Origin

by
Emina Korić
1,*,
Violeta Milutinović
2,
Alma Hajrudinović-Bogunić
3,
Faruk Bogunić
3,
Tatjana Kundaković-Vasović
2,
Irma Gušić
1,
Jelena Radović Selgrad
2,
Kemal Durić
1 and
Haris Nikšić
1
1
Faculty of Pharmacy, University of Sarajevo, Zmaja od Bosne 8, 71000 Sarajevo, Bosnia and Herzegovina
2
Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia
3
Faculty of Forestry, University of Sarajevo, Zagrebačka 20, 71000 Sarajevo, Bosnia and Herzegovina
*
Author to whom correspondence should be addressed.
Plants 2025, 14(1), 119; https://doi.org/10.3390/plants14010119
Submission received: 30 September 2024 / Revised: 10 December 2024 / Accepted: 26 December 2024 / Published: 3 January 2025
(This article belongs to the Special Issue Phytochemistry and Pharmacological Properties of Medicinal Plants)
Browse Figures
Versions Notes

Abstract

:
The genetic, morphological and taxonomic diversity of the genus Sorbus is due to homoploid and polyploid hybridisation, autopolyploidy and apomixis, which also influence the production and diversity of secondary metabolites, especially flavonoids. The aim of this study was to investigate the relationships and variations of flavonoids in terms of hybrid origin and ploidy level between the parental species and their hybrid derivatives. The sampling design included leaf material of the following Sorbus accessions from ten natural localities: parental taxa (di-, tri- and tetraploids of S. aria; diploid S. torminalis and S. aucuparia) and their di-, tri- and tetraploid hybrid derivatives from crosses of S. aria × S. torminalis (subg. Tormaria) as well as the tetraploid S. austriaca and S. bosniaca, which originate from crosses of S. aria × S. aucuparia (subg. Soraria). We analysed the flavonoid profiles from the leaf fractions by LC-MS. A total of 23 flavonoids were identified, including apigenin and luteolin derivatives, which distinguish the hybrid groups from each other. This profiling highlights the distinctiveness of the Tormaria and Soraria accessions and emphasises the potential of the subg. Tormaria for further research on bioactive compounds in biological studies.

1. Introduction

Natural plant extracts contain different chemical profiles and corresponding biological activities with great application potential. In this regard, there is a growing interest in the discovery and production of plant extracts used in the pharmaceutical, cosmetic and food industries due to their bioactive compounds [1]. Since the biosynthetic pathway secondary metabolite formation varies within the same species due to different factors, a major challenge is to define all possible variability factors that may influence the composition of secondary metabolites [2]. The genus Sorbus L. is recognised as a highly valued source of bioactive compounds for the development of pharmaceutical, cosmetic and nutraceutical products [3] but has not yet been sufficiently explored.
The genus Sorbus (whitebeams, rowans and service trees) comprises tree and shrub species characterised by enormous genetic and morphological diversity. This diversity is the result of polyploidisation, hybridisation and apomixis [4,5]. Recurrent hybridisation between diploid species of S. aria (L.) Crantz, S. aucuparia L., S. chamaemespilus (L.) Crantz and S. torminalis (L.) Crantz and backcrossing of hybrid derivatives with their parental species resulted in about 190–200 recognised species inhabiting Europe [6,7]. The genus is traditionally divided into five subgenera: (subg.) Aria, Chamaemespilus, Cormus, Sorbus and Torminaria [8]. However, phylogenetic analyses recognise at least two monophyletic groups (Sorbus, Cormus; Aria, Torminaria and Chamaemespilus) [9,10]. Due to the obvious polyphyletic nature of the genus and extensive hybridisation between the subgenera members, the Sorbus genus is divided into independent genera [7], but the traditional name is still in use.
Most species are polyploid, triploid and tetraploid and reproduce by facultative apomixis [4,5,8,11]. Most polyploid species are endemics with a narrow geographic distribution, whereas the parental species are much more widely distributed throughout Europe [12]. While many aspects of Sorbus taxonomy [6,8,11], genetics [4,13] and reproduction [5,14], including phytochemical diversity, have been thoroughly studied in different parts of Europe, they are still understudied in the Balkans.
The remarkable genetic, morphological and physiological variability within the genus Sorbus is also reflected in the production and diversity of secondary metabolites [15]. Polyploidy and hybridisation are considered triggers that cause increased productivity of secondary metabolites and their diversity [16]. In particular, hybridisation is a driving mechanism that facilitates the production of secondary metabolites and restores their combinations [17,18].
Recent studies have revealed a great diversity and variation in the phytochemical composition of the different Sorbus species [19,20,21]. The genus is rich in flavonoids including anthocyanins [21], phenolic acids [22,23], triterpenes/tannins [24] and various other phytochemicals known for their antioxidant properties [25,26], anti-inflammatory [27], antimicrobial [28], antihyperlipidemic [29], antidiabetic [30], neuroprotective [31], hepatoprotective [24] and cardioprotective effects [32].
Flavonoids, as a group of secondary metabolites, are useful indicators of hybridisation and, in some cases, polyploidisation [33,34], but recent studies do not support flavonoids as indicators of polyploidy. In previous phytochemical studies, well-known flavonols such as quercetin and kaempferol and their glycosides, along with their methyl derivatives and less common C-flavonoids, have been detected in various parts or organs of Sorbus taxa, including the species examined in this study: S. aria, S. torminalis, S. aucuparia, and S. austriaca [19,23,35,36,37,38]. Many studies in the literature have focused on the flavonoid content in Sorbus fruits or inflorescences [23,36,37,38,39], with fewer studies on the bark and even more limited research on the leaves [22,25]. It is interesting that leaf material is the least used in flavonoid research, even though leaves contain a much higher content of polyphenolic components compared to flowers and fruits [22,25]. Most studies investigated the phytochemical composition of crude methanolic extracts from fruits, inflorescences and leaves, while only two reported detailed phytochemical analysis of their fractions [21,39]. In most studies, the plant material used for the investigation of polyphenolic profiles also originated from cultivated Sorbus accessions from botanical gardens and parks [39,40]. Numerous studies dealing with the qualitative and quantitative analysis of Sorbus the polyphenolic compounds were based on LC-UV/VIS spectroscopy [39] and the Folin–Ciocalteu method [41]. In contrast, LC-MS [21,22], UPLC-QTOF-MS [19,42] and UPLC-ESI_MS [3] have been used in a few studies to determine the phytochemical profile of Sorbus species.
The aim of this study is to provide the first insights into the leaf flavonoid profiles of Sorbus species from the Balkans (Bosnia and Herzegovina) using liquid chromatography-mass spectrometry (LC-MS). We analysed the diploid S. aria (subg. Aria Pers.), S. aucuparia (subg. Sorbus) and S. torminalis (subg. Torminaria) as well as the members of the hybridogenous subg. Soraria Májovský and Bernátova (S. aucuparia × S. aria) and Tormaria Májovský and Bernátova (S. torminalis × S. aria). We included tetraploid S. austriaca (Beck.) Hedl. and S. bosniaca Hajrudinović, Frajman, Schönswetter and Bogunić from the subg. Soraria), as well as di-, tri- and tetraploids from the subg. Tormaria. In addition, we also included tri- and tetraploid samples of S. aria. In particular, our aim was to (1) identify flavonoid markers that distinguish Sorbus accessions; (2) investigate the influence of hybridisation on the variation in the qualitative composition of flavonoids; and (3) determine the relationships between the ploidy level and the qualitative and quantitative content of flavonoid compounds.

2. Results

2.1. Ploidy Level

The ploidy level of 17 individuals from 10 locations was determined (See Material and Methods). The absolute genome size revealed three ploidy levels. Diploid values (2C = 2x) were found in S. aucuparia (2C = 1.43 pg), S. aria (2C = 1.44 pg), S. torminalis (2C = 1.44 pg) and S. torminalis × S. aria (2C = 1.43 pg). Triploids (2C = 3x) of S. aria had 2C= 2.11 pg and S. torminalis × S. aria had 2C = 2.18 pg. Tetraploid cytotypes (2C = 4x) were observed in S. aria (2C = 2.80 pg), S. austriaca (2C = 2.79 pg), S. bosniaca (2C = 2.80 pg) and S. torminalis × S. aria (2C = 2.74 pg). (See Material and Methods).

2.2. Mass Spectrometric Identification of Flavonoid Compounds

The structures of 23 flavonoids (1–23) were investigated using LC-MS by analysing 17 different ethyl acetate (EtOAc) fractions from methanol (MeOH) leaf extracts of various Sorbus accessions, including parental and hybrid individuals with different ploidy levels. The spectral data and the results of qualitative analyses are listed in Table 1, and the selected chromatograms are shown in Figure 1A,B. Ten compounds were identified after comparison with commercial standards: apigenin 6,8-di-C-glucoside (vicenin—2), apigenin-6-C-glucoside-8-C-arabinoside (schaftoside—4), quercetin 3-O-rutinoside (rutin—7), quercetin 3-O-galactoside (hyperoside—8), quercetin-3-O-glucoside (isoquercitrin—9), luteolin-7-O-glucoside (cynaroside—10), luteolin-7-O-glucuronide (11), kaempferol 3-O-glucoside (astragalin—16), quercetin 3-O-rhamnoide (quercitrin—17), apigenin 7-O-glucuronide (21). The structures of the other detected compounds were characterised based on similarities of the mass and UV spectra with level 3 data in the literature, according to Schymanski et al. [43].

2.2.1. Flavones

Compounds 2, 4 and 21 were assigned to apigenin glycosides based on their UV spectra (Table 1). The mass spectra (MS) of apigenin 7-O-glucuronide (21; [M–H]− at m/z 445) contained signal corresponding to the aglycone at m/z 269 formed by the neutral loss of 176 Da, whereas flavonoids 2 and 4 showed different fragmentation patterns of C-flavonoid glycosides ([M–H]− at m/z 593, and 563, respectively). As the C–C glycosidic bonds are more stable than O-glycosidic bonds, those flavonoids cannot yield fragments due to the loss of neutral sugar, but fragments resulting from the breakdown of the C–C bonds within the sugar moiety and total cross-ring cleavage [47,48]. In all three cases, the reference compounds confirmed the identity. The UV and MS spectra of 10, 11 and 20 corresponded to luteolin glycosides (the MS spectra contained a fragment ion at m/z 285 corresponding to the aglycone). Compounds 10 and 20 showed a luteolin hexoside ([M–H]− at m/z 447, fragment ion at 285), whereas 11 had a luteolin hexuronide structure ([M–H]− at m/z 461, fragment ion at 285). The compounds identified as luteolin 7-O-glucoside (10) and luteolin 7-O-glucuronide (11) were confirmed with standard compounds.

