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Article

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

by
Daniil N. Olennikov
1,*,
Vladimir V. Chemposov
2 and
Nadezhda K. Chirikova
2
1
Laboratory of Medical and Biological Research, Institute of General and Experimental Biology, Siberian Division, Russian Academy of Science, 670047 Ulan-Ude, Russia
2
Department of Biology, Institute of Natural Sciences, North-Eastern Federal University, 677027 Yakutsk, Russia
*
Author to whom correspondence should be addressed.
Plants 2021, 10(11), 2525; https://doi.org/10.3390/plants10112525
Submission received: 4 November 2021 / Revised: 13 November 2021 / Accepted: 18 November 2021 / Published: 20 November 2021
(This article belongs to the Special Issue Phytochemicals for Plant and Human Health)
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Abstract

:
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.

1. 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 are used less, due to the lack of scientific data and poor awareness about their positive properties. The promising northern species of wild rose include R. acicularis, R. amblyotis, R. davurica and R. oxyacantha, which inhabit large natural areas and are distinguished by high productivity [3].
The largest territories of Siberia are occupied by Rosa acicularis Lindl., or prickly rose, which is a short shrub (up to 2 m) with brown branches that are densely covered with horizontally spaced spines and bristles [4]. Their bright green leaves consist of oblong–ovoid leaves seeded with glands, and the dark-pink single flowers have a characteristic pleasant smell (Figure 1). Elongated oval dark-red fruits ripen by the middle to the end of August and are harvested by the native population of the Siberian regions. This rose species is grown in mixed, sparse dark coniferous pine and birch forests, in meadows, along river banks, in woodlands and forest–tundra in all regions of Western, Central and Eastern Siberia, as well as the Far East, Central Asia, Mongolia, North China and North America [5]. Along with other rose species, such as R. majalis and R. davurica, the prickly rose forms the densely forested areas in Siberia, with a high fruit productivity (2.5–6 tonns/ha), and industrial harvesting is carried out for its fruits [6].
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.

2. Results and Discussion

2.1. 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.

2.2. 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.

2.2.1. Leaves

Chromatograms of R. acicularis leaf samples collected in three developmental stages, May (the beginning of vegetation), July (blossom stage) and September (senile stage), showed the maximal diversity (86 compounds) in the July samples (Figure 2, Table 2). Ellagic acid and hexosides, ellagitannins, gallotannins, catechins, catechin oligomers, hydroxycinnamates and flavonoids (incl. quercetin glycosides, kaempferol glycosides and dihydroquercetin glycosides) were detected after comparing UV, mass spectral patterns and chromatographic behavior with reference standards and literature data [24,38,39,40,41,42,43,44,45,46,47,48,49].
Free ellagic acid (29), a common component of roses [2], was found in all samples of R. acicularis leaves, with the content of it rising from May (0.25 mg/g) to September (4.29 mg/g). Two non-methylated hexosides, 1-O-ellagoyl-β-D-glucopyranoside (28) and ellagic acid tri-O-hexoside (6), and six derivatives of ellagic acid methyl esters—monohexosides of ellagic acid O-methyl ester (30, 31), ellagic acid di-O-methyl ester (36), di-ellagoyl O-methyl ester (39), di-ellagoyl di-O-methyl ester (43) and ellagic acid tri-O-methyl ester (44)—were identified as trace compounds. Some glycosides of ellagic acid were mentioned previously for R. rugosa and R. canina [2]. The total content of ellagic acid and its hexosides in R. acicularis leaves was lowest in the May samples (0.51 mg/g), rising in July (4.74 mg/g) and then showing a slight reduction in September (4.43 mg/g).
Leaf extracts of R. acicularis contained 12 ellagitannins, and the identity of 10 compounds were confirmed as tellimagrandin I1 (14), I2 (19), II (21; isomeric 23), rugosin A (26), B1 (16), B2 (22), D (27), E1 (24) and E2 (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 II1 and tellimagrandin II2, 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].
Gallic acid (2) was found in the leaves of R. acicularis in the free state and in the glycosidic form with different galloyl contents: one—1-O-galloyl-glucopyranoside or glucogallin (1); two—1,6-di-O-galloyl glucose (13) and isomer 12; three—1,3,6-tri-O-galloyl glucose (18) and isomers 17 and 20; four—1,2,3,6-tetra-O-galloyl glucose (33) and isomer 32; five—1,2,3,4,6-penta-O-galloyl glucose (47) and isomer 46; and six—hexa-O-galloyl hexoses 48, 49 and 50. Galloylated glycosides are still rarely described in rose plants containing mono-, di- and three-galloyl hexoses, such as R. rugosa and R. canina [2,55]. The main gallotannin of R. acicularis leaves was 1,2,3,6-tetra-O-galloyl glucose, with the highest level (10.54 mg/g) found in July samples. The accumulation pattern of all gallotannins in R. acicularis leaves was close to that of the ellagitannins: the increase period (spring–summer) bringing a sharp drop in autumn, owing to the destruction of polygalloylated glycosides to free gallic acid, which showed the rapid increase in content from 3.68 mg/g in July to 10.39 mg/g in September.
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 MS2 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-3-O-glucuronoide (71), kaempferol-3-O-arabinoside (juglanin; 74), kaempferol-3-O-(6″-O-p-coumaroyl)-glucoside (tiliroside; 75) and kaempferol-3-O-(6″-O-galloyl)-glucoside (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].
The glycoside moiety of the non-acylated kaempferol glycosides were hexuronic acid and hexose, in ratios of 1:3 (58), 2:2 (60), 1:2 (62) and 1:1 (69), and hexuronic acid and pentose in a ratio of 1:1 (70). There are some known analogues of kaempferol glycosides with a hexuronic acid:hexose ratio of 1:1, such as kaempferol-3-O-glucoside-7-O-glucuronide from Tulipa gesneriana (Liliaceae) [56] and Allium microdicityon [57] in addition to kaempferol-3-O-glucuronide-7-O-glucoside from Euphorbia sanctae-catharinae (Euphorbiaceae) [58], as well as kaempferol glycosides with a hexuronic acid:hexose ratio of 1:2, such as kaempferol 3-O-sophoroside-7-O-glucuronide from Allium cepa (Alliaceae) [59] and kaempferol 3-O-gentiobioside-7-O-glucuronide from Tulipa gesneriana [56]. Compounds 58 and 60 have no possible structure candidates among the known flavonoids.
Acylated kaempferol glycosides showed specific UV absorbance, allowing us to determine the presence of acylation units linked with the flavonoid molecule (Figure 3). Moreover, all spectra of AlCl3 complexes included longwave maxima at 400 ± 4 nm, typical for 3-O-substituted kaempferol glycosides with free 5-O-, 7-O- and 4′-O-positions [60].
Mass spectral behavior indicated various combinations of fragments in the non-aglycone 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:p-coumaroyl in a ratio of 1:2 (84) and hexuronic acid:p-coumaroyl in ratios of 1:2 (85) and 1:3 (86) (Figure 4).
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 [M-H]−→947 for 59; 613 [M-H]−→461 for 77), followed by the loss of hexose (m/z 947 [M-H-galloyl]−→785, 623, 461 for 59) and finally hexuronic acid (m/z 461→285 for 59 and 77). The provisional structures of 59 and 77 were found to be kaempferol 3-O-(O-galloyl-tri-O-hexosyl)-hexuronoside and kaempferol 3-O-(O-galloyl)-hexuronoside, respectively. Both compounds have no analogues among the known kaempferol glycosides.
The UV pattern of compound 82 was close to the known coumaroylated flavonoid kaempferol-3-O-(6’´-O-p-coumaroyl)-glucoside (tiliroside; 75), with the hyperchromed band II indicating the presence of an additional galloyl substituent [60]. The mass spectra of 82 showed the loss of galloyl (m/z 759 [M-H]−→607) and coumaroyl (m/z 759 [M-H]−→613) fragments, and was determined to be the unknown kaempferol 3-O-(O-p-coumaroyl-O-galloyl)-hexuronoside.
The absorption spectra of compounds 83, 84 and 85 saved the kaempferol-3-O-glycoside spectral features, including band II (267 nm) and I (as a shoulder at 354 nm), but the intensity of the band at 310 nm—caused by the presence of the coumaroyl fragment—was much greater than mono-coumaroylated tiliroside. In the mass spectra, we found the loss of two fragments with m/z 146 (coumaroyl) for all three compounds: m/z 915 [M-H]→469, 623 for 83; 739 [M-H]−→593, 447 for 84; and 753 [M-H]−→607, 461 for 85. Taking into account the spectral data, the possible structures of 83, 84 and 85 were kaempferol 3-O-(di-O-p-coumaroyl-O-hexosyl)-hexuronoside, kaempferol 3-O-(di-O-p-coumaroyl)-hexoside and kaempferol 3-O-(di-O-p-coumaroyl)-hexuronoside, respectively. Only flavonoid 84 has possible analogues in characterized structures such as kaempferol 3-(2″,6″-di-O-p-coumaroyl)-glucoside from Quercus canariensis (Fagaceae) [47] or kaempferol 3-(3″,6″-di-O-p-coumaroyl)-glucoside from Aerva lanata (Amaranthaceae) [61].
Compound 86 has a UV pattern with a single maximum at 311 nm and two shoulders at 270 nm and 352 nm that look similar to the absorption spectra of mono- and di-coumaroylated kaempferol glycosides. The gradual loss of three fragments with m/z 146 was detected (m/z 899 [M-H]−→753, 607 and 461), which suggested that the flavonoid was tri-coumaroylated kaempferol glycoside or kaempferol 3-O-(tri-O-p-coumaroyl)-hexuronoside, but this is still unknown.
Fifteen quercetin derivatives were detected in R. acicularis leaves, such as the known compounds quercetin-3-O-rutinoside (rutin; 65), quercetin-3-O-glucuronide (miquelianin; 68), quercetin-3-O-rhamnoside (quercitrin; 72) and quercetin-3-O-(6″-O-p-coumaroyl)-glucoside (helichrysoside; 73). The remaining quercetins were non-acylated and acylated glycosides. In the glycosidic moiety of non-acylated glycosides, hexuronic acid and hexose were found in ratios of 2:3 (52), 1:3 (54), 2:2 (57), 1:2 (61) and 1:1 (66), as well as hexuronic acid and pentose in a ratio of 1:1 (67).
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 AlCl3 spectra were characterized by a maximum longwave of 410 ± 4 nm, typical for quercetin 3-O-glycosides [60] (Figure 5).
The UV spectrum of glycoside 56 was similar to the UV spectrum of non-acylated miquelianin (68), but with a hyperchromed band II (Figure 5). The mass spectral primary loss of the fragment with m/z 152 (m/z 1115 [M-H]−→963) signaled the presence of a galloyl substituent influence on the UV pattern of 56. The additional removal of three hexose (m/z 963 [M-H-galloyl]−→801, 639 and 477) and one hexuronic moieties (m/z 477→301) pointed to the structure probably being quercetin 3-O-(O-galloyl-tri-O-hexosyl)-hexuronoside (Figure 6).
The absorption spectra of compounds 78 and 79 included the broad bands I (350 nm) and II (262 nm), and a new middle band at 305 nm, common to coumaroyl esters. In the mass spectra of 78 and 79, the loss of fragments with m/z 152 and 146 was registered (m/z 761 [M-H]−→615, 609 for 78; 775 [M-H]−→632, 623 for 79), indicating the presence of both substituents. The final identification gave the structures of 78 and 79 as quercetin 3-O-(O-p-coumaroyl-O-galloyl)-hexoside and quercetin 3-O-(O-p-coumaroyl-O-galloyl)-hexuronoside, respectively.
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-O-(di-O-p-coumaroyl)-hexoside (80) and quercetin 3-O-(di-O-p-coumaroyl)-hexuronoside (81).
Compounds 65, 68 and 72 had already been described earlier in R. canina and R. rugosa [2], and the tentatively identified quercetins have the only analogue of 80 among the known flavonoids: quercetin 3-(3″,6″-di-O-p-coumaroyl)-glucoside from Pinus sylvestris (Pinaceae) [62].
In addition to flavonols, five non-acylated dihydroflavonol glycosides with a specific UV pattern (λmax 290 ± 2 nm) and dihydroquercetin (taxifolin) fragment in mass spectrum (m/z 303) were identified as dihydroquercetin di-O-hexuronide-tri-O-hexoside (51), O-hexuronide-di-O-hexoside (53), O-hexuronide-O-hexoside (55), O-hexoside (63) and O-hexuronide (64). Compound 63 may be the known O-galactoside or O-glucoside of taxifolin, with substitution of the 3, 5, 7, 3′ or 4′ position of aglycone [63]. The possible analogues of other taxifolins are unknown.
The basic flavonoids of R. acicularis leaves were quercetin-3-O-glucuronide (68), kaempferol-3-O-glucuronide (71) and kaempferol-O-hexuronide-O-pentoside (70), which had a seasonal content variation of 10.63–21.16 mg/g, 7.03–15.20 mg/g and 3.12–10.65 mg/g, respectively, with a maximal level in July samples for all quantified 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].
Catechin (11), epicatechin (15) and epigallocatechin (9) were detected in R. acicularis leaves using reference standards, and two types of catechin oligomers were found, epicatechin/catechin trimers (34, 35, 37 and 38) and tetramers (40–42, 45). Compounds 11 and 15 were described earlier in R. canina and R. sempervirens [2], as were di-, tri- and tetrameric catechins in R. rugosa and R. canina [2]. The maximal seasonal level of catechins and catechin oligomers was revealed as 25.04 mg/g and 4.35 mg/g, respectively, in July. Additionally, 2-pyrone-4,6-dicarboxylic acid (8) and three hydroxycinnamates—1-O-caffeoylquinic acid (3), 5-O-caffeoylquinic acid (10) and caffeic acid O-hexoside (7)—were the components of R. acicularis leaves in all seasonal stages.

