Study of the Chemical Composition of Rosa beggeriana Schrenk’s Fruits and Leaves

Rosa species are widely used in folk medicine in different countries of Asia and Europe, but not all species are studied in-depth. For instance, Rosa beggeriana Schrenk, a plant which grows in Central Asia, Iran, and some parts of China, is little described in articles. Column and thin-layer chromatography methods were used to isolate biologically active substances. From a study of fruits and leaves of Rosa beggeriana Schrenk, a large number of compounds were identified, seven of which were isolated: 3β,23-dihydroxyurs-12-ene (1), β-sitosterol (2), betulin (3), (+)-catechin (4), lupeol (5), ethyl linoleate (6), and ethyl linolenoate (7). Their structures were elucidated by 1H, DEPT and 13C NMR spectroscopy, mass spectrometry, and GC-MS (gas chromatography–mass spectrometry). The study also identified the structures of organic compounds, including volatile esters and acids. Consequently, comprehensive data were acquired concerning the chemical constitution of said botanical specimen.


Introduction
The genus Rosa holds big commercial significance and is renowned in the domain of folk medicine.Numerous wild species within this genus have played a pivotal role in the development of valuable and economically viable cultivars of ornamental roses [1].Despite the relative under-examination of certain rose species, several of them possess significant potential due to the presence of rosehip fruits.Commercially traded rosehip fruit is derived from several different species.They are long-lived woody perennials found mainly on forest margins and in disturbed habitats, such as roadsides and open fields.The genus Rosa (Rosaceae) has around 150-200 species [1][2][3].Roses have also been cultivated since ancient times as medicinal plants in many countries across Europe and Asia.Rosehips contain many pharmacologically active compounds, such as organic acids, vitamin C and E, flavonoids, carotenoids, and tannins.Therapeutic properties and benefits of rosehips are their nourishing, mild laxative, mild diuretic, mild astringent, diuretic, ophthalmic and tonic effects [1,[4][5][6].Rosa extracts derived from these plants are also widely used in cosmetics, promising antioxidant and moisturizing effects [1,[5][6][7][8].All parts of this wild rose have been used in Asian folk medicine [4,5,[9][10][11].
The intrinsic value of rosehip fruit has been acknowledged for centuries; however, efforts have only recently been made to domesticate and cultivate wild roses specifically for their fruit and to advance agronomic techniques in this regard.This shift in focus can be attributed to an enhanced comprehension of the pivotal role that dietary fruits play in Plants 2023, 12, 3297 2 of 14 enhancing human health and mitigating disease risks [12].The Rosaceae family is one of the most employed as a consolidated source of phytoproducts with functional properties [13].Within this family, the genus Rosa provides various species, and their essential oils possess a wide range of applications as flavor, fragrance, and additive in cosmetic and toiletries [14].In addition to their aromatic composition, many Rosa species from all over the word have been evaluated for their food-related biological properties and multiple functional uses have been suggested [15][16][17][18][19].For example, teas made from the fruits of Rosa canina have mild laxative and diuretic tendencies [12].Rosehips have a longstanding history of utilization in folk medicine spanning centuries, primarily for the prevention and treatment of various ailments such as the common cold, influenza-like infections, fever, infectious diseases, vitamin C deficiency, general exhaustion, gastritis and gastric ulcers prevention, diarrhea, gallstones and gallbladder discomforts, urinary tract diseases and discomforts, as well as for their potential anti-inflammatory, anti-obesity, anticancer, and diabetes management properties.Furthermore, rosehips have been employed to address inadequate peripheral circulation concerns [5,6,[15][16][17][18]20,21].Mixed with small amount of vinegar, rosehips were used as an antidote for the treatment of iron toxicity [20,22].For nutritional purposes, fruits are used for the production of different products like tea, marmalade, jam, stewed fruit, wine, and juices [23].Ground in a hand mill and cooked with milk, they could be used as children's snack and baby food as reported by the latter authors.The functional properties of some Rosa species are attributable to a wide range of bioactive ingredients, such as minerals, flavonoids, tannins, anthocyanin, organic acids, phenolic compounds fatty acids, volatile oils, ascorbic acid, phenols, and sugar.Through the examination of a species within the same taxonomic family as the subject of investigation, co-occurring within the identical geographical region, it becomes feasible to assess the potential anticancer properties of compounds derived from Begger's rosehip [5,[24][25][26][27][28].
Rosa beggeriana Schrenk is an indigenous species predominantly distributed in Central Asia (Kazakhstan, Kyrgyzstan), China (Xinjiang Uygur Autonomous Region), and Iran [29][30][31][32][33].It has been identified as an essential resource for hybridization purposes, particularly in the development of cold-resistant germplasm, when combined with contemporary rose varieties [29].Begger's rosehip contains a large number of compounds with antioxidant activity, including activity against cancer cells.Rosehip hips have a high concentration of the carotenoid lycopene, which is considered a compound with a powerful antioxidant effect and is used as a therapeutic and prophylactic agent for various diseases, including cancer.Begger's rosehip extracts display cytotoxicity and antiproliferative properties against human liver and breast cancer cells, which might be associated with the presence of polyphenols in it [34].
According to the studies mentioned earlier, we can tell that Begger's rosehips have not been studied sufficiently even though other species are very well known in the folk medicine of different countries.