2.2.2. Flavonols

The UV spectra of flavonoids 1, 3, 5–9, 12–15, 16, 17, and 22 indicated the 3-substituted flavonol structure [49]. All mentioned flavonoids (except 12, 16 and 22) had MS spectra containing the signal of deprotonated quercetin molecule at m/z 301 (formed after sugar losses), and fragment ions from its RDA reactions [50]. Quercetin 3-O-rutinoside (7; [M–H]− at m/z 609), quercetin 3-O-galactoside (8; [M–H]− at m/z 463), quercetin 3-O-glucoside (9; [M–H]− at m/z 463), and quercetin 3-O-rhamnoside (17) with the signal of the deprotonated molecule at m/z 447 were identified from standard compounds. Additional quercetin diglycosides 3, 6 and 13 showed fragmentation patterns corresponding to quercetin dihexoside (3; [M–H]− at m/z 625), quercetin deoxyhexosyl hexoside (6; [M–H]− at m/z 609), and quercetin hexosylpentoside (13; [M–H]− at m/z 595), respectively. The fragment ions were generated by the loss of one or more hexose units (162 Da), deoxyhexose (146 Da), or pentose moiety (132 Da).
The structure of compound 5 was assigned to quercetin trideoxyhexoside, while 14 was characterised as quercetin acetylhexoside (with signals of [M–H]− at m/z 505, fragment ions at m/z 463 and 301), and 15 as quercetin pentoside ([M–H]− at m/z 433, fragment ion at m/z 301). The kaempferol glycosides (12, 16, 22) were identified from the standard compound in the case of kaempferol 3-O-glucoside (16; [M–H]− at m/z 447), or by analyzing their mass spectra. Therefore, 12 was characterised as kaempferol deoxyhexosylhexoside ([M–H]− at m/z 593, fragment ion at m/z 285), and 22 ([M–H]− at m/z 489) as acethylhexoside of kaempferol, based on the neutral loss of 42 Da corresponding to the acetyl group and the loss of hexose.

2.2.3. Methylated Flavonols

Flavonoid glycosides 18, 19 and 23 ([M–H]− at m/z 477 and 519) showed mass spectra related to threehydroxy-methoxy-flavone-3-ol with fragment ions at m/z 316 (corresponding to aglycone) originating from neutral losses of hexose (in 18 and 19) and acethylhexose (23); 301 (additional neutral loss of methyl group), 285 (additional loss of methoxy group).

2.3. Quantitative Analysis of Flavonoids

The quantification of the 10 identified flavonoids was performed using the external standard method with authentic commercial standards. Due to structural similarities, five of these standards were also used for the quantification of the remaining 13 identified flavonoids. The regression equations of the calibration curves, their correlation coefficients (r2), concentration ranges, LODs and LOQs are shown in Table S1. Secondary metabolites present in only one individual were not considered in the analysis. The amounts of flavonoids identified (in g/100 g DE) are listed in Table 2. Analysis of the ethyl acetate fractions revealed that quercetin flavonoids were the predominant component of the flavonoid profile in almost all studied Sorbus accessions (accounting for up to 75.34% of the total flavonoids detected in triploid S. aria from Mt. Igman). Luteolin flavonoids and apigenin flavonoids were detected only in samples of the subg. Tormaria group accounted for 6.5–17.8% and 1.7–60% of the total flavonoids identified, respectively.
Kaemferol flavonoids ranged between 1.3 and 21.8% and 2.0 and 3.5% of the total flavonoids detected in the subg. Soraria and Tormaria, respectively. Within the subg. Soraria group, a single triploid cytotype of S. aria from Umoljani is characterised by the trace/absence of kaempferol derivatives (0% for compounds 21 and 25 and a trace amount for compound 33). Within the Tormaria group, a single triploid cytotype of S. torminalis × S. aria from Koznik was characterised by the complete absence of kaempferol derivatives.

2.4. Pattern of Flavonoid Variation and Relationships Among the Studied Sorbus Samples

Phytochemical variation based on the quantitative data was complex and principal component analysis revealed eight significant principal components (Eigenvalues ≥ 1) (two components are shown). They accounted for 49.3% of the total variance (PC1 = 31.2%, PC2 = 18.1%; Figure 2A) and showed moderate correlation with most of the corresponding flavonoid compounds (Table S2). The following compounds contributed most strongly to PC1 (schaftoside, isoquercitrin, luteolin 7-O-glucoside and luteolin hexoside; luteolin 7-O-glucuronide, kaempferol deoxyhexosylhexoside, astragalin and methylquercetin hexoside isomer 2 contributed to PC2 (Table S2). The PCA ordination diagram showed the pattern in which S. torminalis and all Tormaria representatives along PC1 clearly diverged from the other Sorbus accessions (Figure 2A). The position of S. bosniaca was also divergent (Figure 2A). Along PC2, S. austriaca overlapped with a single diploid and two triploid S. aria samples separated from S. aucuparia and the remaining of S. aria samples, including all tetraploids (Figure 2A).
The PCoA analysis of all individuals showed a similar pattern as the PCA but with a higher resolution among the studied accessions (Figure 2B). Sorbus torminalis and all Tormaria members, as well as the diploids (Bijela gora and Gradac) and triploids (Bijela gora and Umoljani) of S. aria, S. austriaca and S. bosniaca are clearly distinct along PCo1 from S. aucuparia and all tetraploids of S. aria as well as the diploids and triploids of Mt. Igman. Sorbus aucuparia was separated from different S. aria cytotypes along PCo2 (Figure 2B). The members of the Soraria subg. (S. austriaca and S. bosniaca) and the cytotypes of S. aria were separated along PCo2 from the members of S. torminalis and Tormaria (Figure 2B).
PCoA ordination of Tormaria members and putative parental species showed a clear position of S. torminalis and Tormaria cytotypes compared to S. aria cytotypes (Figure 3A). The cytotypes of S. torminalis × S. aria were clearly closer to S. torminalis than to S. aria (Figure 3A).
The ordination of Soraria samples (S. austriaca and S. bosniaca) showed an interesting pattern of spatial distribution between PCo1 and PCo2 (Figure 3B). Along PCo1, S. bosniaca and the diploid S. aria from Bijela gora diverged the most; the triploids of S. aria (Umoljani and Bijela gora) and the diploid S. aria from Bijela gora intermingled with S. austriaca compared to the tetraploids of S. aucuparia and S. aria and the diploids and triploids from Mt. Igman (Figure 3B). Sorbus aucuparia had an intermediate position along the PCo2 compared to the di-, tri- and tetraploid cytotypes of S. aria and S. austriaca. Along PCo2, S. bosniaca was clearly separated from the cytotypes of S. austriaca and S. aria (Figure 3B).
The most intriguing pattern was observed in the cytotypes of the S. aria (Figure 3C). The diploids of S. aria were the most divergent and separated along PCo1 and PCo2 (Figure 3C). While the tetraploid S. aria cytotypes represented a uniform group, the triploids segregated along PCo1. No specific pattern was recognisable for the S. aria cytotypes.
The UPGMA cluster analysis revealed two main clusters (Figure 4A). The first cluster contained S. torminalis and its hybrids S. torminalis × S. aria. The second cluster contained two subclusters, one of which contained S. bosniaca, S. austriaca, S. aria diploids (Bijela gora and Gradac) and triploids (Umoljani and Bijela gora), while the second contained S. aucuparia and a group consisting of exclusively S. aria samples (Figure 4A).
The NMDS plot was consistent with the results of the previous multivariate analyses but showed a clearer separation between the Sorbus samples analysed (stress value S = 0.202 (Figure 4B). The relatively moderate stress value (S = 0.202) among the flavonoid compounds in the original data matrix is well illustrated in the ordination diagram (Figure 3B). The samples of Sorbus torminalis, S. aucuparia, S. bosniaca, the diploid S. aria from Bijela gora and the hybrid S. torminalis × S. aria differed significantly from each other. Di-, tri- and tetraploid S. aria cytotypes from Mt. Igman, Bijela gora and Umoljani were grouped together, while S. austriaca overlapped with the triploids S. aria (Figure 4B). Both the qualitative and quantitative values of the luteolin and apigenin derivatives clearly distinguished the Tormaria group, especially between the parental species S. torminalis and the closest cluster, which comprised hybrid derivatives of S. torminalis × S. aria. The active components that grouped di, tri and tetraploid individuals of S. aria from Mt. Igman, Bijela and Umoljani into a unique cluster were quercetin and kaempferol derivatives. In the differentiation of S. aucuparia, S. bosniaca, the diploid S. aria from Bijela gora, S. austriaca and the triploids of S. aria, predominantly quercetin derivatives were detected as discriminating metabolites.
The results of the Mantel test showed a weak and significant correlation between the Bray–Curtis distances (r = 0.285, p ≤ 0.004) for all studied accessions. In contrast, no correlation was found for the members of the subgenus Aria (r = 0.044, p ≤ 0.354).

3. Discussion

3.1. Novel Flavonoid Compounds in the Leaves of Sorbus Accessions

Our study reports the detection of 23 flavonoids in the EtOAc fractions from methanol (MeOH) leaf extracts of all Sorbus accessions examined, including 14 quercetin derivatives and three derivatives of apigenin, luteolin and kaempferol. This study is the first to analyse samples of triploid and tetraploid Tormaria accessions, S. bosniaca, and diploid and polyploid cytotypes of S. aria from the Balkan region. From the point of view of phytochemical research, this study demonstrated for the first time the presence of 14 flavonoids (Table 2) that had not been previously detected in the leaves of the Sorbus accessions studied. Among them, the rare flavonoids C-glycosides (apigenin 6,8-di-C-glucoside, apigenin-6-C-glucoside-8-C-arabinoside) were identified for the first time in representatives of the subg. Tormaria. Hydroxyquercetin deoxyhexosyl hexoside (1), quercetin trideoxyhexoside (5), quercetin deoxyhexosyl hexoside (6), quercetin hexosylpentoside (13), methylquercetin acetylhexoside (23), kaempferol deoxyhexosylhexoside (12) and kaempferol acetylhexoside (20) were found for the first time in S. aria and S. austriaca, while methylquercetin hexoside isomer 2 (19) was found exclusively in S. aria and quercetin pentoside in S. austriaca. For the first time, the presence of luteolin-7-O-glucoside (10), luteolin-7-O-glucuronide (11), luteolin hexoside (20) and methylquercetin hexoside isomer 2 (19) was detected in leaves of S. torminalis. In addition, this study is the first to show the flavonoid profile of leaves of different S. torminalis × S. aria cytotypes from the Tormaria subg. as well as the endemic species S. bosniaca from the Soraria subg. (Table 2).
Compared to the EtOAc fractions of S. aria leaves from the cultivars analysed in the study by Olszewska and Michael [51], the EtOAc fractions of S. aria leaves from the Bosnia and Herzegovina region contained higher amounts of the active components hyperoside (trace amounts in the study by Olszewska and Michael [51], up to 0.568 ± 0.006 g/100 g in our analysed dry extract) and rutin (1.37 ± 0.03 g/100 g in the samples reported by Olszewska and Michael [51], up to 7.504 ± 0.019 g/100 g in our analysed samples). On the other hand, the analysed EtOAc fractions contained lower amounts of astragalin and isoquercitrin compared to the EtOAc fractions of the cultivars from the study by Olszewska and Michael [51]. The EtOAc fractions of S. torminalis leaves from the natural habitat and the EtOAc fractions of S. torminalis leaves from Bosnia and Herzegovina analysed by Gunes AK et al. [21] are characterised by a high concentration of hyperoside (1.226 ± 0.014 g/100 g DE and 1.284 ± 0.009 g/100 g DE, respectively). Although quercitrin was not detected in the EtOAc fractions of S. torminalis analysed in our study, it was identified in the fractions analysed in the study by Gunes AK et al. [21]. In our study, methylquercetin hexoside isomer 2 (19) was detected for the first time in the EtOAc fraction of S. torminalis leaves at a concentration of 1.669 ± 0.015 g/100 g dry extract (DE).

