A New Iridoid Glycoside from the Rhizomes of Cyperus rotundus

Experimental Section

Reagents and Instruments. UV spectra were recorded on a Hewlett-Packard HP-845 UV-VIS spectrophotometer. Specific rotation measurements were recorded on a Perkin- Elmer 242 MC polarimeter. IR spectra were recorded on a Nicolet 470 spectrometer and MS on a Varian MAT-212 mass spectrometer and a Shimadzu GC-MS model QP2010 Plus spectrometer, respectively. NMR spectra were recorded on a Bruker DRX-500 (500 MHz for 1H NMR) using standard Bruker pulse programs. Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as internal standard. The chemiluminescence value was recorded by BPCL-1-G-C Ultraweak Luminescence Analyzer (Beijing Institutes for Biophysics, Chinese Academy of Science). RPMI-1640, Phorbol 12-myristate 13-acetate (PMA) and fetal calf serum (FCS) were obtained from GIBCO (USA), respectively. Column chromatography separations were carried out onsilica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd, Qingdao, P.R. China), ODS (50 mesh, AA12S50, YMC), and Diaion HP-20 (Pharmacia, Peapack, New Jersey, USA). All other chemicals used were of biochemical reagent grade.

Plant Material. The rhizomes of Cyperus rotundus were collected in Zhanjiang, Guangdong Province of China in September 2009, and were identified by Wen-qing Yin (School of Chemistry & Chemical Engineering of Guangxi Normal University, Ministry of Education Key Laboratory of Chemistry and Molecular Engineering of Medicinal Resource, Guilin). A voucher specimen (No. 20090903) has been deposited in the authors’ laboratory.

Extraction and Isolation. The dry rhizomes of Cyperus rotundus (10 kg) were extracted three times under reflux with 95% aqueous ethanol (150 L × 2 h). After removing the solvent under reduced pressure, the residue was suspended in water and then sequentially extracted with petroleum ether, CH2Cl2, EtOAc and n-BuOH. The n-BuOH extract (152 g) was submitted through a column chromatography (CC) of high porous absorption resin (Diaion HP-20), eluting with H2O and CH3OH. The methanol fraction (98 g) was repeatedly CC over normal and reverse phase silica gel to afford four fractions (Frs.1-4). Fr.3 was subjected to ODS CC eluting with CH3OH-H2O (0:1-1:0 and silica gel with CHCl3-MeOH-H2O (8:2:0.2-7:3:0.3) to afford compounds 1 (31 mg). Fr.4 was subjected to ODS CC eluting with CH3OH-H2O (6:4) and silica gel with CHCl3-MeOH-H2O (8:2:0.2-7:3:0.3) to give compounds 2 (25 mg) and 3 (19 mg).

Macrophages Respiratory Burst Inhibitory Activity. The murine macrophage-like cell line RAW 264.7 was routinely cultivated at 37 ℃, 5% CO2 in RPMI-1640 supplemented with 10% FCS (Hyclone, America), 100 μg/mL streptomycin, 118 μg/mL ampicillin, and 2 mg/mL sodium bicarbonate. After 72 h, the macrophage cellsformed a confluent monolayer. The monolayer cells were digested with trypsin. After washed with PBS without Ca2+ and Mg2+, the deposited cells were suspended with RPMI-1640 without FCS (approximately 2 × 106 cell/mL) in the vitreous culture flask.10,11

The details of chemiluminescence assay procedure were according to the method described in the literature.12 Tested compounds were prepared as 10 mM top stocks, dissolved in DMSO, and stored at 4 ℃. Phorbol 12-myristate 13-acetate (PMA) was applied as triggering agent. Data were collected at a frequency of 6 s/min and chemiluminescence was recorded for up to 30 min. The IC50 values were obtained bylinear regression analysis of the dose response curves, which were plots of % inhibition versus concentration.13

Rotunduside C (1). White amorphous powder. −77.1° (c 1.0, MeOH); IR νmax (KBr): 3212-3449, 2931, 1699 and 1603 cm−1. HRESIMS m/z 779.2373 [M+Na]+, Calcd. for C34H44O19Na, 779.2349. 1H NMR (500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N) data see Table 1.

Pungenin (2). White powder, EI-MS m/z 314 [M]+. 1H-NMR (CD3OD, 500 MHz) δ 2.49 (3H, s, -COCH3), 4.88 (1H, d, J = 7.8 Hz, H-1′), 6.95 (1H, d, J = 8.4 Hz, H-3), 7.68 (1H, dd, J = 8.4 Hz, 2.1 Hz, H-4), 7.86 (1H, d, J = 2.1 Hz, H- 6); 13C-NMR (CD3OD, 125 MHz) δ 144.8 (C-1), 155.3 (C- 2), 114.6 (C-3), 124.7 (C-4), 131.9 (C-5), 112.8 (C-6), 197.3 (-COCH3), 26.8 (CH3), 101.4 (C-1′), 74.8 (C-2′), 77.8 (C-3′), 70.9 (C-4′), 78.1 (C-5′), 62.1 (C-6′).

Salidroside (3). White needle, ESI-MS m/z 323 [M+Na]+. 1H-NMR (CD3OD, 500 MHz) δ 7.07 (2H, d, J = 8.4 Hz, H- 2, 6), 6.68 (2H, d, J = 8.4 Hz, H-3, 5), 2.81 (2H, m, H-7), 3.64 (2H, m, H-8), 4.31 (1H, t, J = 6.0 Hz, H-1′), 3.09-4.02 (6H, m, H-2′-6′); 13C-NMR (CD3OD, 125 MHz) δ 130.6 (C- 1), 130.9 (C-2, 6), 116.1 (C-3, 5), 156.9 (C-4), 36.3 (C-7), 72.3 (C-8), 104.3 (C-1′), 75.1 (C-2′), 78.0 (C-3′), 71.4 (C-4′), 78.2 (C-5′), 62.6 (C-6′).

Wieffering Field Test. Field tests involving chemical color tests include heating with dilute hydrochloric acid or acetous copper sulfate. Color reaction in conjunction with thin-layer chromatography (TLC), as developed by wieffering, is currently used.14 Sample was tested by TLC and heated with acetous copper sulfate. In general, a compound responds positively to Wieffering field test, giving blue colouration, which indicates it as an iridoid.