Fragaria × ananassa cv. Senga Sengana Leaf: An Agricultural Waste with Antiglycation Potential and High Content of Ellagitannins, Flavonols, and 2-Pyrone-4,6-dicarboxylic Acid

Strawberry leaves are considered a valuable waste material; so far, mainly due to their antioxidant properties. Since the annual production of this crop is high, our study aimed to thoroughly examine the chemical composition and antidiabetes-related bioactivity of Fragaria × ananassa leaf of its popular and productive cultivar Senga Sengana. Leaves from three different seasons, collected after fruiting, were extensively analyzed (UHPLC-qTOF-MS/MS, HPLC-DAD). Some individual components were isolated and quantified, including specific flavonol diglycosides (e.g., 3-O-[β-xylosyl(1‴→2″)]-β-glucuronosides). The separated quercetin glycosides were tested in an antiglycation assay, and their methylglyoxal uptake capacity was measured. In addition, the biodegradable polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC) was confirmed at relatively high levels, providing further opportunity for strawberry leaf utilization. We want to bring to the attention of the food, pharmaceutical, and cosmetic industries the Senga Sengana strawberry leaf as a new botanical raw material. It is rich in PDC, ellagitannins, and flavonols—potent glycation inhibitors.


Introduction
Plant polyphenols provided to the human body with herbs, vegetables, or fruits significantly influence the digestive system, internal organs, and tissue activity. However, their bio-accessibility is diverse and conditioned by their chemical structure. The available epidemiological and interventional studies featuring healthy volunteers and patients show that polyphenols with antioxidative, antiglycative, and antiphlogistic properties reduce the risk of some chronic noncommunicable diseases, such as diabetes, metabolic syndrome, fatty liver disease, cardiovascular disease, and some cancers. Undoubtedly, in the etiology of those illnesses, an important role is played by pathogenic oxidative and carbonyl stress and ongoing inflammation [1][2][3]. In this regard, much attention has been paid to the properties of commonly consumed polyphenols [4,5]. There is also ongoing research on the interactions between polyphenols and gut microbiota to elucidate their health benefits. The data on flavonoids and ellagitannins are of particular interest [6,7].
In traditional medicine, leaves of species providing berry fruits (such as blackberry, raspberry, or wild strawberry) were used to cure gastroenteritis and mild diarrhea, strengthen the heart and circulatory system, and in metabolic disturbances to 'improve metabolism' and for 'blood purification'. The effectiveness of those recommendations is not sufficiently documented; therefore, their therapeutic application is currently limited. For example, the wild strawberry leaf is included in several multi-component food supplements and traditional herbal medicines (traditional botanical drugs) in the EU, intended to relieve non-specific diarrhea or enhance diuresis and metabolism. Similarly, it is used as a component of notified and licensed products in the USA and Canada [8]. Herbal medicines containing wild strawberry   To identify minor Senga Sengana components, we performed UHPLC-qTOF-MS/MS profiling with co-chromatography (Table 1). Figure 1 shows a typical HPLC-DAD chromatogram of 50% water-methanol extract from leaves of F. ananassa cv. Senga Sengana. The structures of main ellagitannins identified in the analyzed leaves are presented in Figure 2, those of flavonoids in Figure 3, while other are depicted in Figure 4.
Molecules 2022, 27,0 To identify minor Senga Sengana components, we performed UHPLC-qTOF-M profiling with co-chromatography (Table 1). Figure 1 shows a typical HPLC-DAD matogram of 50% water-methanol extract from leaves of F. ananassa cv. Senga Sengan structures of main ellagitannins identified in the analyzed leaves are presented in Fig those of flavonoids in Figure 3, while other are depicted in Figure 4.