2.2.2. 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), known ellagic acid derivatives 6, 28, 30, 31, 36, 39, 43 and 44 and ellagic acid-O-hexoside (90) were only found in trace amounts or as low-content compounds in flowers, with a total concentration of 4.61 mg/g. Flower ellagitannins included tellimagrandin I1 (14), I2 (19), II (21), rugosin A (26), B1 (16), D (27), E1 (24), E2 (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, as the main phenolic group in R. acicularis flowers, with a total level of 66.72 mg/g, were basically the quercetin (43.43 mg/g) and kaempferol derivatives (20.08 mg/g), with a trace content of dihydroquercetin glycosides. The known quercetin glycosides found in flowers were quercetin-3-O-rutinoside (rutin; 65) and quercetin-3-O-glucuronide (miquelianin; 68), and compounds with possible structures were quercetin O-hexuronide-di-O-hexoside 61, quercetin O-hexuronide-O-hexoside 66, quercetin O-galloyl-O-p-coumaroyl-O-hexoside 78, quercetin di-O-p-coumaroyl-O-hexoside 80 and quercetin di-O-p-coumaroyl-O-hexuronide 81. Compounds 61 (16.78 mg/g), 65 (15.25 mg/g) and 78 (11.19 mg/g) amounted to over 60% of the total flavonoid content. Seven kaempferol derivatives included kaempferol-3-O-glucuronoide (71), kaempferol-3-O-(6″-O-p-coumaroyl)-glucoside (tiliroside; 75), kaempferol-3-O-(6″-O-galloyl)-glucoside (76), kaempferol di-O-hexuronide-di-O-hexoside 60, kaempferol O-hexuronide-O-hexoside 69, kaempferol O-hexuronide-O-pentoside 70 and kaempferol O-galloyl-O-hexuronide 77. The acylated flavonoid 77 was the main kaempferol derivative (11.19 mg/g) of R. acicularis flower samples, while peonidin 3,5-di-O-glucoside (93) was the main anthocyanin in R. acicularis flowers (3.21 mg/g) and other rose species, such as R. rugosa [48], R. canina and R. chinensis [65].
Three monomeric catechins (9, 11 and 15) and seven catechin oligomers (34, 37, 38, 40, 42, 45 and 91) gave total contents in flowers of 23.99 mg/g and 18.07 mg/g, respectively. The basic compounds were catechin (19.26 mg/g) and epicatechin/catechin tetramers 40 (8.76 mg/g) and 91 (7.53 mg/g). Mention should also be made of the presence of 2-pyrone-4,6-dicarboxylic acid (8) and traces of hydroxycinnamates 3, 7 and 10 in R. acicularis flowers.