3.2. Potential Discrimination of Subgenera and Lower Taxa in Sorbus: Influence of Flavonoid Profile on Hybridisation and Ploidy Level

Our analysis based on flavonoid profiles revealed intriguing relationships between the Sorbus accessions studied. Analyses based on quantitative data (PCA) and analyses based on binary data (PCoA, CA and NDMS) generally showed a similar pattern, although the latter provided a clearer resolution. The discrimination of the parental species S. aucuparia and S. torminalis is clear, in contrast to S. aria, which showed a remarkable divergence within the intragroup regardless of ploidy level and geographical origin (Figure 2A,B). The discrimination of subg. Aria and the hybrid subg. Tormaria and Soraria showed different patterns. While the members of subg. Tormaria are clearly diverge from each other and are more related to S. torminalis (Figure 2A,B and Figure 3A), the relationships in subg. Aria and Soraria are less clear (Figure 2A,B and Figure 3B,C). Indeed, the number of distinctive flavonoids detected in Tormaria was notably higher than in Soraria and Aria samples (Table 2). Moreover, certain compounds were only detected in S. torminalis and its hybrid cytotypes (Table 2), compared to the members of Aria (including all cytotypes) and Soraria. The large divergence between S. torminalis and Tormaria is primarily due to the presence of ‘discriminating’ compounds (apigenin 6,8-di-C-glucoside, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, luteolin hexoside, apigenin 7-O-glucuronide).
The relationships between the parental species and the derived hybrids within the subg. Tormaria and Soraria are even clearer (Figure 3B,C). Sorbus torminalis acts as the maternal parent, as in most hybridogen taxa of the subg. Tormaria [4,13,52,53], suggesting a maternal effect in the inheritance of flavonoid compounds. The strong parental differentiation between S. torminalis on the one hand and S. aria on the other is due to the ‘discriminating’ compounds that are completely absent in the subgenus Aria (Table 2). These are derivatives of luteolin: luteolin-7-O-gluco-side (10), luteolin-7-O-glucuronide (11) and luteolin hexoside (20), characteristic compounds for the EtOAc fraction of S. torminalis × S. aria and the corresponding parental species S. torminalis. On the other hand, the derivatives of apigenin: apigenin-7-Oglucuronide (21) and apigenin-6-C-glucoside-8-C-arabinoside (4) are also characteristic compounds only for the EtOAc fraction of S. torminalis × S. aria and the corresponding parental species S. torminalis. The apigenin 6,8-di-C-glucoside (2) is exclusively present in the ethyl acetate fraction of S. torminalis × S. aria, but not in the parental species S. torminalis. In contrast to the previously mentioned observation, kaempferol 3-O-glucoside (16) is exclusively detected in the parental lineage S. torminalis, but is completely absent in the individuals of the hybrid S. torminalis × S. aria. The presence or absence of apigenin and luteolin derivatives allowed a clear distinction between the studied groups Aria, Tormaria and Soraria. These flavonoids, known for their biological activity, can serve as markers for the differentiation of the various Sorbus accessions. Within Tormaria, a complete absence of methylquercetin acethylhexoside (23) and quercetin pentoside (15) was found. Although the Tormaria accessions share most flavonoid compounds with the parental species, they also possess two exclusively novel compounds: (21) methylquercetin hexoside isomer 1 and (1) apigenin 6,8-di-C-glucoside (Table 2). Feulner et al. [53] found that the German Tormaria samples contained mixtures of the parental compounds; however, novel compounds were not detected.
The relationships between the Soraria samples and their parental species, particularly S. austriaca and S. bosniaca, were less clear (Figure 3B and Figure 4A,B). The samples of S. austriaca were more closely related to S. aucuparia, but surprisingly distinct from S. bosniaca (Figure 2A, Figure 3B and Figure 4A,B). The flavonoid composition of S. austriaca and S. bosniaca had only six common compounds (rutin, hyperoside, isoquercitrin, quercetin hexosylpentoside and kaempferol acetylhexoside, Table 2), whereas S. bosniaca also contains the three compounds found in S. torminalis, Tormaria subg. and S. aria cytotypes (quercetin deoxyhexosyl hexoside (18), methylquercetin hexoside isomer 1 (21) and methylquercetin hexoside isomer 2 (22), Table 2). In addition, the compound quercetin dihexoside (3) is found exclusively in S. austriaca and S. aucuparia, whereas it is completely absent in the subg. Aria and S. bosniaca. While the component quercetin acetylhexoside (15) is uniquely present in both samples of S. austriaca, quercetin trideoxyhexoside (5) and methylquercetin acethyhexoside (23) are detected in a single individual of S. austriaca (Mt. Umoljani). The absence of these compounds in the second S. austriaca sample is intriguing, as most Balkan populations represent a single genetic clone due to apomixis [54]. This finding suggests a possible independent origin of these two S. austriaca samples. In addition, a triploid S. aria sample (Mt. Umoljani) shares these two compounds with S. austriaca (Table 2), suggesting a possible hybridisation between the two species. The most common way to form Sorbus polyploids is by crossing a polyploid formation, whereas tetraploid apomict is crossed with a sexual diploid to produce a triploid apomict [5,55,56,57]. Therefore, such a scenario is likely, as the Umoljani site represents a large sympatric community of S. aria cytotypes and S. austriaca, which has been well studied in terms of ploidy dynamics and reproductive mode [57].
Similar patterns with respect to the presence or absence of quercetin derivatives are shown for methylquercetin acethyhexoside (23), quercetin deoxyhexosyl hexoside (6) and quercetin hexosylpentoside (13) in S. austriaca and S. bosniaca (Table 2). Such a variable composition of flavonoids in two closely related species that share at least one parental species may be due to several factors. First, both species originated from hybridisation between subg. Aria and S. aucuparia and represent the allotetraploid lineages, but their exact origin remains unclear [54,57]. In most taxa of the subg. Soraria, S. aucuparia is the maternal parent and the pollen donors are various members of the subg. Aria, whose identity remains unknown in most cases [4,14,52,55,57]. Therefore, the significantly different flavonoid profiles of these two species are not surprising (Table 2), which is due to the unknown paternal parent as well as the allopolyploid origin of these taxa. Each polyploid hybridisation is an independent event that can significantly restructure the genome, alter gene expression and induce physiological, phenotypic and biochemical changes [58,59]. Second, the diversity in flavonoid composition could be attributed to the parental influence of the subg. Aria members in hybridisation events, but this should be interpreted with caution and requires further research.
The effects of hybridisation on the occurrence of flavonoids differ within the subg. Soraria and Tormaria. The presence/absence of quercetin derivatives is associated with the subg. Soraria, whereas the variation of luteolin, apigenin and kaempferol derivatives is associated with the subg. Tormaria. In addition, S. aria as the paternal parent has a significant influence on the qualitative variation within the Soraria group in both subg., while S. torminalis plays a more important role in the subg. Tormaria. This phenomenon is probably also related to the biosynthetic pathway of the flavonoids. The pathway starts with p-coumaroyl-CoA and malonyl-CoA, which are condensed by chalcone synthase (CHS) to 2,4,6,4-tetrahydroxy chalcone. Calchone is then converted to naringenin by chalcone isomerase (CHI) [60]. From this point on, naringenin becomes a precursor for all the different biosynthetic pathways. The different biosynthetic pathways depend on the action of key enzymes such as flavone synthase (FSN) and flavonol synthase (FLS). When FSN is active, narigenin is converted into flavones. When FLS is active, narigenin is hydroxylated to form dihydroflavonols, which are precursors of flavanols [61]. Based on the above results and the absence of flavone derivatives in the Soraria samples, it can be concluded that the expression of the FSN enzyme is either repressed or inhibited, in contrast to the Tormaria samples. The question of whether the enzyme has been lost in the Soraria species or whether they have ever acquired it has not yet been clarified [62].
Certain individuals, both in the subg. Soraria and in the subg. Tormaria, produced novel compounds compared to the parental individuals. This is considered a rare example of hybridisation and polyploidy influencing the qualitative variation of secondary metabolites. Inheritance of secondary metabolites often conforms to Mendelian principles, where the presence of a secondary metabolite occurs when at least one parent produces it, resulting in complementary chemical patterns. However, deviations from this pattern are due to changes in the biosynthetic pathway [63]. These changes may include the addition or loss of genes or alleles that lead to new biochemical properties [17,63]. Blockages in the metabolic pathway can lead to the accumulation of intermediates, or unique interactions between enzymes and genes from each parent can lead to novel compounds [64]. Mutations in regulatory genes during hybridisation can also alter where or how secondary metabolites are produced, leading to greater biochemical diversity. On the other hand, the quantitative characteristics of secondary metabolites are controlled by multiple genes and influenced by dominant, recessive, additive or epistatic interactions [65]. F1 hybrids generally exhibit intermediate levels of secondary metabolite expression, with additive inheritance being the most common pattern. Additive effects account for 56% of the variation in secondary metabolite expression [17,65]. This variation can result from allelic differences affecting enzymes in the biosynthetic pathway or regulatory genes [17].

3.3. No Polyploid Effect on Flavonoid Profiles in S. aria Cytotypes

The production of secondary metabolites associated with ploidy is still a poorly understood aspect of plant biology. Polyploidy can lead to the differential regulation of biosynthetic pathways, affecting both the concentrations of biosynthetic products and the presence of certain metabolites. Studies indicate that tetraploids accumulate higher concentrations of phenylpropanoid compounds compared to diploid progenitors [66,67,68]. However, the concentrations of individual metabolites in polyploids can sometimes be lower than expected [68]. Our results, based on the Mantel test, generally showed a weak association of flavonoid profiles with the increase in ploidy in the Sorbus samples analysed. This pattern has been previously confirmed in other species [69,70,71,72,73]. On the other hand, no correlation was found between the increase in ploidy and the flavonoid profiles in samples of the subg. Aria. The Aria samples in this study exhibited a wide diversity of flavonoid profiles, regardless of ploidy level and geographical origin (Figure 3C and Figure 4A,B). The highest divergence among the diploid samples is particularly surprising. This pattern suggests that geographically determined flavonoid diversity is due to geographic origin [74]. Triploids and tetraploids showed less divergence (Figure 3C and Figure 4A,B), with the latter being the most homogeneous group within the analysed Aria group. The polyploids of the subg. Aria originated by hybridisation or polyploidy from diploid S. aria s. str. interacting with polyploids [8,75]. Therefore, the autopolyploid versus the allopolyploid origin of the Aria polyploids is probably also an important factor in shaping the flavonoid diversity within this group. The grouping pattern of S. aria tetraploids in this study was found to be the most homogeneous group in terms of flavonoid compounds (Figure 3C and Figure 4A; Table 2), suggesting an autopolyploid origin. It has been observed that the concentration of secondary metabolites increases in autopolyploids, probably due to a gene dosage effect [76,77], but this is not the case in the tetraploid samples.
In allopolyploids, it is assumed that the combination of biosynthetic complements from two progenitor species increases the variation in the resulting production of secondary metabolites [77]. Allopolyploidisation events can also lead to an organism that can produce all enzymes and metabolites of the progenitor species [78]. A high degree of divergence was observed in triploid S. aria samples (Figure 3C and Figure 4A,B), suggesting a possible allotriploid origin for at least two of them. This is particularly indicative of triploids of S. aria from Umoljani and Bijela gora, which intermix with S. austriaca (Figure 4A,B). Triploids of S. aria are most likely products of pollen exchange from apomictic tetraploids to sexual diploids, a pathway of triploid formation via the tetraploid bridge [5,9,57]. In the case of the two triploid S. aria, the potential pollen donor may be the tetraploid S. austriaca or S. aria [59]. The triploid S. aria from Mt. Igman is in the clade with the diploid and tetraploid S. aria samples from Mt. Igman (Figure 4A), indicating a probable autotriploid origin. The discovery of novel compounds in series of polyploid S. aria (methylquercetin acethylhexoside (23) in the diploid; hydroxyquercetin deoxyhexosyl hexoside (1) in the triploid; kaempferol 3-O-glucoside (16) and methylquercetin hexoside isomer 2 (19) in the tetraploid) suggests an allopolyploid origin of the triploids and tetraploids [69]. The comparison of the flavonoid profiles of S. aria from Mt. Igman shows that eight flavonoid compounds occur in all three ploidy levels. Of these, only one, quercetin-3-O-glucoside (9), shows an increase in the tetraploid sample, while other flavonoids do not follow the pattern of correlation between ploidy level and flavonoid content. In addition, a novel compound, quercetin 3-O-galactoside (8), was identified in the tetraploids. Of these, only one compound, quercetin 3-O-rutinoside (7), shows an increase in the tetraploid sample of S. aria from Bijela gora, while other flavonoids do not follow the pattern of correlation between ploidy level and flavonoid content. In addition, a novel compound, methylquercetin acethylhexoside (23), was found in the diploid S. aria, hydroxyquercetin deoxyhexosyl hexoside (1) in the triploid S. aria and kaempferol 3-O-glucoside (16), methylquercetin hexoside isomer 2 (19) in the tetraploid S. aria were identified.

