Triterpenoid saponins from the seeds of Celosia argentea and their anti-inflammatory and antitumor activities

Article abstract of DOI:10.1248/cpb.59.666

Three new triterpenoid saponins, named celosin E (1), celosin F (2) and celosin G (3), together with a known compound cristatain (4), were isolated from the seeds of Celosia argentea L. (Amaranthaceae). All the isolated compounds were obtained for the first time from this plant. The structures of new compounds were characterized on the basis of extensive NMR experiments and mass spectrometry data. The antitumor and anti-inflammatory activities of the four compounds were tested in vitro.

Full text of DOI:10.1248/cpb.59.666

6
66
Note
Chem. Pharm. Bull. 59(5) 666—671 (2011)
Vol. 59, No. 5
Triterpenoid Saponins from the Seeds of Celosia argentea and Their
Anti-inflammatory and Antitumor Activities
Qingbin WU, Yan WANG, and Meili GUO*
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University; No. 325 Guohe Road, Yangpu
District, Shanghai 200433, P. R. China.
Received December 3, 2010; accepted February 1, 2011; published online February 22, 2011
Three new triterpenoid saponins, named celosin E (1), celosin F (2) and celosin G (3), together with a known
compound cristatain (4), were isolated from the seeds of Celosia argentea L. (Amaranthaceae). All the isolated
compounds were obtained for the first time from this plant. The structures of new compounds were character-
ized on the basis of extensive NMR experiments and mass spectrometry data. The antitumor and anti-inflamma-
tory activities of the four compounds were tested in vitro.
Key words Amaranthaceae; Celosia argentea; triterpenoid saponin; antitumor; anti-inflammatory
Celosia L., one category of Amaranthaceae, is widely dis- for treatment of hepatitis, caligo corneae, hypertention and
2)
tributed in Africa, America, the subtropical zone and temper- sarcoptidosis. The crude EtOH extract of Semen celosiae
1)
ate zone of Asia. There are three species in China. They are possesses antipyretic, antispasmodic, anticancer, diuretic,
3)
Celosia argentea L., Celosia cristata L. and Celosia taitoen- anti-inflammatory and antibacterial activities. However,
sis HAYATA. We have been studying for the seeds of two there wasn’t any report about the seeds of C. cristata. In our
species C. argentea and C. cristata in our laboratory. Semen laboratory research for bioactive compounds from Semen
celosiae, the seeds of C. argentea, has been traditionally used celosiae and the seeds of C. cristata, b-sitosterol, palmitic
Fig. 1. Structures of Compounds 1—4
∗
To whom correspondence should be addressed. e-mail: mlguo@smmu.edu.cn
© 2011 Pharmaceutical Society of Japan

May 2011
667
Fig. 2. The Main HMBC Correlations of Compounds 1—3
acid, stigmasterol, daucosterol, oleanolic acid and four heteronuclear multiple bond connectivity (HMBC) spectrum
saponins named celosin A, celosin B, celosin C and celosin (Fig. 2), the methyl proton at d 1.38 (H-24) showed a long-
D were isolated from Semen celosiae and it was demon- range correlation with the carbonyl carbon at d 182.2 (C-23),
strated that the saponins’ hepatoprotective effect was signifi- a methine proton at d 2.76 (H-9) correlation with the car-
4
—6)
cant
; meanwhile, 4-hydroxyphenethyl alcohol, kaempferol, bonyl carbon at d 181.8 (C-26) and a methine proton at d
quercetin, b-sitosterol, 2-hydroxyoctadecanoic acid, stigmas- 4.27 (H-4ꢁ) correlation with the carbonyl carbon at d 175.5
terol and a hepatoprotective saponin named cristatain were (C-6ꢁ).
separated from the seeds of C. cristata. In this paper, the
7,8)
On acid hydrolysis with 2 M trifluoroacetic acid (TFA), 1
isolation and characterization of three new and one known afforded sugar moieties that identified as D-glucuronopyra-
triterpenoid saponins (Fig. 1) from Semen celosiae and their nose (GlcA) based on the GC-MS analysis of the aldononi-
9)
antitumor and anti-inflammatory activities in vitro are re- trile peracetate and the data of the literature. The saccharide
ported.