2.2.3. 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).
Epigallocatechin (9; 20.45 mg/g), catechin (11; 17.22 mg/g), epicatechin (15; 8.23 mg/g) and epicatechin gallate (111; 0.14 mg/g) were found in all samples of R. acicularis roots, together with numerous catechin oligomers. Seven identified catechin dimers were procyanidin B1 (98), B2 (105), B3 (97) and B4 (100) as well as their gallic acid esters, procyanidin B2 3-O-gallate (106), procyanidin B2 3″-O-gallate (107) and procyanidin B2 3,3″-di-O-gallate (109). The tentative identification of the remaining compounds showed their nature, as epicatechin/catechin tetramers gave an [M-H]− ion with 1153 a.m.u. (40–42, 45 and 114–117) and pentamers gave an [M-H]− ion with 1441 a.m.u. (118–123). 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 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 galloyl-hexahydroxydiphenoyl-di-O-hexosides, 4 and 5, and 2-pyrone-4,6-dicarboxylic acid (8).

2.2.4. Fruits

The fruit samples of R. acicularis were poor in phenolics, as confirmed by HPLC-PDA-ESI-tQ-MS/MS data (Figure 9, Table 2). Only 14 metabolites were identified, among them the known compounds glucogallin (1), gallic acid (2), epicatechin (15), tellimagrandin II (21), rugosin A (26), rugosin D (27), ellagic acid (29) and eschweilenol C (113), and six compounds with tentative structures of galloyl-hexahydroxydiphenoyl-di-O-hexoside (4), ellagic acid methyl ester O-hexoside (30), epicatechin/catechin trimer (37) and tetramer (42), di-ellagoyl methyl ester O-hexoside (39) and ellagic acid di-O-desoxyhexoside (112). The quantifiable phenolics of R. acicularis fruits were gallic acid (1.03 mg/g), eschweilenol C (0.52 mg/g), ellagic acid di-O-desoxyhexoside 112 (0.40 mg/g), ellagic acid (0.38 mg/g), epicatechin (0.20 mg/g) and ellagic acid methyl ester O-hexoside 30 (0.08 mg/g). The remaining compounds were in very low or trace amounts.
The early study of rose plants showed a wide variation in the contents of fruits with regard to basic phenolics, such as gallic acid in R. canina (0.02–0.14 mg/g) [51], R. rugosa (0.03–0.85 mg/g) [68], R. dumalis in addition to R. hirtissima (7.70–12.93 μg/g) [69] and R. arvensis (47.9–170.0 μg/g) [70], catechins in R. canina in addition to R. hirtissima (7.18–50.46 μg/g) [69] and R. arvensis (10.4–61.5 μg/g) [70], procyanidins in R. canina in addition to R. dumalis (7.54–54.41 μg/g) [69] and ellagitannins in R. canina (0.43–1.26 mg/g) [51]. The phenolic composition of R. acicularis fruits has advantages compared to well-known species such as R. canina, R. rugosa and others, with their greater phenolic content.
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 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.

2.3. 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), 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.
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).
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 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 α-amylase 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 α-amylase 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.

3. Materials and Methods

3.1. Plant Material and Chemicals

Samples of Rosa acicularis (334 samples in total) were collected in the model population in Republic Sakha Yakutia, Lena River left bay, Khangalasskii Ulus, Elanka village (61°17′33.5″ N 128°00′53.8″ E, 720 m a.s.l.; plant density 50–54 plant/100 m2) in the pre-flowering period (25–30.V.2020; leaf samples, n = 35), the flowering period (15–20.VI.2020; leaf samples, n = 81; flower samples, n = 45), the fruiting period (28–29.VIII.2020; fruit samples, n = 104) and the post-fruiting period (18–22.IX.2020; leaf samples, n = 57; root samples, n = 12). One sample consisted of 10–15 leaves, 5–7 flowers, 2–3 roots and 30–40 fruits collected from one bush. The species was authenticated by Dr. N.I. Kashchenko (IGEB SB RAS, Ulan-Ude, Russia). For desiccation of the plant material, a ventilated heat oven was used (40 °C, 10–20 days) and dry plants were stored at 3–4 °C before manipulation. The voucher specimens were deposited in the herbarium of the Laboratory of Medical and Biological Research, Institute of General and Experimental Biology (Ulan-Ude, Russia. Specimen No. YA/ROS-0520/14-12–YA/ROS-0520/14-19, YA/ROS-0620/18-24–YA/ROS-0620/18-33, YA/ROS-0820/23-15–YA/ROS-0820/23-22 and YA/ROS-0920/09-14–YA/ROS-0920/09-18).
The reference compounds were purchased from ALB Materials Inc. (Henderson, NV, USA); BioBioPha (Kunming, Yunnan, China); BOC Sciences (Shirley, NY, USA); Carbosynth Ltd. (Compton, Great Britain, UK); ChemFaces (Wuhan, Hubei, China); Extrasynthese (Lyon, France); MCE Med Chem Express (Monmouth, NJ, USA); Sigma-Aldrich (St. Louis, MO, USA); Toronto Research Chemicals (North York, ON, Canada); TransMIT GmbH (Gießen, Germany); or isolated in our laboratory [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), Nα-benzoyl-L-arginine-7-amino-4-methylcoumarin hydrochloride (Cat. No. B7260; Sigma-Aldrich) and 4-methylumbelliferyl heptanoate (Cat. No. M2514; Sigma-Aldrich).

3.2. Chemical Composition Analysis

Spectrophotometric assays were used for the quantitative analysis of flavonols (as mg/g miquelianin equivalents) [95], dihydroflavonols (as mg/g taxifolin equivalents) [96], catechins (as mg/g (+)-catechin equivalents) [97], hydroxycinnamates (as mg/g 5-O-caffeoylquinic acid equivalents) [98], proanthocyanins (as mg/g procyanidin B1 equivalents) [99], ellagitannins (as mg/g ellagic acid equivalents) [100], gallotannins (as mg/g gallic acid equivalents) [101], water-soluble polysaccharides (as mg/g glucose equivalents) [102] and carotenoids (as mg/g β-carotene equivalents) [103] by a UV–Vis spectrophotometer, SF-200 (OKB Spectr, Saint Petersburg, Russia), in quartz cells (1 cm). Ascorbic acid was measured by an ascorbic acid assay kit (Cat. No. ab65656; Abcam plc, Cambridge, Great Britain, UK). A titration assay was applied for the quantitative analysis of titratable acids (as mg/g malic acid equivalents) [104], and total lipids were assayed by the Bligh–Dyer method [105]. All the analyses were carried out five times and the data were expressed as mean values ± standard deviation (S.D.).

3.3. Plant Extracts Preparation

To prepare the HPLC samples (plant extracts), the ground plant material (particle size 0.125 μm; weight 200 mg) was treated with 70% methanol (2 mL) three times by sonication (ultrasonic bath, 30 min, 45 °C, ultrasound power 100 W, frequency 35 kHz). The liquid extracts were centrifuged (6000× g, 10 min) and the supernatant was filtered through 0.22 μm syringe filters into the measuring flask (10 mL). The final volume reached 10 mL with 70% methanol. The liquid methanol extract was stored at 1°C before manipulation. Before analysis, the following internal standards (methanol solutions) were added to samples in a ratio of 1:1: leaf extracts—3′,4′-di-O-acetyl-cis-khellactone (50 μg/mL); flower extracts—isovitexin-7,2″-di-O-glucoside (100 μg/mL) and isojaceoside (60 μg/mL) mixture in a ratio of 1:1; root extracts—6-O-sinapoyl catalpol (60 μg/mL); and fruit extracts—neomangiferin (50 μg/mL).
The extracts of R. acivularis leaves (July sample), flowers, roots and fruits for the biological study were prepared from the milled samples (60 g), treated by the 70% methanol in an ultrasonic bath (30 min, 45 °C, ultrasound power 100 W, frequency 35 kHz), filtered, reduced in a vacuum until dryness and cold-stored (−20 °C) before analysis. The yields of R. acivularis extracts were 32.2% for the leaf sample, 38.4% for the flower sample, 18.61% for the root sample and 32.4% for the fruit sample.