4. Materials and Methods

4.1. Plant Material

Leaf material of Sorbus accessions (species and cytotypes) from 10 locations in Bosnia and Herzegovina was collected in July 2022 and prepared for phytochemical and cytometric analyses (Table 3). The identity of the studied taxa was confirmed by Faruk Bogunić and Alma Hajrudinović (Faculty of Forestry, University of Sarajevo, Bosnia and Herzegovina). The sampling design included the following Sorbus accessions: parental species (di-, tri- and tetraploids of S. aria; diploid S. torminalis and S. aucuparia) and their di-, tri- and tetraploid hybrid derivates from S. torminalis × S. aria crosses (S. subg. Tormaria) and tetraploid S. austriaca and S. bosniaca originated from S. aria × S. aucuparia crosses (S. subg. Soraria) (Table 3). The nomenclature system for the classification of subgenera followed Májovsky and Bernátová [79]. For this study, polyploid S. aria and S. torminalis × S. aria individuals were treated as cytotypes, as these entities have not yet been circumscribed. Detailed information of the geographic origin, genome size (GS) and ploidy level of the Sorbus accessions analysed is provided in Table 3. Fresh leaves were used for the cytometric analyses, while the remaining collected raw material was dried at a temperature of 20–25 °C and stored under dark, dry conditions.

4.2. Determination of Ploidy Level

Ploidy determination was preformed according to the protocol of Hajrudinović et al. [59] using flow cytometry. In brief, fresh leaves of selected individuals from 10 populations (Table 3) were co-chopped with fresh leaves of the internal standard, Medicago truncatula Gaertn. cv. R108-1 (0.98 pg, Marie and Brown [80], in 600 mL of cold Gif nuclear buffer [81]. The suspension was filtered through a 50 μm nylon sieve (CellTrics, Partec), and RNase (Roche) was added to 25 U mL−1. The cell nuclei were stained with propidium iodide (Sigma-Aldrich) at a final concentration of 50 mg mL−1 and incubated on ice for 5 to 10 min before analysis. The fluorescence of approximately 3000 cell nuclei was recorded for each sample using either a Partec CyFlow SL3 (Partec, Münster, Germany) 532 nm laser cytometer or a CyFlow Ploidy Analyser (Sysmex Europe SE) 532 nm laser. The 2C DNA values were determined and the DNA ploidy level [82] were derived by comparing these values with those of individuals with known chromosome number [83].

4.3. Sample Preparation for LC-MS Analysis

Dried leaves of Sorbus accessions were pulverized and extracted with methanol, which served as a suitable extraction solvent at 20 °C, with two cycles of 20 min each using ultrasound. The drug-to-solvent ratio was 1:40. The methanolic extracts were centrifuged at 4000 rpm for 20 min at 4 °C. The prepared methanolic extract was evaporated to dryness, reconstituted in water at a 1:100 ratio, and then subjected to successive liquid–liquid extractions with solvents of increasing polarity: (a) hexane, (b) diethyl ether and (c) ethyl acetate. The ethyl acetate fractions (EtOAc) were collected, evaporated to dryness, and dissolved in 1 mL of HPLC-grade methanol prior to analysis [84].

4.4. LC-MS Analysis

LC-MS analysis of EtOAc fractions of Sorbus leaves obtained from MeOH extracts was performed on an Agilent LC/MS system 1260/6130 (Agilent Technologies, Waldbronn, Germany) with diode array detector (DAD) and single quadrupole API-ESI MSD. A Zorbax SB-Aq column (150 × 3.0 mm; 3.5 μm particle size, Agilent Technologies), and a binary mobile phase (A—formic acid, 0.1%, v/v; B—acetonitrile) were used for chromatographic separation, and the gradient program, column temperature, flow rate, injection volume and DAD and MSD parameters were determined based on previous work [85]. The obtained dry fraction/extracts were dissolved in MeOH (final working concentrations were 5 mg/mL) and filtered through a cellulose membrane filter (0.45). The recorded raw data were processed using ChemStation (Rev. B.04.03-SP1) software. The external standard method was used for quantitative analysis and the levels of the detected compounds were calculated based on the peak areas obtained by DAD (at 350 nm) using calibration curves obtained for ten commercial standards (regression equations, correlation coefficients (r2), linear ranges, LODs and LOQs in μg/mL are given in the Table S1. Following the guidelines of the International Conference on Harmonization (ICH, 2005), the LODs and LOQs were determined using the signal-to-noise ratio as described in the European Pharmacopoeia (Ph. Eur. 11.0) [73,86]. The analysis was performed in triplicate. The amounts of the detected compounds were calculated as gram equivalents of the standards used per 100 g of dry fraction (dw) due to their high structural similarity.

4.5. Chemicals and Reagents

LC-MS-grade formic acid and acetonitrile, as well as standards (of HPLC purity): Rutin, Isoquercitrin, Quercitrin, Luteolin 7-O-glucoside, Luteolin 7-O-glucuronide, Astragalin, Apigenin 6,8-di-C-glucoside (Vicenin), Apigenin-6-C-glucoside-8-C-arabinoside (Schaftoside) and propidium iodide (Sigma-Aldrich, Merck Group, Merck KGaA, Darmstadt, Germany), Quercetin 3-O-galactoside (Carl Roth, Karlsruhe, Germany), Apigenin 7-O-glucuronide from HWI Analytik (Ruelzheim, Germany).

4.6. Data Analysis

Descriptive statistics (mean and standard deviation for each of the analysed compounds at the individual level), univariate statistics (one-way ANOVA followed by a Tukey HSD test) and multivariate techniques, including Principal Component Analysis (PCA), Principal Coordinate Analysis (PCoA), non-metric multidimensional scaling (nMDS) and Cluster Analysis (CA) were applied to assess the variation of flavonoid compounds. Triplicate measurements were averaged for each individual to create a data matrix. To reduce the magnitude of differences in the original values (the amounts of compounds expressed as relative percentages) and to avoid making assumptions about the data distribution, the data were coded (using categorical scale) following Milutinović et al. [87]. The aim of the PCA was to reveal a general pattern of variations and relationships between all individuals. To hypothetically reduce the influence of environmental factors on the production of flavonoid compounds in studied samples, we generated a binary data matrix for the presence/absence of compounds. We then performed separate PCoAs for all individuals, for the Tormaria and Soraria groups, including their putative parental species and their hybrid members (species and cytotypes), and for the Aria group. The PCoAs based on Jaccard distances were used to visualise the relationships between individuals. The cluster analysis by the unweighted pair-group method using arithmetic averages (UPGMAs) using Jaccard distances was performed to verify the affinities between samples. Finally, NMDS using the Bray–Curtis pairwise distance matrix was used to order the studied samples. The Mantel test [88] was used to check whether the quantitative composition of the flavonoid components is affected by the level of ploidy for all accessions, particularly for the Aria subgenus. The Bray–Curtis distances for raw phytochemical data and ploidy data were used for calculation. All analyses were performed using PAST 3.14 [89].

5. Conclusions

The results of HPLC-MS and flow cytometry proved valuable for the analysis of ploidy-dependent plant systems and the identification of flavonoid profiles in a panel of Sorbus accessions, including three diploid parental species and polyploid derivatives of the subg. Tormaria, Soraria and Aria from the region of Bosnia and Herzegovina. Of the 23 flavonoids identified, 14 were newly reported, improving the understanding of the phytochemical profiles of Sorbus species. We have shown that certain fractions of flavonoids are reliable indicators of hybridisation in certain Sorbus groups (Tormaria and Soraria). Correlation with ploidy levels reveals a non-statistically significant relationship with flavonoid profiles in a range of S. aria polyploids. Flavonoid profiling highlights the Tormaria group as target accessions rich in bioactive compounds for further biological studies.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants14010119/s1, Table S1. Calibration curves regression equations, correlation coefficients (r2), linear ranges, LODs and LOQs of reference compounds, Table S2. Principal components (PC) revealed by Principal Component Analysis (PCA) for the studied Sorbus accessions.

Author Contributions

Conceptualisation, H.N., A.H.-B., F.B., K.D. and T.K.-V.; methodology E.K., A.H.-B., F.B., V.M. and H.N.; software F.B., V.M., and E.K.; validation V.M. and J.R.S.; formal analysis, E.K., V.M. and F.B; investigation, E.K.; resources F.B., H.N., T.K.-V. and K.D.; data curation, E.K., A.H.-B. and F.B.; writing—original draft preparation, E.K., F.B., V.M., I.G. and A.H.-B.; writing—review and editing, E.K., F.B., V.M., I.G. and A.H.-B.; visualisation, A.H.-B., V.M. and E.K.; supervision H.N. and F.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Ministry of Education, Science and Youth of Sarajevo Canton to H.N. (grant No. 27-02-11-41250-1/21) and A.H.B (grant No. 27-02-35-37082-30/23), and the Ministry of Science, Technological Development and Innovation of Republic of Serbia (contract Nos: 451-03-65/2024-03/200161 and 451-03-66/2024-03/200161).

