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Metabolites of Prickly Rose: Chemodiversity and Digestive-Enzyme-Inhibiting Potential of Rosa acicularis and the Main Ellagitannin Rugosin D

Prickly rose (Rosa acicularis Lindl.) is the most distributed rose species in the Northern Hemisphere, used by indigenous people for various food purposes. The lack of detailed information about the chemical composition of R. acicularis has led us to study the phytochemical composition and metabolic profile of prickly rose extracts using chromatographic techniques. Many groups of phenolic and non-phenolic compounds were quantified in the leaves, flowers, roots and fruits of R. acicularis. Phenolic compounds were the dominant phytochemicals in the aerial parts and roots of R. acicularis. A precise study by high-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection showed the presence of 123 compounds, among which ellagic acid derivatives, ellagitannins, gallotannins, catechins, catechin oligomers, hydroxycinnamates and flavonoid glycosides of kaempferol, quercetin and dihydroquercetin were all identified for the first time. The most abundant phenolic compounds were ellagitannins and flavonoid glycosides, with a maximal content of 70.04 mg/g in leaves and 66.72 mg/g in flowers, respectively, indicating the great ability of R. acicularis organs to accumulate phenolic compounds. By applying a standardized static, simulated gastrointestinal digestion method, we found the inhibitory potential of the leaf extract against digestive α-amylases. A pancreatic α-amylase activity-inhibiting assay coupled with HPLC microfractionation demonstrated high inhibition of enzyme activity by ellagitannin rugosin D, which was later confirmed by a microplate reaction with mammalian α-amylases and the simulated digestion method. This study clearly demonstrates that R. acicularis leaf extract and its main component, ellagitannin rugosin D, strongly inhibit digestive α-amylase, and may be a prospective antidiabetic agent.


Introduction
Rosa is one of the largest genera of the Rosaceae family, and is an amazing plant genus that has found practical application by humans since ancient times. In a botanical sense, the genus includes about 400 species in the form of shrubs and semi-shrubs, widespread mainly in the Northern Hemisphere [1]. Disregarding the decorative value of the rose species, it should be noted that they are of great importance as medicinal and food plants. Well-known rose species include R. canina, R. damascena, R. majalis and R. rugosa, amongst others, which are sources of bioactive ellagitannins, flavonoids, triterpenoids, carotenoids and fatty oils that have antioxidant, antitumor, anti-inflammatory, gastroprotective and antiatherogenic activity [2]. It is not difficult to notice that the largest amount of scientific information on the chemical composition and biological activity of the rose species was created for the southern plants. Northern species growing in the territory of Siberia and the Far East A wide distribution of R. acicularis has contributed to its use as a food and medicinal plant, and all the organs (leaves, flowers, roots and fruits) of the plant are of practical importance. The most common way to use R. acicularis is to brew it as a tea, the taste of which varies depending on the part of the plant used, from sweet and sour to tart and herbal [7]. Jams, syrups and compotes are prepared from the prickly rose fruits, characterized by good gelling properties. In the medical systems of Siberian and Asian peoples, rosehip medicines are used to treat diseases of the stomach and intestines, as an appetizing and anti-inflammatory agent, as well as in remedies to restore health after long-term illnesses [8]. Yakut traditional nomad medicine recommends the ripe fruits of R. acicularis to strengthen the gums, and an unripe fruit decoction is used to treat cardiac problems [9]. The decoction of the twigs of fresh bushes is a prophylactic remedy against diarrhea and intestinal diseases, and the tea of the leaves is used as a diuretic [10]. The Buryat lamas use the fruits of R. acicularis to treat diseases of bile and to suppress wind [11], as well as to destroy poisons and contribute to the growth of teeth [12]. The stem bark is applied as an antidote and used to cure lymphatic system diseases [13].
In official medical practice, rosehip is applied as a source of ascorbic-acid-containing concentrates and syrups and carotene-rich oils and creams. The most commonly used roses for commercial purposes are R. rugosa, R. canina and R. majalis, as A wide distribution of R. acicularis has contributed to its use as a food and medicinal plant, and all the organs (leaves, flowers, roots and fruits) of the plant are of practical importance. The most common way to use R. acicularis is to brew it as a tea, the taste of which varies depending on the part of the plant used, from sweet and sour to tart and herbal [7]. Jams, syrups and compotes are prepared from the prickly rose fruits, characterized by good gelling properties. In the medical systems of Siberian and Asian peoples, rosehip medicines are used to treat diseases of the stomach and intestines, as an appetizing and anti-inflammatory agent, as well as in remedies to restore health after long-term illnesses [8]. Yakut traditional nomad medicine recommends the ripe fruits of R. acicularis to strengthen the gums, and an unripe fruit decoction is used to treat cardiac problems [9]. The decoction of the twigs of fresh bushes is a prophylactic remedy against diarrhea and intestinal diseases, and the tea of the leaves is used as a diuretic [10]. The Buryat lamas use the fruits of R. acicularis to treat diseases of bile and to suppress wind [11], as well as to destroy poisons and contribute to the growth of teeth [12]. The stem bark is applied as an antidote and used to cure lymphatic system diseases [13].