3.4. High-Performance Liquid Chromatography with Photodiode Array Detection and Electrospray Ionization Triple Quadrupole Mass Spectrometric Detection (HPLC-PDA-ESI-tQ-MS) Metabolite Profiling

High-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS) was used for the quantitative profiling of R. acicularis extracts. A liquid chromatograph, LC-20 Prominence, coupled with a photodiode array detector, SPD-M30A (wavelength range of 200–600 nm), triple-quadrupole mass spectrometer, LCMS 8050 (all Shimadzu, Columbia, MD, USA), and GLC Mastro column (2.1 × 150 mm, 3 μm; Shimadzu, Kyoto, Japan) showed better separation of the compounds. The two-component eluent system of eluent A (0.45% CH3COOH in water) and B (0.45% CH3COOH in MeCN) used the gradient program for the separation: 0–3 min 3%, 3–4 min 3–4%, 4–9 min 4–9%, 9–20 min 9–14%, 20–22 min 14–16%, 22–30 min 16–37%, 33–38 min 37–69%, 38–44 min 69–89% and 44–55 min 89–3%. The injection volume was 1 μL and the flow rate was 120 μL/min. The UV–Vis spectra were recorded by an SPD-M20A photodiode detector (spectral range of 200–600 nm) equipped with a post-column derivatization reactor. The spectra of flavonoid compounds were analyzed before and after the addition of shift reagent (5% AlCl3 in acetonitrile). The negative electrospray ionization was used for the mass spectrometric detection of ellagic acid derivatives, ellagitannins, gallotannins, catechins, catechin oligomers, hydroxycinnamates and flavonoid glycosides, and the positive electrospray ionization was used for anthocyanines. The used temperature levels were 300 °C in the ESI interface, 250 °C in the desolvation line and 400 °C in the heat block. The flow values were 3 L/min for the nebulizing gas (N2), 10 L/min for the heating gas (air) and 0.3 mL/min for the collision-induced dissociation gas (Ar). The source voltage of mass spectra was 3 kV and the collision energy was −10–35 eV (negative ionization) and +20 (positive ionization) by the scanning range of m/z 80–1900. The LC-MS system was managed by LabSolution’s workstation software equipped with the inner LC-MS library. The integrated analysis of retention time, ultraviolet and mass spectra data after comparison with the reference standards and literature data was used for the identification of metabolites.

3.5. HPLC-ESI-tQ-MS Metabolite Quantification

To quantify 123 metabolites of R. acicularis organs, the HPLC-PDA-ESI-tQ-MS conditions used (Section 3.4) and optimized MRM transition MS peak area was used for calculation (Table S2). In total, 34 reference standards were separately weighed (10 mg) and dissolved in the methanol–DMSO mixture (1:1) in volumetric flasks (10 mL) preparing the stock solution (1000 µg/mL) used for the calibration curve building. The calibration solution (1–100 µg/mL) chromatographed in known HPLC-PDA-ESI-tQ-MS conditions and mass spectral data was used to create ‘concentration–mass spectrometric peak area’ correlation. The principal validation criteria, including correlation coefficients (r2), standard deviation (SYX), limits of detection (LOD), limits of quantification (LOQ) and linear ranges, were found using the known method [27] (Table S3). Five HPLC runs were sufficient for the quantitative analyses, and the results were expressed as mean values ± standard deviation (S.D.).

3.6. Simulated Gastrointestinal Digestion

For simulation, the known standardized static gastrointestinal digestion method of Minekus et al. [77] was used with no variation. The artificial substrate mixture was prepared with ethylidene-p-nitrophenyl-α-D-maltoheptaoside (0.2% solution in a DMSO–water mixture with a ratio of 2:8), Nα-benzoyl-L-arginine-7-amino-4-methylcoumarin hydrochloride (0.1% solution in a DMSO–methanol–water mixture with a ratio of 1:1:8) and 4-methylumbelliferyl heptanoate (0.1% solution in a DMSO–water mixture with a ratio of 3:7) in a ratio of 1:1:1. An aliquote of the artificial substrate (0.5 mL) was mixed with 0.5 mL of plant extract solution in a DMSO–water mixture (1:9), a pure DMSO–water mixture (1:9; control sample), a standard inhibitor solution (acarbose, trypsin-chymotrypsin inhibitor or orlistat) or rugosin D, 7.5 mL of simulated gastric fluid electrolyte stock solution, 1.6 mL of porcine pepsin solution (25 000 units/mL), 5 mL of CaCl2 (0.3 M), 0.2 mL of HCl (1 M) and 0.7 mL of water. The probe was thermostated for 2 h at 37 °C, followed by mixing with 11 mL of simulated intestine fluid electrolyte stock solution, 5.0 mL of an enzyme mixture (porcine trypsin, 100 units/mL; bovine chymotrypsin, 25 units/mL; porcine pancreatic amylase, 200 units/mL; porcine pancreatic lipase, 2000 units/mL; and porcine pancreatic colipase, 1/2 of lipase mass), 2.5 mL of bile (160 mM), 40 mL of CaCl2 (0.3 M), 0.15 mL of NaOH (1 M) and 1.31 mL of water, and the probe was then thermostated again for 2 h at 37 °C. Finally, 1 mL of the mixture was mixed with 2 mL of acetonitrile, vortexed (30 s) and then centrifuged (6000× g, 10 min). The supernatant was analyzed using an HPLC-DAD assay (Section 3.7) to quantify the level of markers as p-nitrophenol, 7-amino-4-methylcoumarin and 4-methylumbelliferone formed from ethylidene-p-nitrophenyl-α-D-maltoheptaoside, Nα-benzoyl-L-arginine- 7-amino-4-methylcoumarin hydrochloride and 4-methylumbelliferyl heptanoate, respectively. The level of markers content in the control group (pure DMSO–water mixture with a ratio of 1:9) was 100% of enzyme activity.

3.7. Quantification of Gastrointestinal Digestion Markers by HPLC-DAD

Microcolumn high-performance liquid chromatography with diode array detection (HPLC-DAD) was used to separate the markers formed after gastrointestinal digestion of the artificial substrate mixture. The assay was equipped with a MiLiChrom A-02 microcolumn chromatograph (Econova; Novosibirsk, Russia) coupled with a diode array detector and a ProntoSIL-120-5-C18 AQ column (1 × 60 mm, particle diameter 1 μm; Metrohm AG, Herisau, Switzerland). A two-eluent gradient system with 4.1 M of LiClO4 in 0.1 M of HCLO4 as eluent A and 0.1 M of HCLO4 in acetonitrile as eluent B was used to separate samples, and the gradient program was 0–2 min 10–45% B, 2–5 min 45–56% B, 5–10 min 45–100% B and 10–15 min 100–10% B. The parameters of column temperature, injection volume and elution rate were 35 °C, 1 μL and 150 μL/min, respectively. Chromatograms were recorded at 210 nm. The solutions of reference standards (p-nitrophenol, 7-amino-4-methylcoumarin and 4-methylumbelliferone) were used to compare the sample chromatograms with a standard chromatogram (Figure S1).

3.8. 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 N2. 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.

3.9. 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 IC50 (50% inhibition concentration) in μg/mL, estimated graphically after building the ‘concentration–inhibitory percentage’ correlation.

3.10. 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.

Author Contributions

Conceptualization, D.N.O.; methodology, D.N.O. and N.K.C.; software, D.N.O.; validation, N.K.C.; formal analysis, N.K.C. and V.V.C.; investigation, D.N.O., N.K.C. and V.V.C.; resources, D.N.O. and N.K.C.; data curation, V.V.C.; writing—original draft preparation, D.N.O.; writing—review and editing, N.K.C.; visualization, D.N.O.; supervision, N.K.C.; project administration, N.K.C.; funding acquisition, D.N.O., N.K.C. and V.V.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Education and Science of Russia, grant numbers 121030100227-7 and FSRG-2020-0019; the World-class Interregional Scientific and Educational Center “Baikal”: 2021 and the Russian Foundation of Basic Research, grant number 19-09-00361.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors acknowledge the Buryat Research Resource Center for the technical support given for chromatographic and mass spectrometric research, in addition to the students of the North-Eastern Federal University (Yakutsk, Russia) for helping to collect plant samples. The authors also are very thankful to Petr Kosachev for providing the photo of flowering Rosa acicularis (https://www.inaturalist.org/observations/90397362).

