Spirostanol Sapogenins and Saponins from Convallaria majalis L. Structural Characterization by 2D NMR, Theoretical GIAO DFT Calculations and Molecular Modeling

Two new spirostanol sapogenins (5β-spirost-25(27)-en-1β,2β,3β,5β-tetrol 3 and its 25,27-dihydro derivative, (25S)-spirostan-1β,2β,3β,5β-tetrol 4) and four new saponins were isolated from the roots and rhizomes of Convallaria majalis L. together with known sapogenins (isolated from Liliaceae): 5β-spirost-25(27)-en-1β,3β-diol 1, (25S)-spirostan-1β,3β-diol 2, 5β-spirost-25(27)-en-1β,3β,4β,5β-tetrol 5, (25S)-spirostan-1β,3β,4β,5β-tetrol 6, 5β-spirost-25(27)-en-1β,2β,3β,4β,5β-pentol 7 and (25S)-spirostan-1β,2β,3β,4β,5β-pentol 8. New steroidal saponins were found to be pentahydroxy 5-O-glycosides; 5β-spirost-25(27)-en-1β,2β,3β,4β,5β-pentol 5-O-β-galactopyranoside 9, 5β-spirost-25(27)-en-1β,2β,3β,4β,5β-pentol 5-O-β-arabinonoside 11, 5β-(25S)-spirostan-1β,2β,3β,4β,5β-pentol 5-O-galactoside 10 and 5β-(25S)-spirostan-1β,2β,3β,4β,5β-pentol 5-O-arabinoside 12 were isolated for the first time. The structures of those compounds were determined by NMR spectroscopy, including 2D COSY, HMBC, HSQC, NOESY, ROESY experiments, theoretical calculations of shielding constants by GIAO DFT, and mass spectrometry (FAB/LSI HR MS). An attempt was made to test biological activity, particularly as potential chemotherapeutic agents, using in silico methods. A set of 12 compounds was docked to the PDB structures of HER2 receptor and tubulin. The results indicated that diols have a higher affinity to the analyzed targets than tetrols and pentols. Two compounds (25S)-spirosten-1β,3β-diol 1 and 5β-spirost-25(27)-en-1β,2β,3β,4β,5β-pentol 5-O-galactoside 9 were selected for further evaluation of biological activity.


