1
36
Vol. 57, No. 2
Table 3. Inhibition of PMN-CL by Compounds 1—10 from the Leaves of Aquilaria sinensis (nꢃ3)
Samples
1
2
3
4
5
6
7
8
9
10
IC50 (mmol/l)
MeanꢆS.D.
8
9.92ꢆ1.07 61.25ꢆ0.21 52.59ꢆ7.71 50.34ꢆ1.80 293.06ꢆ9.06
n.d.
n.d.
2.03ꢆ0.24 265.41ꢆ2.78 0.80ꢆ0.13
PMNs were stimulated by PMA. Chemiluminescence was measured with luminol.
Experimental
General Experimental Procedures Optical rotations were measured on
a JASCO P-1020 polarimeter. NMR experiments were performed with a
Brucker AV-500 spectrometer. ESI-MS spectra were recorded by an Agilent
1100 LC API MSD system. HR-ESI-MS was measured on a Bruker Dalton-
ics, APEX III Instrument. Column chromatography was carried out with sil-
ica gel (200—300 mesh, Qingdao Marine Chemistry Company, People’s Re-
public of China). Sephadex LH-20 was from Pharmacia (Sweden). The
chemiluminescence values were recorded by BPCL-1-G-C Ultra-weak Lu-
minescence Analyzer (Beijing Institutes for Biophysics, Chinese Academy
of Science). Male SD rats weighing 200—250 g each were obtained from the
Experimental Animal Center of Southeast University, Nanjing, China. Phor-
bol 12-myristate 13-acetate (PMA) and fetal calf serum (FCS) were obtained
from Promega (U.S.A.) and Hyclone (U.S.A.), respectively.
Fig. 3. Selected HMBC Correlations (H→C) for Compound 2
7
5.9, 73.7, 70.1 and 61.1, suggesting that 2 was a verti-
aflavone glycoside. The presence of the b-D-glucopyranosyl
1
3
moiety was supported by the C-NMR data and further con-
Plant Material Leaves of Aquilaria sinensis (LOUR.) GILG were col-
firmed by the acid hydrolysis of 2, which resulted in a release lected from Dianbai County, Guangdong province, China in October 2005
and was authenticated by Dr. Zenglai Xu (Nanjing Zhongshan Arboretum,
of D-glucose by HPLC and GC in comparison with an au-
Jiangsu province, China). The voucher specimen (BYY051020) was de-
thentic sugar sample. The configuration of the glucopyra-
posited at the Herbarium of China Pharmaceutical University.
nosyl was assigned to be b-D based on the coupling constant
Extraction and Isolation Dried leaves of Aquilaria sinensis (10 kg)
of the anomeric proton H-1ꢄ (d 4.75, d, Jꢃ7.6 Hz). The glu- were extracted with hot EtOH–H O (1 : 1) under refluxing (3 hꢅ5), followed
H
2
cosyl residue was located at the 7-O-position of the aglycon by removal of the solvent in vacuum, to yield a dried EtOH–H
O extract
2
(
848 g). The EtOH extract (848 g) were suspended in H O and extracted suc-
vertiaflavone by the appearance of HMBC cross peaks of the
2
cessively with Petroleum Ether, EtOAc and n-BuOH. The n-BuOH fraction
200 g) was subjected to silica gel CC (1000 g) eluted successively with
CHCl –CH OH (95 : 5), (9 : 1), (8.5 : 1.5), (8 : 2), (7 : 3), (6 : 4) and (1 : 1) to
glucosyl anomeric proton H-1ꢄ (dH 4.75, d, Jꢃ7.6 Hz) with
(
the carbon signal at d 158.3 (C-7). In addition, HMBC cor-
C
3
3
relations (Fig. 3) of the methoxy group protons d 3.90 (3H, afford a total of eight fractions (Fr-1—Fr-8). Fr-3 (6 g) was further chro-
H
s) with C-5 (d 163.6) and of the hydroxyl group protons d
matographed over silica gel, eluting with CHCl –CH OH (95 : 5), and
3 3
C
H
Sephadex LH-20, eluting with MeOH, to give 1 (10 mg). Fr-5 (20 g) was ap-
1
0.26 with C-3ꢀ, 5ꢀ (d 116.0) further confirmed the above
C
plied to a silica gel column eluting with CHCl –CH OH gradients, and then
3
3
assignments. As a consequence, the structure of 2 was deter-
mined as 7-b-D-glucoside of 5-O-methylapigenin.