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Plants 10 02525 g001 550
Figure 1. Prickly rose (Rosa acicularis) in its natural habitat, in the flowering stage (A) (Republic Buryatia, Selenginskii District) and the fruiting stage (B) (Republic Buryatia, Mukhorshibirskii District).
Figure 1. Prickly rose (Rosa acicularis) in its natural habitat, in the flowering stage (A) (Republic Buryatia, Selenginskii District) and the fruiting stage (B) (Republic Buryatia, Mukhorshibirskii District).
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Plants 10 02525 g002 550
Figure 2. High-performance liquid chromatography with photodiode array detection (HPLC-PDA) chromatogram (270 nm) of R. acicularis leaf extract (July sample). Compounds are numbered as listed in Table 2. IS-1—3′,4′-di-O-acetyl-cis-khellactone (25 μg/mL).
Figure 2. High-performance liquid chromatography with photodiode array detection (HPLC-PDA) chromatogram (270 nm) of R. acicularis leaf extract (July sample). Compounds are numbered as listed in Table 2. IS-1—3′,4′-di-O-acetyl-cis-khellactone (25 μg/mL).
Plants 10 02525 g002
Plants 10 02525 g003 550
Figure 3. Ultraviolet absorption spectra of compounds 59 (kaempferol 3-O-hexuronoside-tri-O- hexoside-O-gallate), 82 (kaempferol 3-O-hexuronoside-O-p-coumarate-O-gallate), 83 (kaempferol 3-O-hexuronoside-O-hexoside-di-O-p-coumarate), 86 (kaempferol 3-O-hexuronoside-tri- O-p-coumarate), 75 (tiliroside), and 71 (kaempferol-3-O-glucuronide) before (black line) and after the addition of AlCl3 (blue line).
Figure 3. Ultraviolet absorption spectra of compounds 59 (kaempferol 3-O-hexuronoside-tri-O- hexoside-O-gallate), 82 (kaempferol 3-O-hexuronoside-O-p-coumarate-O-gallate), 83 (kaempferol 3-O-hexuronoside-O-hexoside-di-O-p-coumarate), 86 (kaempferol 3-O-hexuronoside-tri- O-p-coumarate), 75 (tiliroside), and 71 (kaempferol-3-O-glucuronide) before (black line) and after the addition of AlCl3 (blue line).
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Plants 10 02525 g004 550
Figure 4. Mass spectra (MS2) of acylated kaempferol glycosides 59, 77 and 82–86.
Figure 4. Mass spectra (MS2) of acylated kaempferol glycosides 59, 77 and 82–86.
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Plants 10 02525 g005 550
Figure 5. Ultraviolet absorption spectra of compounds 56 (quercetin 3-O-hexuronoside-tri-O-hexoside-O-gallate), 78 (quercetin 3-O-hexuronoside-O-p-coumarate-O-gallate), 81 (quercetin O-hexuronoside-di-O-p-coumarate), 73 (helichrysoside) and 68 (miquelianin) before (black line) and after the addition of AlCl3 (blue line).
Figure 5. Ultraviolet absorption spectra of compounds 56 (quercetin 3-O-hexuronoside-tri-O-hexoside-O-gallate), 78 (quercetin 3-O-hexuronoside-O-p-coumarate-O-gallate), 81 (quercetin O-hexuronoside-di-O-p-coumarate), 73 (helichrysoside) and 68 (miquelianin) before (black line) and after the addition of AlCl3 (blue line).
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Plants 10 02525 g006 550
Figure 6. Mass spectra (MS2) of acylated quercetin glycosides 56 and 78–81.
Figure 6. Mass spectra (MS2) of acylated quercetin glycosides 56 and 78–81.
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Plants 10 02525 g007 550
Figure 7. HPLC-PDA chromatogram (270 nm) of R. acicularis flower extract. Compounds are numbered as listed in Table 2. Internal standards (IS): IS-2—isovitexin-7,2″-di-O-glucoside (25 μg/mL); IS-3—isojaceoside (15 μg/mL).
Figure 7. HPLC-PDA chromatogram (270 nm) of R. acicularis flower extract. Compounds are numbered as listed in Table 2. Internal standards (IS): IS-2—isovitexin-7,2″-di-O-glucoside (25 μg/mL); IS-3—isojaceoside (15 μg/mL).
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Plants 10 02525 g008 550
Figure 8. HPLC-PDA chromatogram (270 nm) of R. acicularis root extract. Compounds are numbered as listed in Table 2. Internal standard: IS-4—6-O-sinapoyl catalpol (30 μg/mL).
Figure 8. HPLC-PDA chromatogram (270 nm) of R. acicularis root extract. Compounds are numbered as listed in Table 2. Internal standard: IS-4—6-O-sinapoyl catalpol (30 μg/mL).
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Plants 10 02525 g009 550
Figure 9. HPLC-PDA chromatogram (270 nm) of R. acicularis fruit extract. Compounds are numbered as listed in Table 2. Internal standard: IS-5—neomangiferin (25 μg/mL).
Figure 9. HPLC-PDA chromatogram (270 nm) of R. acicularis fruit extract. Compounds are numbered as listed in Table 2. Internal standard: IS-5—neomangiferin (25 μg/mL).
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Plants 10 02525 g010 550
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.
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.
Plants 10 02525 g010
Plants 10 02525 g011 550
Figure 11. Pancreatic-α-amylase-inhibiting activity of HPLC fraction of R. acicularis leaf extract. The grey bars show the starch content (as percentage of initial) in the probe after reaction with pancreatic amylase. The HPLC-PDA profile of R. acicularis leaf extract is a blue chromatogram with active compounds numbered as 1—glucogallin; 2—gallic acid; 9—epigallocatechin; 11—catechin; 13—1,6-di-O-galloyl glucose; 19—tellimagrandin I2; 21—tellimagrandin II; 23—tellimagrandin II isomer; 27—rugosin D; 33—1,2,3,6-tetra-O-galloyl glucose; 46—penta-O-galloyl hexose; 50—hexa-O-galloyl hexose; 66—quercetin O-hexuronoside-O-hexoside; 67—quercetin O-hexuronoside-O-pentoside; and 68—miquelianin.
Figure 11. Pancreatic-α-amylase-inhibiting activity of HPLC fraction of R. acicularis leaf extract. The grey bars show the starch content (as percentage of initial) in the probe after reaction with pancreatic amylase. The HPLC-PDA profile of R. acicularis leaf extract is a blue chromatogram with active compounds numbered as 1—glucogallin; 2—gallic acid; 9—epigallocatechin; 11—catechin; 13—1,6-di-O-galloyl glucose; 19—tellimagrandin I2; 21—tellimagrandin II; 23—tellimagrandin II isomer; 27—rugosin D; 33—1,2,3,6-tetra-O-galloyl glucose; 46—penta-O-galloyl hexose; 50—hexa-O-galloyl hexose; 66—quercetin O-hexuronoside-O-hexoside; 67—quercetin O-hexuronoside-O-pentoside; and 68—miquelianin.