of 1 was confirmed by the presence of an anomeric proton
signal at d 4.44 (d, Jꢂ7.5 Hz) [with d, Jꢂ7.5 Hz, indicating
1
Results and Discussion
its b configuration] in the H-NMR spectrum and assigned to
1
1
Compound 1 was obtained as a white amorphous powder, D-GlcA. The H– H correlation spectroscopy (COSY) exper-
and its molecular formula, C H O , was deduced from the iment allowed the sequential assignment of most of the reso-
3
6
54 12
high resolution-electrospray ionization-mass spectra (HR- nances for the sugar ring, staring from the easily distin-
ꢀ
ESI-MS): m/z 679.3675 [MꢀH] (Calcd 679.3694), indicat- guished signals due to anomeric proton. The assignment of
ing the presence of ten unsaturation degrees. There were 36 their corresponding carbons was made by heteronuclear sin-
1
3
carbon signals in C-NMR, among which 30 carbon signals gle quantum coherence (HSQC) spectrum. The sugar was at-
were assigned to the aglycone, and 6 carbon signals to the tached to C-3 of the oleanane residue, which was deduced
1
saccharide moiety. The H-NMR of 1 indicated that the agly- from the HMBC correlations between H-1 (d 4.44) of GlcA
cone contained six methyl protons at d 0.65, 0.72, 0.76, 0.96, unit and C-3 (d 87.0). The relative stereochemistry of 1 was
1
5
2
.12, and 1.38 (each 3H, s), and one olefinic proton at d established with aid of a nuclear Overhauser effect spec-
.11. Correspondingly, six methyl carbons at d 18.0, 33.9, troscopy (NOESY) experiment. Thus, 1 was elucidated as 2-
4.4, 26.9, 17.4, and 14.3 and two olefinic carbons at d 123.6 hydroxy-12,13-ene-23,26-dicarboxy-3-O-[b-D-glucuronopy-
1
3
and 145.5 exhibited in the C-NMR spectrum, respectively. ranosyl]-oleanene and named celosin E.
Additionally, three carbonyl carbons at d 175.5, 181.8, and Compound 2 was obtained as a white amorphous powder,
82.2 exhibited in the C-NMR spectrum, respectively. The and its molecular formula, C H O , was deduced from the
1
3
1
3
5
50 12
ꢃ
presence of the 2-OH group was indicated by a signal at d HR-ESI-MS: m/z 661.3255 [MꢃH] (Calcd 661.3224), indi-
1
3
8)
70.9 in the downfield region of the C-NMR spectrum. In cating the presence of eleven unsaturation degrees. There

6
68
Vol. 59, No. 5
1
13
