Two New Chiratane-Type Triterpenoids from Swertia kouitchensis

Two rare new chiratane-type triterpenoids, kouitchenoids A and B (1, 2), together with oleanolic acid (3) and ursolic acid (4), were isolated from ethanol extract of Swertia kouitchensis. The new structures were determined by the analysis of MS and NMR data. In addition, compounds 1–4 showed moderate inhibitory activity against the α-glucosidase (with IC50 values ranging from 1,812 to 2,027 μM).


Introduction
Swertia kouitchensis Franch. (Gentianaceae), widely distributed in China, has been used for the treatment of hepatitis and diabetes [1,2]. Previous study revealed that the ethanol extract of S. kouitchensis showed α-glucosidase inhibitory effect [3]. Thus, we initiated a study on the subject. As a result, two rare chiratane-type triterpenoids, kouitchenoids A and B (1 and 2), along with oleanolic acid (3) [4] and ursolic acid (4) (Figure 1) [5] were isolated and identified. All of these compounds were evaluated for their inhibitory activities against α-glucosidase. Described herein are the isolation, structure elucidation, and biological activities of these compounds.

Results and Discussion
The 95% ethanol extract of S. kouitchensis whole plants was suspended in water and successively partitioned with petroleum ether, CH 2 Cl 2 , EtOAc and n-butanol. The CH 2 Cl 2 fraction was subjected to column chromatography and partitioned as described in the Experimental section to afford two new triterpenoids 1 and 2, along with two known compounds 3 and 4.
Compound  [6,7], except for the appearance of a carboxyl carbon signal at δ C 181.0 instead of a methyl (C-24) signal of chiratenol, and the downfield shift (+10.8 ppm) for C-4. The position of this carboxyl group was confirmed by HMBC spectrum (Figure 2), in which cross-peaks were observed between H-3 (δ H 3.41) and C-2, C-4, C-23, and C-24, so that the carboxyl group is assigned to C-24. And also, H-3 was assigned as α-orientation by the NOESY correlation ( Figure 2) between H-3 and H-5. Therefore, 1 was deduced to be 3β-hydroxy-chirat-16-en-24-oic acid, and named kouitchenoid A.
Compound 2 was obtained as white amorphous powder. Its molecular formula was assigned to be C 30  IR spectrum of 2 exhibited absorptions at 3,448 and 1,701 cm −1 , assignable to hydroxyl and carboxyl functions, respectively. Its 1 H-NMR and 13 C-NMR shifts for the B, C, D, and E rings were almost the same as those of compound 1, while those of the A ring differed. The main differences in the A ring between these two compounds were that the oxygenated proton H-3 at δ H 3.41 (1H, dd, J = 12.0, 4.4 Hz) in 1 was changed into δ H 4.75 (1H, brs, W 1/2 = 5.7Hz) in 2 and the oxygenated C-3 carbon at δ C 78.7 in 1 was shifted upfield to δ C 71.1 in 2, suggesting that the H-3 in a β-orientation in 2. This assignment could be further supported by the missing correlation between H-3 and H-5 in the NOESY spectrum of 2. Together with its 1 H-1 H COSY and HMBC spectra, which also were similar to those of 1, compound 2 was determined to be 3α-hydroxy-chirat-16-en-24-oic acid and named kouitchenoid B.  Figure 2. Key HMBC, and NOESY correlations of compound 1.

General Procedures
Optical rotations were measured on an AA10R digital polarimeter. IR Spectra were detected on Avater-360 spectrophotometer with KBr pellets, and are reported in cm −1 . 1D and 2D NMR spectra (all in C 5 D 5 N) were recorded on a Bruker AV-400 spectrometer, and chemical shifts are expressed in δ (ppm) and referenced to the solvent peaks at δ H (8.74, 7.59, 7.22) and δ C (150.3, 135.9, 123.9) for C 5 D 5 N, respectively, and coupling constants are in Hz. HR-ESI-MS were determined on a Agilent 6520 Q-TOF LC-MS mass spectrometer. Semi-Preparative HPLC was performed on a Hitachi Spectra Series HPLC system equipped with an L-2130 pump and a UV L-2400 detector in a YMC-ODS column (10 mm × 250 mm, 5 μm; flow rate at 2.0 mL/min; wavelength detection at 208 nm; retention time 34.2 min for 1, 38.0 min for 2, 18.7 min for 3, and 20.1 min for 4). Column chromatography (CC) was performed on SiO 2 (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Toyopearl HW-40C (Tosoh Bioscience Shanghai Co., Ltd., Shanghai, China). Analytical TLCs were run on silica gel plates (GF 254 , Yantai Institute of Chemical Technology, Yantai, China). Fractions were monitored by TLC, and spots were visualized by heating TLC sprayed with 10% H 2 SO 4 .

Plant Material
The whole plant of S. kouitchensis was collected in Enshi, Hubei province, China, in October 2010, and identified by Prof. Jiachun Chen (Tongji Pharmaceutical School of HUST, Wuhan, China). A voucher specimen (S.k-2010-1010) has been deposited in the University herbarium for future reference.

Extraction and Isolation
The chopped, dried whole plants of S. kouitchensis (15 kg) were refluxed twice with 120 L of 95% (v/v) EtOH-H 2 O, two hours each time. After filtration, the filtrate was concentrated under reduced pressure to yield a brownish residue (3.0 kg). Part of the residue (2.5 kg) were suspended in water and partitioned successively with petroleum ether, CH 2 Cl 2 , EtOAc, and n-butanol to afford five fractions. The CH 2 Cl 2 -soluble part (about 400 g) was subjected to CC (SiO 2 , 200-300 mesh, 3.0 kg, 12 × 100 cm, petroleum ether/acetone 100:0→0:100) to yield five fractions A-E. Fraction B (87.4 g) was subjected to HW 40C (CHCl 3 /MeOH 1:1) to give four subfractions B 1-4 . B 2 was subjected to CC