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Plants 10 02525 g012 550
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.
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.
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Table
Table 1. Phytochemical composition of Rosa acicularis organs, mg/g dry weight (S.D.) 1,2.
Table 1. Phytochemical composition of Rosa acicularis organs, mg/g dry weight (S.D.) 1,2.
Phytochemical GroupLeaves (n = 53) 3Flowers (n = 42) 3Roots (n = 6) 4Fruits (n = 104) 5
Phenolic compounds
Flavonols41.48 (3.31)67.39 (4.98)0.11 (0.00)<0.01
Dihydroflavonols0.52 (0.04)0.73 (0.06)<0.01<0.01
Catechins27.37 (2.46)28.86 (2.40)43.04 (1.24)0.76 (0.10)
Hydroxycinnamates1.47 (0.08)0.20 (0.02)<0.01<0.01
Proanthocyanidins8.22 (0.57)16.56 (1.82)26.04 (1.04)1.05 (0.14)
Anthocyanins<0.015.34 (0.64)<0.01<0.01
Ellagitannins73.69 (6.63)33.14 (3.64)12.83 (0.64)<0.01
Gallotannins21.23 (2.01)8.53 (0.51)3.16 (0.06)1.22 (0.15)
Total phenolic compounds173.98160.7585.183.03
Non-phenolic compounds
Water-soluble
polysaccharides		41.88 (3.35)65.14 (5.86)21.76 (0.65)58.39 (4.08)
Ascorbic acid5.37 (0.42)1.14 (0.07)<0.0156.12 (6.17)
Titratable acids8.23 (0.74)11.27 (1.01)2.03 (0.08)38.26 (3.07)
Carotenoids0.43 (0.01)<0.01<0.012.33 (0.21)
Lipids11.07 (1.28)5.67 (0.45)2.53 (0.07)65.12 (7.16)
1 All samples were collected in one model population of R. acicularis in 2020. 2 n—number of samples analyzed. 3 Collection time: flowering period (15–20.VI.2020). 4 Collection time: post-fruiting period, 18–22.IX.2020. 5 Collection time: fruiting period, 28–29.VIII.2020.
Table
Table 2. Retention times (tR) and mass spectral data of compounds 1–123 were found in R. acicularis leaves, flowers and roots, in addition to their content in the May, July and September samples.
Table 2. Retention times (tR) and mass spectral data of compounds 1–123 were found in R. acicularis leaves, flowers and roots, in addition to their content in the May, July and September samples.
NotR, minCompound a (Ref.)[M-H]−, m/z bMS/MS, m/zContent, mg/g of Dry Plant Weight ± S.D.
Leaves: May
(n = 35) c		Leaves: July
(n = 81) c		Leaves: September
(n = 57) c		Flowers:
July
(n = 45) c		Roots:
September
(n = 12) c
11.261-O-Galloyl glucose S (glucogallin) [24]3311690.93 (0.11)1.58 (0.14)0.24 (0.01)0.43 (0.03)0.23 (0.03)
21.48Gallic acid S [24]169 1.04 (0.09)3.68 (0.44)10.39 (1.45)2.27 (0.25)4.27 (0.59)
31.791-O-Caffeoylquinic acid S [38]353191, 179, 173 and 1350.36 (0.02)0.53 (0.04)Traces0.14 (0.01)n.d.
41.95Galloyl-hexahydroxydiphenoyl-di-O-hexoside L [39]795633, 481, 463 and 301TracesTracesTraces0.10 (0.10)0.29 (0.03)
52.11Galloyl-hexahydroxydiphenoyl-di-O-hexoside L [39]795633, 481, 463 and 301TracesTracesTracesTraces0.37 (0.04)
62.41Ellagic acid tri-O-hexoside L [39,40]787625, 463 and 301TracesTracesTracesTraces1.26 (0.14)
72.73Caffeic acid O-hexoside L [38]341179, 165TracesTracesTracesTracesn.d.
82.982-Pyrone-4,6-dicarboxylic acid S [41]183139, 1111.44 (0.12)2.50 (0.34)1.86 (0.22)1.43 (0.15)0.95 (0.11)
94.71Epigallocatechin S [24]305179, 1372.34 (0.22)5.39 (0.54)3.77 (0.40)4.73 (0.42)20.45 (1.84)
105.085-O-Caffeoylquinic acid S [38]353191, 179 and 165Traces0.28 (0.03)TracesTracesn.d.
115.48Catechin S [24]289205, 13712.56 (1.01)18.63 (2.60)16.24 (1.78)19.26 (2.11)17.22 (1.89)
125.83Di-O-galloyl hexose L [24]483331, 169 and 1250.04 (0.00)0.64 (0.81)TracesTracesn.d.
136.581,6-Di-O-galloyl glucose S [24]483331, 169 and 1251.20 (0.14)4.26 (0.53)0.92 (0.10)4.98 (0.45)n.d.
148.27Tellimagrandin I1 S [41,42]785, 392 *3010.12 (0.01)0.89 (0.09)0.05 (0.00)0.09 (0.00)n.d.
158.53Epicatechin S [24]289205, 1370.89 (0.93)1.29 (0.09)0.46 (0.52)Traces8.23 (0.74)
169.08Rugosin B1 S [41,42]953, 476 *785, 597 and 3010.08 (0.00)0.40 (0.04)0.02 (0.00)0.76 (0.06)n.d.
179.38Tri-O-galloyl hexose L [24]635483, 331, 169 and 125Traces0.44 (0.05)Tracesn.d.n.d.
189.591,3,6-Tri-O-galloyl glucose S [24]635483, 331, 169 and 125Traces1.29 (0.11)TracesTracesn.d.
199.82Tellimagrandin I2 S [41,42]785, 392 *3010.65 (0.04)2.93 (0.33)0.59 (0.04)2.14 (0.19)n.d.
2010.02Tri-O-galloyl hexose L [24]635483, 331, 169 and 1250.26 (0.02)2.35 (0.28)0.14 (0.01)Tracesn.d.
2110.48Tellimagrandin II1 S [41,42]937, 468 *3012.96 (0.34)8.98 (0.95)4.22 (0.51)6.03 (0.58)n.d.
2210.72Rugosin B2 S [41,42]953, 476 *785, 597 and 3010.82 (0.08)3.86 (0.42)1.59 (0.17)2.37 (0.19)n.d.
2310.88Tellimagrandin II isomer S [41,42]937, 468 *3012.50 (0.22)8.29 (0.91)5.26 (0.73)n.d.n.d.
2411.01Rugosin E1 S [41,42]860 *937, 785, 597 and 3010.73 (0.06)1.89 (0.26)0.56 (0.06)2.85 (0.40)n.d.
2511.10Rugosin E2 S [41,42]860 *937, 785, 597 and 3010.50 (0.05)1.12 (0.14)0.27 (0.03)0.39 (0.03)n.d.
2611.48Rugosin A S [41,42]1105, 552 *3010.28 (0.02)0.53 (0.06)0.10 (0.01)0.11 (0.01)n.d.
2712.03Rugosin D S [41,42]936 *, 623 **917, 851, 765, 749 and 30118.35 (1.46)41.15 (4.93)32.67 (3.25)4.06 (0.44)n.d.
2812.421-O-Ellagoyl glucose S [39,40]463301Traces0.62 (0.05)TracesTracesn.d.
2912.78Ellagic acid S [39,40]301 0.25 (0.03)2.37 (0.33)4.29 (0.58)Traces4.25 (0.51)
3013.05Ellagic acid methyl ester O-hexoside L [39,40]477315, 301Traces0.36 (0.02)TracesTracesn.d.
3113.42Ellagic acid methyl ester O-hexoside L [39,40]477315, 301Traces0.45 (0.05)Traces0.62 (0.05)n.d.
3213.52Tetra-O-galloyl hexose L [24]787635, 483, 331 and 1697.33 (0.87) d10.54 (1.14) d4.27 (0.51) dn.d.n.d.
3313.821,2,3,6-Tetra-O-galloyl glucose S [24]787635, 483, 331 and 1690.97 (0.10)n.d.
3414.48Epicatechin/catechin trimer L [24]865577, 451, 407 and 287TracesTracesTracesTracesn.d.
3514.67Epicatechin/catechin trimer L [24]865577, 451, 407 and 287TracesTracesTracesn.d.n.d.
3615.21Ellagic acid dimethyl ester O-hexoside L [39,40]491329, 301TracesTracesTracesTracesn.d.
3715.82Epicatechin/catechin trimer L [24]865577, 451, 407 and 287Traces2.14 (0.22)Traces0.35 (0.02)n.d.
