September 2010
1251
13
Table 4. The C-NMR Data for Sugar Moieties of Compounds 1—4 Table 5. Cytotoxic Activity of Compounds 1—4 against Three Human
a,b)
a)
(400 MHz, Pyridine-d5)
Cancer Cell Lines
Carbon
1
2
3
4
IC50 (mM)
Sample
Ara-1
2
3
4
104.3
79.4
72.5
78.1
63.1
104.9
76.1
78.1
71.7
78.0
62.9
104.1
79.2
72.5
77.9
62.7
104.7
75.9
77.9
70.3
77.8
62.7
104.1
79.4
72.5
78.0
63.0
104.8
75.9
78.0
71.4
77.8
62.6
104.4
80.0
72.8
78.3
63.7
105.0
76.0
78.1
71.0
75.0
64.8
20.9
171.0
103.8
81.0
85.8
69.5
78.0
62.0
105.4
75.0
78.0
70.4
66.8
103.6
72.2
72.6
73.8
70.6
18.5
Hela
EJ
BCG-823
1
2
3
4
14.1Ϯ1.5
Ͼ100
Ͼ100
28.2Ϯ4.1
6.8Ϯ1.2
8.2Ϯ3.7
15.4Ϯ4.4
6.9Ϯ2.1
Ͼ100
Ͼ100
5
GluI-1
2
3
4
5
6
COCH3
COCH3
GlcII-1
2
54.7Ϯ6.8
Ͼ100
a) Values were expressed as meanϮS.D. of three independent determinations.
103.8
81.1
85.7
69.5
78.0
62.0
105.5
75.2
78.0
70.7
66.9
103.6
72.3
72.6
73.8
70.5
18.5
103.6
80.9
85.4
69.3
77.8
61.8
105.4
75.0
77.9
70.5
66.7
103.5
72.1
72.5
73.7
70.3
18.4
103.6
80.9
85.2
69.3
77.8
61.8
104.9
72.0
72.5
73.6
70.2
103.4
72.0
72.5
73.6
73.8
18.4
104.6
73.8
77.9
71.3
78.2
62.2
3
4
5
6
Xyl-1
2
3
4
Fig. 2. Selected HMBC Correlations for Compound 2
1
00 MHz) are given in Tables 1—4.
5
Acid Hydrolysis of Compounds 1—4 and Determination of Absolute
Configuration of Monosaccharides Each compound (5 mg) was heated
in 2 M HCl (5 ml) at 90 °C for 4 h. The reaction mixture was extracted with
CHCl (5 mlϫ3). Each remaining aqueous layer was concentrated to dryness
to give a residue. The residue was dissolved in pyridine (0.1 ml), to which
.08 M D-cysteine methyl ester hydrochloride in pyridine (0.15 ml) was
added. The mixture was kept at 60 °C for 1.5 h. After the reaction mixture
was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsily-
limidazole (0.1 ml) for 2 h. The mixture was partitioned between hexane and
H O (0.3 ml each), and the hexane extract was analyzed by GC-MS under
the following conditions: capillary column, DB-17 MS (30 mϫ0.25 mm i.d.,
.25 mm film), ion source temperature, 250 °C; interface temperature,
00 °C, Carrier, Helium gas (1.16 ml/min). In the acid hydrolysate of 1, L-
Rha-1
2
3
4
5
6
3
0
GlcIII-1
2
3
4
5
6
2
0
3
a) Assignments based on TOCSY, HMQC and HMBC experiments. b) Over-
lapped signals are reported without designating multiplicity.
rhamnose, D-glucose, L-arabinose and D-xylose were confirmed by compari-
son of the retention times of their derivatives with those of the derivatives of
L-rhamnose, D-glucose, L-arabinose and D-xylose prepared in a similar way,
which showed retention times of 5.29, 7.33, 5.22 and 6.10 min, respectively.
The constituent sugars of compounds 2—4 were also identified by the same
method.
Extraction and Isolation The dried and cut rhizome parts of A. gigan-
tifolia (13.5 kg) was percolated with 60% ethanol. The 60% ethanol extract
1.54 kg) was partitioned successively between water and petroleum, ethyl
acetate, n-butanol, respectively. After removing the solvent, the n-butanol
layer (600 g) was subjected to a macroporous resin D101 column and eluted
(
Cytotoxicity Assay Hela, EJ and BCG-823 cells were seeded in 96-well
4
plates at adensity of 1ϫ10 cells/well and incubated for 24 h. Test samples
were dissolved in dimethyl sulfoxide (DMSO) and added to the medium.
