Three new oleanane-type triterpenoid saponins from the seeds of Celosia cristata L.

Article abstract of DOI:10.1080/14786419.2017.1343317

Phytochemical investigation of the 1-butanol soluble fraction of 60% ethanol extract of the seeds of Celosia cristata L. led to the identification of three new oleanane-type triterpenoid saponins. Using ¹D and ²D NMR experiment metho

Full text of DOI:10.1080/14786419.2017.1343317

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Three new oleanane-type triterpenoid saponins
from the seeds of Celosia cristata L.
Xin Liu, Jifa Zhang, Kai Guo, Airong Jia, Miansong Zhang, Yaping Shi,
Changheng Liu, Linlin Xiao & Zhenliang Sun
To cite this article: Xin Liu, Jifa Zhang, Kai Guo, Airong Jia, Miansong Zhang, Yaping
Shi, Changheng Liu, Linlin Xiao & Zhenliang Sun (2017): Three new oleanane-type
triterpenoid saponins from the seeds of Celosia cristata L., Natural Product Research, DOI:
10.1080/14786419.2017.1343317
To link to this article: http://dx.doi.org/10.1080/14786419.2017.1343317
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Date: 30 June 2017, At: 04:29

Natural Product research, 2017
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1343317
ꢀ
Three new oleanane-type triterpenoid saponins from the
seeds of Celosia cristata L.
a1
b1
a
a
a
a
Xin Liu , Jifa Zhang , Kai Guo , Airong Jia , Miansong Zhang , Yaping Shi ,
a
b
b
Changheng Liu , Linlin Xiao and Zhenliang Sun
ꢆ
Kꢇy lꢆbꢄꢅꢆꢁꢄꢅy fꢄꢅ Biꢄꢂꢇnꢂꢄꢅꢂ ꢄf sꢀꢆnꢃꢄng Pꢅꢄvinꢈꢇ, Biꢄꢉꢄgy Inꢂꢁiꢁꢊꢁꢇ ꢄf sꢀꢆnꢃꢄng aꢈꢆꢃꢇmy ꢄf sꢈiꢇnꢈꢇꢂ,
b
Jinꢆn, cꢀinꢆ; Fꢇngxiꢆn hꢄꢂpiꢁꢆꢉ, sꢄꢊꢁꢀꢇꢅn Mꢇꢃiꢈꢆꢉ univꢇꢅꢂiꢁy, sꢀꢆngꢀꢆi, cꢀinꢆ
ABSTRACT
ARTICLE HISTORY
Phytochemical investigation of the 1-butanol soluble fraction of 60%
ethanol extract of the seeds of Celosia cristata L. led to the identification
rꢇꢈꢇivꢇꢃ 10 Nꢄvꢇmbꢇꢅ 2016
aꢈꢈꢇpꢁꢇꢃ 27 Mꢆy 2017
1
2
of three new oleanane-type triterpenoid saponins. Using D and D
NMR experiment methods, ESI-MS analysis and acid hydrolysis, their
structures were identified as 3-O-[β-D-xylopyranosyl-(1 → 3)-β-D-
glucuronopyranosyl]-2β-hydroxy-oleanolic acid-28-O-β-D-glucopy
ranoside (1), 3-O-[β-D-xylopyranosyl-(1 →3)-β-D-glucuronopyranosyl]-
KEYWORDS
Celosia cristata; ꢈꢀꢇmiꢈꢆꢉ
ꢈꢄnꢂꢁiꢁꢊꢇnꢁ; ꢁꢅiꢁꢇꢅpꢇnꢄiꢃ
ꢂꢆpꢄnin
2
3
β, 23-dihydroxy-oleanolic acid-28-O-β-D-glucopyranoside (2) and
-O-[β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl]-2-hydroxyl-
medicagenic acid-28-O-β-D-glucopyranosyide (3), respectively.
1
. Introduction
Celosia cristata is a member of the genus Celosia, and is commonly known as cockscomb
because the flower looks like the head on a rooster. It belongs to the class Mangoliopsida,
order Caryophyllales and family Amaranthaceae. Other than its ornamental value due to
vibrant coloured inflorescence, C. cristata is also known for its activities for the treatment of
CONTACT Zꢀꢇnꢉiꢆng sꢊn
Xin liꢊ ꢆnꢃ Jifꢆ Zꢀꢆng ꢈꢄnꢁꢅibꢊꢁꢇꢃ ꢇqꢊꢆꢉꢉy ꢁꢄ ꢁꢀiꢂ wꢄꢅk.
ꢀꢄpꢇ1126@ꢀꢄꢁmꢆiꢉ.ꢈꢄm
1
sꢊppꢉꢇmꢇnꢁꢆꢉ ꢃꢆꢁꢆ fꢄꢅ ꢁꢀiꢂ ꢆꢅꢁiꢈꢉꢇ ꢈꢆn bꢇ ꢆꢈꢈꢇꢂꢂꢇꢃ ꢆꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1343317.
©
2017 Infꢄꢅmꢆ uK limiꢁꢇꢃ, ꢁꢅꢆꢃing ꢆꢂ tꢆyꢉꢄꢅ & Fꢅꢆnꢈiꢂ Gꢅꢄꢊp