Table 1. H-NMR (500 MHz) and C-NMR (125 MHz) Data for Compounds 1, 2 and 3 (1 and 2 in CD OD; 3 in C D N. d in ppm and J in Hz)
3
5
5
1
2
3
Positon
dH (J in Hz)
1.93 (d, 13.5)
4.27 (s)
4.07 (s)
dC
dH (J in Hz)
1.18 (d, 6.5)
4.18 (s)
3.98 (s)
dC
dH (J in Hz)
dC
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
45.1
70.9
87.0
53.6
53.3
29.0
30.9
37.6
42.9
41.1
24.8
123.6
145.5
43.2
21.9
47.4
47.7
49.8
35.1
31.8
33.8
34.0
182.2
14.3
18.0
181.8
26.9
17.4
33.9
24.4
44.9
71.3
86.7
53.6
53.3
21.9
31.1
37.7
43.0
41.2
24.4
124.3
144.8
43.3
30.7
39.3
47.9
49.1
43.0
149.9
33.8
42.9
182.1
13.9
17.3
181.9
17.8
181.1
26.7
107.5
1.35 (s), 2.36 (d, 12.5)
2.11 (m)
4.80 (d, 3.5)
44.6
32.5
86.3
53.1
52.8
21.2
34.1
40.3
48.9
37.0
23.8
122.9
144.9
42.4
23.5
32.5
47.2
42.3
46.5
31.1
34.4
28.4
180.3
14.3
17.0
17.6
26.2
176.8
33.4
23.9
1.51 (d, 8.5)
1.65 (m), 0.83 (d, 14)
1.07 (s)
1.48 (m)
1.06 (s), 1.58 (m)
2.03 (m)
2.15 (m)
1.86 (m), 1.72 (m)
1.30 (m)
2.77 (dd, 13.5, 2.5)
2.43 (t, 13.5)
1.82 (m)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1.77 (m)
5.11 (s)
1.62 (m)
5.23 (s)
1.00 (s), 2.17 (m)
5.50 (s)
1.52 (s)
1.50 (s)
1.18 (d, 6.5)
1.44 (m), 1.73 (m)
1.99 (s)
2.11 (m)
2.62 (dd, 18.0, 4.0)
2.43 (t, 13.5)
3.19 (dd, 14.5, 4.0)
1.82 (m), 1.32 (s)
1.17 (s)
1.41 (s)
1.18 (s), 1.43 (m)
2.75 (d, 11.0), 1.95 (m)
1.49 (d, 9.0)
2.17 (m), 1.35 (s)
1.38 (s)
0.65 (s)
1.30 (s)
1.18 (s)
2.07 (s)
1.65 (s)
1.07 (s)
1.33 (s)
0.96 (s)
1.12 (s)
0.72 (s)
0.76 (s)
0.73 (s)
1.09 (s)
4.51 (s)
1.35 (s)
1.00 (s)
3-O-GlcA
1
2
3
4
5
6
4.44 (d, 7.5)
3.32 (t, 8.0)
3.45 (m)
4.27 (s)
3.55 (m)
105.5
75.3
77.9
70.9
73.9
175.5
3-O-Xyl
1
2
3
4
5
4.31 (d, Jꢂ7.5)
3.19 (m)
3.25 (m)
3.39 (m)
3.45 (t, 6.5)
105.5
75.0
77.5
73.5
62.9
3-O-Glc
1
2
3
4
5
6
5.28 (d, 7.5)
4.01 (m)
4.24 (m)
4.02 (m)
4.26 (m)
4.32 (m)
106.2
75.0
77.7
78.6
71.1
67.6
28-O-Fuc
1
2
3
4
5
6
6.11 (d, 8.0)
4.71 (m)
4.24 (m)
4.02 (m)
4.77 (m)
1.65 (s)
94.9
74.3
76.7
73.3
72.5
17.0
Fuc-2-Rha
1
2
3
4
5
6
6.47 (br s)
4.91 (s)
4.77 (m)
4.40 (m)
4.85 (d, 3.0)
1.77 (d, 6.0)
101.5
71.9
72.5
85.5
70.9
18.7
Rha-4-glcA
1
2
3
4
5
6
5.10 (d, 7.5)
4.13 (d, 5.5)
4.01 (m)
4.26 (m)
4.13 (dd, 14.5, 5.5)
107.8
76.4
75.0
71.1
78.9
170.7

May 2011
669
1
3
11,12)
were 35 carbon signals in C-NMR, among which 30 car- the literature.
