Isolation and identification of a new saponin from Cephalaria aytachii

Abstract A new hederagenin-type triterpenoid glycoside (1) named Aytachoside A, along with eight known triterpene glycosides, were isolated from the aerial parts of Cephalaria aytachii Gokturk and Sumbul (Dipsacaceae). The structures of compounds 1–9 were determined by spectroscopic (1D and 2D NMR, HRESIMS) and chemical examinations. The antimicrobial effect of compound 1 was found considerably active against Escherichia coli, Pseudomonas aeruginosa and especially Salmonella typhimurium microorganisms using the MIC method. Although compound 1 was found not to have a remarkable toxic effect at a concentration lower than 300 μg/mL, cytotoxic activity tests demonstrated that prosapogenin 1a exhibits a significant cytotoxic activity against HeLa cell lines using the MTT assay for the first time.

The coupling constants showed two β-glycosidic linkages for two glucose units and three α-glycosidic linkages for two rhamnose and one arabinose units. This fact is also confirmed by way of acidic hydrolysis and GC-MS analysis. Acid hydrolysis and GC-MS analysis revealed that compound 1 has two D-glucose, one L-arabinose and two L-rhamnose units. The HMBC correlations between H-1 of arabinose at δ H 4.31 and C-3 of aglycone at δ C 79.8 and between carbonyl carbon at δ C 175.7 and H-1 of glucose II at δ H 5.20, indicated that the glycosidic chains were located at C-3 and C-28 of aglycone, respectively. The other HMBC correlations between H-1 of rhamnose I at δ H 5.09 and C-2 of arabinose at δ C 74.5, between H-1 of glucose I at δ H 4.32 and C-3 of rhamnose I at δ C 81.9, between H-1 of rhamnose II at δ H 5.01 and C-4 of glucose I at δ C 81.2 proved all the linkaging points between sugar to sugar units. After the alkaline hydrolysis of 1, the 1 H NMR data of prosapogenin (1a) also verified the structure as 3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl hederagenin 28-O-β-D-glucopyranosyl ester, namely Aytachoside A (Figure 1).

General
optical rotations of pure compounds were measured at 23 °C using a Rudolph Research Analytical Autopol I automatic polarimeter fitted with a sodium lamp with 1 mL cell. IR spectra were obtained on ATI Mattson 1000 Genesis Series FTIR instrument using KBr discs. NMR experiments were performed on Varian AS 400 MHz and Varian ASP 600 MHz instruments in DMSo-d 6 and in pyridine-d 5, respectively. All chemical shifts (δ) were given in ppm units with reference to tetramethylsilane (TMS) as an internal standard, and the coupling constants (J) were in Hz. HRESIMS analyses were carried out using a Bruker LC micro-Q-ToF mass spectrometer. GC-MS analysis was performed by a Shimadzu GC-MS QP 2010 plus instrument with Rtx-CLP2 cat-42302 apolar column (20 m-0.18 mm ID-0.14 μm df ). Medium pressure liquid chromatography (MPLC) was run using a Buchi system (Buchi C-605 pumps, coupled to a uV detector) with Buchi glass column (26/920). Lichroprep RP-18 (25-40 μm; Merck) and silica gel 60 (0.063-0.200 mm; Merck) were used both for column chromatography and MPLC studies. Thin-layer chromatography (TLC) was performed on F254 (Merck) and RP-18 F254s (Merck) precoated aluminium sheets.

Plant material
The aerial parts of C. aytachii Gokturk & Sumbul (Dipsacaceae) were collected from Eskisehir, Sivrihisar-Afyon highway, at 942 m in altitude, in August 2010 and identified by Prof. H. Sumbul and Prof. R. S. Gokturk (Department of Biology, Faculty of Art and Science, Akdeniz university). Voucher specimens (R. S. Gokturk 7483) were kept in the Herbarium Research and Application Centre of Akdeniz university, Antalya in Turkey.

Extraction and isolation
Air-dried and powdered plant material of C. aytachii (whole plant, 1130 g) was extracted with MeoH (4 × 4L) at room temperature overnight. The methanolic solution was then evaporated to dryness under reduced pressure at ~40 °C and gave 135 g of a dark residue. This residue was extracted with (3 × 400 mL) n-BuoH:H 2 o (1:1) solvent system. After the separation of n-BuoH and H 2 o phases, the n-BuoH fraction was defatted with n-hexane (10 × 50 mL) to remove the apolar and oily substances.
The n-BuoH extract (60.5 g) was subjected to vacuum liquid chromatography (VLC) using reversed-phase silica gel (Lichroprep RP-18) employing MeoH:H 2 o solvent system with a gradient from 0% to 100% MeoH to give 11 main fractions.

Acid hydrolysis and sugar analysis
The configurations of the sugar units were determined using GCMS analyses comparing with authentic samples. In this method, firstly compound 1 (5 mg) was hydrolysed with 1N HCl for 6 h at 95 °C. After the extraction with CHCl 3 , the aqueous layer was evaporated to dryness and kept in drying oven for 48 h at 25 °C. After that the sugars residue and the standard sugar samples were dissolved in anhydrous pyridine (1 mL), and then 1 mL of HMDS-TMCS (hexamethyldisilazane-trimethylchlorosilane, 1:1) was added. Then the mixtures were stirred at 70 °C for 1 h., concentrated under N 2 stream, solved with n-hexane (1 μL) and analysed by GCMS (Programme: initial temp. 100 °C rising 10 o C/min. Hold time 2 min. Then rising 30 °C up to 280 °C, hold time 3 min. Total programme time 17.67 min) (Kirmizigul & Anil 1994).While the standard silylated sugar peaks of L-arabinose, L-rhamnose and D-glucose were detected at tR (min) 5.70, 5.76 and 8.40, these peaks were observed at 5.61, 5.71 and 8.30 min for 1, respectively.

Antimicrobial activity studies
In vitro antimicrobial activity tests of the new compound 1 was evaluated using Minimum Inhibitory Concentration ( Gentamycin (Sigma) and Clotrimazole (Sigma) were used as positive and negative controls, respectively (Atlas et al. 1995).

Cytotoxic activity studies
Cytotoxic activities of the samples (1 and 1a) were tested using the MTT assay with minor modifications (onay-ucar et al. 2012), (see supplementary material). The half maximal inhibitory concentration (IC 50 ) of the extracts on HeLa cells were calculated from a graph of cell viability versus the sample concentrations.
Data are given as mean values ± SD with 'n' denoting the number of experiments. Statistical comparisons were made using one-way analysis of variance (ANoVA) module of GraphPad Prism 5. Difference in mean values were considered significant when p < 0.05.

Conclusion
one new and eight known glycosides have been isolated from C. aytachii. The aglycones of all compounds were identified as hederagenin. Antimicrobial and cytotoxic activity tests of Aytachoside A were examined by MIC and MTT methods, respectively. Comparing the antimicrobial activity results with standards, compound 1 was found considerably active against E. coli, P. aeruginosa and especially S. typhimurium micro-organisms (Table 1). Prosapogenin 1a was found as slightly active compound in literature (Kayce et al. 2014). Cytotoxic activity results of compounds 1 and prosapogenin 1a, demonstrated that prosopagenin 1a is the most active compound on HeLa cells. Compound 1 did not show a remarkable toxic effect on HeLa cells, at a concentration lower than 300 μg/mL (Table 2, Figure S22).