Identification of the Isolated Compounds from Leaves of Rosa beggeriana Schrenk
Utilizing column chromatography, a total of seven distinct substances were successfully isolated from both the fruits and leaves of the wild rose species known as Begger (Rosa sp.).The isolated substances encompassed a diverse range of chemical classes, including triterpenoids, catechins, and fatty acid esters.Remarkably, 3β,23-dihydroxyurs-12-ene had not been previously reported in this particular plant species.Additionally, no NMR characterization data were available for this newly isolated substance [35][36][37].Apart from this novel isolate, the triterpenoids, catechins, and a mixture of fatty acid esters were also successfully identified and isolated from the aforementioned plant material.

GC-MS Data
GC-MS analysis was used to obtain data on the fatty acid composition of the leaves (Table 1) and fruits (Table 2) of Rosa beggeriana Schrenk.When comparing the two tables, a richer composition of the fatty acids in fruits can be observed (Tables 1 and 2).NMR data for Compound 1 were not found in literature.Hence, the analysis of the NMR spectra (Figures S1-S4) and the comparison of the spectroscopic data (Table 3) with those compounds that have a similar structure and described in the literature [35][36][37] allowed the identification of the compound 1.The mass spectra also allowed us to identify compound 1.Also, the melting point for compound 1 was 226-229 • C. droxyurs-12-ene (1) [35][36][37] (21 mg) was isolated.The chemical structures are shown in Figure 1.
NMR data for Compound 1 were not found in literature.Hence, the analysis of the NMR spectra (Figures S1-S4) and the comparison of the spectroscopic data (Table 3) with those compounds that have a similar structure and described in the literature [35][36][37] allowed the identification of the compound 1.The mass spectra also allowed us to identify compound 1.Also, the melting point for compound 1 was 226-229 °C.No. 13
For compound 4 (m.p.175-177 °C, optical rotation [α]D +17.2°), a detailed analysis of the NMR data led to the proposed structure, confirmed by the analysis of the spectra and Compound 3 (betulin) was identified by its characteristic 1     (6), and ethyl linolenoate (7) were isolated from the ethanol extract (35 g) of Rosa beggeriana Schrenk (400 g).The chemical structures are shown in Figure 3.  Compound (2) was identified as β-sitosterol (Figures S5 and S6) according to the literature [44,45].