In official medical practice, rosehip is applied as a source of ascorbic-acid-containing concentrates and syrups and carotene-rich oils and creams. The most commonly used roses for commercial purposes are R. rugosa, R. canina and R. majalis, as evidenced by the good level of knowledge regarding them [2,14,15]. Information about the chemical composition and bioactivity of R. acicularis is limited. The early study of R. acicularis leaves showed the good Fe-reducing power and antioxidant potential of the extract in radical scavenging assays against free radicals such as DPPH, ABTS and superoxide anion, caused by the presence of phenolic compounds (126 mg/g), flavonoids (8 mg/g) and flavanols (1 mg/g) in the plant [16]. Various R. acicularis extracts were effective as inhibitors of lipase activity [17] and HIV-1 protease activity [18], as well as antimicrobial agents [19]. The known scientific information about R. acicularis chemistry includes data on the organ-specific distribution of nine microelements [20], the essential oil and fatty acid composition [21] in addition to the total level of flavonoids, phenols and procyanidins in leaf isolates extracted by different solvents [22]. So far, however, there has been no precise study of R. acicularis metabolite composition, nor any LC-MS-based investigations of prickly rose extracts. Our earlier study of Siberian plants demonstrated the high potential of R. acicularis extracts to inhibit α-glycosidase, indicating the promising antidiabetic potential of prickly rose extracts [23], especially given the ethnopharmacological data about the use of R. acicularis decoction and tincture to treat diabetes in traditional medicine of Siberian nomads [13].
As part of an ongoing study on plant antidiabetic metabolites [23][24][25][26][27][28][29], and based on the preliminary information available concerning rose metabolites, we performed qualitative and quantitative chromatographic analyses of phenolic compounds for the first time in the leaves, flowers, roots and fruits of R. acicularis by means of high-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS/MS). The total extracts of R. acicularis organs were bioassayed by in vitro methods for their ability to inhibit digestive enzymes, followed by an HPLC-based bioassay, which allowed metabolites with the greatest inhibitory potential to be found. Finally, rugosin D was found to be the main inhibitor of α-amylase in a simulated gastrointestinal digestion model.

Metabolites of Rosa acicularis: Distribution of Phytochemicals in Organs
The known ethnopharmacological data refer to the use of the whole plant of R. acicularis for medicinal and dietary purposes [17][18][19][20][21][22]. A preliminary study of the general phytochemical composition of R. acicularis showed the varying content of phenolic and non-phenolic compounds in different organs (Table 1). The leaves accumulated ellagitannins (73.69 mg/g of dry weight), gallotannins (21.23 mg/g) and hydroxycinnamates (1.47 mg/g), while high levels of flavonoids (67.39 mg/g as flavonols and 0.73 mg/g as dihydroflavonols), anthocyanins (5.34 mg/g) and water-soluble polysaccharides (65.14 mg/g) were found in flowers, and high levels of catechins (43.04 mg/g) and proanthocyanidins (26.04 mg/g) were found in root samples. The fruits were able to store free organic acids (42.59 mg/g; measured as titratable acids), ascorbic acid (56.12 mg/g), carotenoids (2.33 mg/g) and lipids (65.12). The total phenolic content of R. acicularis organs varied from 3.03 mg/g in fruits and 85.18 mg/g in roots to 160.75 mg/g in flowers and 173.98 mg/g in leaves. All this points to the organ-specific accumulation of phytochemicals in the whole R. acicularis plant. Early data of R. acicularis phytochemicals showed a lower level of total phenolics (74 mg/g), flavonoids (24 mg/g) and proanthocyanidins (13 mg/g) in leaf extracts of Chinese origin [22]. The total phenolic content and flavonoid content of leaf extracts of the Turkish species R. sempervirens were 17-203 mg/g and 10-96 mg/g, respectively [30]. Analysis of leaves of 17 Polish Rosa species revealed variations in total phenolic content, from 5.7% (R. rugosa) to 15.2% (R. canina var. dumalis), and flavonoids, from 5.6 mg/g (R. vosagiaca) to 19.01 mg/g (R. gallica) [31]. Four Hungarian rosehips (R. canina, R. gallica, R. rugosa and R. spinosissima) contained 255.9 to 766.0 mg/100 g of total phenolics [32], and the fruits of four Lithuanian roses (R. rugosa, R. pimpinellifolia, R. multiflora and R. canina) showed a variation of 15-50 mg/g of total phenolics and 0.5-5 mg/g of flavonoids [33].