This compound occurred as a C-3 and C-
bons were assigned to the aglycone, and 5 carbons to the sac- 28 bisdesmosidic triterpene glycoside. In the NMR spectrum
1
charide moiety. The H-NMR of 2 indicated that the agly- of compound 3, four anomeric proton [dH 5.28 (d, Jꢂ
cone contained four methyl protons at d 0.72 (H-27), 1.09 7.5 Hz), 6.11 (d, Jꢂ8.0 Hz), 6.47 (br s), 5.10 (d, Jꢂ7.5 Hz)]
(
H-29), 1.18 (H-25) and 1.29 (H-24) (each 3H, s), two and four anomeric carbon (d 106.3, 94.9, 101.5, 107.8) sig-
C
olefinic protons at d 5.23 (H-12) and 4.51 (H-30). Corre- nals were observed, corresponding to D-glucose, D-fucose, L-
spondingly, four methyl carbons were at d 13.9 (C-24), 17.3 rhamnose and D-glucuronopyranosyl. In the HMBC spec-
(
C-25), 17.8 (C-27) and 26.7 (C-29), four olefinic carbons at trum, the main correlations from H-1 of glucose to C-3 and
d 107.5 (C-30), 124.3 (C-12), 144.8 (C-13) and 149.9 (C- from H-1 of fucose to C-28 confirmed that the sugar units
2
0). Additionally, three carbonyl carbons at d 181.2 (C-28), were located at C-3 and C-28 of the aglycon, respectively.
13
1
81.9 (C-26) and 182.1 (C-23) exhibited in the C-NMR Other correlations were observed between signals at d 4.71
H
spectrum (Table 1), respectively. The presence of the 2-OH (H-2 of Fuc) and d 101.5 (C-1 of Rha) and between d 5.10
group was indicated by a signal at d 71.3 in the downfield re- (H-1 of GlcA) and d 85.5 (C-4 of Rha). The relative stereo-
C
H
C
1
3
gion of the C-NMR spectrum. In HMBC spectrum, the chemistry of 3 was established with aid of a NOESY experi-
methyl proton at d 1.29 (H-24) showed long-range correla- ment. Therefore, compound 3 was assigned as 23-carboxy-
tion with the carbonyl carbon at d 182.2 (C-23), a methylene 3-O-[b-D-glucopyranosyl]-28-O-[b-D-glucuronopyranosyl-
proton at d 2.03 (H-7) was correlated with the carbonyl car- (1→4)-a-L-rhamnopyranosyl-(1→2)-b-D-fucopyranosyl]-
bon at d 181.9 (C-26) and a methylene proton at d 2.74 (H- oleanolic acid and named celosin G.
2
2
2) was correlated with the carbonyl carbon at d 181.1 (C-
Compounds 1, 2, 3 and 4 were tested for their antitumor
8). The methylene proton at d 2.74 (H-22) was also corre- activities toward five human cancer cell lines (SHG44,
lated with the carbonyl carbon at d 149.9 (C-20) and 107.5 HCT116, CEM, MDA-MB-435 and HepG2) by the 3-(4,5-
C-30). It indicated that the aglycone might be one type of dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(
10)
ursolic acid. The sugar moiety of 2 were determined to be (MTT) assay (Table 2).
D-xylose which was analyzed by GC-MS with the aldonon-
11)
The four compounds were also detected for their ability to
1
1
itrile peracetate of standard sugar. A H– H COSY experi- inhibit nitric oxide (NO) production by LPS-induced RAW
ment allowed analysis of their spin systems and assignments 264.7 cells. The inorganic free radical NO, synthesized by a
of their proton resonances. This was also confirmed by the family of enzymes termed NO-synthase (NOS), acts as a
monosaccharide anomeric proton signals at d 4.31 (d, host defense mechanism by damaging pathogenic DNA and
Jꢂ7.5 Hz) [with d, Jꢂ7.5 Hz, indicating its b configuration], also acts as a regulatory molecule with homeostatic activi-
showed correlation with the aglycone carbon at d 86.7 (C-3) ties. However, excess production of NO, due to the reaction
in the HMBC spectrum. The relative stereochemistry of 2 with superoxide in biological systems, gives rise to various
was established with aid of the NOESY experiment. Based diseases such as inflammation, carcinogenesis, and athero-
13)
14)
on the above results, 2 was elucidated as 2-hydroxy-20,29- sclerosis. Therefore, down-regulation of NO production
ene-23,26-dicarboxy-3-O-[b-D-xylopyranosyl]-ursolic acid and may be of therapeutic benefit in various diseases induced by
named celosin F.
