Three New Steroidal Glycosides from the Roots of Cynanchum stauntonii

Three new steroidal glycosides, named as stauntosides L, M, and N (1–3), along with one known C21 steroidal glycoside, anhydrohirundigenin monothevetoside (4), were isolated from the 95% ethanol extract of the roots of Cynanchum stauntonii. The structures of these new compounds were elucidated on the basis of extensive spectroscopic analyses, mainly 1D and 2D NMR, HRESI-MS, and chemical methods.


Introduction
Cynanchum stauntonii (Decne.) Schltr. ex Levl. is a perennial medicinal herb from the family of Asclepiadaceae, which is widely distributed in south-central region of China. e dried-up roots of C. stauntonii, along with that of another species of the same genus, C. glaucescens (Decne.) Hand.-Mazz., has been used as antitussives and expectorants to treat diseases in the history of China [1]. Both of which are given the name of "Bai-qian" in traditional Chinese medicine (TCM) [2]. e main chemical constituents isolated from Cynanchum species are steroids, especially the steroidal saponins with aglycones assignable to either the normal four-ring C 21 steroid skeleton or the aberrant 13,14 : 14,15disecopregnane-type skeleton or the equally abnormal 14,15secopregnane-type skeleton, respectively [3,4]. It is known that C 21 steroids and their glycosides are of considerable bioactivities, such as hypolipidemic and antitumor activities. However, chemical investigation into the title plant is very rare up to now with, to the best of our knowledge, only three papers have reported several steroids, including four ones by our group eight years ago [1]. e ongoing investigations in our group intend to enrich the information about the chemical constituents and their bioactivities of this plant which has led to the isolation and elucidation of some known and new steroidal glycosides [5]. In this paper, we describe three new steroidal glycosides (1-3) and one known analogue, anhydrohirundigenin monothevetoside (4) (Figure 1), from the roots of C. stauntonii. e isolated new steroidal glycosides contained steroid aglycones with either the 13,14 : 14,15-disecopregnane-type skeleton or the 14,15secopregnane-type skeleton and were given the trivial names stauntosides L-N, respectively.

General Methods.
Optical rotations were measured on a Perkin-Elmer 241 digital polarimeter at 20 ∘ C. IR spectra were recorded on a Nicolet 5700 spectrometer. 1D and 2D NMR spectra were taken on a Varian INOVA-500 spectrometer or a Varian NMR System-600 NMR spectrometer with tetramethylsilane as internal standard. ESIMS and HRESIMS were obtained using an Agilent 1100 series LC/MSD Trap SL mass spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD system equipped with a SPD-10A detector, and a reversed-phase C18 column (YMC-Pack ODS-A U 20×250 mm, 10 m) was employed. Column chromatography (CC) was undertaken over silica gel (200-300 mesh conducted on an Agilent 7890A instrument. Reference compounds, glaucogenin C 3-O--L-cymaropyranosyl-(1 → 4)--D-digitoxopyransyl-(1 → 4)--D-canaropyranoside, cynatratoside B, and glaucogenin C mono-D-thevetoside which were used to identify the monosaccharides obtained in the acid hydrolysis, including their absolute con�guration, were isolates from the title plant in our previous work [5]. Acetonitrile used in preparative HPLC procedure was in HPLC grade, and other solvents were of analytical grade.
e known compound anhydrohirundigenin monothevetoside (4) [1] was identi�ed by comparison of their spectroscopic data ( 1 H and 13 C NMR, MS) with the literature values. �.�. �e�ermina�i�n �� �he ��s��u�e C�n��ura�i�ns �� ��n�sac� charides. e absolute con�gurations of D-digitoxose, Lcymarose, and D-thevetose were determined as per the method published by Hara et al. [6]. e monosaccharides obtained on acid hydrolysis, as described above, were dissolved in pyridine and reacted with L-cysteine methyl ester hydrochloride at 60 ∘ C for 1 h. Equal volume of acetic anhydride was added and heating was carried out for another 1 h. Acetylated thiazolidine derivatives were injected into GC system. e absolute con�gurations of the sugars were determined by comparing the retention times with those of acetylated thiazolidine derivatives synthesized from the known sugars obtained through acid hydrolysis of the reference compounds. (Also, the retention times of D-digitoxose, L-cymarose, and D-thevetose were determined by interactive comparison. GC conditions in the test: column, HP-5, 30 m × 0.25 mm, 0.25 m; detection FID; carrier gas, N 2 ; injection temperature, 250 ∘ C, detection temperature, 280 ∘ C, column temperature, 150 ∘ C (0 min), 10 ∘ C/min to 250 ∘ C (20 min). tR D-digitoxose 13.09 min, tR L-cymarose 13.46 min, and tR Dthevetose 16.07 min). e D-cymarose involved in this paper was not detected by GC method because of the lack of reference sugars, but, from the results of the typical monosaccharides, it can be concluded that the absolute con�gurations of the monosaccharides composed of the sugar units can be really determined by comparison of their spectroscopic data with those reported in the literature. is determination is also because of the very common kind of D-cymarose in the case of the Cynanchum species.

