Phytoprofiling of Sorbus L. Inflorescences: A Valuable and Promising Resource for Phenolics

The leaves and fruits of various Sorbus L. genotypes have long ethnopharmacological and food-usage histories, but inflorescences are still underutilized and neglected materials with scarce phytochemical scientific evidence. The aim of this study was to determine the phenolic profiles of inflorescence extracts of 26 Sorbus species, genotypes, and cultivars. HPLC and UPLS with MS detection were applied, and coupled data revealed unique phytochemical phenolic profiles. Neochlorogenic and chlorogenic acids were the key compounds, reaching up to 5.8 mg/g of dw. Rutin, isoquercitrin, quercetin 3-O-malonylglucoside, isorhamnetin 3-O-rutinoside, sexangularetin derivative, and kaempferol acetyl hexoside were detected in all Sorbus inflorescence samples. Overall, high quantitative heterogeneity across the various Sorbus genotypes was found by profiling. Phenolic fingerprint profiles and sexangularetin derivatives could serve as markers in authenticity studies and quality control schemes. The species S. amurensis, S. arranensis, S. commixta, and S. discolor and the cultivars ‘Chamsis Louing’, ‘Coral Beauty’, and ‘Edulis’ could be used as target genotypes for production of smart and innovative inflorescence matrix-based ingredients.


Introduction
Increasing consumers' awareness about safe, health-promoting foods-and, specifically, the search for new safe and natural plant origin extracts and compounds that could be used with versatility in various industries for the production of cosmeceuticals, nutraceuticals, and pharmaceuticals-raises challenges and scientific uncertainties [1]. Furthermore, the increasing demand for innovative and functional food products with clear and clean labeling requires natural, safe, and simple ingredients that can be used conventionally [2]. Plant origin materials contain bodies of specialized metabolites that, when present in the extracts, exert health-promoting or pharmacological effects, as well as synergistic and additive effects [3]. In addition to the well-known plant materials with long ethnopharmacological histories, there are certain underutilized and neglected plants that can provide various raw materials that are extremely rich in particular groups of compounds of interest [4][5][6].
The genus Sorbus L. has a highly complicated systematics encompassing numerous subgenera and sections that determine its diversity sensu lato [7]. Furthermore, Sorbus' extraordinary apomixis, cross-breeding, and intergeneric hybridization abilities have led to a great expansion of Sorbus genotypes, cultivars, hybrids, and varieties [8,9]. Almost all parts of the Sorbus plants have a historical record of usage in various traditional medicinal systems, as well as in food, fodder, beauty, health, and wellness-promoting products [10][11][12][13][14]. The fruits have been included in pharmacopoeias as polyvitaminic substances and in daily nutrition, but, due to their tart and specific taste, they are neglected or only used occasionally, although the ascorbic acid content exceeds the amount in oranges [4,15,16]. Nevertheless, their fruit powders could be incorporated as an ingredient in various processed foods and new products [4,17]. Extensive breeding and hybridization programs have led to several sweet-tasting rowanberries (Sorbus L. cultivars) that have potential as functional food ingredients containing amounts of carotenoids equivalent to commonly known sources, such as carrots [16,[18][19][20]. Furthermore, due to their unique pattern of sugars and sugar alcohols, they could be incorporated in specialized food production [21,22]. Recent research has been devoted to elucidating the phytochemical richness and variability of phenolic profiles in the leaves of Sorbus species and varieties, as they contain more than fivefold higher amounts of phenolic compounds than commonly used fruits [23][24][25]. Although all Sorbus raw materials-namely, bark, leaves, and fruits-have been extensively investigated, the inflorescences are still one of the least studied raw materials. Sorbus plants contain decorative corymbs that cluster the dense flowers [26]. Scientific knowledge regarding the phytochemical composition of inflorescences is still limited. Olszewska et al. determined the phytochemical compositions of inflorescences of S. aucuparia, S. commixta, S. decora, S. gracilis, S. koehneana, S. torminalis, S. pohuashanensis, S. sitchensis, S. intermedia, and S. aria [23][24][25][27][28][29]. Recently, they produced a standardized inflorescence extract from S. aucuparia and determined its detailed phytochemical profile, as well as antioxidant and other biological properties [27]. However, the detailed profiling of inflorescences of other Sorbus species and cultivars has not yet been performed. Preliminary data suggest that Sorbus inflorescences contain significantly higher amounts of phenolic compounds than the fruits and even leaves [23][24][25]30]. Therefore, it is extremely important to determine their qualitative and quantitative profiles and define the analytical markers. Furthermore, the inflorescences of Sorbus contain the specific compound sexangularetin and its derivatives, which have not been detected in the fruits or leaves. These compounds could serve as authentication markers ensuring the quality of extracts [19].
Being climate-and environment-resilient, Sorbus plants are particularly attractive species for growth in standardized plantations, as they can withstand poor soils and harsh environments [31]. Sustainable and no-waste technologies could be applied for smart collection and preparation of Sorbus extracts from various raw materials rich in particular compounds [4,32].
The aim of this study was to determine the phenolic profiles of inflorescence extracts of 26 Sorbus L. species, genotypes, and cultivars. To the best of our knowledge, the profiles for the inflorescences of most species are reported for the first time. The research was focused on the phenolic profiles of the inflorescences, utilizing chemometric analysis to elucidate the chemophenetic differences and link the genotype with the pattern of phenolic acids and flavonoids. The lack of research on the phytochemical patterns of inflorescences of different Sorbus species highlights the need to elucidate the patterns and markers of the phenolic profiles. Phenolic compounds could act as chemical markers for the coupling or differentiation of species and substantiation of their parental origin. The peculiarities of the phenolic profiles could provide significant information for chemophenetic studies of this remarkably diverse genus of Sorbus.