2
X. LIU ET AL.
haematemesis, abnormal uterine bleeding, haematochezia, haemorrhoidal bleeding, leuc-
orrhea, chronic dysentery with persistent diarrhoea, redness of the eye and dizziness due to
excessive heat of fire in the liver (Cai et al. 2003). Semen C. cristata is the seeds of C. cristata
L. named‘Ji Guan Hua Zi’in Chinese, and used as a traditional Chinese medicine for removing
‘
liver-heat ’, improving eyesight, clearing wind-heat and as an anti-inflammatory agent (Wang
et al. 2010). It has long been used as a folk medicine for the treatment of hypertension, palsy,
cataract, keratitis, diabetes, fatigue, atherosclerosis, leucorrhoea and osteoporosis (Wang
et al. 2010; Sun et al. 2011). Recent phytochemical studies reported the chemical constituents
of flavonoids, phenolic glycosides, coumaronochromones and saponins in Semen C. cristata
(
Wen et al. 2006; Wang et al. 2010; Sun et al. 2011; Yan and Zhang 2016). Modern pharmaco-
logical research has demonstrated that C. cristata possesses antiviral, antioxidant, anthelmintic
and hepatoprotective activities (Balasubrahmanyam et al. 2000; Gholizadeh et al. 2004; Rubini
et al. 2012). Therefore, its health benefits have aroused increasing attention interest.
Triterpenoid saponins, as an important class of natural products, are distributed widely
in the plant kingdom. Their pharmacological effects, such as anticancer, antibacterial, anti-
mutagenesis, anti-inflammatory and inhibition of cardio-cerebral vascular diseases, have
been reported recently (Soetan et al. 2014). Triterpenoid saponins are also known to be
important bioactive constituents in Celosia plants. Previous phytochemical and pharmaco-
logical studies on Celosia genus plants have resulted in the isolation of several triterpenoid
saponins which have been reported to possess anti-inflammatory, antitumour and hepato-
protective activities (Wen et al. 2006; Wang et al. 2010; Sun et al. 2011; Wu et al. 2013).
Therefore, the present study focused on the isolation and structure elucidation of three new
triterpenoid saponins from the 1-butanol soluble fraction of 60% ethanol extract of Semen
1
2
C. cristata. Their structures were identified by extensive use of D and D NMR experiments
along with HR-ESIMS analysis and acid hydrolysis.
2
. Results and discussion
Three new triterpenoid saponins were isolated and identified from the 1-butanol fraction
1
13
by chromatographic methods and spectrometric data. The optical rotation, H and C NMR
and mass spectra analysis were used to identify these compounds (Figure 1).
2
5
Compound 1, obtained as a white amorphous powder, m.p., 280 ~ 282 °C;[ꢀ] , +20.8 (c
D
0
.25, MeOH), positive to Liebermann–Burchard reaction. The IR spectrum showed the pres-
−1
−1
ence of hydroxyl group (3418 cm ) and carbonyl group (1735 cm ). The HR-ESI-MS of 1
Figure 1. cꢀꢇmiꢈꢆꢉ ꢂꢁꢅꢊꢈꢁꢊꢅꢇꢂ ꢄf ꢈꢄmpꢄꢊnꢃꢂ 1–3 iꢂꢄꢉꢆꢁꢇꢃ fꢅꢄm ꢁꢀꢇ ꢂꢇꢇꢃꢂ ꢄf C. cristata.

