Three New Sesquiterpene Glycosides from the Rhizomes of Trillium tschonoskii

Three new sesquiterpene glycosides, possessing a rare aglycone with a sulfonyl between C-1 and C-15 positions, named 3-(3′E-7′R,8′-dihydroxy-4′,8′-dimethyl-3′-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside (1), 3-(3′E-7′R,8′-dihydroxy-4′,8′-dimethyl-3′-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside (2), and 3-(3′E-7′R,8′-dihydroxy-4′,8′-dimethyl-3′-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-6′-O-acetyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside (3), respectively, were isolated from the rhizomes of Trillium tschonoskii. Their structures were established on the basis of spectroscopic data, including HR-ESI-MS, IR, 1D and 2D NMR. The cytotoxic properties of the three compounds were investigated using human hepatic L02 cells.


Introduction
Trillium tschonoskii Maxim is perennial herb, mainly distributed in Hubei, Shanxi and Anhui provinces of China at an altitude of 1600-3200 m [1]. The rhizomes of T. tschonoskii named Yan Ling Cao, have been used as a traditional Chinese medicine (TMC) for the treatment of headache, traumatic injury, and neurasthenia [2]. Recent pharmacological studies have shown that Yan Ling Cao possesses anti-tumor, anti-inflammatory, analgesic and blood coagulation activities [3][4][5][6][7]. Previous phytochemical studies on the Trillium reveals that steroidal saponins constitute the main chemical components [6,[8][9][10][11][12]. Besides, it also contains a few phenylpropanoid glycosides [13] and sesquiterpenoid glycosides [3,13,14]. In this study, a chemical investigation on T. tschonoskii led to the isolation of three new sesquiterpene glycosides that have a rare aglycone with a sulfonyl between C-1 and C-15 positions. Their structures were identified using the spectroscopic techniques of HR-ESIMS, IR, and NMR. The cytotoxic activity of the three compounds were evaluated against L02 cells.

Plant Material
The rhizomes of T. tschonoskii were collected from the Shennongjia of Hubei province, were identified by Professor Bao-Lin Guo (Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences). A voucher specimen (No. 151010) was deposited in the author s laboratory in the Beijing Institute of Radiation Medicine.

Extraction and Isolation
The rhizomes of T. tschonoskii

Acid Hydrolysis and GC-MS Analysis
Compound 1 (1.4 mg), 2 (1.6 mg), and 3 (1 mg) were hydrolyzed with 2 N aq. CF 3 COOH (5 mL) for 5 h at 95 • C, respectively. After extraction with CH 2 Cl 2 (3 × 5 mL), the aq. layer was repeatedly evaporated to dryness with EtOH until neutral, and then analyzed by TLC over silica gel (CHCl 3 :MeOH:H 2 O, 8:5:1) by comparison with authentic samples. Furthermore, the residue of sugars was dissolved in anhydrous pyridine (2 mL), and L-cysteine methyl ester hydrochloride (3 mg) was added. The mixture was stirred at 60 • C for 1 h, then 3 mL of HMDS-TMCS (hexamethyldisilazane:trimethylchlorosilane, 2:1) was added, and the mixture was stirred at 60 • C for 30 min. The precipitate was centrifuged off, and the supernatant was analyzed by GC-MS (Agilent Technologies 5977A MSD). The absolute configurations were determined by comparing the retention times with derivatives prepared in a similar way from standard D-glucose (Sigma-Aldrich). Identification of D-glucose was carried out for compounds 1-3, giving two peaks at 3.75 min and 4.15 min which were two silylated derivatives (Supplementary Material, Figure S25).

Cytotoxicity Assay
The cytotoxic activity was measured by MTT assay [18]. L02 Cells were seeded in 96-well plates and treated 24 h later with various (100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM) concentrations of compounds 1-3. After 24 h of incubation, MTT was added to all wells. Plates were incubated for another 24 h, and cell viability was measured by observing absorbance at 492 nm.

Conclusions
In conclusion, three new sesquiterpene glycosides were isolated from the rhizomes of T. tschonoskii. Their structures were elucidated by extensive analysis of spectroscopic methods including 1D and 2D NMR experiments (HSQC, HMBC, 1 H-1 H COSY), IR, and HR-ESI-MS. The aglycone of the compounds found in this study was a rare aglycone which contains a sulfonyl between C-1 and C-15 positions. Compounds 1-3 were investigated for their cytotoxic activity against L02 cells, and no obvious cytotoxic activity was found.