purified on Sephadex LH-20 eluting with CHCl –CH OH (1 : 1) to give
3
3
compounds 7 (10 mg), 2 (20 mg) and 6 (38 mg). Same treatment for Fr-6
The structures of the known compounds were identified (21 g) as Fr-5 afforded pure 4 (100 mg) and 5 (40 mg). The EtOAc fraction
7
)
8)
(107 g) was subjected to silica gel CC (800 g) eluting successively with
as iriflophenone (3), mangiferin (4), 5-O-xylosylglycoside
9,10)
CHCl –CH OH (100 : 0), (98 : 2), (95 : 5), (9 : 1), (8 : 2), (7 : 3) and (1 : 1) to
3
3
of 7-O-methylapigenin (5),
5-O-xylosylglucoside of 7,4ꢀ-
5-b-D-glucoside of 7,3ꢀ-di-O-
afford a total of eleven fractions (Fr-1—Fr-11). Fr-5 (15 g) was applied to a
silica gel column eluted with Petroleum Ether–EtOAc gradients, and then
purified by recrystallization to give compounds 8 (15 mg), 9 (2 g) and 10
11)
di-O-methylapigenin (6),
1
2)
13)
14)
methylluteolin (7), luteolin (8), genkwanin (9), hy-
15)
droxygenkwanin (10), respectively, by comparison their (13 mg). Fr-7 (1.8 g) was further chromatographed over silica gel, eluting
with CHCl –CH OH (95 : 5), and Sephadex LH-20, eluting with MeOH, to
NMR data with those reported in literatures.
Polymorphonuclear neutrophils (PMNs) are important
cells involved in the bactericidal host defense system through
3
3
give 3 (18 mg).
25
Aquilarinoside A (1): Amorphous white powder, mp 140—142 °C. [a]D
ꢂ138.77° (cꢃ0.05, MeOH). UV lmax (MeOH) nm (log e): 229 (sh), 309
the respiratory burst. PMNs respiratory burst plays a critical (4.28). IR (KBr): 3373, 1640, 1608, 1509. NMR data are shown in Table 1;
ꢁ
ꢁ
role in the immune-inflammatory processes. Inhibition of ESI-MS m/z: 391 [MꢁH] ; HR-ESI-MS m/z: 391.1015 [MꢁH] (Calcd for
C H O : 391.1023).
neutrophils respiratory burst has been one of the well-docu-
mented methods for the evaluation of anti-inflammatory ac-
tivity for various synthetic compounds and natural prod-
19 19
9
7
-b-D-Glucoside of 5-O-Methylapigenin (2): Pale yellow powder, mp
25
1
(
81—183 °C. [a]D ꢂ81.20° (cꢃ0.05, MeOH). UV lmax (MeOH) nm
log e): 261 (3.90), 332 (4.36). IR (KBr): 3311, 1643, 1611, 1517, 1497.
1
6—18)
ꢁ
ucts.
In the present study, we chose neutrophils respira- NMR data are showed in Table 2; ESI-MS m/z: 447 [MꢁH] ; HR-ESI-MS
ꢁ
tory burst assay as a model to evaluate the compounds iso- m/z: 447.1274 [MꢁH] (Calcd for C22
H O10: 447.1285).
23
Acidic Hydrolysis of Compound 2 A solution of compound 2 (2 mg),
lated from the leaves of Aquilaria sinensis for their activity.
The biological test results of the isolated compounds are
shown in Table 3. The compounds 1, 2, 3, 4, 8 and 10
in 1 M HCl (dioxane–H O, 1 : 1, 2 ml) was refluxed for 4 h. After removing
2
dioxane, the solution was diluted with H O and extracted with EtOAc
2
(1 mlꢅ3) to separate the aglycone. The water layer was neutralized by pass-
showed significant inhibitory activity against neutrophils res- ing through an Amberlite IRA 400 column, eluting with water and concen-
trated in vacuum. Portion of the residue was examined by HPLC analysis
comparing to standard sample [condition: column, Cosmosil carbohydrate
piratory burst stimulated by PMA with IC50 values ranging
from 0.80 to 89.92 mmol/l, whereas compounds 5, 9 only
showed marginal inhibition activity.
analysis column (4.6ꢅ250 mm, 5 mm); solvent, CH CN–H O (85 : 15); flow
3
2
rate, 1 ml/min; detector, Alltech ELSD 500 detector; drift tube temperature,
90 °C; retention time, D-glucose (15.7 min)]. The absolute configuration of
the glucose was determined by GC after converted to its thiazolidine deriva-