Data Availability Statement

All data generated during this study are included in this article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Anunciato, T.P.; da Rocha Filho, P.A. Carotenoids and polyphenols in nutricosmetics, nutraceuticals, and cosmeceuticals. J. Cosmet Dermatol. 2012, 11, 51–54. [Google Scholar] [CrossRef] [PubMed]
  2. Verma, N.; Shukla, S. Impact of various factors responsible for fluctuation in plant secondary metabolites. J. Appl. Res. Med. Aromat. Plants. 2015, 2, 105513. [Google Scholar] [CrossRef]
  3. Zymone, K.; Raudone, L.; Raudonis, R.; Marksa, M.; Ivanauskas, L.; Janulis, V. Phytochemical profiling of fruit powders of twenty Sorbus L. cultivars. Molecules 2018, 23, 2593. [Google Scholar] [CrossRef]
  4. Robertson, A.; Rich, T.C.; Allen, A.M.; Houston, L.; Roberts, C.A.; Bridle, J.R.; Harris, S.A.; Hiscock, S.J. Hybridization and polyploidy as drivers of continuing evolution and speciation in Sorbus. Mol. Ecol. 2010, 19, 1675–1690. [Google Scholar] [CrossRef]
  5. Lepší, M.; Koutecký, P.; Nosková, J.; Lepší, P.; Urfus, T.; Rich, T.C. Versatility of reproductive modes and ploidy level interactions in Sorbus s.l. (Malinae, Rosaceae). Bot. J. Linn. Soc. 2019, 191, 502–522. [Google Scholar] [CrossRef]
  6. Kurtto, A.K.; Sennikov, A.N.; Lampinen, R.E. Distribution of vascular plants in Europe: 17. Rosaceae (Sorbus s. lato). In Atlas Florae Europaeae, 1st ed.; The Committee for Mapping the Flora of Europe & Societas Biologica Fennica Vanamo: Helsinki, Finland, 2018. [Google Scholar]
  7. Sennikov, A.N.; Kurtto, A. A phylogenetic checklist of Sorbus s.l. (Rosaceae) in Europe. Memo. Soc. Fauna Flora Fenn. 2017, 93, 1–78. [Google Scholar]
  8. Rich, T.C.G.; Houston, L.; Robertson, A.; Proctor, M.C.F. Whitebeams, rowans and service trees of Britain and Ireland: A monograph of British and Irish ’Sorbus’ L. In A monograph of British and Irish Sorbus L., 1st ed.; B.S.B.I. Handbook No. 14; Botanical Society of the British Isles: London, UK, 2010. [Google Scholar]
  9. Lo, E.Y.Y.; Donoghue, M.J. Expanded phylogenetic and dating analyses of the apples and their relatives (Pyreae, Rosaceae). Mol. Phylogen. Evol. 2012, 63, 230–243. [Google Scholar] [CrossRef]
  10. Potter, D.; Eriksson, T.; Evans, R.C.; Oh, S.; Smedmark, J.E.E.; Morgan, D.R.; Kerr, M. Phylogeny and classification of Rosaceae. Plant Syst. Evol. 2007, 266, 5–43. [Google Scholar] [CrossRef]
  11. Meyer, N.; Meierott, L.; Schuwerk, H.; Angerer, O. Beiträge zur Gattung Sorbus in Bayern; Sonderband der Berichte der Bayerischen Botanischen Gesellschaft: München, Germany, 2005. [Google Scholar]
  12. Kurtto, A. Rosaceae (pro parte majore). In Euro+Med Plantbase—The Information Resource for Euro-Mediterranean Plant Diversity. 2009. Available online: http://www.europlusmed.org (accessed on 26 December 2024).
  13. Hamston, T.J.; De Vere, N.; King, R.A.; Pellicer, J.; Fay, M.F.; Cresswell, J.E.; Stevens, J.R. Apomixis and hybridization drives reticulate evolution and phyletic differentiation in Sorbus L. Implications for conservation. Front. Plant. Sci. 2018, 9, 1796. [Google Scholar] [CrossRef]
  14. Hajrudinović, A.; Siljak-Yakovlev, S.; Brown, S.C.; Pustahija, F.; Bourge, M.; Ballian, D.; Bogunić, F. When sexual meets apomict: Genome size, ploidy level and reproductive mode variation of Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina. Ann. Bot. 2015, 116, 301–312. [Google Scholar] [CrossRef]
  15. Sołtys, A.; Galanty, A.; Podolak, I. Ethnopharmacologically important but underestimated genus Sorbus: A comprehensive review. Phytochem. Rev. 2020, 19, 491–526. [Google Scholar] [CrossRef]
  16. Kumar, S. Impact of ploidy changes on secondary metabolites productions in plants. In Evolutionary Diversity as a Source for Anticancer Molecules; Academic Press: Cambridge, MA, USA, 2021; pp. 29–46. [Google Scholar] [CrossRef]
  17. Cheng, D.; Vrieling, K.; Klinkhamer, P.G. The effect of hybridization on secondary metabolites and herbivore resistance: Implications for the evolution of chemical diversity in plants. Phytochem. Rev. 2011, 10, 107–117. [Google Scholar] [CrossRef] [PubMed]
  18. Ćavar Zeljković, S.; Siljak-Yakovlev, S.; Tan, K.; Maksimović, M. Chemical composition and antioxidant activity of Geranium macrorrhizum in relation to ploidy level and environmental conditions. Plant Syst. Evol. 2020, 306, 18. [Google Scholar] [CrossRef]
  19. Raudonė, L.; Raudonis, R.; Gaivelytė, K.; Pukalskas, A.; Viškelis, P.; Venskutonis, P.R.; Janulis, V. Phytochemical and antioxidant profiles of leaves from different Sorbus L. species. Nat. Prod. Res. 2015, 29, 281–285. [Google Scholar] [CrossRef] [PubMed]
  20. Raudonis, R.; Raudonė, L.; Gaivelytė, K.; Viškelis, P.; Janulis, V. Phenolic and antioxidant profiles of rowan (Sorbus L.) fruits. Nat. Prod. Res. 2014, 28, 1231–1240. [Google Scholar] [CrossRef]
  21. Ak, G.; Tüfekci, E.F.; Mustafa, A.M.; Caprioli, G.; Altunoglu, Y.C.; Baloglu, M.C.; Cakılcıoglu, U.; Polat, R.; Darendelioglu, E.; Zengin, G. Exploring Sorbus torminalis leaves: Unveiling a promising natural resource for diverse chemical and biological applications. Chem. Biodivers. 2024, 21, e202400233. [Google Scholar] [CrossRef]
  22. Tian, Y.; Liimatainen, J.; Alanne, A.L.; Lindstedt, A.; Liu, P.; Sinkkonen, J.; Kallio, H.; Yang, B. Phenolic compounds extracted by acidic aqueous ethanol from berries and leaves of different berry plants. Food Chem. 2017, 220, 266–281. [Google Scholar] [CrossRef]
  23. Rutkowska, M.; Owczarek-Januszkiewicz, A.; Magiera, A.; Gieleta, M.; Olszewska, M.A. Chemometrics-driven variability evaluation of phenolic composition, antioxidant capacity, and α-glucosidase inhibition of Sorbus aucuparia L. fruits from Poland: Identification of variability markers for plant material valorization. Antioxidants 2023, 12, 1967. [Google Scholar] [CrossRef]
  24. Yin, Y.; Zhang, Y.; Li, H.; Zhao, Y.; Cai, E.; Zhu, H.; Li, P.; Liu, J. Triterpenoids from fruits of Sorbus pohuashanensis inhibit acetaminophen-induced acute liver injury in mice. Biomed. Pharmacother. 2019, 109, 493–502. [Google Scholar] [CrossRef]
  25. Sarv, V.; Venskutonis, P.R.; Bhat, R. The Sorbus spp.—Underutilized plants for foods and nutraceuticals: Review on polyphenolic phytochemicals and antioxidant potential. Antioxidants 2020, 9, 813. [Google Scholar] [CrossRef]
  26. Mrkonjić, Z.; Nadpal, J.D.; Beara, I.; Aleksić-Sabo, V.S.; Cetojević-Simin, D.D.; Mimica-Dukić, N.; Lesjak, M. Phenolic profiling and bioactivities of fresh fruits and jam of Sorbus species. J. Serb. Chem. Soc. 2017, 82, 651–664. [Google Scholar] [CrossRef]
  27. Kim, C.S.; Oh, J.; Subedi, L.; Kim, S.Y.; Choi, S.U.; Lee, K.R. Two new phenolic glycosides from Sorbus commixta. Chem. Pharm. Bull. 2018, 66, 839–842. [Google Scholar] [CrossRef] [PubMed]
  28. Kavak, D.D.; Akdeniz, B. Sorbus umbellata (Desf.) Fritsch var. umbellata leaves: Optimization of extraction conditions and investigation antimicrobial, cytotoxic, and β-glucuronidase inhibitory potential. Plant Foods. Human Nutr. 2019, 74, 364–369. [Google Scholar] [CrossRef]
  29. Forino, M.; Tenore, G.C.; Tartaglione, L.; Novellino, E.; Ciminiello, P. (1S, 3R, 4S, 5R) 5-O-Caffeoylquinic acid: Isolation, stereo-structure characterization and biological activity. Food Chem. 2015, 178, 306–310. [Google Scholar] [CrossRef] [PubMed]
  30. Shang, N.; Saleem, A.; Musallam, L.; Walshe-Roussel, B.; Badawi, A.; Cuerrier, A.; Arnason, J.T.; Haddad, P.S. Novel approach to identify potential bioactive plant metabolites: Pharmacological and metabolomics analyses of ethanol and hot water extracts of several Canadian medicinal plants of the Cree of Eeyou Istchee. PLoS ONE 2015, 10, e0135721. [Google Scholar] [CrossRef]
  31. Kim, C.S.; Suh, W.S.; Subedi, L.; Kim, S.Y.; Choi, S.U.; Lee, K.R. Neuroprotective fatty acids from the stem bark of Sorbus commixta. Lipids 2016, 51, 989–995. [Google Scholar] [CrossRef]
  32. Yu, X.; Wang, Z.; Shu, Z.; Li, Z.; Ning, Y.; Yun, K.; Bai, H.; Liu, R.; Liu, W. Effect and mechanism of Sorbus pohuashanensis (Hante) Hedl. flavonoids protect against arsenic trioxide-induced cardiotoxicity. Biomed. Pharmacother. 2017, 88, 1–10. [Google Scholar] [CrossRef]
  33. Marin, P.D. Biohemijska i Molekularna Sistematika Biljaka; NNK International: Toronto, ON, Canada, 2003. [Google Scholar]
  34. López-Caamal, A.; Tovar-Sánchez, E. Genetic, morphological, and chemical patterns of plant hybridization. Rev. Chil. Hist. Nat. 2014, 87, 16. [Google Scholar] [CrossRef]
  35. Radović Selgrad, J.; Milutinović, V.; Suručić, R.; Samardžić, S.; Kopanja, Đ.; Kundaković-Vasović, T. Enzyme inhibitory activities, phenolic profile, and in silico studies of Sorbus torminalis tree bark methanol extract. Chem. Biodivers. 2024, 21, e202401459. [Google Scholar] [CrossRef]
  36. Karakousi, C.V.; Xanthippi, B.; Theano, S.; Eugene, K. Phytochemical analysis, antioxidant and ALR2 Inhibitory activity of Sorbus torminalis (L.) fruits at different maturity stages. Fitoterapia 2024, 175, 105863. [Google Scholar] [CrossRef]
  37. Rutkowska, M.; Kolodziejczyk-Czepas, J.; Owczarek, A.; Zakrzewska, A.; Magiera, A.; Olszewska, M.A. Novel insight into biological activity and phytochemical composition of Sorbus aucuparia L. fruits: Fractionated extracts as inhibitors of protein glycation and oxidative/nitrative damage of human plasma components. Food Res. Int. 2021, 147, 110526. [Google Scholar] [CrossRef] [PubMed]
  38. Šavikin, K.P.; Zdunić, G.M.; Krstić-Milošević, D.B.; Šircelj, H.J.; Stešević, D.D. Sorbus aucuparia and Sorbus aria as a source of Antioxidant Phenolics, Tocopherols, and Pigments. Chem. Biodivers. 2017, 14, e1700329. [Google Scholar] [CrossRef] [PubMed]
  39. Olszewska, M.A.; Presler, A.; Michel, P. Profiling of phenolic compounds and antioxidant activity of dry extracts from the selected Sorbus species. Molecules 2012, 17, 3093–3113. [Google Scholar] [CrossRef]
  40. Olszewska, M.A.; Nowak, S.; Michel, P.; Banaszczak, P.; Kicel, A. Assessment of the content of phenolics and antioxidant action of inflorescences and leaves of selected species from the genus Sorbus sensu stricto. Molecules 2010, 15, 8769–8783. [Google Scholar] [CrossRef]
  41. Gaivelyte, K.; Jakstas, V.; Razukas, A.; Janulis, V. Variation of quantitative composition of phenolic compounds in rowan (Sorbus aucuparia L.) leaves during the growth season. Nat. Prod. Res. 2014, 28, 1018–1020. [Google Scholar] [CrossRef]
  42. Bujor, A.; Miron, A.; Luca, S.V.; Skalicka-Wozniak, K.; Silion, M.; Ancuceanu, R.; Dinu, M.; Girard, C.; Demougeot, C.; Totoson, P. Metabolite profiling, arginase inhibition and vasorelaxant activity of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts. Food Chem. Toxicol. 2019, 133, 110764. [Google Scholar] [CrossRef]
  43. Schymanski, E.L.; Jeon, J.; Gulde, R.; Fenner, K.; Ruff, M.; Singer, H.P.; Hollender, J. Identifying small molecules via high resolution mass spectrometry: Communicating confidence. Environ. Sci. Technol. 2014, 48, 2097–2098. [Google Scholar] [CrossRef]
  44. Mohammed, H.A.; Al-Omar, M.S.; Khan, R.A.; Mohammed, S.A.A.; Qureshi, K.A.; Abbas, M.M.; Al Rugaie, O.; Abd-Elmoniem, E.; Ahmad, A.M.; Kandil, Y.I. Chemical Profile, Antioxidant, Antimicrobial, and Anticancer Activities of the Water-Ethanol Extract of Pulicaria undulata Growing in the Oasis of Central Saudi Arabian Desert. Plants 2021, 10, 1811. [Google Scholar] [CrossRef]