Quantitative Phenolic Profiling of Sorbus L. Species and Cultivars
Analysis of the qualitative and quantitative composition of the phenolic compounds in the raw plant material samples of rowan inflorescences was performed. The chromatogram of ethanol extracts from the inflorescence samples of Sorbus plants is presented in Figure  S1. The results of qualitative analysis of phenolic compounds are presented in Table S1. Neochlorogenic, chlorogenic, and cryptochlorogenic acids, as well as dicaffeoylquinic acid derivative 1 and dicaffeoylquinnic acid derivative 2, were identified in all inflorescence samples tested (Table 1). Caffeoylshikimic acid was not detected only in inflorescences of S. aria and S. arranensis, and coumaroylquinic acid derivative was detected only in certain inflorescence samples. The concentrations of these caffeoylquinic acids varied signifi-cantly among the tested samples, with differences of up to twentyfold. Chlorogenic acid was the predominant component in most of the inflorescence samples from the various species and cultivars, although the predominant component in the inflorescence samples of S. arranensis and S. lancifolia was neochlorogenic acid, while quercetin 3-O-malonylglucoside was the predominant component in the inflorescences of 'Edulis'. The greatest amount of chlorogenic acid was found in the inflorescence samples of S. commixta (31,539 ± 2503 µg/g), S. amurensis (29,733 ± 1465 µg/g), and 'Chamsis Louing' (25,867 ± 1557 µg/g) ( Table 1). The greatest concentrations of neochlorogenic acid (18,712 ± 1740 µg/g, 17,739 ± 2442 µg/g, and 17,011 ± 441 µg/g) were found in the inflorescence samples of 'Coral Beauty', 'Carpet of Gold', and S. amurensis, respectively (Table 1). Significant variation in the flavonol profiles depending on the Sorbus species/cultivar was determined (Tables 2 and 3

