Oleanolic Acid Glycosides from Scabiosa caucasica and Scabiosa ochroleuca: Structural Analysis and Cytotoxicity

In the field of research on medicinal plants from the Armenian flora, the phytochemical study of two Scabiosa L. species, S. caucasica M. Bieb. and S. ochroleuca L. (Caprifoliaceae), has led to the isolation of five previously undescribed oleanolic acid glycosides from an aqueous-ethanolic extract of the roots: 3-O-α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid, 3-O-β-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. Their full structural elucidation required extensive 1D and 2D NMR experiments, as well as mass spectrometry analysis. For the biological activity of the bidesmosidic saponins and the monodesmosidic saponin, their cytotoxicity on a mouse colon cancer cell line (MC-38) was evaluated.


Introduction
As a developing country, Armenia still faces difficulty in combatting major diseases such as cancer, diabetes, and heart disease. According to the reports of the World Health Organization and the International Agency for Research on Cancer, the crude cancer incidence rate in 2020 was estimated to be 312.5 cases per 100,000 individuals in Armenia [1].
The complete structural analysis is detailed below for the five glycosides isolated from S. caucasica and S. ochroleuca.
The mass spectrum of compound 1 in ESIMS (positive mode) revealed a quasi-molecular ion peak at m/z 1683.7553 [M + Na] + , in agreement with the molecular formula, C76H124O39. This suggests a molecular mass of 1660.
The complete structural analysis is detailed below for the five glycosides isolated from S. caucasica and S. ochroleuca.  (Figures 1 and 2).
The mass spectrum of compound 2 in ESIMS (positive mode) showed a quasi-molecular ion peak at m/z 1815.8003 [M + Na] + , suggesting a molecular formula of C 81 H 132 O 43 , and a molecular mass of 1792. The difference of 132 u between 1 and 2 is related to a supplementary pentose moiety in the oligosaccharidic chain. The assignments of all the NMR signals of the molecule 2 revealed a structure almost superimposable to 1, except for the Glc II. In fact, the chemical shifts of the positions 2 and 4 were deshielded at δ C /δ H 76.7/3.44 and 76.9/3.74, respectively. HMBC correlations between δ H /δ C 4.38 (d, J = 7.8 Hz) (Xyl II H-1) and 76.7 (Glc II C-2), and between δ H /δ C 5.37 (s) (Rha II H-1) and 76.9 (Glc II C-4) proved the 2,4 disubstitution of the Glc II. Therefore, the structure of 2 is established as For compound 3, a quasi-molecular ion peak at m/z 1491.6949 [M + Na] + in ESIMS (positive mode) revealed a molecular formula of C 69 H 112 O 33 and a molecular mass of 1468. The main difference with 2, in the NMR spectra, is the value of δ C 180.2 ppm of the carbonyl function at C28, which suggests a carboxylic group instead of an ester group in compound 2. This was confirmed by the lack of NMR signals corresponding to the gentiobiose unit, 28-Oβ-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, which led to the conclusion of a monodesmosidic saponin,  13 C-and 1 H-NMR data of the aglycon part in compounds 1-5 in CD 3 OD *.   The saponins 1-5, found in S. caucasica and S. ochroleuca, have structural similarities with those found in S. semipapposa [10], S. stellata [11], and S. tschiliensis [12]. They share the same aglycone, an oleanolic acid with a gentiobiose unit attached to the C28 of oleanolic acid for 1, 2, 4, 5, and also share similar sets of the first three osidic units in the oligosaccharide chains attached to the C3 position, such as the sequence 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. Surprisingly, these structures are close to those isolated from Weigela species, such as W. stelzneri and W. × "Bristol Ruby" [4,5]. The Scabiosa and Weigela genera belong to the same Caprifoliaceae family of the Dipsacales order, but their subfamily is different-Dipsacoideae for Scabiosa and Diervilloideae for Weigela. From a chemotaxonomic point of view, we must continue our investigations to determine whether this type of sequence could represent a chemotaxonomic marker of these two subfamilies. Table 2. 13 C-and 1 H-NMR data of the osidic part, in compounds 1-5 in CD 3 OD *.  The cytotoxicity of the bidesmosidic saponins, 1, 2, 5, and the monodesmosidic, 3, was evaluated on a mouse colon cancer cell line (MC-38). Only compound 3 showed potent cytotoxicity, with an IC 50 of 4.37 µM, compared to 5-FU with an IC 50 of 11.7 µM. These results highlight the key role of the lack or presence of an esterification of the C-28 position (Figure 4). The cytotoxicity of saponins is often related to their ability to interact with membranes. This interaction seems to usually be lowered when the saponin possess a second sugar chain [6]. Among the tested molecules, compound 3 is the only monodesmosidic saponin. This would explain why compound 3 is active, compared with the bidesmosidic 2, which has the same structure but, with an additional sugar chain at the C-28 position. The other structural differences between the compounds are the number of sugars linked to the genin at the C3 position (6 or 7 sugars), and the nature and the linkage of the last three sugars of the oligosaccharidic chain. Those structural elements do not seem to influence their activity, as none of them are cytotoxic. The saponins 1-5, found in S. caucasica and S. ochroleuca, have structural similarities with those found in S. semipapposa [10], S. stellata [11], and S. tschiliensis [12]. They share the same aglycone, an oleanolic acid with a gentiobiose unit attached to the C28 of oleanolic acid for 1, 2, 4, 5, and also share similar sets of the first three osidic units in the oligosaccharide chains attached to the C3 position, such as the sequence 3-O-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. Surprisingly, these structures are close to those isolated from Weigela species, such as W. stelzneri and W. x "Bristol Ruby" [4,5]. The Scabiosa and Weigela genera belong to the same Caprifoliaceae family of the Dipsacales order, but their subfamily is different-Dipsacoideae for Scabiosa and Diervilloideae for Weigela. From a chemotaxonomic point of view, we must continue our investigations to determine whether this type of sequence could represent a chemotaxonomic marker of these two subfamilies.
The cytotoxicity of the bidesmosidic saponins, 1, 2, 5, and the monodesmosidic, 3, was evaluated on a mouse colon cancer cell line (MC-38). Only compound 3 showed potent cytotoxicity, with an IC50 of 4.37 µM, compared to 5-FU with an IC50 of 11.7 µM. These results highlight the key role of the lack or presence of an esterification of the C-28 position (Figure 4). The cytotoxicity of saponins is often related to their ability to interact with membranes. This interaction seems to usually be lowered when the saponin possess a second sugar chain [6]. Among the tested molecules, compound 3 is the only monodesmosidic saponin. This would explain why compound 3 is active, compared with the bidesmosidic 2, which has the same structure but, with an additional sugar chain at the C-28 position. The other structural differences between the compounds are the number of sugars linked to the genin at the C3 position (6 or 7 sugars), and the nature and the linkage of the last three sugars of the oligosaccharidic chain. Those structural elements do not seem to influence their activity, as none of them are cytotoxic.
3 mm triple resonance inverse and 3 mm dual broadband probe heads. Spectra were recorded in methanol-d 4 , and all spectra were recorded at T = 308.15 K. Pulse sequences were taken from the Varian pulse sequence library (gCOSY, gHSQCAD, and gHMBCAD with adiabatic pulses, CRISIS-HSQC and CRISIS-HMBC). TOCSY spectra were acquired using a DIPSI spin-lock and 150 ms mixing time.  8)). The spray reagent for saponins was vanillin reagent (1% vanillin in EtOH/H 2 SO 4 , 50:1). The HPLC was performed on an Agilent 1260 instrument, equipped with a degasser, a quaternary pump, a sample changer, and a UV detector (210 nm). The chromatographic separation for the analytical part was carried out on a C18 column (250 mm × 4.6 mm internal diameter, 5 µm; Phenomenex LUNA, Torrance, CA, USA) at room temperature and protected by a guard column. The mobile phase consists of (A) 0.01% (v/v) aqueous trifluoroacetic acid, and (B) acetonitrile delivered at 1 mL/min, going from 30% to 80% B in 30 min. The injection volume was 10 µL.