Introduction
Convallaria majalis from the family Liliaceae (lily of the valley) is widely distributed in Europe whereas Convallaria keisukei grows in East Asia. C. majalis is a known source of cardiac glycosides. However, non-cardiac substances, such as steroidal saponins, are also of pharmaceutical interest [1]. Steroidal saponins have hemolytic, insecticidal, antiparasitic, antifungal, antibacterial, antiviral, anti-inflammatory, antihyperlipidemic, antidiabetic and antitumor properties, among others [2]. Additionally, steroidal glycosides have a wide variety of commercial uses such as surfactants, foaming agents and precursors for the industrial production of pharmaceutical drugs [3].
It is worth noting that a pair of compounds with spirosten or spirostan moiety, such as 1 and 2, is probably present in the plant naturally. Yet, it cannot be excluded that spirostanol-type sapogenin came from the cyclization of the original glycoside with a furost-26-ol moiety during extraction and separation procedures. The chromatographic separation of steroid sapogenins bearing the same number of OH groups and separation of neoand iso-isomers (with a different configuration of the C-25 methyl group) is difficult in a mixture [24]. Tschesche et al. [25] reported in 1961 that 1 convallamarogenin and 2 dihydroconvallamarogenin are characterized by the same Rf values (0.50) in TLC, and dihydromarogenin was obtained by catalytic hydrogenation of convallamarogenin. Similarly, it was not easy to perform chromatographic isolation of pure spirostens or spirostans, which were present as admixtures in small quantities (of a few percent). Therefore, pure dihydro-derivatives (such as 4) were obtained by hydrogenation of the respective spirostens (on PtO2, for 2 days, at room temperature).
Compounds 1-12 were obtained as colorless crystals; unfortunately, our attempts to prepare single crystals suitable for X-ray diffraction studies were unsuccessful.
The LSI mass spectrum of 1 was consistent with the molecular formula C27H42O4, MS of 2 showed an [M + H] + ion at m/z = 433, which is higher than that of 1 by two mass units. The presence of two more hydrogens was suggestive of a saturated fragment (spirostan) and molecular formula C27H44O4. The melting points and the values of [α]D are in accordance with the data given by Tschesche et al. [25] for convallamarogenin 1 and dihydroconvallamarogenin 2.
It is worth noting that a pair of compounds with spirosten or spirostan moiety, such as 1 and 2, is probably present in the plant naturally. Yet, it cannot be excluded that spirostanol-type sapogenin came from the cyclization of the original glycoside with a furost-26-ol moiety during extraction and separation procedures. The chromatographic separation of steroid sapogenins bearing the same number of OH groups and separation of neo-and iso-isomers (with a different configuration of the C-25 methyl group) is difficult in a mixture [24]. Tschesche et al. [25] reported in 1961 that 1 convallamarogenin and 2 dihydroconvallamarogenin are characterized by the same R f values (0.50) in TLC, and dihydromarogenin was obtained by catalytic hydrogenation of convallamarogenin. Similarly, it was not easy to perform chromatographic isolation of pure spirostens or spirostans, which were present as admixtures in small quantities (of a few percent). Therefore, pure dihydroderivatives (such as 4) were obtained by hydrogenation of the respective spirostens (on PtO 2 , for 2 days, at room temperature).
Compounds 1-12 were obtained as colorless crystals; unfortunately, our attempts to prepare single crystals suitable for X-ray diffraction studies were unsuccessful.
The LSI mass spectrum of 1 was consistent with the molecular formula C 27 Table 1). The 2D HETCOR spectra allowed the identification of quaternary carbons (C- 10,13,22), directly coupled with 1 H-13 C pairs and axial/equatorial hydrogens of most methylene groups. The ambiguities, due to the number of protons with very similar chemical shifts (particularly in the region δ1.1 to 2.1 ppm) could be resolved by observation of the connectivities in the COSY and HMBC spectra. The 1 H and 13 C signals of 2 were observed essentially in the same positions as those of 1, except for the signals due to the E-and F-ring (Table 2).     [11]. The crosspeaks between C-22 and H-26 signals in the HMBC spectrum confirm the presence of ring F at C-22 (i.e., it is spirost-25(27)-en, not the furost-25(27)-en structure). The (25S)-spirosten-1β,3β-diol 1 has not been characterized yet by 1 H and 13 C NMR spectroscopy, whereas 13 C NMR spectrum of (25S)-spirostan-1β,3β-diol 2 (25,27-dihydro-convallamarogenin, also named rhodeasapogenin) was assigned by Tori et al. [13].  6 . The number of oxygen atoms, besides oxygens in the furan and spirostan rings, suggested that this compound should have four OH groups. In the 1 H NMR spectra the H-27 signal appeared at 4.76/4.79 ppm, and the signals of three methyl groups: Me-18, Me-19 and Me-21 (doublet) can be observed. The 13 C NMR spectrum of 3 included 27 intense signals: 4× CH 3 , 9× CH 2 and 10× CH, according to DEPT experiment; standard 13 C spectra showed a double bond between C-25 (δ142.9) and C-27 (δ108.2). The order of methyl carbons was unusual since C-19 resonance appeared at 12.3 ppm, whereas in the spectra of 1 and 2-at 18.9 ppm. A strong shielding effect (6.6 ppm) resulted from nearby β-hydroxyl groups [26,27]. The stereochemistry at C-5 and other carbons of ring A was confirmed by the NOESY spectrum. The cross-peaks H-1/H-11, H-2/H-9 or H-9/H-4 indicate the cis-fusion of the A/B rings. Using the 1 H-13 C correlation (HETCOR), the signals which appeared between 65 and 77 ppm were assigned to the carbons bearing the OH groups. The configuration of hydroxyl groups at C-1 and C-3 was determined to be β (axial) from the multiplicity of the H-1 (a broad singlet), H-2 (triplet-like, 3J < 3.5 Hz) and H-3 (a broad singlet). The strong cross peak in the NOESY spectrum between H-2 and H-9α shows that H-2 is on the α-side. The cross peak H-9α and H-4 at 2.39 ppm (and the absence of the peak H-9α/H-4 δ1.85) can be understood assuming a reverse assignment of the H-4 axial/equatorial pair, since H-4α, axial and H-9α are proximal.