Following a 48 h incubation, the wells were incubated with the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (100 ml/well
concentrated at 5 mg/ml) at 37 °C for 4 h. The supernatant was aspired, and
the 200 ml of DMSO was added to redissolve the formazan crystals. The op-
tical density was measured by an enzyme-linked immunosorbent assay plate
reader at 570 nm (PerkinElmer 1420-012, China). The experiments were
repeated three times and cytotoxicity was expressed as the IC50 value, which
reduces the number of viable cells by 50%.
with H O and 30%, 50%, 70%, and 95% EtOH. The 70% EtOH eluate
2
(
(
52 g) was chromatographed over a silica gel column with CHCl –MeOH
3
3 : 1) to give six fractions. The fraction 5 (22 g) was further separated by
preparative HPLC at a flow rate of 15.2 ml/min to afford 2 (58 mg,
t ϭ49.6 min, 68% MeOH, 200 nm) and 3 (82 mg, t ϭ54.9 min, 68% MeOH,
R
R
2
00 nm). The fraction 6 (10 g) was subjected to a silica gel column with
CHCl –MeOH–H O (65 : 35 : 10), which gave rise to 1 (745 mg). The 50%
3
2
EtOH eluate (50 g) was chromatographed over a silica gel column with a
gradient of CHCl –MeOH (3 : 1, 2 : 1, 1 : 1) to give three fractions 5-1, 5-2
3
and 5-3. The fraction 5-2 was further subjected to a silica gel H column with
Acknowledgment We are grateful to Mr. Li-Ping Kang for the NMR
and HR-EI-MS measurement in the Instrumentation Center of the Academy
of Military Medical Sciences.
CHCl –MeOH–H O (5 : 2 : 0.1) to afford 4 (395 mg).
3
2
2
6
Compound 1: White amorphous powder; [a] Ϫ21.5 (cϭ0.10, MeOH);
ESI-MS m/z 1191 [MϪH] . HR-ESI-TOF-MS m/z 1191.6279 [MϪH]
Calcd for C H O ). H-NMR (C D N, 400 MHz) and C-NMR (C D N, References
D
Ϫ
Ϫ
1
13
(
58 95 25 5 5 5 5
1
00 MHz) are given in Tables 1—4.
1) Jiangsu New Medicinal College, “Dictionary of Chinese Drugs,”
Shanghai Scientific and Technological Press, Shanghai, 2001, p. 1097.
2) Liu D. L., Zhang X. M., Wang N. L., Yao X. S., Shenyang Pharma
Univ., 21, 394—400 (2004).
2
6
Compound 2: White amorphous powder; [a] Ϫ19.8 (cϭ0.10, MeOH);
D
Ϫ
Ϫ
ESI-MS m/z 1249 [MϪH] . HR-ESI-TOF-MS m/z 1249.6344 [MϪH]
1
13
(Calcd for C H O ). H-NMR (C D N, 400 MHz) and C-NMR (C D N,
60 97 27 5 5 5 5
1
00 MHz) are given in Tables 1—4.
3) Guang Y. T., Wang M. T., Zhao F. Z., Hong S. H., Chin. Tradit. Herb.,
18, 338—341 (1987).
4) Machocho A., Kiprono P., Crinberg S., Bittner S., Phytochemistry, 62,
573—577 (2003).
2
6
Compound 3: White amorphous powder; [a] Ϫ30.8 (cϭ0.10, MeOH);
D
Ϫ
Ϫ
ESI-MS m/z 1353 [MϪH] . HR-ESI-TOF-MS m/z 1353.6787 [MϪH]
1
13
(Calcd for C H O ). H-NMR (C D N, 400 MHz) and C-NMR (C D N,
64 105 30 5 5 5 5
1
00 MHz) are given in Tables 1—4.66
5) Luo J. G., Ma L., Kong L. Y., Bioorg. Med. Clrenr., 16, 2912—2920
(2008).
6) Mimaki Y., Yokosuka A., Hamanaka M., Sakuma C., Yamori T.,
Sashida Y., J. Nat .Prod., 67, 1511—1516 (2004).
2
6
Compound 4: White amorphous powder; [a] Ϫ21.1 (cϭ0.09, MeOH);
D
Ϫ
Ϫ
ESI-MS m/z 1233 [MϪH] . HR-ESI-TOF-MS m/z 1233.6385 [MϪH]
1
13
(Calcd for C H O ). H-NMR (C D N, 400 MHz) and C-NMR (C D N,
60 97 26 5 5 5 5