Hierarchical Cluster Analysis of Phenolic Compounds in Sorbus L. Species and Cultivars
Hierarchical cluster analysis was performed for the samples of Sorbus inflorescences, using the concentrations of identified biologically active compounds as clustering variables. The investigated Sorbus inflorescence samples were grouped into seven significant clusters ( Figure 1).  The inflorescences of Sorbus were clustered into four groups. Inflorescence samples of group I were closely clustered along the negative side of PC1 due to the high total concentration of quercetin diglycosides and low total concentrations of dicaffeoylquinic acids, isorhamnetin derivatives, and kaempferol derivatives. Inflorescences of group III were located on the positive side of PC1. The total concentrations of dicaffeoylquinic acids, isorhamnetin derivatives, and kaempferol derivatives that scored highly in PC1 ranged from the mean values to the highest in the corresponding samples. In contrast, the total concentration of quercetin diglycosides, which had a negative loading in PC1, was determined to be low in these inflorescence samples. Inflorescence samples of group I and group III were situated near the zero point of PC2. The concentration of caffeoylshikimic acid and total concentration of caffeoylquinic acids determined in these samples were close to the mean values. Group II was distinguished by a high concentration of caffeoylshikimic acid and a total concentration of caffeoylquinic acids that scored highly in PC2. Inflorescence samples of group IV differed from the others in their low concentration of caffeoylshikimic acid and total concentration of caffeoylquinic acids, which had positive loadings in PC2. Inflorescence samples of 'Edulis' were distanced from all the others and were grouped on the positive side of the PC3. This could be explained by the high concentration of sexangularetin derivative and total concentration of quercetin monoglycosides. The inflorescences of Sorbus were clustered into four groups. Inflorescence samples of group I were closely clustered along the negative side of PC1 due to the high total concentration of quercetin diglycosides and low total concentrations of dicaffeoylquinic acids, isorhamnetin derivatives, and kaempferol derivatives. Inflorescences of group III were located on the positive side of PC1. The total concentrations of dicaffeoylquinic acids, isorhamnetin derivatives, and kaempferol derivatives that scored highly in PC1 ranged from the mean values to the highest in the corresponding samples. In contrast, the total concentration of quercetin diglycosides, which had a negative loading in PC1, was determined to be low in these inflorescence samples. Inflorescence samples of group I and group III were situated near the zero point of PC2. The concentration of caffeoylshikimic acid and total concentration of caffeoylquinic acids determined in these samples were close to the mean values. Group II was distinguished by a high concentration of caffeoylshikimic acid and a total concentration of caffeoylquinic acids that scored highly in PC2. Inflorescence samples of group IV differed from the others in their low concentration of caffeoylshikimic acid and total concentration of caffeoylquinic acids, which had