The study of the distribution of water-soluble sugars in R. rugosa organs found 0.2% in achenes, 0.4% in leaves, 0.8% in petals and 15% in fruits [34]. The ascorbic acid content was 274-1157 mg/100 g in Iranian rosehips [35] and 121-360 mg/100 g in Transylvanian R. canina fruits [36]. The level of carotenoids in fruits of Swedish species, such as R. dumalis, R. rubiginosa and R. spinosissima, was 0.3-1 mg/g [37]. In comparing the phytochemical composition of prickly rose with other Rosa species, we can deduce the remarkable level of valuable phenolics and non-phenolic compounds in R. acicularis.

Metabolites of Rosa acicularis: LC-MS Profile and Organ-Specific Distribution
The study of metabolite diversity in R. acicularis was realized using high-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS/MS) in four plant organs: leaves, flowers, roots and fruits.
Leaf extracts of R. acicularis contained 12 ellagitannins, and the identity of 10 compounds were confirmed as tellimagrandin I 1 (14), I 2 (19), II (21; isomeric 23), rugosin A (26), B 1 (16), B 2 (22), D (27), E 1 (24) and E 2 (25) using reference standards. Isomeric compounds 4 and 5 were described as ellagitannins due to their mass spectral patterns, with m/z 795, 633, 481, 463 and 301 typical for the galloyl-hexahydroxydiphenoyl-di-O-hexosides [41,42]. Ellagitannins with hexahydroxydiphenoyl and valoneoyl substituents are known tannins of the Rosaceae family [50] and some Rosa species (R. canina [51], R. chinensis [52] and R. rugosa [53]). The dominant ellagitannins of R. acicularis leaves were rugosin D, tellimagrandin II 1 and tellimagrandin II 2 , with the highest levels of 41.15 mg/g, 8.98 mg/g and 8.29 mg/g, respectively, in summer samples. An increase in ellagitannin accumulation was found in R. acicularis leaves from spring to summer, followed by a decrease in the autumn. The latter involves the possibility of a seasonal destruction of polymeric ellagitannins by a specific tannase, resulting in the release of the simpler compound [54]. Ellagic acid, as a final product of ellagitannin cleavage, showed a marked increase in autumn samples of R. acicularis leaves, which supports this hypothesis. The total content of ellagitannins in R. acicularis leaves varied from 26.99 mg/g in May to 70.04 mg/g in July, and was much more than those in the leaves of R. canina (1.11 mg/g), R. glauca (1.05 mg/g), R. sempervirens (1.09 mg/g) and R. rubiginosa (4.81 mg/g) [55].
Flavonoids were the largest phenolic group of R. acicularis leaves, supplied by derivatives of kaempferol, quercetin and dihydroquercetin, all in the form of glycosides, and gave aglycone ions in the MS 2 spectra with m/z 285, 301 and 303, respectively. The mass spectrometric analysis demonstrated the loss of carbohydrate fragments of glucose/hexose (162 a.m.u.), glucuronic acid/hexuronic acid (176 a.m.u.) and arabinose/pentose (132 a.m.u.), as well as acyl fragments of p-coumaric acid (146 a.m.u.) and gallic acid (152 a.m.u.).
The group of kaempferol glycosides included known compounds, such as kaempferol- (76), which were identified by comparison with reference standards as well as with 12 compounds with tentative structures. Flavonoids 71, 74 and 75 were described in R. canina and R. rugosa [2].