  45. Candela, L.; Formato, M.; Crescente, G.; Piccolella, S.; Pacifico, S. Coumaroyl Flavonol Glycosides and More in Marketed Green Teas: An Intrinsic Value beyond Much-Lauded Catechins. Molecules 2020, 25, 1765. [Google Scholar] [CrossRef]
  46. Mikulic-Petkovsek, M.; Krska, B.; Kiprovski, B.; Veberic, R. Bioactive components and antioxidant capacity of fruits from nine Sorbus genotypes. J. Food Sci. 2017, 82, 647–658. [Google Scholar] [CrossRef]
  47. Kachlicki, P.; Piasecka, A.; Stobiecki, M.; Marczak, Ł. Structural characterization of flavonoid glycoconjugates and their derivatives with mass spectrometric techniques. Molecules 2016, 21, 1494. [Google Scholar] [CrossRef] [PubMed]
  48. Wang, Y.; Gu, M.; Mao, J.; Liu, J.; Fan, S.; Zhang, H.; Liu, Q. Phytochemical study: Fragmentation patterns of flavonoid-C-glycosides in the enriched flavonoids from corn silk using high-efficiency ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry. Sep. Sci. Plus 2024, 7, 2300156. [Google Scholar] [CrossRef]
  49. Mabry, T.J.; Markham, K.R.; Thomas, M.B.; Mabry, T.J.; Markham, K.R.; Thomas, M.B. The Systematic Identification of Flavonoids; Springer: Berlin/Heidelberg, Germany, 1970. [Google Scholar] [CrossRef]
  50. Cuyckens, F.; Claeys, M. Mass spectrometry in the structural analysis of flavonoids. J. Mass Spectrom. 2004, 39, 1–15. [Google Scholar] [CrossRef] [PubMed]
  51. Olszewska, M.A.; Michel, P. Activity-guided isolation and identification of free radical-scavenging components from various leaf extracts of Sorbus aria (L.) Crantz. Nat. Prod. Res. 2012, 26, 243–254. [Google Scholar] [CrossRef]
  52. Chester, M.; Cowan, S.C.; Fay, M.; Rich, C.G.T. Parentage of endemic Sorbus L. (Rosaceae) species in the British Isles: Evidence from plastid DNA. Bot. Linn. Soc. 2007, 154, 291–304. [Google Scholar] [CrossRef]
  53. Feulner, M.; Pointner, S.; Heuss, L.; Aas, G.; Paule, J.; Dötterl, S. Floral scent and its correlation with AFLP data in Sorbus. Org. Divers. Evol. 2014, 14, 339–348. [Google Scholar] [CrossRef]
  54. Hajrudinović-Bogunić, A.; Frajman, B.; Schönswetter, P.; Siljak-Yakovlev, S.; Bogunić, F. Apomictic mountain whitebeam (Sorbus austriaca, Rosaceae) comprises several genetically and morphologically divergent lineages. Biology 2023, 12, 380. [Google Scholar] [CrossRef]
  55. Robertson, A.; Newton, A.C.; Ennos, R.A. Multiple hybrid origins, genetic diversity and population genetic structure of two endemic Sorbus taxa on the Isle of Arran, Scotland. Mol. Ecol. 2004, 13, 123–134. [Google Scholar] [CrossRef]
  56. Ludwig, S.; Robertson, A.; Rich, T.C.; Djordjevic, M.; Cerovic, R.; Houston, L.; Harris, S.A.; Hiscock, S.J. Breeding systems, hybridization and continuing evolution in Avon Gorge Sorbus. Ann. Bot. 2013, 111, 563–575. [Google Scholar] [CrossRef]
  57. Hajrudinović, A.; Frajman, B.; Schönswetter, P.; Silajdžić, E.; Siljak-Yakovlev, S.; Bogunić, F. Towards a better understanding of polyploid Sorbus (Rosaceae) from Bosnia and Herzegovina (Balkan Peninsula), including description of a novel, tetraploid apomictic species. Bot. J. Linn. 2015, 178, 670–685. [Google Scholar] [CrossRef]
  58. Hegarty, M.J.; Hiscock, S.J. Genomic clues to the evolutionary success of polyploid plants. Curr. Biol. 2008, 18, R435–R444. [Google Scholar] [CrossRef] [PubMed]
  59. Weiss-Schneeweiss, H.; Emadzade, K.; Jang, T.S.; Schneeweiss, G.M. Evolutionary consequences, constraints and potential of polyploidy in plants. Cytogenet. Genome Res. 2013, 140, 137–150. [Google Scholar] [CrossRef] [PubMed]
  60. Liga, S.; Paul, C.; Peter, F. Flavonoids: Overview of Biosynthesis, Biological Activity, and Current Extraction Techniques. Plants 2023, 12, 2732. [Google Scholar] [CrossRef] [PubMed]
  61. Nabavi, S.M.; Šamec, D.; Tomczyk, M.; Milella, L.; Russo, D.; Habtemariam, S.; Suntar, I.; Rastrelli, L.; Daglia, M.; Xiao, J.; et al. Flavonoid biosynthetic pathways in plants: Versatile targets for metabolic engineering. Biotechnol. Adv. 2020, 38, 107316. [Google Scholar] [CrossRef] [PubMed]
  62. Sanderson, S.C.; Ge-Ling, C.; McArthur, E.D.; Stutz, H. Evolutionary loss of flavonoids and other chemical characteristic in the Chenopodiaceae. Biochem. Syst. And Ecol. 1988, 16, 143–149. [Google Scholar] [CrossRef]
  63. Orians, C.M. The effects of hybridization in plants on secondary chemistry: Implications for the ecology and evolution of plant-herbivore interactions. Am. J. Bot. 2000, 87, 1749–1756. [Google Scholar] [CrossRef]
  64. Reiseberg, L.H.; Ellstrand, N.C.; Arnold, M. What Can Molecular and Morphological Markers Tell Us About Plant Hybridization? Crit. Rev. Plant Sci. 1993, 12, 213–241. [Google Scholar] [CrossRef]
  65. Hallgren, P.; Ikonen, A.; Hjalten, J.; Roininen, H. Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: Implications for herbivore responses to hybrid plants. J. Chem. Ecol. 2003, 29, 1143–1158. [Google Scholar] [CrossRef]
  66. Caruso, I.; Lepore, L.; De Tommasi, N.; Piaz, F.D.; Frusciante, L.; Aversano, R.; Garramone, R.; Carputo, D. Secondary metabolite profile in induced tetraploids of wild Solanum commersonii Dun. Chem. Biodivers. 2011, 8, 2226–2237. [Google Scholar] [CrossRef]
  67. Gaynor, M.L.; Lim-Hing, S.; Mason, C.M. Impact of genome duplication on secondary metabolite composition in non-cultivated species: A systematic meta-analysis. Ann. Bot. 2020, 126, 363–376. [Google Scholar] [CrossRef]
  68. McCarthy, E.W.; Berardi, A.E.; Smith, S.D.; Litt, A. Related allopolyploids display distinct floral pigment profiles and transgressive pigments. Am. J. Bot. 2017, 104, 92–101. [Google Scholar] [CrossRef] [PubMed]
  69. Yu, Y.; Qu, Y.; Wang, S.; Wang, Q.; Shang, X.; Fu, X. An integrative analysis of metabolome and transcriptome reveals the molecular regulatory mechanism of the accumulation of flavonoid glycosides in different Cyclocarya paliurus ploidies. Forests 2023, 14, 770. [Google Scholar] [CrossRef]
  70. De Jesus-Gonzalez, L.; Weathers, P.J. Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids. Plant Cell Rep. 2003, 21, 809–813. [Google Scholar] [CrossRef] [PubMed]
  71. Chung, H.H.; Shi, S.K.; Huang, B.; Chen, J.T. Enhanced agronomic traits and medicinal constituents of autotetraploids in Anoectochilus formosanus Hayata, a top-grade medicinal orchid. Molecules 2017, 22, 1907. [Google Scholar] [CrossRef] [PubMed]
  72. Huang, X.; Ouyang, K.; Luo, Y.; Xie, G.; Yang, Y.; Zhang, J. A comparative study of characteristics in diploid and tetraploid Anoectochilus roxburghii. Front. Nutr. 2022, 9, 1034751. [Google Scholar] [CrossRef]
  73. International Conference on Harmonisation (ICH). ICH Harmonised Tripartite Guideline. In Validation of Analytical Procedures: Text and Methodology Q2 (R1); ICH: Geneva, Switzerland, 2005. [Google Scholar]
  74. Chen, J.; Ning, S.; Lu, X.; Xiang, W.; Zhou, X.; Bu, Y.; Li, L.; Huang, R. Variation in flavonoid and antioxidant activities of Pyrrosa petiolosa (Christ) Ching from different geographic origins. Front. Plant Sci. 2023, 14, 1173489. [Google Scholar] [CrossRef]
  75. Velebil, J.; Lepší, M.; Nosková, J.; Lepši, P. Taxonomic assessment of Sorbus subgenus Aria in the Malé Karpaty Mountains. Preslia 2022, 94, 305–334. [Google Scholar] [CrossRef]
  76. Lavania, U.C.; Srivastava, S.; Lavania, S.; Basu, S.; Misra, N.K.; Mukai, Y. Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation. Plant J. 2012, 71, 539–549. [Google Scholar] [CrossRef]
  77. Parida, B.P.; Misra, B.B. Is a plant’s ploidy status reflected in its metabolome? J. Postdr. Res. 2015, 1, 11. [Google Scholar] [CrossRef]
  78. Ranney, T.G. Polyploidy: From evolution to new plant development. Comb. Proc. Int. Plant Propagators’ Soc. 2006, 56, 137–142. [Google Scholar]
  79. Májovský, J.; Bernátová, D. Nové hybridogénne podrody rodu Sorbus L. emend. Crantz. Acta Horticulturae et Regiotecturae 2001, 4, 20–21. [Google Scholar]
  80. Marie, D.; Brown, S.C. A cytometric exercise in plant DNA histograms, with 2C values for 70 species. Biol. Cell. 1993, 78, 41–51. [Google Scholar] [CrossRef] [PubMed]
  81. Bourge, M.; Brown, S.C.; Siljak-Yakovlev, S. Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment. Genet. Appl. 2018, 2, 1–2. [Google Scholar] [CrossRef]
  82. Suda, J.; Krahulcová, A.; Trávníek, P.; Krahulec, F. Ploidy level versus DNA ploidy level: An appeal for consistent terminology. Taxon 2006, 55, 447–450. [Google Scholar] [CrossRef]
  83. Siljak-Yakovlev, S.; Pustahija, F.; Šolić, E.M.; Bogunić, F.; Muratović, E.; Bašić, N.; Catrice, O.; Brown, S.C. Towards a genome size and chromosome number database of Balkan flora: C-values in 343 taxa with novel values for 242. Adv. Sci. Lett. 2010, 3, 190–213. [Google Scholar] [CrossRef]
  84. Turumtay, H.; Midilli, A.; Turumtay, E.A.; Demir, A.; Selvi, E.K.; Budak, E.E.; Er, H.; Kocaimamoglu, F.; Baykal, H.; Belduz, A.O.; et al. Gram (−) microorganisms DNA polymerase inhibition, antibacterial and chemical properties of fruit and leaf extracts of Sorbus aucuparia and Sorbus caucasica var. yaltirikii. Biomed. Chromatogr. 2017, 31, e3901. [Google Scholar] [CrossRef]
  85. Elferjane, M.R.; Milutinović, V.; Jovanović Krivokuća, M.; Taherzadeh, M.J.; Pietrzak, W.; Marinković, A.; Jovanović, A.A. Vaccinium myrtillus L. leaf waste as a source of biologically potent compounds: Optimization of polyphenol extractions, chemical profile, and biological properties of the extracts. Pharmaceutics 2024, 16, 740. [Google Scholar] [CrossRef]
  86. EDQM. European Pharmacopoeia 11.0; European Directorate for the Quality of Medicine & Health Care of the Council of Europe (EDQM): Strasbourg, France, 2023. [Google Scholar]
  87. Milutinović, V.; Niketić, M.; Krunić, A.; Nikolić, D.; Petković, M.; Ušjak, L.; Petrović, S. Sesquiterpene lactones from the methanol extracts of twenty-eight Hieracium species from the Balkan Peninsula and their chemosystematic significance. Phytochem 2018, 154, 19–30. [Google Scholar] [CrossRef]
  88. Mantel, N. The detection of disease clustering and a generalized regression approach. Cancer Res. 1967, 27, 209–220. [Google Scholar]
  89. Hammer, Ø.; Harper, D.A. Past: Paleontological statistics software package for educaton and data analysis. Palaeontol. Electron. 2001, 4, 1–9. Available online: https://www.researchgate.net/publication/259640226 (accessed on 26 December 2024).
Plants 14 00119 g001
Figure 1. Liquid chromatography–mass spectrometry chromatograms (wavelength, 350 nm) of 17 analysed ethyl acetate fractions (A). The chromatograms are supplemented with localities for each analysed accession. and liquid chromatography–mass spectrometry chromatograms (a—diode array detector, b—mass selective detector) of the ethyl acetate fraction of Sorbus bosniaca leaves (B). Compound numbers are in circles and correspond to those in Table 1. The chromatograms are supplemented with localities for each analysed accession.
Figure 1. Liquid chromatography–mass spectrometry chromatograms (wavelength, 350 nm) of 17 analysed ethyl acetate fractions (A). The chromatograms are supplemented with localities for each analysed accession. and liquid chromatography–mass spectrometry chromatograms (a—diode array detector, b—mass selective detector) of the ethyl acetate fraction of Sorbus bosniaca leaves (B). Compound numbers are in circles and correspond to those in Table 1. The chromatograms are supplemented with localities for each analysed accession.
Plants 14 00119 g001
Plants 14 00119 g002
Figure 2. PCA and PCoA ordination ((A)—PC1 vs. PC2; (B)—PCo1 vs. PCo2) of the flavonoid compounds of the investigated Sorbus accessions. The ordination diagrams are supplemented with the localities for each studied accession. Compound numbers in circles (A) correspond to those in Table 1.