GC-MS Data
The GC-MS analysis uncovered a heterogeneous chemical composition encompassing various classes of volatile compounds, which has been meticulously documented and organized in Tables 1 and 2. Considering the paucity of scholarly investigations on the Plants 2023, 12, 3297 8 of 14 phytochemical composition of R. beggeriana, the GC-MS data were juxtaposed with data obtained from other species belonging to the Rosa genus, as cited in [49][50][51][52].The comparative analysis of compositions, specifically the leaves and fruits, revealed a higher degree of complexity in the composition of the latter.It is noteworthy that this study represents the inaugural examination of the fatty acids' profiles pertaining to this plant species.
Upon comprehensive analysis of the collective findings presented in Tables 2 and 3, it becomes evident that both the leaves and fruits of R. beggeriana exhibited a substantial abundance of saturated and unsaturated acids, terpenoids, and various other substances.These results collectively indicate a highly diverse and rich composition within the examined plant components.

Isolation and Identification of Individual Compounds
Identification of the isolated compounds (1-5) was based on spectroscopic analyses ( 1 H, DEPTQ, DEPT135, 13 C NMR) compared with the data published in the literature.
In this study, the presence of 3β,23-dihydroxyurs-12-ene (compound 1-white and crystalline) in the leaves of R. beggeriana was established and confirmed using NMR data analysis, coupled with relevant literature sources [35][36][37].A structurally similar compound, 3β,24-dihydroxyurs-12-ene, had been previously isolated from Protium heptaphyllum.By comparing the 1 H-and 13 C-NMR spectra of the two compounds, it was observed that all peaks were identical, except for those corresponding to C-23 and C-24.For 3β,23dihydroxyurs-12-ene, the chemical shifts for C-23 and C-24 were 63.0 and 15.6 ppm, respectively, whereas for 3β,24-dihydroxyurs-12-ene, these values were reversed [35].The presence of an oxygen moiety at C-23 in 3β,23-dihydroxyurs-12-ene was deduced based on the chemical shift at 63.0 ppm.The 1 H-NMR spectrum displayed signals corresponding to seven methyl groups: 1.25 (H-24), 0.98 (H-25), 1.02 (H-26), 1.08 (H-27), 0.81 (H-28), 0.80 (H-29), and 0.81 (H-30).The 13 C-NMR spectrum exhibited two peaks at 124.4 (C-12) and 139.5 (C-13), indicating the presence of a double bond in the ring.Overall, the number of observed peaks suggested the presence of 30 carbon atoms in compound 1.Importantly, this study represents the first isolation of 3β,23-dihydroxyurs-12-ene from the Rosa genus.Compound 1 was further compared to 3β,28-dihydroxyurs-12-ene, which contained a Plants 2023, 12, 3297 9 of 14 -CH 2 OH group at the 28th carbon atom, resulting in a chemical shift of 69.20 ppm.Consistent correlations were observed in the 1 H-NMR spectra, supporting the structural analysis.High-resolution mass spectrometry (HR MS), as depicted in Figure S4, furnishes valuable insights pertaining to the molecular attributes of the compound under investigation.Specifically, it elucidated a molecular weight of 442 m/z, thereby affording a comprehensive breakdown of constituent particles within this compound.For instance, it is reasonable to deduce the presence of two highly mobile hydrogen atoms (m/z 440).Furthermore, the observation of protonation events at 424 [M + H 2 O] + and 406 [M + H 2 O] + suggests the existence of two hydroxyl (-OH) groups.
In addition, the HR MS data imitate the potential stability of a fragment with an m/z value of 273, indicative of a cleavage point that partitions the molecule into two relatively stable subunits.Moreover, the ensuing particles predominantly originated from the cleavage of the molecule at the central region of its third ring, yielding fragments with m/z values of 133, 189, 203, and 234.These findings contribute to a more intricate understanding of the compound's structural composition and fragmentation pattern.Notably, structurally related compounds (although not identical) [37,43,53,54] have been identified within the Rosa family, specifically in the fruits of R. multiflora and the roots of R. taiwanensis, and have been associated with anti-inflammatory activity.Based on the available data, it is plausible to postulate that 3β,23-dihydroxyurs-12-ene may possess similar activities to those of the structurally related compounds mentioned earlier.However, further investigation and experimental studies are necessary to validate and ascertain its potential biological activities.This compound have potential for use in medicine because there many studies of almost structurally identical compounds that have different types of activities [37,55,56].