Flavonoids
Seventeen flavonoid glycosides were recognized in extracts from Senga Sengana leaves (Table 1, Figure 3). Five compounds were derivatives of quercetin, five of

Phenolic and Carboxylic Acids
UHPLC-qTOF-MS/MS analysis of F. ananassa cv. Senga Sengana leaf extracts showed intense peaks of several phenolic acid derivatives, as well as carboxylic acids. Among them, 1 was identified as 2-pyrone-4,6-dicarboxylic acid (PDC, Figure 4). This heterocyclic dicarboxylic acid is considered as a chemotaxonomic marker of Rosoideae and was formerly reported in a concentration of 0.1-2% in the following genera: Alchemilla, Agrimonia, Duchesnea, Filipendula, Fragaria, Geum, Potentilla, Rosa, Rubus, Sanguisorba, and Waldsteinia [25]. PDC is also known as a low molecular product of catabolism (biodegradation) of plant polyphenols (phenolic acids, lignans, and lignin) by some microorganisms, e.g., by the soil bacterium Sphingomonas paucimobilis [49]. The biological origin of 2-pyran-4,6-dicarboxylic acid in strawberry leaves has not been definitively elucidated. In the examined raw plant material, the PDC content was in the range of 1-1.7% dry matter (Table 2). Carboxylic acid 2, obtained from the same fraction as PDC, was identified as a cyclic polyol named quinic acid. The presence of citric acid was also confirmed in Senga Sengana leaves (Table 1).
Compound 4a was separated as an off-white crystalline powder from fraction  [50], corresponding to a dihydroxybenzoic acid residue after the loss of pentose followed by decarboxylation. The ions formed by the homolytic cleavage were dominant, which suggested the presence of two free phenolic groups in the benzene ring. In the 1 H-NMR spectrum of 4, a dominant anomeric signal (4a) was accompanied by an additional small one (4b). The 13 C-NMR spectrum of 4a showed signals corresponding to β-Xylp of protocatechuic acid. Shifts of glycone carbons were compared positively with literature data [51]. Esterification with protocatechuic acid was confirmed by simple acidic and alkaline hydrolysis, as Markham described [51]. Our results indicate that 4a should be 1-O-protocatechuoyl-β-xylose ( Figure 4). As an impurity of 4a, protocatechuic acid-3-O-β-glucoside (4b) was deduced.   Jonsok [42]. In addition, several ions derived from isomers of cis/trans-caffeic and p-coumaric acid glycosides or esters were also observed [21,43].