Figure 2. PCA and PCoA ordination ((A)—PC1 vs. PC2; (B)—PCo1 vs. PCo2) of the flavonoid compounds of the investigated Sorbus accessions. The ordination diagrams are supplemented with the localities for each studied accession. Compound numbers in circles (A) correspond to those in Table 1.
Plants 14 00119 g002
Plants 14 00119 g003
Figure 3. PCoA ordination (PCo1 vs. PCo2) of the flavonoid compounds of the investigated Sorbus accessions given for the subgenera Tormaria (A), Soraria (B), and Aria (C). The ordination diagrams are supplemented with the localities for each studied accession.
Figure 3. PCoA ordination (PCo1 vs. PCo2) of the flavonoid compounds of the investigated Sorbus accessions given for the subgenera Tormaria (A), Soraria (B), and Aria (C). The ordination diagrams are supplemented with the localities for each studied accession.
Plants 14 00119 g003
Plants 14 00119 g004
Figure 4. Results of multivariate analyses for 17 analysed Sorbus accessions. (A)—dendrogram showing the flavonoid relationships between the flavonoids of the accessions using the UPGMA method; (B)—non-metric dimensional scaling analysis of the studied accessions. Each figure is supplemented with localities for studied accessions.
Figure 4. Results of multivariate analyses for 17 analysed Sorbus accessions. (A)—dendrogram showing the flavonoid relationships between the flavonoids of the accessions using the UPGMA method; (B)—non-metric dimensional scaling analysis of the studied accessions. Each figure is supplemented with localities for studied accessions.
Plants 14 00119 g004
Table 1. UV and MS spectral data of the detected flavonoids in EtOAc fractions of Sorbus accessions.
Table 1. UV and MS spectral data of the detected flavonoids in EtOAc fractions of Sorbus accessions.
NoRt (min)λmax (nm)MWm/z
[M–H]−		Product IonsCompound
Flavones
2 *15.07–15.94270, 338594593226; 473Apigenin 6,8-di-C-glucoside (Vicenin) st **
418.76–19.12270, 338564563473Apigenin-6-C-glucoside-8-C-arabinoside (Schaftoside) st
2125.44–25.64268, 338446445269Apigenin 7-O-glucuronide st
1022.22–22.48254, 266sh, 350448447285Luteolin 7-O-glucoside (Cynaroside) st
1122.47–22.74254, 266sh, 350462461285Luteolin 7-O-glucuronide st
2024.78–25.08248, 268sh, 340448447285Luteolin hexoside [44]
Flavanols
1 *14.99–15.12252, 266sh, 372626625317Hydroxyquercetin deoxyhexosyl hexoside [45]
317.36–17.76256, 264sh, 354626625301Quercetin dihexoside [23]
519.97–20.19256, 264sh, 354740739447; 593Quercetin trideoxyhexoside
620.15–20.69256, 264sh, 354610609301Quercetin deoxyhexosyl hexoside [45]
720.40–20.73256, 264sh, 354610609301Quercetin 3-O-rutinoside (Rutin) st
821.28–21.57256, 264sh, 354464463269; 301Quercetin 3-O-galactoside (Hyperoside) st
921.50–21.96256, 264sh, 354464463269; 301Quercetin-3-O-glucoside (Isoquercitrin) st
1222.67–23.10266, 348594593285Kaempferol deoxyhexosylhexoside [45]
1322.61–22.96256, 264sh, 354596595301; 433Quercetin hexosylpentoside [46]
1422.51–23.38256, 268sh, 356506505301; 463Quercetin acetylhexoside [22,46]
1523.88–24.08256, 264sh, 354434433301Quercetin pentoside [22]
1624.00–24.23266, 348448447285Kaempferol 3-O-glucoside (Astragalin) st
1723.98–24.30256, 264sh, 354448447301Quercetin 3-O-rhamnoide (Quercitrin) st
2225.80–26.27266, 348490489285; 447Kaempferol acetylhexoside [46]
Methylflavonols
1823.85–24.17256, 268sh, 356478477271; 301; 315Methylquercetin hexoside isomer 1 [46]
1924.26–24.80256, 268sh, 356478477271; 285; 301; 315Methylquercetin hexoside isomer 2 [46]
2325.98–26.30256, 268sh, 356520519285; 301; 316; 447Methylquercetin acethylhexoside [46]
* The numbers of the detected compounds, based on the order of elution; Rt—retention time; ** The references for comparison of the UV and/or MS data of the compounds are given in square brackets; st.—commercial authentic compounds used for identification; sh.—second harmonic wavelength.
Table 2. Quantities (%, g/100 g DE) of detected flavonoids in EtOAc fractions of Sorbus accessions.
Table 2. Quantities (%, g/100 g DE) of detected flavonoids in EtOAc fractions of Sorbus accessions.
TaxonSubgenusLocationDNA Ploidy LevelFlavones
Xsr (%)STDXsr (%)STDXsr (%)STDXsr (%)STDXsr (%)STDXsr (%)STD
2 4 21 10 11 20
S. ariaAriaGrkarica, Mt. Igman2xn.d. n.d. n.d. n.d. n.d. n.d.
Bijela gora, Mt. Orjen2xn.d. n.d. n.d. n.d. n.d. n.d.
Gradac, Posušje2xn.d. n.d. n.d. n.d. n.d. n.d.
Grkarica, Mt. Igman3xn.d. n.d. n.d. n.d. n.d. n.d.
Umoljani, Mt. Bjelašnica3xn.d. n.d. n.d. n.d. n.d. n.d.
Bijela gora, Mt. Orjen3xn.d. n.d. n.d. n.d. n.d. n.d.
Grkarica, Mt. Igman4xn.d. n.d. n.d. n.d. n.d. n.d.
Umoljani, Mt. Bjelašnica4xn.d. n.d. n.d. n.d. n.d. n.d.
Bijela gora, Mt. Orjen4xn.d. n.d. n.d. n.d. n.d. n.d.
S. aucupariaSorbusVeliko polje, Mt. Igman2xn.d. n.d. n.d. n.d. n.d. n.d.
S. austriacaSoraria (Sorbus × Aria)Grkarica, Mt. Igman4xn.d. n.d. n.d. n.d. n.d. n.d.
Umoljani, Mt. Bjelašnica4xn.d. n.d. n.d. n.d. n.d. n.d.
S. bosniacaMt. Krug planina4xn.d. n.d. n.d. n.d. n.d. n.d.
S. torminalis × ariaTormaria (Torminalis × Aria)Pratača, Lokve2x0.226 *0.015trace */2.414 *0.0130.107 *0.0130.464 *0.0050.161 *0.01
Koznik3x0.059 *0.014n.d. 0.617 *0.0920.148 *0.0120.348 *0.0060.007 *0.001
Crne lokve, Posušje4x0.167 *0.015trace */n.d. 0.206 *0.0140.347 *0.0051.224 *0.02
S. torminalisTorminariaTihovići2xn.d. trace */8.9130.1240.186 *0.0131.159 *0.0200.506 *0.007
TaxonSubgenusLocationDNA Ploidy LevelFlavonols
Xsr (%)STDXsr (%)STDXsr (%)STDXsr (%)STDXsr (%)STDXsr (%)STDXsr (%)STD
1 3 5 6 7 8 9
S. ariaAriaGrkarica, Mt. Igman2xn.d. n.d. n.d. n.d. 4.7380.022n.d. 1.7790.011
Mt. Bijela gora2xn.d. n.d. n.d. n.d. 0.5770.012n.d. 2.8470.005
Gradac, Posušje2xn.d. n.d. n.d. trace */1.1270.0120.5680.0063.7380.005
Grkarica, Mt. Igman3xn.d. n.d. n.d. n.d. 7.5040.019n.d. 1.7910.007
Umoljani, Mt. Bjelašnica3xn.d. n.d. 0.878 *0.003trace */3.5550.0080.2490.0071.5860.003
Mt. Bijela gora3x0.083 *0.005n.d. n.d. n.d. 3.7150.0270.3050.0081.2660.014
Grkarica, Mt. Igman4xn.d. n.d. n.d. n.d. 4.1150.0440.1380.0051.8980.016
Umoljani, Mt. Bjelašnica4xn.d. n.d. n.d. n.d. 4.1150.0440.1380.0051.8980.016
Mt. Bijela gora4xn.d. n.d. n.d. n.d. 5.3300.010n.d. 2.0040.007
S. aucupariaSorbusVeliko polje, Mt. Igman2xn.d. 0.3460.004n.d. n.d. 0.1960.0130.4600.0140.6410.004
S. austriacaSoraria (Sorbus × Aria)Grkarica, Mt. Igman4x0.377 *0.009n.d. n.d. trace */3.1750.0230.4690.0151.0430.008
Umoljani, Mt. Bjelašnica4xn.d. trace trace */n.d. 1.9600.0290.2230.0111.3450.017
S. bosniacaMt. Krug planina4xn.d. n.d. n.d. 0.179 *0.0363.695 *0.0202.658 *0.0123.102 *0.018
S. torminalis × ariaTormaria (Torminalis × Aria)Pratača,Mt. Igman2xn.d. n.d. n.d. 0.2230.0100.2670.0211.8960.0141.8830.011
Koznik3xn.d. n.d. trace/0.0980.0031.5940.0201.5920.0140.9390.009
Crne lokve, Posušje4xn.d. n.d. n.d. 0.2650.0082.5200.0091.6130.0092.0630.003
S. torminalisTorminariaTihovići2xn.d. n.d. n.d. n.d. 0.2360.0101.2840.0090.6420.01
12 13 14 15 16 17 22
S. ariaAriaGrkarica, Mt. Igman2x0.886 *0.005n.d. 2.5820.015n.d. 0.6980.005n.d. 0.366 *0.003
Mt. Bijela gora2xn.d. 0.167 *0.01n.d. n.d. n.d. 2.7360.0100.108 *0.003
Gradac, Posušje2xn.d. 0.131 *0.0070.6500.003n.d. 0.3080.016n.d. n.d.
Grkarica, Mt. Igman3x0.459 *0.002n.d. 2.3800.010n.d. 0.2630.003n.d. 0.244 *0.005
Umoljani, Mt. Bjelašnica3xn.d. 0.435 *0.0100.7830.004n.d. n.d. 1.8530.003trace */
Mt. Bijela gora3x0.330 *0.0480.500 *0.0121.0470.132n.d. n.d. 1.6510.000n.d.
Grkarica, Mt. Igman4x0.958 *0.012n.d. 1.3230.031n.d. 1.0560.015n.d. 0.231 *0.009
Umoljani, Mt. Bjelašnica4x0.209 *0.007n.d. 0.7740.013n.d. 0.1360.006n.d. trace */
Mt. Bijela gora4x1.033 *0.015n.d. 0.6680.009n.d. 0.6240.008n.d. trace */
S. aucupariaSorbusVeliko polje, Mt. Igman2x0.2880.022n.d. 0.3310.005n.d. n.d. n.d. trace/
S. austriacaSoraria (Sorbus × Aria)Grkarica, Mt. Igman4xn.d. 0.281 *0.0062.1030.0210.384 *0.003n.d. n.d. 0.153 *0.009
Umoljani, Mt. Bjelašnica4xn.d. 0.128 *0.0031.6090.0120.222 *0.015n.d. n.d. 0.082 *0.001
S. bosniacaMt. Krug planina4xn.d. 0.590 *0.01n.d. n.d. n.d. n.d. 0.152 *0.000
S. torminalis × ariaTormaria (Torminalis × Aria)Pratača, Mt. Igman2xn.d. n.d. 0.3670.006n.d. n.d. n.d. 0.1980.001
Koznik3xn.d. 0.3530.0130.7800.010n.d. n.d. n.d. n.d.
Crne lokve, Posušje4x0.3460.039n.d. 0.4310.004n.d. n.d. n.d. n.d.
S. torminalisTorminariaTihovići2xn.d. n.d. n.d. n.d. 0.3390.008n.d. n.d.
TaxonSubgenusLocationDNA Ploidy LevelMethylflavonols
Xsr (%)STDXsr (%)STDXsr (%)STD
18 19 23
S. ariaAriaGrkarica, Mt. Igman2xn.d. 1.016 *0.0060.444 *0.005
Mt. Bijela gora2xn.d. n.d. 0.134 *0.002
Gradac, Posušje2xn.d. 0.476 *0.009n.d.
Grkarica, Mt. Igman3xn.d. 0.724 *0.0030.205 *0.007
Umoljani, Mt. Bjelašnica3xn.d. n.d. n.d.
Mt. Bijela gora3xn.d. n.d. n.d.
Grkarica, Mt. Igman4xn.d. 0.496 *0.0210.087 *0.008
Umoljani, Mt. Bjelašnica4xn.d. 0.172 *0.002n.d.
Mt. Bijela gora4xn.d. 0.173 *0.003n.d.
S. aucupariaSorbusVeliko polje, Mt. Igman2xn.d. n.d. n.d.
S. austriacaSoraria (Sorbus × Aria)Grkarica, Mt. Igman4xn.d. n.d. n.d.
Umoljani, Mt. Bjelašnica4xn.d. n.d. trace */
S. bosniacaMt. Krug planina4x0.881 *0.0070.186 *0.003n.d.
S. torminalis × ariaTormaria (Torminalis × Aria)Pratača, Mt. Igman2x0.8330.0140.7380.014n.d.
Koznik3x0.7100.0080.5250.021n.d.
Crne lokve, Posušje4x0.4270.0080.3860.007n.d.
S. torminalisTorminariaTihovići2xn.d. 1.6690.015n.d.
Xsr—mean value of triplicates; STD—standard deviation; DE—dry extract. The names of the secondary metabolites correspond to their numerical values, which are given by Table 1: UV and MS spectral data of the detected flavonoids in EtOAc fractions of Sorbus accessions. Newly identified compounds from the leaves of Sorbus accessions are highlighted using bold font and marked with asterisk (*).
Table 3. Geographic origin, genome size and ploidy level of studied Sorbus accessions.
Table 3. Geographic origin, genome size and ploidy level of studied Sorbus accessions.
TaxonSubgenusGenome Size (2C pg)DNA Ploidy LevelLocationVoucher NumberNorthEastAltitude (m)
S. ariaAria1.472xGrkarica, Mt. IgmanSARA (54276)43.73916718.2913891350
1.412xMt. Bijela goraSARA (54280)42.67777818.475833730
1.412xGradac, PosušjeSARA (54278)42.42517.3925720
2.123xGrkarica, Mt. Igman SARA (51415)43.73916718.2913891350
2.113xUmoljani, Mt. BjelašnicaSARA (51412)43.66416718.2261111300
2.113xMt. Bijela goraSARA (54277)42.67777818.475833730
2.824xGrkarica, Mt. IgmanSARA (51416)43.73916718.2913891350
2.804xUmoljani, Mt. BjelašnicaSARA (51413)43.66416718.2261111300
2.784xMt. Bijela goraSARA (54279)42.67777818.475833730
S. aucupariaSorbus1.432xVeliko polje, Mt. IgmanSARA (51417)43.74527818.2751210
S. austriacaSoraria (Sorbus × Aria)2.814xGrkarica, Mt. IgmanSARA (51418)43.73916718.2913891350
2.774xUmoljani, Mt. BjelašnicaSARA (51414)43.66416718.2261111300
S. bosniaca2.804xMt. Krug planinaWU (080424)43.84222217.1997221300
S. torminalis × ariaTormaria (Torminalis × Aria)1.402xPratača, Mt. IgmanSARA (54281)43.76361118.19138915
2.183xKoznikSARA (54282)43.712517.9675900
2.744xCrne lokve, PosušjeSARA (54283)43.442517.464444700
S. torminalisTorminaria1.442xTihovićiSARA (54284)43.92388918.377778910
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Korić, E.; Milutinović, V.; Hajrudinović-Bogunić, A.; Bogunić, F.; Kundaković-Vasović, T.; Gušić, I.; Radović Selgrad, J.; Durić, K.; Nikšić, H. Phytochemical Characterisation of Sorbus Species: Unveiling Flavonoid Profiles Related to Ploidy and Hybrid Origin. Plants 2025, 14, 119. https://doi.org/10.3390/plants14010119