Results and Discussion
All three new compounds were obtained as white lamellae or amorphous powder and showed up positive Liebermann-Burchard and Keller-Kiliani reactions, suggesting their glycosidic steroidal category with 2-deoxysugar units existing in their sugar moieties [7]. Hz, H-6 �� ). In addition, two characteristic ole�nic proton signals at 5.32 (1H, d, 5 Hz, H-6) and 6.47 (1H, s, H-18) and two methoxyls at 3.44 (3H, s) and 3.94 (3H, s) were also determined in the 1 H NMR spectrum, the later ole�nic signal was obviously deshielded and the two methoxyls were compatible with two methylated deoxypyranoses when examining the 13 C and DEPT NMR data which exhibited forty-one carbon signals, with seven methyls, nine methylenes, twenty methines, and �ve quaternary carbons being categorized (Table 2). With the exception of the 13 C and DEPT NMR signals assignable to three monosaccharides, the remaining resonances were very similar to those of glaucogenin C, a known steroidal aglycone isolated previously from C. atratum [8]. e main differences were observed for glycosidation shis at C-2 (−2.3), C-3 (+7.1), and C-4 (−4.0) in aglycone moiety of 1, so the oligosaccharide chain was determined to link with the C-3 hydroxyl of 1, which was also con�rmed, with the aid of HSQC spectrum for determining the direct carbonproton linkages, by the long-range 1 H-13 C correlation of the signal of H-1 � with the signal of C-3 in the HMBC spectrum. Aer the anomeric protons were identi�ed, the 1 H-1 H COSY experiment, coupled with the HSQC spectrum, was very effective in determining the spin systems within the sugar moieties because of the handsome differences of the chemical shis and the relatively large coupling constants theoretically (Figure 1). One -D-thevetopyranose, one -D-digitoxopyranose, and one -D-cymaropyranose in the very three sugar units were further speculated by comparing the 1 H and 13 C NMR spectroscopic data of 1 with those of stauntoside J [5], which were supported by the splitting patterns and coupling constants of the above-mentioned anomeric proton signals. ese conclusions about the absolute con�gurations of the monosaccharides were con-�rmed by acid hydrolysis as described above in Acid Hydrolysis of Reference Compounds and Compounds 1-3 and Determination of the Absolute Con�gurations of Monosaccharides, which not only gave one D-thevetopyranose, one D-digitoxopyranose, and another kind of sugar unit, but also con�rmed that the absolute con�gurations of the monosaccharides determined by comparison of their spectroscopic data with those reported are really consistent with reality. Also, this determination is because of the very common kind of D-cymarose in the case of the Cynanchum species. Because of the lack of reference substance, D-cymaropyranose units could not be determined in the GC test. e sugar sequence e IR spectrum showed the absorption bands for hydroxy (3482 cm −1 ), carbonylic (1733 cm −1 ), and ole�nic (1652 cm −1 ) groups. A detailed comparison between compounds 2 and 1 indicated that they have the consistent 1 H-and 13 C-NMR spectroscopic data from their aglycone moieties (see experimental and Table 2), which was con�rmed to be glaucogenin C by detailed analysis of 2D NMR spectra (Figure 2. Complete data not shown). With the exception of the aglycone signals, the 1 H NMR spectrum of 2 revealed the diagnostic signals of four sugar units by four anomeric proton signals at 4.82 (1H, d, 8 Hz, H-1 � ), 5.52 (1H, br d, 1 Hz, H-1 �� ), 5.13 (1H, br d, 5 Hz, H-1 ��� ), and 5.19 (1H, d, 3 5 Hz, H-1 ���� ), which correlated to the corresponding anomeric carbon signals at C 102.3 (C-1 � ), 99.0 (C-1 �� ), 99.6 (C-1 ��� ), and 101.2 (C-1 ���� ), respectively, in the HSQC spectrum, and four secondary methylic signals at 1.46 (3H, d, 6 Hz, H-6 � ), 1.42 (3H, d, 6 Hz, H-6 �� or H-6 ���� ), 1.30 (3H, d, 6 5 Hz, H-6 ��� ), and 1.56 (3H, d, 6 5 Hz, H-6 ���� or H-6 �� ). e 1 H, 1 H-COSY experiment, coupled with the HSQC spectrum, established the spin systems within the sugar moiety ( Figure 1). By comparing the 1 Hand 13 C-NMR spectroscopic data of 2 with those of 1 and stauntoside H [5], the structures of the four sugar units were suggested, that is, one -D-thevetopyranose, one -D-digitoxopyranose, one -D-cymaropyranose, and one -L-cymaropyranose, which were further supported by the splitting patterns of the above-mentioned anomeric proton signals. Compound 2 was subjected to acid

Conclusions
In recent years, only several papers have described phytochemical investigations of C. stauntonii and led to a small amount of steroidal glycosides being reported. In the present work, we reported on three new steroidal glycosides, named as stauntosides L, M, and N, from C. stauntonii. Here, the structure elucidation, mainly undertaken by means of spectroscopic and chemical evidence, provided unambiguous information about the aglycone skeletons and structures, the position of the glycosidic linkage, and the sequence of the monosaccharides in the sugar moiety. In addition, it should be emphasized that the main and active ingredients of Cynanchum species are steroidal glycosides [10]. In conclusion, this study has enriched the information about the compounds of the title plant and further established that C. stauntonii is a signi�cant source of steroidal glycosides.