Mass spectral behavior indicated various combinations of fragments in the nonaglycone part of the molecule, such as hexuronic acid:hexose:galloyl in a ratio of 1:3:1 (59), hexuronic acid:galloyl in a ratio of 1:1 (77), hexuronic acid:p-coumaroyl:galloyl in a ratio of 1:1:1 (82), hexuronic acid:hexose:p-coumaroyl in a ratio of 1:1:2 (83), hexose:pcoumaroyl in a ratio of 1:2 (84) and hexuronic acid:p-coumaroyl in ratios of 1:2 (85) and 1:3 (86) (Figure 4).  1, 10, 2525 12 of 29  Compounds 59 and 77 demonstrated the hypsochromic shift of the kaempferol glycoside shoulder, from 287 nm to 284 nm, and the hyperchromic shift of the shortwave band, signaling the galloyl moiety attachment [46]. In mass spectra, we found the primary loss of the particle with m/z 152 related to the galloyl particle (m/z 1099    Compounds 59 and 77 demonstrated the hypsochromic shift of the kaempferol glycoside shoulder, from 287 nm to 284 nm, and the hyperchromic shift of the shortwave band, signaling the galloyl moiety attachment [46]. In mass spectra, we found the primary loss of the particle with m/z 152 related to the galloyl particle (m/z 1099  Compounds 59 and 77 demonstrated the hypsochromic shift of the kaempferol glycoside shoulder, from 287 nm to 284 nm, and the hyperchromic shift of the shortwave band, signaling the galloyl moiety attachment [46]. In mass spectra, we found the primary loss of the particle with m/z 152 related to the galloyl particle The acylated quercetin derivatives included hexuronic acid, hexose and gallic acid (ratio of 1:3:1; 56); hexose, gallic acid and p-coumaric acid (ratio of 1:1:1; 78); hexuronic acid, gallic acid and p-coumaric acid (ratio of 1:1:1; 79); hexose and p-coumaric acid (ratio of 1:2; 80); and hexuronic acid and p-coumaric acid (ratio 1:2; 81) in the glycosidic fragments. As in the case of acylated kaempferol glycosides, the UV patterns of quercetin glycosides varied with the type of acylation group, and the AlCl 3 spectra were characterized by a maximum longwave of 410 ± 4 nm, typical for quercetin 3-O-glycosides [60] (Figure 5).    The spectral behavior of compounds 80 and 81 was similar to mono-coumaroylated quercetin glycoside helichrysoside (73), but the UV spectral bands at 310 nm were more intensive, as is the case for di-coumaroyl esters [62]. This was confirmed by the mass spectral loss of two coumaroyl fragments, indicating the probable structures of these flavonoids as being quercetin 3

Flowers
In total, 67 compounds were identified in R. acicularis flower samples (Figure 7, Table 2), most of which were previously described in leaves. Ellagic acid (29) (25) and three compounds, 87, 88 and 89, isomeric to tellimagrandin II, rugosin A and rugosin D, respectively. The principal ellagitannin of R. acicularis flowers was tellimagrandin II, which showed a concentration level of 6.03 mg/g, and the total ellagitannin content in flowers was 24.10 mg/g, which was close to the ellagitannin content in leaves collected in May and slightly lower than the July and September leaves. Gallic acid (2) and gallotannins 1, 12, 13, 18, 20, 33, 46, 48 and 92 were also found in R. acicularis flowers, and gave a total content of 9.42 mg/g. flavonoids. The total flavonoid content in R. acicularis leaves changed from 15.35 mg/g in May to 40.51 mg/g in July and finally to 17.52 mg/g in September. This pattern of change in flavonoid accumulation was previously revealed in other rosaceous plants, such as Agrimonia asiatica [23] and Rubus matsumuranus [64].

Roots
The root samples of R. acicularis showed no presence of hydroxycinnamates or flavonoids, but high levels of catechins (46.04 mg/g), catechin oligomers (21.80 mg/g) and derivatives of ellagic acid (12.52 mg/g) (Figure 8, Table 2). Additional O-hexosides of catechin dimers 95, 96, 99 and 101-104, demonstrating the characteristic loss of a hexose fragment with m/z 162, were found in R. acicularis roots, as well as the epicatechin/catechin dimer O-gallate 108, isomeric to 106 and/or 107. The basic catechin oligomer was dimeric procyanidin B2, which showed a content level of 7.62 mg/g, and the total content of catechin oligomers in roots of R. acicularis was more than in other organs, at about 21.80 mg/g. The presence of dimeric and trimeric procyanidins in the Rosa species was demonstrated in R. rugosa [66], R. canina, R. glutinosa, R. rubiginosa, R. multiflora and R. spinosissima [67], but teramers and pentamers were found were found in R. acicularis roots, as well as the epicatechin/catechin dimer O-gallate 108, isomeric to 106 and/or 107. The basic catechin oligomer was dimeric procyanidin B 2, which showed a content level of 7.62 mg/g, and the total content of catechin oligomers in roots of R. acicularis was more than in other organs, at about 21.80 mg/g. The presence of dimeric and trimeric procyanidins in the Rosa species was demonstrated in R. rugosa [66], R. canina, R. glutinosa, R. rubiginosa, R. multiflora and R. spinosissima [67], but teramers and pentamers were found in the genus for the first time. Ellagic acid (29), ellagic acid 4-O-rhamnoside (escheweilenol C; 113) and preliminarily identified ellagic acid tri-O-hexoside (6) as well as ellagic acid di-O-desoxyhexoside (112) were found in R. acicularis roots, along with two galloylhexahydroxydiphenoyl-di-O-hexosides, 4 and 5, and 2-pyrone-4,6-dicarboxylic acid (8).