Inspection of NMR data shows that 1 H and 13 C chemical shifts for 3 and 4 are the same for the A, B and C rings (Tables 1 and 3); this is also true of other spirosten/spirostan pairs. On the other hand, spirostans exhibit similar spectra within fragments assigned to the D, E and F rings, especially the pattern of long-range correlations in the HMBC spectra. These correlations (Table 3) are sufficient to identify the spirostanol structure of the F-ring.
The 25S configuration of 4 can be deduced from the analysis of 13 C chemical shifts of C-25 to C-27. These values are in agreement with assignments of the F-ring carbon signals of 25S-spirostans reported earlier [13,28].
The  (27)-spirosten structure. However, the analysis of NMR spectra clearly showed that the hydroxylation pattern was different from that of 3 or 4. The connectivities observed in the 1 H-1 H COSY and 1 H-13 C HMBC spectra allowed the identification of four carbons bearing hydroxyl groups as C1, C3, C4 and C5 whereas C2 was a methylene carbon. Thus, the structure of 5 was determined as 5β-spirost-25 (27)-en-1β,3β,4β,5β-tetrol.
The 13 C NMR spectrum of 6 was very similar to that of 5 except the signals due to the F-ring (C22-C27, see Table 4). The resonance of Me-27 (3H doublet in the 1 H spectrum at 10.6 ppm) at 15.9 ppm and the resonances of C23 and C24 at ca. 26 ppm evidenced 25Sspirostanol configuration. Compound 6 was found to be 25S-spirostan-1β,3β,4β,5β-tetrol, identical with convallagenin-B (III) isolated from C. keisukei [5].   Mass spectrometry data suggested that the molecular formula of 7 and 8 was higher by one oxygen atom than that of 3-6. The 1 H spectrum of 7 showed three signals of methyl groups (Me-18, Me-19 and Me-21) and four signals of H1-H4 from methine groups. In the 13 C spectra five signals of oxygenated carbons (including quaternary one at δ 78.7) were observed between 67 and 80 ppm. This suggested that there are five hydroxyl groups in the A-ring part. Characteristic signals of a spirost-25 (27)-en at δ144.2 (C25) and 108.9 (C27) and the analysis of COSY and HMBC correlations indicated that the F-ring does not have a furostane-type structure. Compound 7 was finally assigned as 5β-spirost-25 (27)-en-1β,2β,3β,4β,5β-pentol; previously isolated from the underground part of Aspidistra elatior BLUME (Liliaceae) by Hirai [14].
Unambiguous assignments of 1 H and 13 C NMR signals of 8 were made using COSY and HMBC correlations ( Table 2). Chemical shifts of the A-E rings are the same as for 7, except those belonging to the F-ring. The 1 H spectrum showed four methyl signals, including a doublet of Me-27. In the HMBC spectrum carbon signals at δ26.6 (C24), 28.2 (C25) and 65.6 (C26) were correlated with Me-27. 13 C chemical shifts of C-22 (δ110.5) and C-27 (δ16.5) showed clearly that it was a 25S spirostanol derivative. Comparing the NMR data of 8 with those of a known compound neopentologenin, the 13 C chemical shifts were found to be identical or very similar, suggesting that they had the same configuration of the F-ring (Table 4). On the basis of those data, 8 was identified as (25S)-spirostan-1β,2β,3β,4β,5β-pentol; it was isolated from Aspidistra elatior by Konishi [15].
The fraction (I) which was subjected to a series of chromatographic separations without hydrolysis gave compounds 9-12. The NMR spectra of those saponins were measured in pyridine-d5 to minimize the signal overlap. The molecular formula of compound 9 was determined as C 33 H 52 O 12 by the pseudo-molecular peak at m/z = 663.3 [M + Na] + in the positive FAB MS. Acid hydrolysis and TLC analysis confirmed that the sugar was galactose. The 1 H NMR spectrum showed signals for two angular methyl groups at δ 0.82 ppm and 1.72 ppm (singlets), a doublet at 1.07 ppm (Me-21), and one anomeric proton signal at δ 5.23 ppm, suggesting 9 to be a spirostanol glycoside. The signals of four oxygenated carbons appear in the range 67-78 ppm and that of C5, linked to sugar, at 88 ppm (Table 5). This means that the aglycone is pentahydroxylated spirostanol 7. In the HMBC spectrum, the correlation between the anomeric proton of galactose and the quaternary carbon signal at 87.9 ppm showed that the sugar was linked to C5. The signal of C5 was shifted by 9.2 ppm downfield and the C1, C4, C6 carbon signals were shifted by 0.7-5.4 ppm upfield in comparison with those of 7, in agreement with the glycosidation shifts. The 1 H and 13 C NMR data of sugar moiety were similar to the data determined previously [29]. Compound 10 was identified to be (25S)-pentahydroxyspirostanol 5-β-D-galactoside. The FAB-MS showed an [M + Na] + ion at 665.3; for C 33 H 54 O 12 Na. The 1 H NMR spectrum showed signals for two angular methyl groups, two doublets (Me-21 and Me-27) and one anomeric proton signal. The COSY, HMBC and NOESY correlations confirmed the spirostanol structure of the F-ring and penta-hydroxylation of the A-ring.
The molecular formula of 11 was established as C 32 H 50 O 11 by FAB-MS (m/z 633.3 [M + Na] + ). Acid hydrolysis yielded arabinose and a genin, identified as 8. A cross peak between H1 (δ 5.23 ppm) of arabinose and the quaternary C5 signal (δ 86.9) in the HMBC spectrum provided definite evidence for an ester linkage. Interpretation of NMR spectra and comparison with the reported data [23] confirmed the pentahydroxyspirosten structure.
Saponin 12 was assigned the molecular formula C 32 H 52 O 11 by FAB-MS ([M + Na] + at m/z 635.3). The 1 H and 13 C NMR data (Tables 1 and 5) demonstrated that the aglycon of 12 was pentahydroxyspirostanol, similar to that of 10, but bearing a pentose unit.
The chemical shift of C6 is informative-this signal appeared at 34.3 ppm in compounds with methylene carbon C4 (3 and 4), and the substitution with 4β-OH results in a ca. 4 ppm upfield shift (δ30.4 in 7, 8); in saponins 9-12 with sugar linked to C5 a further increase in shielding occurs (δ24.4-25.0) due to the steric hindrance.
Preliminary investigations of biological activity using in silico methods were then performed. Due to potential antiproliferative activity of saponins, two targets were chosen to verify possible mechanisms: HER2 receptor and tubulin. A set of 12 ligands was docked to the PDB structures. Based on the docking results, the compounds were ranked by comparing total score values and each received RANK value according to this order ( Table 6).