pathological levels of NO. In the present study, it was found
Compound 3 was obtained as a white amorphous powder, that the four compounds had inhibiting nitric oxide produc-
and its molecular formula, C H O , was deduced from the tion activity compared with the positive control in-
5
4
84 24
ꢀ
HR-ESI-MS: m/z 1139.5248 [MꢀNa] (Calcd 1139.5250), domethacin. The IC50 values was showed in Table 3. From
indicating the presence of thirteen unsaturation degrees. these data, it can be concluded that the sequence of anti-in-
1
3
There were 54 carbon signals in C-NMR, among which 30 flammatory activities of these compounds was as follows:
carbons were assigned to the aglycone, and 24 carbons to the compound 4ꢄcompound 1ꢄcompound 3ꢄcompound 2ꢄin-
saccharide moiety. Most signals due to the aglycone of 3 domethacin (reference). Cell viability was also determined
6)
were similar to celosin D. On acid hydrolysis with 2 M TFA, by application of the MTT method in order to evaluate
afforded sugar moieties that identified as D-glucose, D-fu- whether inhibition of NO production was due to the cytotoxi-
3
cose, L-rhamnose and D-glucuronopyranosyl in the relative city of these tested compounds. It was found that none of the
proportions of 1 : 1 : 1 : 1 based on the GC-MS analysis of the concentrations used in the experiment was cytotoxic (cell vi-
aldononitrile peracetates of standard sugars. And the absolute ability ꢄ85%). The experiment results supported the follow-
configuration of sugars was further confirmed by consulted ing ideas: 1. It seemed that the structure of the aglycone es-
Table 2. Cytotoxic Activity of Compounds 1—4 against Five Human Cancer Cell Lines
IC50 (mg/ml)
Sample
SHG44
HCT116
CEM
MDA-MB-435
HepG2
1
2
3
4
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
ꢄ100
b)
23.71ꢅ2.96
0.117ꢅ0.024
26.72ꢅ4.11
0.176ꢅ0.015
31.62ꢅ2.66
0.00134ꢅ0.00013
27.63ꢅ2.93
0.0717ꢅ0.0162
28.35ꢅ2.32
0.116ꢅ0.025
a)
DOX
a) Hydroxydaunomycin hydrochloride (DOX) as positive control. b) The data represent the mean plus S.D. of three independent experiments in which each compound con-
centration was tested in two replicate wells.

6
70
Vol. 59, No. 5
Table 3. IC50 Values of Compounds 1—4 and Indomethacin with Differ- tent with the literature.8)
6)
ent Concentration
Process of Acid Hydrolysis
Each saponins (3 mg) was heated in an
ampule with 5 ml of aqueous 2 M CF COOH at 120 °C for 2 h. The aglycone
3
1
2
3
4
Indomethacin
0.371
was extracted with dichloromethane, and the aqueous residue was evapo-
rated under reduced pressure. Then 0.8 ml of pyridine and 2 mg of
IC50 value (mmol/ml)
0.158
0.384
0.278
0.047
NH OH·HCl were added to the dry aqueous residue, and the mixtures were
2
heated at 90 °C for 30 min. After the reaction mixtures were cooled, 0.8 ml
LPS: Negative control; indomethacin: positive control.
of Ac O was added and the mixtures were heated at 90 °C for 1 h. The reac-
2
tion mixtures were evaporated under reduced pressure, and the resulting al-
dononitrile peracetates were analyzed by GC-MS using the aldononitrile
peracetates of standard sugars as reference samples.
pecially the carbonyl group at C-23 and the hydroxyl group
at C-2 played an important role in terms of anti-inflammatory
16)
MTT Assay
Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraa-
activity against RAW 264.7 cells; 2. The anti-inflammatory zolium bromide (MTT assay) procedures were used with the slight modifica-
4
tion. Cells were placed in 96-well microassay culture plates (4ꢆ10 cells per
activities of oleanene-type saponins were more available than
ursanene-type ones. However, more extensive studies are re-
quired before a clear structure-activity relationship can be
reached.