This study demonstrated that the whole R. acicularis plant is characterized by organ-specific accumulation of phenolic metabolites: in particular, the basic compounds in the leaves were ellagitannins and gallotannins, while flavonoids dominated in flowers and catechins, monomers and oligomers were found in roots. The lowest content and diversity of phenolics were in the fruits. This can influence the bioactivity of R. acicularis extracts as possible inhibitors of digestive enzymes, because variation in the activity of different phenolic groups is known [71,72], as well as the influence of different Rosa ex-  Table 2. Internal standard: IS-5-neomangiferin (25 µg/mL).
This study demonstrated that the whole R. acicularis plant is characterized by organspecific accumulation of phenolic metabolites: in particular, the basic compounds in the leaves were ellagitannins and gallotannins, while flavonoids dominated in flowers and catechins, monomers and oligomers were found in roots. The lowest content and diversity of phenolics were in the fruits. This can influence the bioactivity of R. acicularis extracts as possible inhibitors of digestive enzymes, because variation in the activity of different phenolic groups is known [71,72], as well as the influence of different Rosa extracts, based on the α-glucosidase, such as R. damascena flowers [73], R. canina fruits [74], R. roxburghii and R. sterilis fruits [75] and R. acicularis leaves [23], and on the amylase, such as R. canina fruits and flowers [76]. In that regard, it is reasonable to study the interaction with digestive enzymes of extracts from R. acicularis organs and define the inhibiting principles of the most active extract.

Digestive-Enzyme-Inhibiting Potential of R. acicularis Extracts and Rugosin D
To study the inhibitory potential of R. acicularis extracts on the digestive enzymes, we used the standardized static, simulated gastrointestinal digestion method of Minekus et al., based on physiologically relevant conditions of digestion [77]. In brief, the artificial substrate mixture, including ethylidene-p-nitrophenyl-α-D-maltoheptaoside (as a carbohydrate model), N α -benzoyl-L-arginine-7-amino-4-methylcoumarin hydrochloride (as a protein model) and 4-methylumbelliferyl heptanoate (as a lipid model), was digested with extracts of R. acicularis leaves, flowers, roots and fruits (in doses of 100 µg/mL and 1000 µg/mL) by gastric and intestinal enzymatic and electrolyte mixtures (or juices). The gastric phase enzyme was pepsin, and the intestinal phase enzymes included pancreatic amylase, trypsin, chymotrypsin, pancreatic lipase and pancreatic colipase, which allowed for better simulation of physiological digestion enzyme diversity. In a final step, concentrations of specific markers, such as p-nitrophenol, 7-amino-4-methylcoumarin and 4-methylumbelliferone, released from an artificial nutrient mixture, were analyzed by an HPLC-DAD assay. The low level of markers indicated the low enzymatic activity of gastrointestinal juices and the high enzyme-inhibiting activity of the studied sample.
The reference standard enzyme inhibitors demonstrated a high potential against amylase (acarbose, 1000 µg/mL), proteases (trypsin-chymotrypsin inhibitor from Glycine max, 1000 µg/mL) and lipases (orlistat, 1000 µg/mL), reducing the initial enzyme activity to 55%, 12% and 38%, respectively ( Figure 10). The extract of R. acicularis leaves showed a significant reduction in amylase activity, to 84% at the 100 µg/mL dose and to 61% at the 1000 µg/mL dose, with a minor impact on the protease and lipase activity. The extracts of R. acicularis flowers, roots and fruits showed little or no influence on the activity of the digestive enzymes. This means that only R. acicularis leaf extract possessed a notable inhibitory potential on the amylase and needs further investigation of its active principles. et al., based on physiologically relevant conditions of digestion [77]. In brief, the artificial substrate mixture, including ethylidene-p-nitrophenyl-α-D-maltoheptaoside (as a carbohydrate model), Nα-benzoyl-L-arginine-7-amino-4-methylcoumarin hydrochloride (as a protein model) and 4-methylumbelliferyl heptanoate (as a lipid model), was digested with extracts of R. acicularis leaves, flowers, roots and fruits (in doses of 100 μg/mL and 1000 μg/mL) by gastric and intestinal enzymatic and electrolyte mixtures (or juices). The gastric phase enzyme was pepsin, and the intestinal phase enzymes included pancreatic amylase, trypsin, chymotrypsin, pancreatic lipase and pancreatic colipase, which allowed for better simulation of physiological digestion enzyme diversity. In a final step, concentrations of specific markers, such as p-nitrophenol, 7-amino-4-methylcoumarin and 4-methylumbelliferone, released from an artificial nutrient mixture, were analyzed by an HPLC-DAD assay. The low level of markers indicated the low enzymatic activity of gastrointestinal juices and the high enzyme-inhibiting activity of the studied sample.
The reference standard enzyme inhibitors demonstrated a high potential against amylase (acarbose, 1000 μg/mL), proteases (trypsin-chymotrypsin inhibitor from Glycine max, 1000 μg/mL) and lipases (orlistat, 1000 μg/mL), reducing the initial enzyme activity to 55%, 12% and 38%, respectively ( Figure 10). The extract of R. acicularis leaves showed a significant reduction in amylase activity, to 84% at the 100 μg/mL dose and to 61% at the 1000 μg/mL dose, with a minor impact on the protease and lipase activity. The extracts of R. acicularis flowers, roots and fruits showed little or no influence on the activity of the digestive enzymes. This means that only R. acicularis leaf extract possessed a notable inhibitory potential on the amylase and needs further investigation of its active principles. Figure 10. Enzyme activity (percentage of initial) in digestive medium after simulated gastrointestinal digestion of artificial substrate mixture in the presence of R. acicularis extracts of leaves, flowers, roots and fruits (100 and 1000 μg/mL), acarbose (1000 μg/mL), trypsin-chymotrypsin inhibitor (1000 μg/mL) and orlistat (1000 μg/mL). *-p < 0.05 vs. acarbose group; **-p < 0.05 vs. trypsin-chymotrypsin inhibitor group; and ***-p < 0.05 vs. orlistat group.
To find the most active metabolite of the R. acicularis leaf extract, the HPLC microfractionation technique was applied. The probe of the extract was separated by HPLC, and fractions were eluted every 30 sec, collected, dried and mixed with pancreatic α-amylase. The hydrolytic activity of amylase was studied spectrophotometrically using Figure 10. Enzyme activity (percentage of initial) in digestive medium after simulated gastrointestinal digestion of artificial substrate mixture in the presence of R. acicularis extracts of leaves, flowers, roots and fruits (100 and 1000 µg/mL), acarbose (1000 µg/mL), trypsin-chymotrypsin inhibitor (1000 µg/mL) and orlistat (1000 µg/mL). *-p < 0.05 vs. acarbose group; **-p < 0.05 vs. trypsin-chymotrypsin inhibitor group; and ***-p < 0.05 vs. orlistat group.
To find the most active metabolite of the R. acicularis leaf extract, the HPLC microfractionation technique was applied. The probe of the extract was separated by HPLC, and fractions were eluted every 30 sec, collected, dried and mixed with pancreatic α-amylase. The hydrolytic activity of amylase was studied spectrophotometrically using starch azure as a substrate [78]. Some chromatographic zones demonstrated different effectiveness to protect starch against destructive enzyme influence ( Figure 11). The most active was the zone of rugosin D (27), which was capable of protecting 82-95% of starch, and medium activity was found for miquelianin (68), 1,2,3,6-tetra-O-galloyl glucose (33), tellimagrandin II (21) and tellimagrandin II isomer (23).
The dimeric valoneoyl ellagitannin rugosin D and other phenolics were studied previously as Rosa gallica metabolites with inhibitory activity against bacterial α-amylase from Bacillus sp. and fungal α-glucosidase from Saccharomyces sp. [78]. The inhibition of rugosin D on mammalian amylases was not found previously, so we have therefore studied the impact of ellagitannin on porcine pancreas α-amylase, human saliva α-amylase and human pancreas α-amylase (Table 3).

Compound
Porcine The inhibitory potential of rugosin D (IC50 32.09 μg/mL) was comparable to the reference inhibitor acarbose against porcine pancreas α-amylase (IC50 35.67 μg/mL) and exceeded the acarbose activity against both human α-amylases, with the most sensitive to rugosin D being human pancreas α-amylase (IC50 30.84 μg/mL). This was an indication that the bulk ellagitannins of the Rosa genus are effective inhibitors of digestive amylases.
The simulation of digestion processes with an artificial substrate mixture showed the high effectiveness of rugosin D as an amylase inhibitor, in a dose-dependent manner ( Figure 12). The presence of ellagitannin in digestive fluid in doses of 1-1000 μg/mL resulted in 92-38% suppression of amylase activity, while the acarbose demonstrated 99-58% suppression in the same concentration range. The activity of other enzymes, such as proteases and lipases, were not substantially inhibited, indicating the possible selective impact of rugosin on digestive enzymes.  The inhibitory potential of rugosin D (IC 50 32.09 µg/mL) was comparable to the reference inhibitor acarbose against porcine pancreas α-amylase (IC 50 35.67 µg/mL) and exceeded the acarbose activity against both human α-amylases, with the most sensitive to rugosin D being human pancreas α-amylase (IC 50 30.84 µg/mL). This was an indication that the bulk ellagitannins of the Rosa genus are effective inhibitors of digestive amylases.
The simulation of digestion processes with an artificial substrate mixture showed the high effectiveness of rugosin D as an amylase inhibitor, in a dose-dependent manner ( Figure 12). The presence of ellagitannin in digestive fluid in doses of 1-1000 µg/mL resulted in 92-38% suppression of amylase activity, while the acarbose demonstrated 99-58% suppression in the same concentration range. The activity of other enzymes, such as proteases and lipases, were not substantially inhibited, indicating the possible selective impact of rugosin on digestive enzymes. The plant-supporting therapy of diabetes is commonly based on ethnopharmacological data of the application of some extracts, such as hypoglycaemic remedies in traditional medicines [79][80][81]. Among flora, plants of the Rose genus are famous antidiabetic medicines with an inhibitory influence on digestive enzymes (α-glucosidase, Figure 12. Enzyme activity inhibition (percentage of initial) in digestive fluid after simulated gastrointestinal digestion of artificial substrate mixture in presence of rugosin D (1, 10, 100 and 1000 µg/mL) and acarbose (1, 10, 100 and 1000 µg/mL). * -p < 0.05 vs. acarbose group 10 µg/mL; ** -p < 0.05 vs. acarbose group 100 µg/mL; and *** -p < 0.05 vs. acarbose group 1000 µg/mL.
The plant-supporting therapy of diabetes is commonly based on ethnopharmacological data of the application of some extracts, such as hypoglycaemic remedies in traditional medicines [79][80][81]. Among flora, plants of the Rose genus are famous antidiabetic medicines with an inhibitory influence on digestive enzymes (α-glucosidase, α-amylase), including R. canina [74,78], R. damascena [73], R. gallica [78], R. roxburghii and R. sterilis [75]. The prickly rose (Rosa acicularis) is no exception, and is used in Tibetan and Siberian traditional medicines to prepare antidiabetic decoctions, extracts and tablets [12,13], although it is still an underestimated plant with poor scientific knowledge in regard to its metabolites and bioactivity. In our study of R. acicularis organs, many phytochemical classes of a phenolic and non-phenolic nature were quantified in the leaves, flowers, roots and fruits, and phenolic compounds were the most substantive. Use of the HPLC-PDA-ESI-tQ-MS/MS technique allowed for the identification of 123 phenolic compounds in R. acicularis, belonging to ellagic acid derivatives, ellagitannins, gallotannins, catechins, catechin oligomers, hydroxycinnamates and flavonoids. The combination of chromatographic and spectrometric data uncovered the variety of new flavonol glycosides, non-acylated and acylated with fragments of gallic acid and p-coumaric acids, as well as unknown galloyl hexoses, epicatechin/catechin tri-, tetra-and pentamers. The main phenolics of the leaves were ellagitannins (26.99-70.04 mg/g) and gallotannins (10.80-30.10 mg/g), whilst we found a high concentration of flavonoids (70.72 mg/g) in flowers; catechins and catechin oligomers accumulated in the roots (23.99 mg/g and 18.07 mg/g, respectively). The general metabolic profile of R. acicularis was typical for Rose plants. The early study of roses found ellagic acid and its glycosides [2], various ellagitannins, such as tellimagrandins in R. laevigata, R. multiflora and R. rugosa [50], rugosins A, B, D and E in R. canina [51] and R. gallica [78], gallic acid and gallotannins in R. gallica [78] and R. rugosa [82], flavonoids of kaempferol and quercetin groups in R. canina, R. glauca, R. rubiginosa and R. sempervirens [55] as well as dihydroflavonols of the taxifolin group in R. rugosa and R. canina [2]. While extensive work has been carried out studying prickly rose phenolics, data about other phytochemicals, such as terpenoids, carbohydrates and primary metabolites are not yet complete, and additional chemical and chromatographic studies are need.