Discussion
Phenolic-rich plant materials are considered as sources for added-value ingredients that could provide positive health and nutritional effects in the frame of chronic degenerative diseases resulting from the features of modern life [33]. Plants synthesize phenolic compounds as specialized metabolites against exogenous and endogenous stress factors. Furthermore, various studies have confirmed their remarkable health effects and pharmacological activities; namely, antioxidant, anti-inflammatory, cardioprotective, neuroprotective, gut microbiota-promoting, and antidiabetic effects [24,28,[33][34][35]. The latter effect is one of the main medicinal effects documented for the raw materials of Sorbus species, with the phenolic compounds being suggested as one of the bioactive fractions [19,36,37]. The majority of ethnopharmacological, phytochemical, and nutritional studies have focused on the leaves and fruits of various Sorbus species and cultivars. This study elucidated the phenolic profiles of inflorescences of Sorbus species and cultivars. High quantitative heterogeneity across various Sorbus genotypes was found in the profiling. However, the prevailing compounds that were common to all Sorbus materials tested were present in a constant manner, with the chlorogenic and neochlorogenic acids being the key components and rutin, isoquercetin, and quercetin malonyl glucoside the prevailing flavonoids. The results are consistent with our previous studies [20,[38][39][40][41] and with the studies by Olszevska et al., which, to the best of our knowledge, are to date the only phytochemical studies on the inflorescences of Sorbus species [23,24,27,30]. Epidemiological and clinical studies show that hydroxycinnamic acid intake is associated with reduced risk of metabolic syndrome, diabetes, and colorectal cancer. Furthermore, chlorogenic acid can modulate colonic microbiota and, via the gut-brain axis, express health benefits [42]. Hydroxycinnamic acids have anti-inflammatory [43], neuroprotective [44], antidiabetic [45], antimicrobial [46], and ultraviolet-protective [47] effects. The amount of neochlorogenic acid we observed in the inflorescence samples was close to the amount identified by Olszewska et al. (0.19-1.98%) [24,30]. Olszewska et al. found 1.78-4.17% chlorogenic acid in their inflorescence samples, and the inflorescences of S. commixta contained significantly higher amounts of chlorogenic acid compared to the leaves (3.92% and 0.79%, respectively) [24,30]. The species and cultivars we analyzed accumulated less chlorogenic acid (0.76-2.25%).
S. commixta is a valuable species with an extraordinarily rich phenolic composition [29]. In our previous research [38][39][40], the fruits and leaves of this species were found to be exclusively separate from other species in the hierarchical cluster and PCA analyses, and they had the highest antioxidant activities. Furthermore, in this study, the inflorescences of S. commixta contained the greatest amounts of chlorogenic acid and, surprisingly, the lowest amounts of neochlogenic acid compared to other Sorbus species. However, the amounts of chlorogenic acid were sevenfold and twofold lower compared to the leaves and fruits, respectively [38,39]. The quantitative composition of phenolic acids in sweet rowanberries of the 'Titan', 'Granatnaja', 'Alaja Krupnaja' cultivars has been previously examined [16]. The greatest amounts of neochlorogenic and chlorogenic acids were in the fruits of 'Granatnaja', while the lowest amounts were determined in the fruits of 'Alaja Krupnaja'. Our study determined corresponding patterns in the inflorescence samples of 'Titan', 'Granatnaja', and 'Alaja Krupnaja'. Kylli et al. (2010) found the greatest amounts of neochlorogenic acid and chlorogenic acid in fruit samples from the cultivars 'Titan' and 'Granatnaja' [48]. We compared the amounts of hydroxycinnamic acids in leaves, fruits, and inflorescences (certain species overlapped in our previous studies), and the results demonstrated the superiority of the leaves and fruits. In contrast, Olszewska et al. (2010) determined that S. commixta inflorescences contained significantly greater amounts of total phenolics and about threefold greater amounts of chlorogenic acid compared to leaves [30]. Olszewska and Michel (2009) determined that the antioxidant activities of S. aucuparia, S. aria, and S. intermedia inflorescences were higher compared to the leaves and fruits. In fact, the phenolic content in the inflorescences and leaves of various Sorbus genotypes are comparable, and superiority strongly depends on geographic, climatic, and edaphic conditions. Further studies could target phytogeographic, phonologic, and ontogenetic profiling. The total phenolic contents in inflorescences and leaves were comparable; however, the profiles of individual flavonoids were highly variable between plant materials [24]. Flavonols are the predominant flavonoids in Sorbus materials [19]. Flavonols demonstrate cardioprotective [49], neuroprotective [50], hepatoprotective [51], anticancer [52], antimicrobial [53], anti-inflammatory [54], and anti-platelet [55] activities. On average, the total amount of identified flavonoids in the tested Sorbus inflorescences was 1.7 mg/g. The individual patterns showed high quantitative heterogeneity. Overall, the amounts of individual flavonoids in inflorescences were lower than in the leaves of the respective species (S. arranensis, S. commixta, S. discolor, S. semi-incisa) [38,40]. Olszewska determined that the total flavonoid content was superior in the inflorescences of S. aucuparia and S. intermedia compared to the leaves, and only S. aria leaves were superior to the inflorescences. In contrast, our results show that the total identified flavonoids in S. aria inflorescences were lower compared to the leaves. Hukkanen et al. (2006), for fruit samples of the 'Titan', 'Granatnaja', and 'Alaja Krupnaja' cultivars, determined that the greatest amount of flavonols was found in the fruits of 'Granatnaja', while the lowest amount was found in the fruits of 'Alaja Krupnaja' [16]. The pattern corresponds with the results of our present study, confirming the phytochemical impact of genotype. Quercetin derivatives have been determined to be the predominant substances in identified flavonoid complexes. The amounts of other flavonoids are significantly lower. These results are consistent with data supplied by other researchers [23]. Upon comparison of the amounts of isorhamnetin derivatives in inflorescence samples from various species and cultivars of Sorbus, we determined that the inflorescence samples of the subgenus Aria contained greater amounts of these flavonoids than the inflorescence samples of the subgenus Sorbus. The same patterns were detected by Polish scientists [23,29]. The amounts of isorhamnetin determined in the inflorescence samples of S. aria during previous research were 31-32% [56] and 44% [23] of the total flavonoid amount. Isorhamnetin could be used as an analytical marker for differentiation of raw plant materials of different subgenera. Sexangularetin is an important analytical marker of Sorbus inflorescence materials. In contrast to leaves, the inflorescences are abundant in sexangularetin derivatives; namely, methoxylated flavonoids [27]. Methoxylated flavonoids could be of particular scientific interest, as they have high metabolic stability and bioavailability [57]. The pattern for methoxylated flavonoids highlights the uniqueness of inflorescences compared to leaves and fruits. Our results showed that S. discolor had the greatest amount of sexangularetin derivatives. The genotype determined the pattern of the phenolic compounds, and the chemometric analysis distinguished two principal groups of Sorbus inflorescences; namely, the Rossica cultivar group (Table 4) and the group constituted by S. x arnoldiana cultivars and sect. Sorbus species. S. amurensis, S. aria, S. arranensis, S. commixta, and S. x hostii formed different, distinct groups or separate clusters, confirming their unique and particular phenolic profiles. The cultivars 'Chamsis Louing', 'Coral Beauty', and 'Edulis' and the species S. amurensis, S. arranensis, and S. commixta, remarkably, had the highest total phenolic compounds determined, ranging from 5.9 to 7.9 mg/g. Plant inflorescences, rich in hydroxycinnamic acids and quercetin derivatives, are potent antioxidants, possess anti-inflammatory activities, and, traditionally, are used as diuretic, diaphoretic, and anti-inflammatory agents [58][59][60][61]. Furthermore, food plants provide various raw materials that contain phytochemical compounds with health benefits. The processing of the raw materials can, depending on the final concentration of compounds, create an interface between food, medicine, and cosmetics [62]. Plant material infusions targeted as daily food intake can be employed as preventive nutrition in restoring and sustaining health and wellness [60]. Sorbus inflorescences are attractive plant matrixes with notable phenolic profiles. Modulation of the stages of genotype selection, extraction, and processing can unlock their potential multi-industrial applications in future preparations. The collected raw plant material samples were dried at room temperature and stored in a dark, dry place. The research results were re-calculated for absolutely dry raw plant material.