Betulin ( 3) and (+)-catechin (4) have been identified in various Rosa species, present not only in leaves, but also in the roots, stems, with (+)-catechin present in the fruits and flowers [43,[57][58][59].The identification of compound 3 (betulin-solid and white crystalline) was accomplished through the analysis of its characteristic 1 H NMR (400 MHz; CDCl 3 ) and 13 C NMR (CDCl 3 ) spectra.The carbon peaks were meticulously examined, revealing the presence of 30 carbon atoms in this compound.Utilizing the DEPT135 method, it was determined that betulin comprises 12 -CH 2 groups, 5 unhydrolyzed carbons (because we can see them in 13 C NMR spectra but not there), 13 -CH 3 and/or -CH groups, two oxygenated carbons (63.06 and 79.01), and one double bond (109.35).Proton magnetic resonance spectra further confirmed these structural features.Specifically, the 1   S10-S14, encompassing 2D nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS) spectra, it is discernible that compound 3 corresponds to betulin.The GC-MS spectra provides essential information such as the retention time and molecular weight of this compound.Additionally, compound (+)-catechin (4) has been identified through a combination of NMR analysis, which was previously elucidated, and mass spectrometry.The mass spectra of compound 4 exhibited a discernible molecular weight of approximately 290 units.This determination is in accordance with our NMR-based assumption of it being catechin.Notably, the mass spectra also revealed distinct ions with molecular weights of 110, 138, and 55 units.These observations are congruent with the structural features of catechin, particularly with regard to the presence of vulnerable chemical bonds in its structure.Betulin was isolated from the roots of Rosa taiwanensis [43], and catechin was reported in the roots of R. taiwanensis [43], rosehips of Rosa canina [57,58].Notably, betulin was reported to have anti-inflammatory and anticancer properties [60].The structural identification of (+)-catechin (4) was established based on the analyses of the 1 H NMR, DEPT135, and 13 C NMR signals.The NMR data of β-sitosterol (2) were subjected to a comprehensive comparison with relevant literature data [44,45,61].
Lupeol ( 5) is a triterpenoid that was found in the fruits of R. beggeriana and identified through comparison with NMR data from the literature data [39,46,47].R. rugosa was also reported to have lupeol [62].Using carbon nuclear magnetic resonance, it was found that the compound had 30 carbon atoms, 10 of which were methylene groups.The presence of a hydroxyl group and a double bond due to the presence of a shift was also established.Also, thanks to proton magnetic resonance, it was possible to establish the structure by calculating the number and area of peaks.
Ethyl linoleate (6) and ethyl linolenoate (7) were isolated together, and structure was elucidated by GC-MS data (Figures S21 and S22) and NMR, which were compared to literature [48].
Regardless of the precise study of several species, like R. canina, R. rugosa, and others, there are many species in the Rosa genus with minimal data.Many species of the Rosa family grow in diverse conditions, hence they can have different chemical composition [1,4,5].

Plant Material
The plant was harvested at a 23-25 • C temperature near Ili River and in the Almaty oblast N44 • 79.3959, E76 • 29.8245 in September 2021, by a biologist employee of the Botanical Garden in Almaty, Madina Ramazanova.Then, the plant was dried in the drying cabinet at 35 • C for 6 h and 48 h at room temperature, And it was deposited at the herbarium collection at the Institute of Botany and Phytointroduction of the Ministry of Higher Education and Science, Almaty, Kazakhstan (0002540).