Discussion
Compounds 1-12 isolated from C. majalis are all polyhydroxylated steroidal saponins with 5β-H, 5β-OH or 5β-O-sugar as the common structural feature.
Steroidal saponins with sugar moiety attached to the angular position of C5 were first found in C. keisukei by  (convallasaponin-B, formulated as convallagenin-B α-L-arabinopyranoside). It is worth noting that in a series of studies on saponins of Japanese Convallaria keisukei no spirost-25(27)-ens were reported. The spirostanol-type sapogenins with two, three and four hydroxyl groups at ring A were isolated from both C. keisukei and C. majalis ( Table 3). The negative Cotton effects were consistent with the A/B-cis ring fusion, and the S configuration at C-25 [30]. All hydroxyl groups, including 5β-OH, were considered to have β-configuration.
The molecules of sapogenins show little conformational freedom, except the reorientation of the OH groups in the A-ring. Theoretical calculations of NMR shielding constants (see Tables 3-5) allowed the assignment of all 13 C resonances in the 13 C NMR spectra by comparing the experimental chemical shift values with the theoretical NMR parameters. It is possible by converting the shielding constants into chemical shift using the formula: δiso = σTMS-σiso, where the calculated value of the shielding constant relates to the tetramethylsilane shielding constant (TMS). Since the interpretation of the A-and F-ring signals is difficult and ambiguous, GIAO DFT calculations were especially helpful; these data may complement or even replace classic assignments using 2D NMR.
This chapter deals with investigations of biological activity using in silico methods. Figure 2 illustrates the mean RANK value calculated for diols, tetrols and pentols. An earlier study of steroidal glycosides from Convallaria majalis showed that the introduction of polar substituents to the steroidal nuclei resulted in reduced cytotoxicity [32]. Our docking results suggest that diols (less polar) have a higher affinity to the analyzed targets than tetrols and pentols (more polar). However, not only the structures of the aglycone moiety but also the sugar sequences in the steroidal glycosides may affect its affinity. All glycosides were 5-hydroxy substituted, 9 and 11 had the same aglycone moiety as 10 and 12. Compound 9 (galactoside) had RANK position from docking to HER2 and tubulin 2 and 1, respectively, whereas compound 10 (also galactoside) had 12 and 15 positions. Two arabinosides 11 and 12 have 6 and 2 RANK and 4 and 6 RANK positions, respectively. It shows that the sugar type in glycosides is also related to the binding effect.
The molecules of sapogenins show little conformational freedom, except the reorientation of the OH groups in the A-ring. Theoretical calculations of NMR shielding constants (see Tables 3-5) allowed the assignment of all 13 C resonances in the 13 C NMR spectra by comparing the experimental chemical shift values with the theoretical NMR parameters. It is possible by converting the shielding constants into chemical shift using the formula: δiso = σTMS-σiso, where the calculated value of the shielding constant relates to the tetramethylsilane shielding constant (TMS). Since the interpretation of the A-and F-ring signals is difficult and ambiguous, GIAO DFT calculations were especially helpful; these data may complement or even replace classic assignments using 2D NMR.
This chapter deals with investigations of biological activity using in silico methods. Figure 2 illustrates the mean RANK value calculated for diols, tetrols and pentols. An earlier study of steroidal glycosides from Convallaria majalis showed that the introduction of polar substituents to the steroidal nuclei resulted in reduced cytotoxicity [32]. Our docking results suggest that diols (less polar) have a higher affinity to the analyzed targets than tetrols and pentols (more polar). However, not only the structures of the aglycone moiety but also the sugar sequences in the steroidal glycosides may affect its affinity. All glycosides were 5-hydroxy substituted, 9 and 11 had the same aglycone moiety as 10 and 12. Compound 9 (galactoside) had RANK position from docking to HER2 and tubulin 2 and 1, respectively, whereas compound 10 (also galactoside) had 12 and 15 positions. Two arabinosides 11 and 12 have 6 and 2 RANK and 4 and 6 RANK positions, respectively. It shows that the sugar type in glycosides is also related to the binding effect. Molecular docking shows that compounds 1 and 9 should be selected for further evaluation of biological activity due to their best RANK values for both molecular targets. Molecular docking shows that compounds 1 and 9 should be selected for further evaluation of biological activity due to their best RANK values for both molecular targets. Inspired by the study of Abd El-kader et al. [33] we analyzed the binding mode of the compounds with HER2 receptor and investigated their similarity to doxycycline.
Compound 1 forms one hydrogen bond with Cys773, whereas 9 does not incorporate H-bond in the active site of HER2 receptor. However, both compounds may show hydrophobic interactions with amino acids residues in the binding pocket, such as Leu694, Val702, Gly692, Phe699, Arg817, Asp776, Leu820 (Figure 3). Our results are in agreement with the results of Abd El-kader et al. [33]. Since their docking results were confirmed by biological experiments, we could expect similar effects. Further evaluation of biological activity of the hydroxylated saponins is in progress.
Compound 1 forms one hydrogen bond with Cys773, whereas 9 does not incorporate H-bond in the active site of HER2 receptor. However, both compounds may show hydrophobic interactions with amino acids residues in the binding pocket, such as Leu694, Val702, Gly692, Phe699, Arg817, Asp776, Leu820 (Figure 3). Our results are in agreement with the results of Abd El-kader et al. [33]. Since their docking results were confirmed by biological experiments, we could expect similar effects. Further evaluation of biological activity of the hydroxylated saponins is in progress.