Chemical Similarity of the Leaves of F. ananassa cv. Senga Sengana and F. vesca
The results obtained from qualitative and quantitative examination of F. ananassa cv. Senga Sengana leaves (FaSS1, FaSS2, FaSS3) were compared with the corresponding data for F. vesca leaves (Fv1 and Fv2). Metabolite profiling showed high similarity between these two species. In woodland strawberry leaves, we observed a comparable PDC content (0.9-1.1% dry matter) and lower levels of ellagitannins and flavonoids. The principal component in both species was agrimoniin (F. vesca 1.48-2.70%). Significant differences were observed in flavonol composition, as the quercetin and kaempferol diglycosides   The identity of other minor ellagitannins (ETs: davuriciin D 2 /fragariin A; laevigatins B/C/F; potentillin; galloyl-HHDP-glucose isomers such as sanguiin H-4; digalloyl-HHDPglucose), as well as gallotannins (3,5-O-digalloylquinic acid; mono-, di-and trigalloylglucose) [27,32,[34][35][36][37] in strawberry leaf extracts and fractions was determined using UHPLC-qTOF-MS/MS (Table 1). Davuriciin D 2 was isolated for the first time from the root of Rosa davurica Pall. [37]. Ellagitannin with MW 2038 Da was also obtained from strawberry fruit pomace under the name fragariin A [32]. Analysis of the published spectroscopic data and structures proposed for these ETs by their authors indicates that they are most likely identical. In addition, four ETs with molecular weights of 934 (two isomers), 1236, and 2020 Da were detected in Senga Sengana leaves. The same compounds were observed in pseudo-fruits, flowers, and leaves of several other F. ananassa cultivars, but their formulas were not elucidated [27,28,30,36]. Fragmentation of those minor strawberry ETs in the negative ion mode provided products typical for HHDP esters (m/z 481 and 301) and an additional ion derived from a low molecular depside at m/z 451 (exactly 450.9950). The same signal has been observed on the MS 2 spectrum of davuriciin , which is a valoneoyl analog of agrimoniin [37]. Therefore, the presence of the m/z 451 ion suggests a structural affinity, so we tentatively propose their chemical formulas starting with davuriciin D 2 /fragariin A (dimeric ellagitannin composed of α-pedunculagin and α-praecoxin A linked by DHDG, Figure 2). Ellagitannin-2020 is possibly a lactone of davuriciin D 2 /fragariin A (in the valoneoyl group) composed of α-pedunculagin and α-praecoxin D coupled by DHDG. The remaining compounds are secondary ellagitannins formed by the neutral loss of monomeric ellagitannin molecules, i.e., davuriciin D 2 /fragariin A lactone without the pedunculagin fragment (ET-1236 → 2020 − 784 = 1236) and davuriciin D 2 /fragariin A lactone without pedunculagin and DHDG coupler (ET-934 isomers → 2020 − 784 − 302 = 934). The latter compounds are probably α-and β-anomers of praecoxin D [31]. The absence of the DHDG lactone-derived ion in MS 2 supports that thesis. As a result of the fragmentation of m-GOG type dimers and certain secondary ellagitannins, besides both ellagic acid (m/z 301) and decarboxylated monolactone of hexahydroxydiphenic acid (LHHDP−CO 2 , m/z 275) ions, a lactone of the dehydrodigallic acid ion (LDHDG, m/z 319) also occurred. Tannins with a valoneoyl group additionally released the diagnostically relevant ion of valoneic acid trilactone (VTL, m/z 451). The reported ETs with molecular weights below 1870 Da were probably products of partial degradation or metabolism of agrimoniin and davuriciin D 2 /fragariin A. D2 content was many times lower than agrimoniin at 0.2-0.8% of dry matter ( Table 2).
The quantitative analysis of ellagitannins in various parts of strawberry cultivars in relation to the development stage was the topic of work of Karlińska and co-workers [46].

Flavonoids
Seventeen flavonoid glycosides were recognized in extracts from Senga Sengana leaves (Table 1, Figure 3). Five compounds were derivatives of quercetin, five of kaempferol, one of isorhamnetin, and six were taxifolin glycosides. In this study, we separated the dominant flavonoids, especially those that have not been further characterized. Three diglycosides (9, 11, and 13), as well as four monoglycosides (8, 12, 14, and 15), were isolated from FaSS-A. Some of the noted flavonoids, including flavonol diglycosides (MW 594, 610, and 640 Da), were described previously in leaves of F. ananassa cv. Polka [27], in flowers of the cultivar Jonsok [28], and in pseudo-fruits of the Japanese cultivar Tochiotome [21]. Nevertheless, the abovementioned authors assigned the structures only tentatively by LC-MS.
The NMR data of flagarin (11) were in accordance with those published data [21]. , of which the shift with higher value was assigned to β-GlcAp. The glycosylation position was deduced similarly as in 11, from C-3 upfield shift from~135 to~132 ppm together with downfield C-4 and C-1 signals. The second sugar of glycone (β-Xylp) was attached to the first one in the same way as in 11. That was proved by 2D-NMR (NOESY: H-1 ↔H-2 ) and downfield of the C-2 signal of the β-GlcAp moiety (from~73 to~81 ppm). The NMR data were closely similar to 11, an analogic glycoside of quercetin [21]. Our results indicate that compound 13 should be 3-O-[β-xylosyl(1 →2 )]β-glucuronoside of kaempferol. This compound is newly described and supplied with NMR data (Appendix B, Supplementary Materials).
All spectroscopic data of 8 were clearly similar to those included in a previous paper [19] and different from those of Pan and Lundgren [47], but the spectrum revealed several overlapping isomer signals. That compound was also found in Tochiotome strawberries [21]. Other taxifolin pentosides and hexosides were also observed in LC-MS as minor components (Table 1). For example, taxifolin-3-O-α-arabinoside was isolated previously from roots of F. ananassa cv. Reikov [26].