Extraction and Isolation of Leaves
The air-dried leaves (553.0 g) were macerated with ethanol 95% (1.5 L × 3 times) at room temperature.The ethanol extracts were combined, and the solvent was distilled under reduced pressure at low temperature to afford a 10.0 g yield.
The ethanol extract was processed using vacuum liquid chromatographic techniques with silica gel in a column (600 g, 0.063-0.200mm; Merck, Darmstadt, Germany).The extract was eluted using a gradient system with n-hexane, DCM, ethyl acetate, and ethanol, with growing polarity as a mobile phase starting with 100% n-hexane and ending with 100% methanol, and 5 fractions were obtained.Fractions were grouped depending on their chemical similarity and monitored using thin-layer chromatography and concentrated using a rotary evaporator.The obtained fractions were L-F1 (1.35 g), L-F2 (0.56 g), L-F3 (0.74 g), L-F4 (1.85 g), and L-F5 (2.37 g).
L-F1, L-F2, and L-F3 were concentrated together to fraction L-F1 according to similar spots on TLC.L-F4 and L-F5 was collected to fraction L-F2.Then, each fraction was separated using chromatographic fractionation in a glass column with silica gel 60 (200 g, 0.063-0.200mm; Merck, Darmstadt, Germany) and Sephadex LH-20 (Lipophilic, 25-100 µm, Sigma).The mobile phase was n-hexane and ethyl acetate, and 12 fractions were obtained.Some fractions that were isolated from fractions L-F1 and L-F2 were studied by GC-MS.To analyze fatty acids by GC-MS, it was prepared by refluxing 20 mg of the isolated fractions with 20 mL CH 3 OH and 2 mL H 2 SO 4 for 4 h.

Extraction and Isolation of Fruits
The air-dried fruits (400.0 g) were macerated with ethanol 95% (1.5 L ×3 times) at room temperature.The ethanol extracts were combined, and the solvent was distilled under reduced pressure at low temperature to afford a 15.0 g yield.
The ethanol extract was processed using vacuum liquid chromatographic techniques with silica gel premium-grade C18 (40-63 µm; 60Å; Sorbtech, Norcross GA, USA) in a column.The extract was eluted using a gradient system with DCM, methanol, and water, with decreasing polarity as a mobile phase starting with 100% water and ending with 100% DCM, and 6 fractions were obtained.
Subsequently, the fractions were separated on Sephadex LH-20 (0.25-0.1 mm, GE Healthcare, Sweden) using methanol as the eluent.As a result, compounds 2, 5, 6, and 7 were obtained.Some fractions that were isolated from fractions B-F1, B-F2, B-F3, B-F4, and B-F5 were studied by GC-MS.To analyze fatty acids by GC-MS, it was prepared by refluxing 20 mg of the isolated fractions with 20 mL CH 3 OH and 2 mL H 2 SO 4 for 4 h.

Conclusions
Previously, this type of rosehip (Rosa beggeriana Schrenk) had not been studied in terms of chemical composition, hence there are no articles on isolated compounds from this plant.However, there are many research articles about species from the genus Rosa, which are very well known in traditional medicine.The fractionation of Rosa beggeriana Schrenk leaves and fruits resulted in the isolation and structural elucidation of seven compounds, including phytosterol, triterpenoids, polyphenol, and mixture of fatty acids.β-sitosterol (2), betulin (3), (+)-catechin (4), lupeol (5), ethyl linoleate (6) have already been isolated from the genus Rosa but not from Rosa beggeriana Schrenk.And compounds like 3β,23dihydroxyurs-12-ene (1) and ethyl linolenoate (7) were isolated for the first time for both Rosa and Rosa beggeriana Schrenk.

Table 1 .
GC-MS data for the leaves of Rosa beggeriana Schrenk.

Table 2 .
GC-MS data for the fruits of Rosa beggeriana Schrenk.