860
Vol. 56, No. 6
which 2 mg of L-cysteine methyl ester hydrochloride was added. The mix-
ture was kept at 60 °C for 2 h and evaporated under N2 stream and dried in
vacuo. The residue was trimethylsilylated with N-trimethylsilylimidazole
(0.2 ml) for 2 h. The mixture was partitioned between n-hexane and H2O
(2 ml each), and the n-hexane extract was analyzed by GC-MS under the fol-
ride and recombinant mouse interferon-g (IFN-g) activated
murine macrophage like cell line RAW 264.7. Compound 1
(IC50ꢂ113.2 mM) showed somewhat weaker inhibition than
quercetin (IC50ꢂ24.8 mM). Compound 2 (IC50ꢅ200 mM) had
no activity. Quercetin is reported to have an inhibitory effect lowing conditions: capillary column, DB-5 (30 mꢆ0.25 mmꢆ0.25 mm); de-
tection, FID; detector temperature, 280 °C; injection temperature, 250 °C;
on the production of NO by LPS stimulated macrophage cell
RAW 264.7.5,6) Compound 1 didn’t exhibit any cytotoxicity.
initial temperature was maintained at 100 °C for 2 min and then raised to
280 °C at the rate of 10 °C/min, and final temperature was maintained for
5 min; carrier, N2 gas. The peak of the hydrolysate of 1 was detected at
15.48 min (D-xylcose) by comparison with authentic sample. The peaks of D-
Experimental
General Melting points were determined using a Fisher Johns apparatus
xylcose (15.48 min) and D-glucose (24.87 min) were detected in the hy-
and are uncorrected. IR spectra were obtained in KBr disks on a Perkin-
drolysate of 2 by the same method. The aglycones were both identified as
Elmer 983G spectrophotometer. NMR spectra were recorded on a INOVA
400 spectrometer. EI-MS, ESI-MS and HR-FAB-MS were recorded in a Mi-
cromass ZabSpec spectrometer. GLC was carried out on a TSQ7000 (Finni-
gan) GC-MS instrument. TLC employed precoated Silica gel plates (5—
7 mm, Qingdao Haiyang). For column chromatography, Silica gel (H, 200—
300 mesh, Qingdao Haiyang), Sephadex LH-20 (Pharmacia) and Macrop-
orous resin D101 (26—60 mesh, Tianjin Haiguang Chemistry Company,
Tianjin, China) were used.
quinovic acid on thin-layer chromatography (TLC) by comparison with the
authentic sample which had been isolated.7)
Detection of the Sulfate Group A 1—2 mg aliquot of each sample was
refluxed with 10% HCl (4 ml) for 4 h and then extracted with Et2O. An
aliquot of the aqueous layer of each was treated with 70% BaCl2 to give a
white precipitate (BaSO4).8)
Inhibitory Activity on NO Production from Activated Macrophages-
Like Cell Line, RAW 264.7 The cells were seeded at 1.2ꢆ106 cells/ml
Plant Material The barks of Zygophyllum fabago L. were collected
onto 96-well flat bottom plate (Sumitomo Bakelite, #8096R, Tokyo) and
from Wulumuqi, Xinjiang Autonomous Region of China in March 2004,
then incubated at 37 °C for 2 h. Next, the test extract was added to the cul-
and identified by Prof. Guo-Qiang Li of Institute of Medicinal Plant Devel-
ture simultaneously with both Escherichia coli LPS (100 ng/ml) and recom-
opment, Chinese Academy of Medical Sciences & Peking Union Medical
College, China, where a voucher sample has been deposited.
Extraction and Isolation The air-dried, powdered barks (4 kg) of the
plant material were successively extracted with 75% EtOH (each 65 lꢆ3)
under reflux. The resultant extract was combined and dried under reduced
pressure to give concentrated extractives (200 g). The latter was subse-
quently suspended in water and partitioned successively with CHCl3, EtOAc
and n-butanol. The 1-butanol part (60 g) was subjected to column chro-
binant mouse IFN-g (0.33 ng/ml). Then cells were incubated at 37 °C for ap-
proximately 16 h and subsequently chilled on ice. One hundred microliters
of the culture supernatant was placed in duplicate in the wells of 96-well flat
bottomed plates. A standard solution of NaNO2 was placed in alternate wells
on the same plate. To quantify nitrite, 50 ml of Griess reagent (1% sulfanil-
amide in 5% H3PO4 and 0.1% N-1-naphthyletyl-enediamide dihydrochlo-
ride) was added to each well. After 10 min the reaction products were colori-
metrically quantified at 550 nm using a Model 3550 Microplate Reader
matography by a combination of D101 macroporous resin, eluted gradiently
(BIO-RAD) and the background absorbance (630 nm) was subtracted. Cyto-
with H2O, 10% EtOH, 30% EtOH, 50% EtOH, 75% EtOH, 95% EtOH, suc-
toxicity was measured by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetra-
cessively. The fraction eluted with 30% EtOH (11 g) was subjected to a sil-
zolium bromide (MTT) assay method.
ica gel column with a CHCl3–MeOH gradient system (1 : 0—0 : 1), affording
20 fractions. Fraction 9 (1.3 g) was purified by repeated ODS column chro-
References
matography [MeOH–H2O (1 : 1—1 : 0)] to afford compound 1 (15 mg). Frac-
1) Jiangsu Institute of Botany, Chinese Academy of Medical Sciences
tion 5 (1 g) was separated on Sephadex LH-20 columns with MeOH, subse-
and Kunming Institute of Botany, “Xinhua bencao gangyao,” Vol. 1,
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Shanghai Science and Technology Press, Shanghai, 1988, p. 272.
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Compound 1: White powder; mp 221—223 °C; [a]D20 ꢀ23.7° (cꢂ0.114,
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Compound 2: White powder; mp 202—204 °C; [a]D20 ꢀ20° (cꢂ0.04,
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985; ESI-MS m/z: 861, 883, 905, 671, 464; HR-ESI-MS m/z: 883.3769
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was obtained, which was finally evaporated and dried in vacuo to furnish a
monosaccharide residue. The residue was dissolved in pyridine (1 ml), to