Phenolic and Carboxylic Acids
UHPLC-qTOF-MS/MS analysis of F. ananassa cv. Senga Sengana leaf extracts showed intense peaks of several phenolic acid derivatives, as well as carboxylic acids. Among them, 1 was identified as 2-pyrone-4,6-dicarboxylic acid (PDC, Figure 4). This heterocyclic dicarboxylic acid is considered as a chemotaxonomic marker of Rosoideae and was formerly reported in a concentration of 0.1-2% in the following genera: Alchemilla, Agrimonia, Duchesnea, Filipendula, Fragaria, Geum, Potentilla, Rosa, Rubus, Sanguisorba, and Waldsteinia [25]. PDC is also known as a low molecular product of catabolism (biodegradation) of plant polyphenols (phenolic acids, lignans, and lignin) by some microorganisms, e.g., by the soil bacterium Sphingomonas paucimobilis [49]. The biological origin of 2-pyran-4,6-dicarboxylic acid in strawberry leaves has not been definitively elucidated. In the examined raw plant material, the PDC content was in the range of 1-1.7% dry matter (Table 2). Carboxylic acid 2, obtained from the same fraction as PDC, was identified as a cyclic polyol named quinic acid. The presence of citric acid was also confirmed in Senga Sengana leaves (Table 1) [50], corresponding to a dihydroxybenzoic acid residue after the loss of pentose followed by decarboxylation. The ions formed by the homolytic cleavage were dominant, which suggested the presence of two free phenolic groups in the benzene ring. In the 1 H-NMR spectrum of 4, a dominant anomeric signal (4a) was accompanied by an additional small one (4b). The 13 C-NMR spectrum of 4a showed signals corresponding to β-Xylp of protocatechuic acid. Shifts of glycone carbons were compared positively with literature data [51]. Esterification with protocatechuic acid was confirmed by simple acidic and alkaline hydrolysis, as Markham described [51]. Our results indicate that 4a should be 1-O-protocatechuoyl-β-xylose ( Figure 4). As an impurity of 4a, protocatechuic acid-3-O-β-glucoside (4b) was deduced.

Chemical Similarity of the Leaves of F. ananassa cv. Senga Sengana and F. vesca
The results obtained from qualitative and quantitative examination of F. ananassa cv. Senga Sengana leaves (FaSS1, FaSS2, FaSS3) were compared with the corresponding data for F. vesca leaves (Fv1 and Fv2). Metabolite profiling showed high similarity between these two species. In woodland strawberry leaves, we observed a comparable PDC content (0.9-1.1% dry matter) and lower levels of ellagitannins and flavonoids. The principal component in both species was agrimoniin (F. vesca 1.48-2.70%). Significant differences were observed in flavonol composition, as the quercetin and kaempferol diglycosides typical for F. ananassa cv. Senga Sengana were absent in F. vesca. Between flavonol derivatives, only 3-O-β-glucuronosides were detected, and their content was slightly higher than in the leaves of the garden strawberry (12, Q3gr, 0.26-0.53%; K3gr, 0.09-0.12%). We also confirmed the occurrence of other minor components. The chemical composition of Fv1 and Fv2 was largely consistent with the scientific literature data [52]. A summary of HPLC chromatograms of F. vesca and F. ananassa cv. Senga Sengana leaf extracts is displayed in Figure 5.

Antiglycative and Anti-MGO Effects of Flavonols
Flavonols such as quercetin and kaempferol are known for their properties of trapping reactive carbonyl species (RCS), including methylglyoxal (MGO) and glyoxal (GO) [53]. These dicarbonyls originate from the metabolism of simple sugars such as fructose and glucose (from fructolysis and glycolysis in the liver), as well as from lipid peroxidation, and induce carbonyl stress. RCS, regardless of their sources of origin, react with the amino, guanidine and thiol groups of proteins, modifying their structure (post-translational modification) and physiological functions. They also react with lipoproteins, and purine bases in nucleic acids. These reactions result in harmful advanced glycation end products (AGEs) [54]. MGO, produced in excess in the liver, is subsequently secreted into the systemic circulation. Higher plasma MGO levels have been confirmed in hyperglycemic and dyslipidemic subjects and have been linked to metabolic dysfunction, insulin resis-

Antiglycative and Anti-MGO Effects of Flavonols
Flavonols such as quercetin and kaempferol are known for their properties of trapping reactive carbonyl species (RCS), including methylglyoxal (MGO) and glyoxal (GO) [53]. These dicarbonyls originate from the metabolism of simple sugars such as fructose and glucose (from fructolysis and glycolysis in the liver), as well as from lipid peroxidation, and induce carbonyl stress. RCS, regardless of their sources of origin, react with the amino, guanidine and thiol groups of proteins, modifying their structure (post-translational modification) and physiological functions. They also react with lipoproteins, and purine bases in nucleic acids. These reactions result in harmful advanced glycation end prod-ucts (AGEs) [54]. MGO, produced in excess in the liver, is subsequently secreted into the systemic circulation. Higher plasma MGO levels have been confirmed in hyperglycemic and dyslipidemic subjects and have been linked to metabolic dysfunction, insulin resistance, type 2 diabetes, diabetic retinopathy and nephropathy, non-alcoholic fatty liver (NAFLD), central obesity, atherosclerosis, gout, and other age-related chronic inflammatory diseases such as cardiovascular disease and disorders of the central nervous system. The high MGO concentration in hepatocytes blocks the allosteric binding of AMP to adenosine monophosphate-activated protein kinase (AMPK). Methylglyoxal can modify three arginines in the gamma subunit of AMPK, resulting in its inactivation. AMPK is an enzyme that controls cellular energy, which functions as an energy sensor for metabolic homeostasis and insulin signaling. When AMPK is inhibited, it thereby favors the anabolic processes, including lipogenesis and insulin resistance, which are related to metabolic syndrome and NAFLD [3]. Therefore, strategies that reduce the MGO level through its uptake (neutralization) are considered appropriate for therapeutic or preventive health care. An example of such an intervention is a randomized, double-blind, placebo-controlled, crossover study with quercetin-3-O-β-glucoside (isoquercitrin, 160 mg/day), which observed an 11% reduction in plasma MGO levels in humans [55]. Cardio-metabolic benefits of quercetin in elderly patients with metabolic syndrome were found by Shatylo et al. [56]. The study of Yi et al. [57] summarized quercetin's therapeutic effects and mechanisms in metabolic diseases.
The antiglycation activity of polyphenols is the result of several processes, among which the ability to trap in situ formed RCS, neutralization of reactive oxygen species, as well as reducing and chelating properties, can be considered important. Quercetin and its glycosides are characterized by potent antiradical and reducing effects, and significant antiglycation activity [13]. The type of sugar forming the glycosidic bond and the site of its substitution with the aglycone seem to be essential for this action. Diglycoside 11 (flagarin) was found to be substantially less active compared to the aglycone, but monoglycosides had comparable glycation inhibitory potency (Figure 6), especially those with sugar at the C-3 position (Q3ga = Q ≥ Q3g ≥ Q3gr ≥ Q4 g > Q3grx). It is known that substituting phenolic groups at positions C-5 and C-7 can lose flavonoid MGO trapping potency. Still, position C-4 (spireoside) only slightly reduces the effect, probably due to the loss of ability to form quinone forms or complexes with metal ions by the catechol group. Interestingly, the 3-O-β-glucuronoside of quercetin (12, miquelianin) retained high glycation inhibitory activity, which gives hope that the antiglycation capacity of flavonol metabolites can be maintained. Indeed, quercetin-3-O-glucuronoside, quercetin-3 -O-sulfate, and isorhamnetin-3-O-glucuronoside (syn. 3 -O-methylquercetin-3-O-glucuronoside) have been identified among the phase II metabolites after oral administration of quercetin glycosides [58,59]. All quercetin monoglycosides better protected BSA from modification compared with metformin. However, it should be noted that these compounds may be less available for different human tissues and organs. After oral intake, plasma concentrations of flavonol metabolites are unfortunately much lower compared to metformin. Neverthe-less, they may play an important role in inhibiting glycation in the gut. There is known evidence for the endogenous formation of AGEs in the gastrointestinal tract [60].
quercetin, hyperoside, spireoside, and miquelianin (Q, Q3ga, Q4′g, and Q3gr) ( Table 3). The flavonol A-ring arrangement provides the possibility of MGO addition at two positions, and the resulting adducts take the structure of hemiketal or hemiacetal [61]. Therefore, in the reaction products, we observed several forms of isomeric mono-and di-adducts with different retention times but identical masses, higher by 72 Da or 144 Da, respectively, compared to the precursor. Figure S6 shows the corresponding ions for Q3gr (12) and Q3grx (11) adducts.
Due to the nature of the tannins (their incompatibility with the BSE proteins), this assay was found to be not suitable to measure their suspected antiglycation potential.   In another in vitro test, we examined the ability of quercetin glycosides to trap MGO. The quercetin aglycone was used as the reference substance. All compounds formed adducts with methylglyoxal in vitro. However, di-adducts were noted only for quercetin, hyperoside, spireoside, and miquelianin (Q, Q3ga, Q4 g, and Q3gr) ( Table 3). The flavonol A-ring arrangement provides the possibility of MGO addition at two positions, and the resulting adducts take the structure of hemiketal or hemiacetal [61]. Therefore, in the reaction products, we observed several forms of isomeric mono-and di-adducts with different retention times but identical masses, higher by 72 Da or 144 Da, respectively, compared to the precursor. Figure S6 shows the corresponding ions for Q3gr (12) and Q3grx (11) adducts.  Due to the nature of the tannins (their incompatibility with the BSE proteins), this assay was found to be not suitable to measure their suspected antiglycation potential.

Discussion
Therapeutic uses of wild strawberry leaf preparations include treating gastrointestinal and urinary tract disorders [8]. Possibly, tannins present in leaves exert an astringent effect on the mucosa and have antimicrobial activity [9,62]. On the other hand, flavonoids are responsible for antioxidant, anti-inflammatory, cytoprotective, and diuretic action [63][64][65][66]. The glycation inhibitory and anti-MGO actions of strawberry flavonols may also be crucial for improving liver metabolism, e.g., quercetin and kaempferol glycosides could protect AMPK against post-translational modification and/or activate AMPK [67]. That effect was described in older scientific sources as 'improving metabolism' and 'blood purification'. Thus, polyphenols identified in the Senga Sengana leaf extracts could contribute to its biological effects. For example, Zhang and coworkers' findings [68] showed that F. ananassa leaves significantly alleviate cognitive and memory dysfunction in a diabetic animal model. The garden strawberry leaf components exhibited a strong antioxidant effect, reduced the blood glucose of diabetic rats, and improved their cognitive function by regulating the inflammatory response and inhibiting the caspase cascade. Similarly, F. ananassa leaf extract significantly decreased blood glucose, plasma creatinine, urea nitrogen, and renal malondialdehyde in diabetic nephropathy of rats [69]. Kashchenko et al. [70] attributed the hypoglycemic effect of agrimoniin to α-glucosidase inhibition. In experiments by D'Urso et al. [52], an aqueous extract of woodland strawberry leaves possessed direct, endothelium-dependent vasodilatation activity, and its potency was similar to that of an aqueous hawthorn extract. Other researchers reported the inhibitory action of F. vesca leaf extract, and the respective ellagitannin-enriched fraction, in Helicobacter pylori isolates with differential virulence, suggesting the potential of this plant material for the development of new medicines [71]. Furthermore, Juergenliemk et al. [72] confirmed in vitro that quercetin-3-O-glucuronoside (miquelianin) could cross the blood-brain barrier and reach the CNS, which determines its antidepressant effects demonstrated in animal model studies.
Therefore, based on our results and the cited scientific data, we conclude that the compounds identified in the leaves of F. ananassa and F. vesca are bioactive components. Their occurrence explains the biological effects observed in the quoted experiments, which may partially be due to the ability of flavonols to trap MGO and inhibit the harmful glycation of biomolecules. However, the antiglycation properties of flavonol glycosides demonstrated in this study are only preliminary and fraught with the inherent limitations of an in vitro model. Their translation to a therapeutic or prophylactic effect in humans requires further studies, including clinical trials.
In recent years, significant emphasis has been placed on the zero-waste way of life. As a result, different branches of industry, farming, and cultivation are forced to be more productive in a green manner. Some of the prominent examples are mainly connected with cereals or fruit processing, including valorization of by-products [73][74][75][76][77] and technological solutions [78,79]; however, more holistic approaches are found, too [80,81]. Our findings show that waste materials resulting from strawberry production should attract the attention of pharmaceutical companies.

Sample Preparation for LC
For the LC analysis, 50% water-methanol extracts (DER 1:100; m/v) were prepared from 0.5 g samples of powdered strawberry leaves and water-methanol mixture (1+1, v/v) in an ultrasonic bath Sonorex Digital 10P (Bandelin, Berlin, Germany). Extraction was performed at an ambient temperature for 15 min [11]. The resulting extracts were transferred to volumetric flasks, brought up to 50 mL with an extraction solvent, and next filtered through Whatman filter papers Grade 1 (Little Chalfont, UK). Fractions, sub-fractions, and isolated compounds were dissolved with water-methanol (1+1, v/v). Samples of all analyzed extracts and solutions were subsequently filtered by syringe filters (Durapore 0.22 µm).

Structure Elucidation Equipment
One-dimensional-and two-dimensional-NMR experiments were performed on Bruker Avance 300 MHz and 500 MHz spectrometers (Bruker BioSpin, Rheinstetten, Germany) using the residual solvent peaks as internal standards. NMR tests for glycosides were conducted in DMSO-d6, ellagitannins in acetone-d6 with D 2 O (1+1, v/v), and acids and esters in CD 3 OD. NMR data were analyzed by MestReNova 12 software (Mestrelab Research, Santiago de Compostela, Spain). Direct MS spectra (in negative mode) were recorded in water-methanol (1+1, v/v) on the ESI-qTOF Compact mass spectrometer (Bruker Daltonics, Bremen, Germany). LC-MS-derived spectra were recorded in an appropriate eluent (water-methanol acidified with formic acid) in negative mode. MS data were managed by Data Analysis 4.2 software (Bruker Daltonics). UV-VIS spectra of isolated compounds were measured in water-methanol (1+1, v/v; 0.01-0.03 mM) on a Cecil CE 3021 spectrometer (Cecil Instruments, Cambridge, UK).
The validated HPLC-DAD method described previously was used to examine the components of strawberry leaf extracts [33]. Separation was achieved on a Hypersil Gold C18 column (250 × 4.6 mm, ∅ 5 µm) with a C18 precolumn (10 × 4.6 mm, ∅ 5 µm) (Thermo Fisher Scientific, Waltham, MA, USA). The linearity of the HPLC-DAD method and quantification of selected polyphenols and PDC was performed based on regression equations determined for the isolated compounds from peak areas and corresponding concentrations. The Ultimate 3000 system (Thermo Fisher Scientific) with a Kinetex C18 column (150 × 2.1 mm, ∅ 2.6 µm for extracts; 100 × 2.1 mm, ∅ 2.6 µm for MGO derivatives; Phenomenex, Torrance, CA, USA) coupled to the ESI-qTOF Compact mass spectrometer (Bruker Daltonics) was used for qualitative UHPLC-qTOF-MS/MS. Equipment, analysis parameters, and gradient were as reported previously [82] (for analysis of extracts), [13] (for analysis of MGO adducts).

Acid Hydrolysis
Solutions of isolated hydrolyzable tannins (∼50 mg in 5 mL of 5% H 2 SO 4 ) were heated (90 • C, reflux, eight hours) according to Tanaka et al. [83]. The hydrolysis products were purified on the octadecyl columns (Isolute C18, 10 g, Biotage, Uppsala, Sweden), then concentrated under reduced pressure and diluted in methanol (5 mL). Phenolic acids and depsides released by hydrolytic degradation were analyzed by TLC (mobile phase P).
Solutions of other polyphenols isolated in small amounts (1 mg in 1 mL 5% H 2 SO 4 ) were heated at 90 • C in sealed vials for four hours. The hydrolysis products were separated by adding ethyl acetate into water residues [51]. Water layers, concentrated and re-dissolved in 1 mL of methanol, were examined for sugars (TLC, mobile phase S). Organic layers, concentrated and re-dissolved in 1 mL of methanol, were investigated for aglycones (TLC, mobile phases X and Z).

Alkaline Hydrolysis
To initially differentiate the 3-O-and 7-O-position of glycosylation, a small quantity of isolated polyphenol (1 mg) was subjected to alkaline hydrolysis in 1 mL of 0.5% (~0.09 M) aqueous KOH in a boiling water bath for three hours [84]. The formation of advanced glycation end-products (AGEs) was measured following a slightly modified method proposed by Liu et al. [85]. In brief, 21.2 µM bovine serum albumin was incubated with methylglyoxal (MGO) at 0.5 mM in 100 mM PBS at pH 7.4 with 0.02% sodium azide (for prevention of microbial growth). The compounds investigated for inhibition of non-enzymatic glycation were added at a final concentration of 1.5 mM. Then, the reaction solution was incubated at 37 • C and shaken at 50 revolutions per minute for seven days in closed vials away from light. Measurement of the fluorescent intensity of total AGEs after incubation was carried out using a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA) at a wavelength of 360 nm for excitation and 460 nm for emission. Data acquisition was obtained with the Gen5 Software (BioTek Instruments Inc., Winooski, VT, USA). The measurements from three experiments were all performed in triplicate, and the percentage inhibition of AGE formation was calculated using the following equation: where Fl 0 is the mean fluorescence intensity of the blank sample and Fls is the mean fluorescence intensity of the sample.

MGO Trapping Assay
Methylglyoxal trapping activity was investigated according to a slightly modified version of the Sang et al. [86] method, as previously described [13]. Briefly, 0.6 mM methylglyoxal was incubated for one hour with 0.2 mM of each compound in 100 mM PBS at pH 7.4 and 37 • C to equate to physiological conditions and shaken at 50 revolutions per minute. The incubation reaction was stopped by adding 2.5 µL of acetic acid and placing the collected samples in an ice-cold water bath. Next, the samples were filtered through hydrophilic syringe filters (Durapore 0.22 µm) and analyzed using UHPLC-qTOF-MS/MS to investigate their ability to form adducts with methylglyoxal. The trapping agent solutions were freshly prepared before each series of experiments was begun, and the pH of the sodium phosphate buffer was determined immediately before use.

Statistical Analysis
All data are presented as means ± standard deviation (SD). Data were analyzed using the Shapiro-Wilk test to assess normality of distribution, followed by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test using the GraphPad Prism 6 software; P values equal to or less than 0.05 were considered significant.

Conclusions
To summarize, a detailed chemical analysis of F. ananassa cv. Senga Sengana leaves revealed the presence of a broad spectrum of polyphenolic metabolites. Their major components were ellagitannins. Galloyl esters of quinic acid and glucuronosides of flavonols constituted the second group. Proanthocyanidins, flavan-3-ols, and phenolic acid derivatives were minor components. Several new polyphenols including kaempferol and quercetin xylosyl-glucuronosides were also identified.
The occurrence of polyphenols together with 2-pyrone-4,6-dicarboxylic acid at relatively high levels further provides the opportunity to utilize this resource in biotechnological processes to obtain PDC. Moreover, strawberry flavonols have demonstrated the capacity to uptake methylglyoxal and inhibit protein glycation in vitro. Due to that, the leaves of Senga Sengana may become a new raw plant material with therapeutic, pro-health, or cosmetic phytoconstituents.