AMA Style

Korić E, Milutinović V, Hajrudinović-Bogunić A, Bogunić F, Kundaković-Vasović T, Gušić I, Radović Selgrad J, Durić K, Nikšić H. Phytochemical Characterisation of Sorbus Species: Unveiling Flavonoid Profiles Related to Ploidy and Hybrid Origin. Plants. 2025; 14(1):119. https://doi.org/10.3390/plants14010119

Chicago/Turabian Style

Korić, Emina, Violeta Milutinović, Alma Hajrudinović-Bogunić, Faruk Bogunić, Tatjana Kundaković-Vasović, Irma Gušić, Jelena Radović Selgrad, Kemal Durić, and Haris Nikšić. 2025. "Phytochemical Characterisation of Sorbus Species: Unveiling Flavonoid Profiles Related to Ploidy and Hybrid Origin" Plants 14, no. 1: 119. https://doi.org/10.3390/plants14010119

APA Style

Korić, E., Milutinović, V., Hajrudinović-Bogunić, A., Bogunić, F., Kundaković-Vasović, T., Gušić, I., Radović Selgrad, J., Durić, K., & Nikšić, H. (2025). Phytochemical Characterisation of Sorbus Species: Unveiling Flavonoid Profiles Related to Ploidy and Hybrid Origin. Plants, 14(1), 119. https://doi.org/10.3390/plants14010119

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop