Flavonoid Glycosides from Endemic Bulgarian Astragalus aitosensis (Ivanisch.)

Astragalus is a very interesting plant genus, well-known for its content of flavonoids, triterpenes and polysaccharides. Its secondary metabolites are described as biologically active compounds showing several activities, e.g., immunomodulating, antibacterial, antiviral and hepatoprotective. This inspired us to analyze the Bulgarian endemic A. aitosensis (Ivanisch.) to obtain deeper information about its phenolic components. We used extensive chromatographic separation of A. aitosensis extract to obtain seven phenolic compounds (1–7), which were identified using combined LC-MS and NMR spectral studies. The 1D and 2D NMR analyses and HR-MS allowed us to resolve the structures of known compounds 5–7 as isorhamnetin-3-O-robinobioside, isorhamnetin-3-O-(2,6-di-O-α-rhamno-pyranosyl-β-galactopyranoside), and alangiflavoside, respectively, and further comparison of these spectral data with available literature helped us with structural analysis of newly described flavonoid glycosides 1–4. These were described in plant source for the first time.


Introduction
Genus Astragalus comprises from more than 2500 species, which makes it the largest genus in the family Fabaceae. Astragalus species are cosmopolitans, widely distributed in dry and semi-dry regions, mainly in the temperate regions of the Northern hemisphere [1]. Certain sources assign genus Astragalus as the largest genus of flowering plants [2]. About 133 species are distributed in Europe [3], and 29 have been identified in Bulgaria [4,5]. Fourteen Astragalus species from the Bulgarian flora are protected by the Bulgarian Biodiversity Act and they are included in the Red List of Bulgaria, as is the species A. aitosensis, the focus in this investigation [4].

Isolation of Compounds
The aerial parts of A. aitosensis were extracted with 80% methanol under reflux. The preliminary chromatographic analysis showed a bunch of signals of flavonoid compounds, with retention times predicting a high degree of glycosylation ( Figure 1). is a low, spiny, tussock-forming shrub with strongly branched stems (30-50 cm in height) [8]. The plant grows in dry stony places (90-550 m alt) with neutral to alkaline soil. It is distributed only in the suburbs of the small Bulgarian town Aytos, which gives rise to its name. From pharmacological point of view, Astragalus species are well-known and widely used as remedies in the traditional folk medicine of different countries, but only about 100 species from the genus are researched for their phytochemical composition and properties. Their activity described in the literature is a result of the presence of flavonoids, saponins, and polysaccharides [9,10], which endue these plants with immunomodulating, antibacterial, antiviral, hepatoprotective and other protective pharmacological effects [1, 9,10].
The aim of this study was to isolate content compounds of the methanolic extract from aerial parts of A. aitosensis and preform their structural elucidation using 1 H, 13 C, COSY, HSQC, HMBC, NOESY, and TOCSY NMR experiments. HR-ESI-MS was used for additional confirmation of the structures revealed by NMR. We report here the isolation and structural elucidation of six isorhamnetin and one kaempferol glycosides 1−7, four of which are new natural glycosides: three tetra-(1-3) and one triglycoside (4). The other three already known structures were determined for the first time in A. aitosensis: with two (5), three (6) and four (7) sugar units, respectively.

Isolation of Compounds
The aerial parts of A. aitosensis were extracted with 80% methanol under reflux. The preliminary chromatographic analysis showed a bunch of signals of flavonoid compounds, with retention times predicting a high degree of glycosylation ( Figure 1).  The crude extract was therefore defatted by liquid−liquid partitioning with chloroform and further fractionated via successive column chromatography with final step of semi-preparative HPLC purification of the isolated compounds ( Figure 2). Their nature (UV spectral properties) and the behavior of these compounds during separation on reversed phase-the polar character of the compounds-gave us the idea of flavonoid glycosides with a high glycosylation pattern. further fractionated via successive column chromatography with final step of semi-preparative HPLC purification of the isolated compounds ( Figure 2). Their nature (UV spectral properties) and the behavior of these compounds during separation on reversed phase-the polar character of the compounds-gave us the idea of flavonoid glycosides with a high glycosylation pattern.

Structural Analysis
To the best of our knowledge, spectral data of four of the isolated compounds (1-4) did not correspond to the data of compounds previously published in the literature. The 1D and 2D-NMR analysis, HR-MS and comparison with literature allowed us to resolve the structures of compounds further fractionated via successive column chromatography with final step of semi-preparative HPLC purification of the isolated compounds ( Figure 2). Their nature (UV spectral properties) and the behavior of these compounds during separation on reversed phase-the polar character of the compounds-gave us the idea of flavonoid glycosides with a high glycosylation pattern.

Structural Analysis
To the best of our knowledge, spectral data of four of the isolated compounds (1-4) did not correspond to the data of compounds previously published in the literature. The 1D and 2D-NMR analysis, HR-MS and comparison with literature allowed us to resolve the structures of compounds

Structural Analysis
To the best of our knowledge, spectral data of four of the isolated compounds (1-4) did not correspond to the data of compounds previously published in the literature. The 1D and 2D-NMR analysis, HR-MS and comparison with literature allowed us to resolve the structures of compounds 5-7 as isorhamnetin-3-O-robinobioside [12], isorhamnetin-3-O-(2,6-di-O-αrhamno-pyranosyl-β-galactopyranoside) [13,14], and alangiflavoside [15], respectively. The further comparison of these spectral data with available literature helped us with structural analysis of newly described flavonoid glycosides 1-4. For a detailed description, please see Supplementary Materials, Figures S1-S77. The aglycones for compounds 1-6 were determined based on HR-ESI-MS and NMR ( 1 H, 13 C, COSY, HSQC and HMBC) spectral analysis. 1 H and 13 C spectra are shown in Table 1. HMBC spectra showed the following significant correlations: proton at C-2 was a doublet with meta coupling and, in the HMBC, it showed strong correlation to C-4 and weak to C-3 . The proton at C-6 was observed as doublet of doublet (ortho and meta coupling) and showed strong correlation in the HMBC to C-4 . The proton at C-5 was a doublet with ortho coupling, displaying in the HMBC the strong correlation to C-3 and weak to C-4 . The methoxy group showed correlation to C-3 , and, therefore, the position of the methoxy is at C-3 and the aglycone of compounds 1-6 was finally identified as 3 -O-methylquercetin, syn. isorhamnetin [16]. Because of the identification procedure, we describe the elucidation of structures of known compounds prior to the new compounds. Detailed 1 H and 13 C chemical shifts for compound 5 are listed in Tables 2 and 3 Tables 2 and 3 respectively. NMR data of compound 7 and mass spectral analysis results show a good accordance with the data already reported for alangiflavoside [15].
Based on the previously described analysis of known compounds, we tried to identify the other isolated compounds, which showed differences from those previously described in the literature. Some of the fragmentation MS/MS results used for the identification of compounds 1-4 are depicted in Figure 4.  Figure 4). After lining up interpretation of NMR spectra (Table 1) with MS analysis, the structure of compound 1 was elucidated to be the isorhamnetin substituted by four sugars. One of them is a 6-deoxyhexose (a methyl group as doublet), two are hexoses (a glucose, a galactose), and one is a pentose. DEPT spectrum of compound 1 showed four methylene groups. Altogether, two of them belong to each hexose (the glucose and galactose), respectively, and since a 6-deoxyhesose (expected rhamnose) does not have a methylene moiety, the two left CH 2 moieties must belong to a pentose. Four anomeric protons are found in HMBC spectrum: δ H 5.57 ppm, (1H, d, J = 7.84 Hz), δ H 5.43 ppm (1H, d, J = 1.55 Hz), δ H 4.50 ppm (1H, d, J = 1.56 Hz) and δ H 5.06 ppm (1H, d, J = 7.53 Hz), corresponding to carbon atoms with δ C 99.8 ppm, δ C 109.2 ppm, δ C 100.5 ppm and δ C 100.1 ppm from the HSQC spectrum. After complete resonance assignments, analyses of coupling constants, intensities, interpretation of cross-peaks in the COSY spectrum, and 13 C-NMR chemical shift values, one hexose moiety was identified as a β-glucosyl unit, the second as a β-galactosyl moiety, the 6-deoxysugar was identified to be the α-rhamnose, and the pentose was recognized as the β-apiose, which contains two of the above-mentioned methylene groups. 13 C values of C-6 indicate that β-glucose residue is not connected to other sugar unit (δ C 61.1 ppm), while the β-galactose moiety is connected at C-6 position (δ C 66.0 ppm-shifted to a higher field). HMBC correlations allowed us to elucidate the precise structure of the sugar chains and the positions of their attachment to the aglycone. The anomeric proton (δ H 5.06 ppm) of the β-glucose moiety showed a three-bond correlation to C-7 (δ C 163.0 ppm) of the aglycone, while a HMBC correlation between the anomeric proton of the galactose (δ H 5.57 ppm) and the carbon at δ C 133.6 ppm indicated that the galactosyl unit is connected at C-3 towards the aglycone. The anomeric proton of α-rhamnose correlated to C-6 in the β-galactose (δ C 66.0 ppm). The anomeric atom of the last pentose sugar (apiose) δ H 5.43 ppm bonded to δ C 109.2 ppm showed a HMBC correlation with C-2 of the β-galactose molecule (δ C 75.1 ppm). The remaining two methylene groups (δ C 74.2 ppm and δ C 65.1 ppm) were recognized as carbons C-4 and C-5 in the apiosyl moiety.   Compound 2 was obtained as a yellow amorphous powder. The spectral analysis showed data very similar to those observed for compound 1 (Table 1), and the only difference found was the presence of a β-glucosyl residue connected to the sugar chain, attached to the 3-O position (particularly C-5 of apiosyl residue); a connection to the 7-O position of the aglycone was not found. Further interpretation of HRESIMS supported this suggestion by observing a lacking of the fragment of a hexose loss (m/z 162.0235), which can be observed in spectra of all compounds possessing glucose attached at 7-O position (compounds 1, 3, 4 and 7).  Table 3). The compound was therefore identified as isorhamnetin Together with an interpretation of NMR spectra (Tables 2 and 3), the structure was predicted to be composed of isorhamentin and four sugar moieties. Two of them were recognized as 6-deoxyhexoses (two methyl groups in the form of the doublet), and the other two were hexoses (possibly a glucose, or a galactose). DEPT spectrum showed their two methylene groups (δ C 61.1 ppm and 65.9 ppm) that belonged to the galactose and the glucose, respectively. We observed four anomeric protons in HSQC spectrum: δ H 5.79 ppm, (1H, d, J = 7.84 Hz), δ H 5.16 ppm (1H, d, J = 1.52 Hz), δ H 4.53 ppm (1H, d, J = 1.53 Hz) and δ H 5.06 ppm (1H, d, J = 7.24 Hz), corresponding to carbon atoms δ C 99.3 ppm, 101.4 ppm, 100.6 ppm and 100.1 ppm, respectively. After complete resonance assignments and analyses of coupling constants, intensities of cross-peaks in the COSY spectrum, and 13 C-NMR chemical shift values, one hexose moiety was identified as a β-glucosyl unit, the other as a β-galactosyl moiety, and the 6-deoxy sugars were found to be α-rhamnosyl moieties. 13 C values indicated that β-glucosyl residue is free at C-6 (δ C 61.1 ppm), while the β-galactose is bonded to C-6 (δ C 65.9 ppm-shifted to a higher field). According to HMBC correlations, structure of the side chains and their attachment to the aglycone were established. Anomeric proton (δ H 5.06 ppm) of the glucose moiety showed a three-bond correlation to C-7 (δ C 163.0 ppm) of the aglycone, while an HMBC correlation between the anomeric proton of the β-galactosyl moiety (δ H 5.79 ppm) and the carbon at δ C 133.3 ppm indicated that the β-galactosyl unit is bonded at C-3 toward the aglycone. The anomeric proton of one of the α-rhamnose residues (δ H 5.16 ppm) was correlated to position C-2 of the β-galactose (δ C 76.4 ppm), while the anomeric proton of the other α-rhamnose showed correlation to C-6 of the β-galactose (δ C 65.9 ppm). Methyl residues of α-rhamnopyranosyl residues were located in the low-field region of 1 H spectrum at δ H 0.89 ppm, (3H, d, J = 6.25 Hz) for the α-rhamnosyl residue attached to C-2 of the β-galactose and at δ H 1.16 ppm (3H, d, J = 6.08 Hz) for the α-rhamnosyl residue attached to C-6 of the β-galactose, respectively. Hence, we identified compound 3 as the new natural product isorhamnetin
Genus Astragalus is one of the largest genera of Fabaceae family. As mentioned, bioactivity of Astragalus plants are connected with a presence of flavonoids, saponins and polysaccharides. The use of Astragalus spp. is mainly connected with immunomodulation, antibacterial and antiviral activity, and hepatoprotection [9,10]. The reviews of Gorai et al. [16], Bratkov et al. [10], and Li et al. [17] show an overview of Astragalus genera and flavonoids isolated, showing the presence of flavones, flavonols, flavanones, flavan-4-ols, isoflavones, isoflavans, pterocarpans and others in 60 different Astragalus species. Their reviews also include isorhamnetin and kaempferol glycosides. Another comprehensive review of Bulgarian Astragalus species, published in 2016 [18], similarly describes the presence of several kaempferol and isorhamnetin glycosides, including alangiflavoside from A. monspessulanus ssp. monspessulanus [18,19].
In recent years, Bulgarian researchers isolated and reported new tri-and tetraglycosides of flavonols, including some new compounds from the rarely-met group of flavo-alkaloids [10,11]. Many species of Astragalus possess in nature the widely-distributed aglycones-kaempferol, quercetin and methylquercetines-in their free and glycosidic forms [10]. A. aitosensis has previously shown presence of rutin, quercetin-3-O-β-D-glucoside and astragalin [7]. As visible from comparison of glycosides isolated from A. aitosensis with the literature, similar compounds-glycosides-were obtained for example from A. monspessulanus ssp. monspessulanus, A. cicer and A. centralpinus [18][19][20]. This may, from chemotaxonomic point of view, confirm their close relationships.