General Experimental Procedures
Optical rotation was measured using a JASCO P-1020 polarimeter at 20 °C. An AMD-604 mass spectrometer was used for LSI-MS and FAB MS. NMR analysis: 1 H and 13 C NMR spectra were recorded on a Bruker DRX-500 spectrometer; the spectra of 1, 2 in CDCl3 and 3-12 in CDCl3 + CD3OD (v:v, 1:1) solution. The spectra of saponins 8-12 were also run for pyridin-d5 solutions. The 2D 1 H-13 C correlations were performed using the phase-sensitive gradient-selected HSQC inverse technique; the HMBC experiment was optimized for J = 5 Hz. Standard pulse programs from Bruker library were used for 1 H-1 H COSY, NOESY and ROESY experiments. The assignment of 13 C and 1 H chemical shifts was performed based on 2 D experiments. Chemical shifts were reported in ppm relative to internal TMS.

General Experimental Procedures
Optical rotation was measured using a JASCO P-1020 polarimeter at 20 • C. An AMD-604 mass spectrometer was used for LSI-MS and FAB MS. NMR analysis: 1 H and 13 C NMR spectra were recorded on a Bruker DRX-500 spectrometer; the spectra of 1, 2 in CDCl 3 and 3-12 in CDCl 3 + CD 3 OD (v:v, 1:1) solution. The spectra of saponins 8-12 were also run for pyridin-d5 solutions. The 2D 1 H-13 C correlations were performed using the phase-sensitive gradient-selected HSQC inverse technique; the HMBC experiment was optimized for J = 5 Hz. Standard pulse programs from Bruker library were used for 1 H-1 H COSY, NOESY and ROESY experiments. The assignment of 13