Sample Preparation
For qualitative and quantitative analysis of flavonoid glycosides and phenolic acids, the rowan inflorescence samples were crushed into particles and passed through a 355 µm sieve. The samples weighed around 0.25 g (accurate sample). The weighed raw plant material sample was then placed into a conical flask with 25 mL of 50% ethanol. The extraction was performed by submerging the materials in a BioSonic UC100 ultrasonic bath (Cuyahoga Falls, Ohio, USA) for 20 min. The obtained extract was filtered through a paper filter into a 25 mL volumetric flask and adjusted accordingly to a volume with 50% ethanol. Before high-performance liquid chromatography (HPLC) analysis, extracts were filtered through a membrane filter with a pore size of 0.22 µm (Carl Roth GmbH, Karlsruhe, Germany).

HPLC Analysis
The analysis of extracts was performed using a previously optimized and validated (Gaivelyte et al., 2014) HPLC method. Briefly, quantitative analysis was performed using a Waters 2695 Alliance system (Waters, Milford, MA, USA) with a Waters 996 photodiode array detector. Separation was performed using an ACE (ACT, Aberdeen, UK) column (C18, 150 mm × 4.6 mm, particle size: 3 µm). The mobile phase of the optimized chromatographic method consisted of eluents A (0.05% trifluoracetic acid) and B (acetonitrile). The gradient variation was as follows: 0-5 min-12% B, 5-50 min-12-30% B, 50-51 min-30-90% B, 51-56 min-90% B, 57 min-12% B. The eluent flow rate was 0.5 mL/min, and the injection volume was 10 µL. The column was temperature-controlled, and the temperature was maintained at 25 • C. Calibration curves of compounds identified in the rowan inflorescence extracts were compiled; these curves were used for quantitative assessment. Quantification of caffeoylshikimic acid and dicaffeoylquinic acid derivatives was performed using a chlorogenic acid calibration curve, quantification of quercetin diglycosides using a rutin calibration curve, quantification of kaempferol derivatives using an astragalin calibration curve, and quantification of isoramnetin and sexangularetin derivatives using an isorhamnetin 3-O-rutinoside calibration curve. Concentrations of phenolic acids were calculated at a wavelength of 325 nm, while the concentrations of flavonoids were calculated at a wavelength of 350 nm.

Statistical Analysis
Phenolic compound content was expressed as the mean ± standard deviation (SD) of three replicates. The statistical data analysis was evaluated by applying ANOVA with a Tukey HSD post hoc test. Statistically significantly different means were marked with different letters. Differences were considered statistically significant when p < 0.05. In accordance with the quantitative composition of the identified compounds, the tested samples were compared using the method of hierarchical cluster analysis with squared Euclidean distances. Principal component analysis was performed taking into account factors with eigenvalues higher than 1. The data were processed using Microsoft Office Excel 2010 (Microsoft, JAV) and SPSS 20 software.

Conclusions
The current study provides a comprehensive analysis of the phenolic profiles of 27 Sorbus genotypes and complements knowledge regarding the patterns of specialized metabolites in Sorbus plant materials. The findings showed that the inflorescence extracts were abundant in hydroxycinnamic acids and flavonol derivatives. The leaves and inflorescences were comparable in phenolic contents, and the significant superiority of inflorescences was not observed. Phenolic fingerprint profiles and sexangularetin derivatives could serve as markers in authenticity studies and quality control schemes. The species S. amurensis, S. arranensis, S. commixta, and S. discolor and cultivars 'Chamsis Louing', 'Coral Beauty', and 'Edulis' could be target genotypes for production of smart and innovative inflorescence matrix-based ingredients.