well) and grown overnight at 37 °C in a 5% CO incubator. Test compounds
2
were then added to the wells to achieve six final concentrations ranging from
ꢃ3
2
1
0
to 10 mg/ml. Control wells were prepared by addition of culture
medium (100 ml). Wells containing culture medium without cells were used
as blanks. The plates were incubated at 37 °C in a 5% CO incubator for
2
Experimental
72 h. Upon completion of the incubation, stock MTT dye solution (20 ml,
5 mg/ml) was added to each well. After 4 h incubation, buffer (100 ml) con-
taining N,N-dimethylformamide (50%) and sodium dodecyl sulfate (20%)
was added to solubilize the MTT formazan. The optical density of each well
was then measured on a microplate spectrophotometer at a wavelength of
570 nm. The IC50 values were determined by plotting the percentage viabil-
ity versus concentration on a logarithmic graph and reading off the concen-
tration at which 50% of cells remain viable relative to the control. Each ex-
periment was repeated three times to get the mean values. Five different
tumor cell lines were the subjects of this study: SHG44 (human glioma
cells), HCT116 (human colon cancer cells), CEM (human leukemia cells),
General Experimental Procedures The IR spectra were obtained on a
Bruker Vector 22 spectrometer with KBr pellets. ESI-MS and HR-ESI-MS
were measured on Micromass Q-TOF and the Agilent 6538 Q-TOF mass
spectrometer, respectively. The melting points were measured on a Yanaco
micromelting point apparatus without correction. Gas chromatography
analysis was done on an HP-5892 II with FID detector, and an HP-20M
(
Carbowx 20M) capillary column (25 mꢆ0.32 mmꢆ0.3 mm) was used. Col-
umn chromatography separations were carried out on D101 macroporous
resin (Chemical Factory of Nankai University, Tianjin, P. R. China), Octa-
1
13
decyl silane (ODS) (50 mesh, AA12S50, YMC). The H-NMR, C-NMR,
distortionless enhancement by polarization transfer (DEPT), H-detected MDA-MB-435 (human breast cancer cells) and HepG2 (human hepatocellu-
1
heteronuclear multiple quantum coherence (HMQC) and HMBC spectra
were recorded on a Bruker DMX-500 NMR spectrometer with tetramethyl-
silane (TMS) as an internal standard. The preparative high-speed counter-
current chromatography instrument (Model TBE-300B, Shanghai Tauto Bio-
logical Co., China), the constant-flow pump (Model TBP5002 Shanghai
Tauto Biological Company, China), Model HX-1050 constant-temperature
controller (Beijing Detianyou Technology, Beijing, China), evaporative light
scattering detection (SEDEX 85 ELSD, France).
lar carcinoma cells).
Inhibition Ability against LPS-Induced NO Production and Cell Via-
bility The inhibition ability against LPS-induced NO production and cell
viability was evaluated according to the literature with some modification.
RAW 264.7 macrophages were seeded at 1ꢆ10 /ml in 96-well microtiter
plates. The cells were co-incubated with the isolated compounds and LPS
(5 mg/ml) for 24 h. The amount of NO was assessed by determining the ni-
trite concentration in the cultured RAW 264.7 macrophage supernatants with
1
7)
6
Plant Material The seeds (Semen celosiae) were collected in Bozhou, Griess reagent (1% sulfanilamide/0.1% naphthylethylendiamine dihydro-
Anhui province of China in October 2004, and authenticated by Prof. Mei-li
Guo, School of Pharmacy, Second Military Medical University (Shanghai,
China). A voucher specimen (SMMU 04063) was deposited at the herbar-
ium of Second Military Medical University of China.