Acid Hydrolysis and Absolute Configuration Determination
An aliquot (150 mg) of a fraction-rich saponin was hydrolyzed with 2N aqueous CF 3 COOH (25 mL) for 3 h at 95 • C. After extraction with CH 2 Cl 2 (3 × 15 mL), the aqueous layer was evaporated to dryness with H 2 O until neutral, to produce the sugar residue (40 mg). Arabinose, glucose, xylose, and rhamnose were identified by comparison with authentic samples by TLC using CH 3 COOEt/CH 3 COOH/CH 3

Bioactivity Assay
MC-38 cells (mouse colon cancer, kerafast ® ENH204-FP) were seeded at the density of 2 × 10 4 cells in 96 well plates (Falcon, Merck), and cultivated for 24 h before treatments in an RPMI 1640 medium (Corning, Corning, NY, USA) with 10% FBS (Dutcher, Limassol, Cyprus). Cells were then treated with the isolated compounds at the concentrations from 1 to 50 µM, for 48 h in an RPMI 1640 medium without FBS (200 µL per well). Viability was measured using an MTS colorimetric assay (Abcam, Waltham, MA, USA), following the manufacturer's recommendations. Briefly, 20 µL of MTS reagent was added into each well, and optical density at 490 nm was read using a microplate reader (Spark ® , Männedorf, Switzerland, Tecan), after 1 h incubation at 37 • C, 5% CO 2 , in the dark. The results were expressed as concentrations of compounds producing 50% toxicity (IC 50 value).
Fluorouracil (5-FU) was used as positive control and exhibited IC 50 values of 11.7 µM. The experiment was conducted in quadruplicate.

Conclusions
The saponins, 1-5, found in S. caucasica and S. ochroleuca, possess an oleanolic acid as aglycon, a gentiobiose unit attached to C28 for 1, 2, 4, 5, and the same first three osidic units in the oligosaccharide chain attached to C3, such as the sequence, 3-O-β-Dxylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. These glycosides have structural similarities with those found not only in other Scabiosa species from the Dipsacoideae subfamily of the Caprifoliaceae, but also with Weigela species, from the Diervilloideae. From a chemotaxonomic point of view, we need to continue our investigations to determine whether this type of sequence could represent a chemotaxonomic marker of these two subfamilies. Compound 3 exhibits potent cytotoxicity on a mouse colon cancer cell line (MC-38), with an IC 50 of 4.37 µM, highlighting the key role of the lack or the presence of an esterification of the C-28 position.