LAUSR

Plants of the genus Swertia (Gentianaceae) comprise about 170 species, of which 79 are present in China [1]. Swertia is a popular medicinal herb, which has been used for the treatment of dyspepsia, urinary infection, and hepatitis diseases in the middle-western ethnic district of China [2]. Iridoids, secoiridoids, xanthones, flavonoids, and triterpenoids have been proved to be the main constituents of the genus Swertia [3]. Swertia bimaculata Hook.f. & Thomson ex C.B. Clarke is a well-known ethnomedicine recorded as Zhangyacai in Jiuhuang Bencao composed of the Chinese Ming Dynasty 600 years ago [4]. Besides the report of several xanthones and oleanolic triterpenes from this species, the chemical composition of S. bimaculata is still unexplored [5]. Therefore, we have performed a phytochemical investigation into the whole plants of S. bimaculata. The whole plants of S. bimaculata were collected from Cang mountain in Dali, Yunnan Province of China in February 2009 and were identified by Prof. Hai-zhou Li (Kunming University of Science and Technology). A voucher specimen of this collection (200909Z) was deposited at the Herbarium of the Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China. Genaral Procedures. Column chromatography (CC): silica gel (200–300 or 100–200 mesh, Qingdao Marine Chemical Factory, P. R. China), MCI gel (75–150 μm, Mitsubishi, Japan), Lichroprep RP-18 (40–63 μm, Merck, Germany), and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Visualization by spraying with 10% H2SO4–EtOH, followed by heating on a hot plate. Semipreparative HPLC: Agilent 1200 system with a Zorbax SB-C18 column (5 μm, 9.4 × 250 mm). 1D NMR spectra: Bruker AM-400, DRX-500, and Avance III-600 instruments, with tetramethylsilane as an internal standard (Bruker BioSpin Group). ESI-MS: API-Qstar-TOF instrument (Allen-Bradley). Extraction and Isolation. The air-dried powder of S. bimaculata (12.0 kg) was extracted with 90% EtOH three times (20 L × 3) at room temperature and concentrated under reduced pressure to give a residue, which was suspended in H2O and successively partitioned with petroleum ether (155.4 g), EtOAc (350.0 g), and n-BuOH (500.8 g). The EtOAc-soluble fraction was separated on a silica gel column using a stepwise gradient of CHCl3–MeOH (from 99:1 to 1:99, each 5 L) to yield five fractions (Frs. 1–5). Fraction 2 (45.1 g) was further separated by silica gel CC (petroleum ether–EtOAc, from 49:1 to 1:1), followed by purification by silica gel (petroleum ether–EtOAc, from 19:1 to 1:1) and Sephadex LH-20 CC (petroleum ether– CHCl3–MeOH, 5:5:1) and semipreparative HPLC (60%, MeOH–H2O) to yield 3,7,8-trimethoxy-8-hydroxyxanthone (1, 18.0 mg) [6], 3,7-dimethoxy-1-hydroxyxanthone (2, 6.8 mg) [7], bellidifolin (3, 16.8 mg) [8], oleanolic acid (4, 26.3 mg) [9], 3α-hydroxy11α-methoxyolean-12(13)-en-28-oic acid (5, 13.5 mg) [10], coniferyl aldehyde (6, 5.5 mg) [11], and sinapaldehyde (7, 7.5 mg) [12]. Fraction 4 (40.5 g) was subjected to CC (RP-18, MeOH–H2O, 30, 50, 70, 90, and 100%) to yield four fractions (Frs. 4-1–4-5). Through repeated silica gel (CHCl3–acetone, from 25:1 to 1:1, each 1 L) and Sephadex LH-20 CC (CHCl3–MeOH, 1:1), (rel-(3R,3′S,4R,4′S)-3,3′,4,4′-tetrahydro-6,6′-dimethoxy[3,3′-bi-2H-benzopyran]-4,4′-diol (8, 4.9 mg) [13], 5-(3′′-hydroxypropyl)7-methoxy-2-(3′,4′-methylenedioxyphenyl)benzofuran (9, 7.4 mg) [14], 5-carbethoxyethenyl-7-methoxy-2-(3,4methylenedioxyphenyl)-benzofuran (10, 5.4 mg) [15], 3-acetoxymethyl-5-[(E)-2-formylethen-1-yl]-2-(4-hydroxy-3methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran (11, 4.4 mg) [16], and (–)-simulanol (12, 5.6 mg) [17] were purified from Fr. 4-2.