3816.04Epicatechin/catechin trimer L [24]865577, 451, 407 and 287TracesTracesTraces0.29 (0.02)n.d.
3916.54Di-ellagoyl methyl ester O-hexoside L [39,40]761477, 315 and 3010.26 (0.03)0.94 (0.11)0.14 (0.01)2.45 (0.29)n.d.
4016.97Epicatechin/catechin tetramer L [24]1153863, 577, 575, 451, 407 and 287Traces1.95 (0.22)0.11 (0.01)8.76 (0.96)0.73 (0.06)
4117.09Epicatechin/catechin tetramer L [24]1153863, 577, 451, 407 and 287TracesTracesTracesn.d.0.07 (0.01)
4217.81Epicatechin/catechin tetramer L [24]1153863, 577, 451, 407 and 287Traces0.26 (0.02)Traces1.14 (0.10)0.05 (0.00)
4318.42Di-ellagoyl dimethyl ester O-hexoside L [39,40]775477, 315 and 301TracesTracesTracesTracesn.d.
4418.80Ellagic acid trimethyl ester O-hexoside L [39,40]505343, 301TracesTracesTraces1.54 (0.18)n.d.
4520.14Epicatechin/catechin tetramer L [24]1153863, 577, 451, 407 and 287TracesTracesTracesTracesTraces
4620.38Penta-O-galloyl hexose L [24]939787, 635, 483, 331 and 169Traces1.80 (0.22)Traces0.77 (0.08)n.d.
4720.451,2,3,4,6-Penta-O-galloyl glucose S [24]939787, 635, 483, 331 and 169Traces0.53 (0.06)Tracesn.d.n.d.
4820.73Hexa-O-galloyl hexose L [24]1091939, 787, 635, 483, 331 and 169TracesTracesTracesTracesn.d.
4921.59Hexa-O-galloyl hexose L [24]1091939, 787, 635, 483, 331, 169TracesTracesTracesn.d.n.d.
5021.98Hexa-O-galloyl hexose L [24]1091939, 787, 635, 483, 331 and 169Traces2.99 (0.33)0.84 (0.73)n.d.n.d.
5123.11Dihydroquercetin di-O-hexuronoside-di-O-hexoside L [43]979817, 655, 479 and 303TracesTracesTracesn.d.n.d.
5223.76Quercetin di-O-hexuronoside-tri-O-hexoside L [39,44,45]1139977, 815, 653, 477 and 301Traces0.12 (0.01)Tracesn.d.n.d.
5324.27Dihydroquercetin O-hexuronoside-di-O-hexoside L [43]803641, 479 and 303TracesTracesTracesn.d.n.d.
5424.80Quercetin O-hexuronoside-tri-O-hexoside L [39,44,45]963801, 639, 477 and 3010.64 (0.05)1.52 (0.12)0.21 (0.2)n.d.n.d.
5525.63Dihydroquercetin O-hexuronoside-O-hexoside L [43]641479, 303TracesTracesTracesn.d.n.d.
5628.44Quercetin O-hexuronoside-tri-O-hexoside-O-gallate L
[39,44,45,46]		1115963, 801, 639, 477 and 301TracesTracesTracesn.d.n.d.
5726.78Quercetin di-O-hexuronoside-di-O-hexoside L [39,44,45]977815, 653, 477 and 301TracesTracesTracesn.d.n.d.
5827.31Kaempferol O-hexuronoside-tri-O-hexoside L [39,44,45]947785, 623, 461 and 285TracesTracesTracesn.d.n.d.
5928.02Kaempferol O-hexuronoside-tri-O-hexoside-O-gallate L
[39,44,45,46]		1099947, 785, 623, 461 and 285TracesTracesTracesn.d.n.d.
6028.76Kaempferol di-O-hexuronoside-di-O-hexoside L [39,44,45]961799, 637, 461 and 285TracesTracesTracesTracesn.d.
6129.01Quercetin O-hexuronoside-di-O-hexoside L [39,44]801639, 477 and 301TracesTracesTraces16.78 (2.01)n.d.
6229.44Kaempferol O-hexuronoside-di-O-hexoside L [39,44,45]785623, 461 and 285TracesTracesTraces7.37 (0.74)n.d.
6329.81Dihydroquercetin O-hexoside L [43]465303TracesTracesTracesTracesn.d.
6430.10Dihydroquercetin O-hexuronoside L [43]4793030.22 (0.03)0.35 (0.03)0.39 (0.04)Tracesn.d.
6530.71Quercetin-O-rutinoside S (rutin) [39,44,45]609463, 3010.10 (0.01)0.28 (0.03)0.06 (0.00)15.25 (1.67)n.d.
6631.09Quercetin O-hexuronoside-O-hexoside L [39,44,45]639477, 3010.16 (0.01)0.88 (0.09)0.12 (0.01)Tracesn.d.
6731.42Quercetin O-hexuronoside-O-pentoside L [39,44,45]609477, 3010.52 (0.04)3.04 (0.28)0.83 (0.09)n.d.n.d.
6831.83Quercetin-3-O-glucuronide S (miquelianin) [24,39,44]4773015.31 (0.42)10.08 (1.22)9.35 (0.94)0.21 (0.02)n.d.
6932.02Kaempferol O-hexuronoside-O-hexoside L [39,44]623461, 2851.11 (0.09)3.16 (0.37)0.51 (0.05)Tracesn.d.
7032.40Kaempferol O-hexuronoside-O-pentoside L [39,44]593461, 2851.57 (0.12)5.38 (0.51)2.21 (0.25)Tracesn.d.
7132.62Kaempferol-3-O-glucuronide S [24,39,44]4612853.61 (0.28)7.63 (0.79)3.37 (0.34)0.12 (0.01)n.d.
7233.24Quercetin-3-O-rhamnoside S (quercitrin) [24,39,44]447301TracesTracesTracesn.d.n.d.
7333.76Quercetin-3-O-(6″-O-p-coumaroyl)-glucoside S (helichrysoside) [24,39,44]609463, 301TracesTracesTracesn.d.n.d.
7434.22Kaempferol-3-O-arabinoside S (juglanin) [24,39,44]4172850.22 (0.02)0.40 (0.04)Tracesn.d.n.d.
7534.97Kaempferol-3-O-(6″-O-p-coumaroyl)-glucoside S
(tiliroside) [24,39,44]		593447, 2851.73 (0.16)2.14 (0.23)0.47 (0.04)0.31 (0.02)n.d.
7635.63Kaempferol-3-O-(6″-O-galloyl)-glucoside S [39,44,45,46]599447, 2850.12 (0.01)5.03 (0.52)Traces0.98 (0.09)n.d.
7735.96Kaempferol O-hexuronoside-O-gallate L [39,44,45,46]613461, 2850.04 (0.00)0.35 (0.03)Traces10.95 (1.28)n.d.
7836.47Quercetin O-hexoside-O-p-coumarate-O-gallate L [39,44,45,46]761615, 609, 463 and 301Traces0.15 (0.01)Traces11.19 (1.01)n.d.
7936.69Quercetin O-hexuronoside-O-p-coumarate-O-gallate L
[39,44,45,46,47]		775632, 623, 477 and 301TracesTracesTracesn.d.n.d.
8036.97Quercetin O-hexoside-di-O-p-coumarate L [24,39,44,47]755609, 463 and 301TracesTracesTracesTracesn.d.
8137.15Quercetin O-hexuronoside-di-O-p-coumarate L [24,39,44,47]769623, 477 and 301TracesTracesTracesTracesn.d.
8237.90Kaempferol O-hexuronoside-O-p-coumarate-O-gallate L
[39,44,45,46,47]		759613, 607, 461 and 285TracesTracesTraces0.35 (0.03)n.d.
8338.09Kaempferol O-hexuronoside-O-hexoside-di-O-p-coumarate L [24,39,44,47]915769, 623, 461 and 285TracesTracesTracesn.d.n.d.
8438.93Kaempferol O-hexoside-di-O-p-coumarate L [24,39,44,47]739593, 447 and 285TracesTracesTracesn.d.n.d.
8539.05Kaempferol O-hexuronoside-di-O-p-coumarate L
[24,39,44,47]		753607, 461 and 285TracesTracesTracesn.d.n.d.
8639.58Kaempferol O-hexuronoside-tri-O-p-coumarate L
[24,39,44,47]		899753, 607, 461 and 285TracesTracesTracesn.d.n.d.
8710.53Hexahydroxydiphenoyl-tri-O-galloyl-hexose
(tellimagrandin II isomer) L [41,42]		937, 468 *301n.d.n.d.n.d.1.95 (0.20)n.d.
8811.80Valoneoyl-tri-O-galloyl-hexose (rugosin A isomer) L [41,42]1105, 552 *301n.d.n.d.n.d.0.52 (0.04)n.d.
8911.98Hexahydroxydiphenoyl-valoneoyl-tetra-O-galloyl- di-O-hexose (rugosin D isomer) L [41,42]936 *, 623 **917, 851, 765, 749 and 301n.d.n.d.n.d.2.73 (0.38)n.d.
9012.81Ellagic acid O-hexoside L [39,40]463301n.d.n.d.n.d.Tracesn.d.
9117.49Epicatechin/catechin tetramer L [24]1153863, 577, 575, 451, 407 and 287n.d.n.d.n.d.7.53 (1.05)n.d.