The great phenolic diversity of R. acicularis implies a wide spectrum of bioactivity, including antidiabetic properties, as in the majority of ellagitannins, flavonoids and gallic acid derivatives [76][77][78]. The model of simulated digestion applied to the artificial mixture demonstrated the specific protection of the carbohydrate substrate against the destructive influence of α-amylase by R. acicularis leaf extract. The HPLC fractions of R. acicularis leaves containing rugosin D were the most active inhibitors of pancreatic α-amylase activity, and experiments with the pure compound later confirmed the inactivating properties of rugosin D on mammalian α-amylases, comparable with the potency of the known αamylase inhibitor acarbose. The inhibitory action of mono-and oligomeric ellagitannins of the Rosaceae family on various α-amylases have already been revealed. Tellimagrandin I and II, rugosins A and D as well as casuarictin from R. gallica petals were the main inhibitors of α-amylase from Bacillus stearothermophilus [78], and rubusuaviins A-F from Rubus suavissimus leaves inhibited human salivary α-amylase [83]. Strawberry extracts rich in ellagitannins with different degrees of polymerization demonstrated a pancreatic αamylase inhibitory activity by decreased postprandial glycaemia of rats [84]. The possible mechanism of α-amylase inactivation may involve the ellagitannin binding of a protein molecule of the digestive enzymes and the formation of insoluble complexes, eventually leading to a reduction in α-amylase activity and the inability to hydrolyze carbohydrate substrates into simple molecules. Rigorous proof of this theory has not been reported in the scientific literature, but the known data about protein-tannin interaction signifies the possibility of protein precipitation by various tannins [85,86]. The protein molecule of α-amylases may precipitate after contact with ellagitannins and inactivation, but of course this fact needs to be proven experimentally. As ellagitannins are often found in the Rosaceae family [50], it can be assumed that the ethnopharmacological evidence of the use of these plants in diabetes is associated with the ability of their extracts to inactivate digestive enzymes by binding to insoluble complexes. The widely distributed rosaceous species R. acicularis is an appropriate source of the α-amylase-inactivating ellagitannin rugosin D, and may be a prospective antidiabetic plant for use in the medical and food industry. Our results suggest that future efforts should be focused on the study of ellagitannin-α-amylase interaction to better understand the nature of the antidiabetic potential of this plant extract.  [87][88][89][90][91][92][93][94] (Table S1). The enzymes used in study were α-amylase from porcine pancreas (PMSF-treated, type I-A, saline suspension, ≥1000 units/mg protein; Cat. No. 6255; SigmaAldrich); α-amylase from human saliva (type IX-A, lyophilized powder, 1000-3000 units/mg protein; Cat. No. 0521; Sigma-Aldrich); α-amylase from human pancreas (>400 units/mL; Cat. No. 120-15; Lee Biosolutions, Inc., Maryland Heights, MO, USA); trypsin from bovine pancreas (≥10,000 units/mg protein; Cat. No. T1426; Sigma-Aldrich, St. Louis, MO, USA); α-chymotrypsin from bovine pancreas (type II; ≥40 units/mg protein; Cat. No. C4129; Sigma-Aldrich); lipase from porcine pancreas (type II; ≥650 units/mg protein; Cat. No. L3126; Sigma-Aldrich); and colipase from porcine pancreas (Cat. No. C3028; Sigma-Aldrich). Artificial substrates were ethylidene-p-nitrophenyl-α-D-maltoheptaoside (Cat. No. EN45922; Carbosynth Ltd., Compton, Great Britain), N α -benzoyl-L-arginine-7-amino-4-methylcoumarin hydrochloride (Cat. No. B7260; Sigma-Aldrich) and 4-methylumbelliferyl heptanoate (Cat. No. M2514; Sigma-Aldrich).

HPLC Microfractionation with Post-Column Pancreatic α-Amylase Inhibition
HPLC-PDA-ESI-tQ-MS conditions (Section 3.4) were used to separate the enlarged volume of R. acicularis leaf extract (July sample; 25 µL). The eluates (60 µL) were collected every 30 s by an automated fraction collector, a Shimadzu FRC-10A, and dried under a stream of N 2 . The eluate residue was dissolved in 50% methanol (50 µL) and added to 50 µL of water, 2.5 mL of Remazol-Brilliant-Blue-R-dyed starch (2% suspension in phosphate buffer, pH of 6.8) and 500 µL α-amylase from the human pancreas (0.4 U/mL), incubated for 50 min (37 • C) and the absorbance was measured at 620 nm. The eluates with the most active α-amylase inhibition prevented the blue color formation instead of the inactive eluates, giving a strong coloration. The value of 100% destruction of Remazol-Brilliant-Blue-R-dyed starch (or 0% content of intact starch compound) was measured for the eluate with a retention time of 0.5-1.0 min.

α-Amylase Inhibitory Activity
The α-amylase-inhibiting potential of rugosin D was studied using a microplate spectrophotometric assay [106] with three mammalian amylases, including α-amylase from porcine pancreas, α-amylase from human saliva and α-amylase from human pancreas. Acarbose was a positive control while water was a negative control. The inhibitory activity was measured as IC 50 (50% inhibition concentration) in µg/mL, estimated graphically after building the 'concentration-inhibitory percentage' correlation.

Statistical Analysis
Statistical analyses were performed by one-way analysis of variance, and the significance of the mean difference was determined by Duncan's multiple range test. Differences at p < 0.05 were considered statistically significant. The results are presented as mean values ± standard deviations (S.D.) of some replicates.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/plants10112525/s1, Table S1: Reference standards and internal standards used for the qualitative and quantitative analysis by HPLC-MS and HPLC-DAD, Table S2: Optimized MRM transitions of compounds 1-123 in HPLC-MS/MS analysis, Table S3: Regression equations, correlation coefficients, standard deviation, limits of detection, limits of quantification and linear ranges for 34 reference standards and Figure S1: HPLC-DAD chromatograms of gastrointestinal digestion markers after digestion of the artificial substrate mixture.