Extraction and Isolation Dried Semen celosiae (10 kg) was ground to
a coarse powder and extracted with 50% ethanol at room temperature after medium containing 2 mg/ml of MTT was added to each well. The cells were
chloride/2.5% H PO ). The absorbance at 540 nm was read using a mi-
3 4
croplate reader (POLARstar). Cell viability was determined using the mito-
chondrial respiration-dependent MTT reduction method. After transferring
the required supernatant to another plate for the Griess assay, the remaining
supernatant was aspirated from the 96-well plates, and 100 ml of fresh
24 h maceration. The extracted liquid was concentrated under reduced pres-
incubated at 37 °C in humidified air with 5% CO . After incubating for 4 h,
2
sure to give an ethanol extract (3.6 kg). A portion of the ethanol extract
the medium was removed and the violet crystals of formazan in viable cells
were dissolved in dimethyl sulfoxide (DMSO). Absorbance at 570 nm was
(3.0 kg) was subjected to chromatographic separation over D101 MR
(
10 kg), eluted sequentially with a gradient of H O, 30%, 60% and 95% measured using a microplate reader.
2
EtOH to give four fractions with the yields of 43.5 g, 55.8 g, 95.4 g and
5.8 g, respectively. The 30% EtOH fraction was chromatographed through
1
Acknowledgements We thank Mr. Xin Dong and Mrs. Liping Shi for
ODS, eluted with a gradient of MeOH–H O (5 : 95→30 : 70→50 : 50, v/v) HR-ESI-MS and NMR measurements.
2
to afford three fractions. The second fraction was separated by high-speed
counter-current chromatography using a solvent system of dichloromethane–
methal–n-butanol–water (4 : 3 : 0.3 : 2, v/v/v/v) with addition of 0.5% glacial
acetic acid to afford compounds 1 (15 mg), 2 (18 mg), 3 (13 mg) and 4
References
1) Chinese Academy of Sciences in China Plant Volunteer Editors Com-
mittee, “Flora Reipublicae Popularis Sinicae,” 1st ed., Vol. 25, Science
Press, Beijing, 1979, p. 200.
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(
10 mg).
2
0
Celosin E (1): White amorphous powder, mp: 235—237 °C, [a] ꢃ5.6
cꢂ0.02, MeOH). The IR spectrum (KBr) showed absorptions at 3418,
944, 1697, 1624, 1263, 1057 cm . HR-ESI-MS (m/z): 679.3675 [MꢀH]
D
1
5)
(
2
ꢃ1
ꢀ
1
13
(Calcd 679.3694). H- and C-NMR: see Table 1.
2
0
Celosin F (2): White amorphous powder, mp: 225—227 °C, [a]D ꢃ7.5
cꢂ0.03, MeOH). The IR spectrum (KBr) showed absorption at 3439, 2927,
694, 1655, 1384, 1084 cm . HR-ESI-MS (m/z): 661.3255 [MꢃH] (Calcd
61.3224). H- and C-NMR: see Table 1.
Celosin G (3): White amorphous powder, mp: 239—241 °C, [a]D ꢃ4.3
cꢂ0.03, MeOH). The IR spectrum (KBr) showed absorption at 3426, 2943,
(
1
6
ꢃ1
ꢃ
1
13
2
0
(
1
ꢃ1
723, 1691, 1629, 1384, 1076 cm
.
HR-ESI-MS (m/z): 1139.5248
ꢀ
1
13
[MꢀNa] (Calcd 1139.5250). H- and C-NMR: see Table 1.
2
0
Cristatain (4): White amorphous powder, mp: 243—245 °C; [a]D ꢃ7.0
cꢂ0.04, MeOH); The IR spectrum (KBr) showed absorption at 3403, 3322,
(
2
ꢃ1
937, 2855, 1781, 1696, 1640, 1025 cm ; HR-ESI-MS (m/z): 661.3591
ꢃ
1
13
[MꢃH] (Calcd 661.3588). The H- and C-NMR spectral data are consis- 10) Reynolds W. F., McLean S., Poplawski J., Enriquez R. G., Escobar L.

May 2011
671
I., Leon I., Tetrahedron, 42, 3419—3428 (1986).
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1
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