9221.50Hexa-O-galloyl hexoside L [24]1091939, 787, 635, 483, 331 and 169n.d.n.d.n.d.Tracesn.d.
9331.12Peonidin 3,5-di-O-glucoside S [48]625 e463, 301 en.d.n.d.n.d.3.21 (0.35)n.d.
942.84Ellagic acid tri-O-hexoside L [39,40]787625, 463 and 301n.d.n.d.n.d.n.d.1.63 (0.19)
956.18Epicatechin/catechin dimer O-hexoside L [24]739577, 451, 407 and 287n.d.n.d.n.d.n.d.1.14 (0.14)
966.57Epicatechin/catechin dimer O-hexoside L [24]739577, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
977.29Procyanidin B3 S [24]577451, 407 and 287n.d.n.d.n.d.n.d.1.29 (0.15)
987.71Procyanidin B1 S [24]577451, 407 and 287n.d.n.d.n.d.n.d.Traces
998.52Epicatechin/catechin dimer O-hexoside L [24]739577, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
1008.82Procyanidin B4 S [24]577451, 407 and 287n.d.n.d.n.d.n.d.0.11 (0.01)
1019.01Epicatechin/catechin dimer O-hexoside L [24]739577, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
1029.11Epicatechin/catechin dimer O-hexoside L [24]739577, 451, 407 and 287n.d.n.d.n.d.n.d.0.10 (0.01)
1039.47Epicatechin/catechin dimer O-hexoside L [24]739577, 451, 407 and 287n.d.n.d.n.d.n.d.3.84 (0.42)
1049.58Epicatechin/catechin dimer O-hexoside L [24]739577, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
10510.00Procyanidin B2 S [24]577451, 407 and 287n.d.n.d.n.d.n.d.7.62 (0.91)
10610.25Procyanidin B2 3-O-gallate S [49]729577, 559, 541, 441 and 289n.d.n.d.n.d.n.d.Traces
10710.41Procyanidin B2 3″-O-gallate S [49]729577, 559, 541, 441 and 289n.d.n.d.n.d.n.d.Traces
10810.52Epicatechin/catechin dimer O-gallate L [49]729577, 541, 441 and 289n.d.n.d.n.d.n.d.Traces
10910.78Procyanidin B2 3,3″-di-O-gallate S [49]881729, 577, 559, 541, 441 and 289n.d.n.d.n.d.n.d.0.96 (0.10)
11011.091-O-Ellagoyl-gentiobiose (amritoside) S [39,40]625463, 301n.d.n.d.n.d.n.d.Traces
11111.41Epicatechin gallate S [24]441 n.d.n.d.n.d.n.d.0.14 (0.01)
11211.72Ellagic acid di-O-desoxyhexoside L [39,40]593447, 301n.d.n.d.n.d.n.d.3.10 (0.43)
11312.05Ellagic acid 4-O-rhamnoside (eschweilenol C) S [39,40]447301n.d.n.d.n.d.n.d.3.91 (0.46)
11416.41Epicatechin/catechin tetramer L [24]1153863, 577, 575, 451, 407, 287n.d.n.d.n.d.n.d.0.31 (0.02)
11517.51Epicatechin/catechin tetramer L [24]1153863, 577, 575, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
11618.10Epicatechin/catechin tetramer L [24]1153863, 577, 575, 451, 407 and 287n.d.n.d.n.d.n.d.0.40 (0.04)
11718.48Epicatechin/catechin tetramer L [24]1153863, 577, 575, 451, 407 and 287n.d.n.d.n.d.n.d.0.46 (0.04)
11818.97Epicatechin/catechin pentamer L [24]14411153, 863, 577, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
11919.50Epicatechin/catechin pentamer L [24]14411153, 863, 577, 451, 407 and 287n.d.n.d.n.d.n.d.1.26 (0.17)
12019.72Epicatechin/catechin pentamer L [24]14411153, 863, 577, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
12120.41Epicatechin/catechin pentamer L [24]14411153, 863, 577, 451, 407 and 287n.d.n.d.n.d.n.d.Traces
12221.01Epicatechin/catechin pentamer L [24]14411153, 863, 577, 451, 407 and 287n.d.n.d.n.d.n.d.1.87 (0.26)
12321.52Epicatechin/catechin pentamer L [24]14411153, 863, 577, 451, 407 and 287n.d.n.d.n.d.n.d.1.59 (0.22)
Total phenolic content,71.24178.09106.41148.4888.10
incl. ellagic acid and hexosides0.514.744.434.6114.15
ellagitannins26.9970.0445.3324.100.66
gallotannins10.8030.1016.809.424.50
catechins15.7925.0420.4723.9946.04
catechin oligomersTraces4.35Traces18.0721.80
hydroxycinnamates0.360.81Traces0.14n.d.
flavonoids,15.3540.5117.5266.72n.d.
incl. quercetin glycosides6.7316.0710.5743.43n.d.
kaempferol glycosides8.4024.096.5620.08n.d.
dihydroquercetin glycosides0.220.350.39Tracesn.d.
anthocyaninsn.d.n.d.n.d.3.21Traces
various compounds1.442.501.861.430.95
a Compound identification was based on the comparison of retention time, UV and MS spectral data with reference standards (S) or the interpretation of UV and MS spectral data in comparison with literature data (L). b * [M-2H]2−. ** [M-3H]3−. c In brackets—number of samples analyzed. d Calculated as a sum of compounds 32 and 33. e Positive ionization. Abbreviations used: Ara—arabinose; pCou—p-coumaroyl; EllA—ellagic acid; Glc—glucose; GlcA—glucuronic acid; Gall—galloyl; Hex—hexose; HexA—hexuronic acid; HHDP—hexahydroxydiphenoyl; Me—methyl; Rha—rhamnose; Rut—rutinose; Val—valoneoyl; n.d.—not detected; and traces—<LOQ (limit of quantification).
Table
Table 3. Inhibitory effects of rugosin D and acarbose against mammalian α-amylases, IC50 (μg/mL ± S.D.).
Table 3. Inhibitory effects of rugosin D and acarbose against mammalian α-amylases, IC50 (μg/mL ± S.D.).
CompoundPorcine Pancreas α-AmylaseHuman Saliva α-AmylaseHuman Pancreas α-Amylase
Rugosin D32.09 ± 1.2167.59 ± 4.12 *30.84 ± 1.23 *
Acarbose35.67 ± 1.4282.33 ± 3.5456.39 ± 2.81
*—p < 0.05 vs. acarbose.
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Olennikov, D.N.; Chemposov, V.V.; Chirikova, N.K. Metabolites of Prickly Rose: Chemodiversity and Digestive-Enzyme-Inhibiting Potential of Rosa acicularis and the Main Ellagitannin Rugosin D. Plants 2021, 10, 2525. https://doi.org/10.3390/plants10112525

AMA Style

Olennikov DN, Chemposov VV, Chirikova NK. Metabolites of Prickly Rose: Chemodiversity and Digestive-Enzyme-Inhibiting Potential of Rosa acicularis and the Main Ellagitannin Rugosin D. Plants. 2021; 10(11):2525. https://doi.org/10.3390/plants10112525

Chicago/Turabian Style

Olennikov, Daniil N., Vladimir V. Chemposov, and Nadezhda K. Chirikova. 2021. "Metabolites of Prickly Rose: Chemodiversity and Digestive-Enzyme-Inhibiting Potential of Rosa acicularis and the Main Ellagitannin Rugosin D" Plants 10, no. 11: 2525. https://doi.org/10.3390/plants10112525

APA Style

Olennikov, D. N., Chemposov, V. V., & Chirikova, N. K. (2021). Metabolites of Prickly Rose: Chemodiversity and Digestive-Enzyme-Inhibiting Potential of Rosa acicularis and the Main Ellagitannin Rugosin D. Plants, 10(11), 2525. https://doi.org/10.3390/plants10112525

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