Plant Material
The roots and rhizomes of C. majalis were collected in Poland, from the garden of the Agriculture University of Warsaw (SGGW, 51.82 • N, 19.90 • E). The voucher specimen is deposited at the Department of Pharmacognosy, The Medical University of Warsaw.
Extraction and separation.
The dried rhizomes and roots (700 g) were powdered, macerated with methanol-water (v:v, 1:1) and extracted twice (1.5 L × 2) for 24 h. The MeOH/water solution was combined, filtered off and concentrated under reduced pressure to 1/3 of its volume. The residue was extracted with CHCl 3 to give (I) and then with n-BuOH + H 2 O (II). The butanolic fraction was evaporated to dryness and the residue (9.6 g) was subjected to acid hydrolysis.

Acid Hydrolysis of the Butanolic Fraction
A solution in 1 M HCl in MeOH (100 mL) was refluxed for 8 h on a boiling water bath. After cooling the reaction mixture was neutralized and submitted to partition into sugar and aglycone fractions.

Catalytic Hydrogenation
The spirosten (e.g., 3) was dissolved in MeOH and hydrogenated at room temperature over PtO 2 for 2 days; the catalyst was removed by filtration and the solution was evaporated to dryness. The presence of spirostane was confirmed by 13 C NMR spectra (lack of C-25 resonance at δ > 140 ppm).

Theoretical Calculations
The molecules were first optimized at the PM3 level. Quantum-chemical calculations were carried out using Gaussian 09 software [34]. Further optimizations were performed using the DFT method (B3LYP 6-31G(d,p)). At the same level of theory, vibrational frequencies and intensities were computed. The final low-energy structures with positive vibrational frequencies were used for calculations of NMR shielding constants.
Then, the experimental chemical shift values (δ iso ) were compared with the theoretical NMR parameters. It was possible by converting the shielding constants (σ iso ) into chemical shift using the formula: δ iso = σ TMS -σ iso , where the calculated value of the shielding constant relates to the tetramethylsilane shielding constant (σ TMS = 191.8 ppm) calculated at B3LYP 6-31G(d,p).
Linear regression was determined for the relationship of the experimental chemical shifts and the calculated theoretical chemical shifts. The correlation between the experimental and computational results is presented in the form of the R 2 correlation coefficient at the bottom of Tables 3-5.
The correlation of the calculated chemical shifts with experimental data in each case is satisfactory because R 2 > 0.99. Therefore, as a second criterion of the quality of calculations was chosen the mean absolute error (MAE) between theoretical and experimental data and enclosed at the bottom of Tables 3-5. The combination of the R 2 coefficient and the MAE error in our previous studies gave satisfactory results in assessing the quality of the theoretically obtained structures in relation to the experimental NMR data [35].
The calculations allowed to verify and confirm the correct assignment of signals based on 2D spectra.

Molecular Docking
The molecular docking of sapogenins and saponins against tubulin protein and HER2 receptor was carried out using Sybyl X 1.2 (Tripos International) software. The structures were drawn in Sybyl-X 1.2 Sketch, then hydrogens were added, and finally, the structures were optimized (Tripos forcefield, gradient 0.05 kcal/mol*A). Molecular target (HER2 receptor and tubulin) was taken from RSC Protein Data Bank (PDB id: 5JEB and 1SA0, respectively). The structures were prepared by removing water, adding hydrogens, and performing the optimization using Sybyl structure preparation tools. Surflex protomols (an idealized active site ligand) were defined based on the ligand position in the crystal structures from PDB. Other parameters of Surflex were used as default values. The best docking poses were chosen according to total score values.

Conclusions
Steroidal saponins have a wide range of biological properties, besides those known for decades, including antimicrobial, anti-inflammatory and anticancer. New spirostanol sapogenins and saponins were isolated from the roots and rhizomes of Convallaria majalis L. New steroidal saponins were found to be tetra-or pentahydroxy 5 β-O-glycosides, with all OH groups on the same side of the A ring, bearing arabinose or galactose unit. Theoretical calculations of NMR shielding constants allowed the assignment of all 13 C resonances in the 13 C NMR spectra. GIAO DFT calculations were especially helpful; these data may complement or even replace classic assignments using 2D NMR. The molecular activity of different saponins is attributed to their structural composition, e.g., the number of OH groups. Our docking results suggest that diols (less polar) have a higher affinity to the analyzed targets than tetrols and pentols (more polar). The sugar moiety contributes to heteropolarity of saponins, and may lead to different bioactivity.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.