NATURAL PRODUCT RESEARCH
3
+
exhibited a [M + Na] ion at m/z 965.4713, indicating a molecular formula of C H O . There
47
74 19
1
3
were 47 carbon signals in the C NMR spectrum, among which 30 carbons were assigned
to the aglycone, with another 17 carbons belonging to the sugar moieties. The H NMR of 1
1
showed that aglycone contained seven methyl protons at δ 0.85, 0.88, 1.11, 1.26, 1.30, 1.36
H
and 1.45 (each 3H, s), one olefinic proton at δ 5.40. Correspondingly, seven methyl carbons
H
at δ 23.6, 33.1, 17.5, 26.1, 29.5, 18.5 and 16.5, two olefinic carbons at δ 123.0 and 144.0,
C
C
1
3
and one carbonyl carbon at δ 176.4 exhibited in the C NMR spectrum, respectively. A
feature of the H NMR spectrum was the signal at δ 5.40 typical of H-12 of ∆ skeleton,
C
1
12
H
which was also indicated by the carbon signals at δ 123.0 and 144.0, due to C-12 and C-13
C
1
3
in the C NMR spectrum. The presence of two oxygenated methines was deduced from the
signals at δ 3.21 (1H, m) and 4.63 (1H, m), which correlated in the HSQC spectrum with the
H
carbon resonances at δ 89.7 and 70.4, respectively. These signals are typical of ring A of a
C
2ß-hydroxy substituted oleanolic acid. The configuration of C-2 and C-3 in the triterpene
structure was confirmed by NOESY experiments, in which a cross peak between H-2 and H-3
was observed, confirming the presence of 2ß,3ß-dihydroxyoleanane skeleton. Furthermore,
in the HMBC spectrum, the methine proton (H-3, δ 3.36) was correlated with an anomeric
H
carbon (δ 106.1) of the sugar moiety. Three anomeric proton signals at δ 5.31 (1H, d,
C
H
J = 7.4 Hz, H-1′), 5.02 (1H, d, J = 7.1 Hz, H-1″), 6.29 (1H, d, J = 8.1 Hz, H-1″′) and three corre-
sponding anomeric carbon signals at δ 106.1, 106.4 and 95.7, were also observed revealing
C
1
13
that compound 1 contained three sugar units (Figure S1). Comparison of H and C NMR
data of 1 with those of celosin H, isolated from the seeds of C. argentea, showed that 1 was
almost identical to celosin H, except for the absence of an aldehyde group and the presence
of a methyl group (Pang et al. 2014).
Acid hydrolysis of 1 followed by GC-MS analysis of its derivative enabled further identi-
fication of the existence of d-glucuronic acid, d-xylose and d-glucose. The β-configurations
of d-glucuronic acid, d-xylose and d-glucose were determined by the J values of 7.4 Hz,
1
,2
1
1
7
.1 Hz and 8.1 Hz, respectively. A H- H COSY experiment allowed analysis of their spin sys-
tems and assignments of their spin systems and proton resonances. The assignment of their
corresponding carbons, made by a HSQC spectrum, indicated that xylose was a terminal
unit. The Xyl-(1 → 3)-GlcA structure of the disaccharide moiety at C-3 of the oleanolic acid
residue was deduced from the HMBC correlations from H-3 (δ 3.36) to C-1′ (δ 106.1) of the
H
C
GlcA unit and from H-3′ (δ 4.33) of the GlcA unit to C-1″ (δ 106.4) of the Xyl unit. The gly-
H
C
cosylation position of glucose was confirmed due to the long-range HMBC correlations from
H-1″′ at δ 6.29 to C-28 at δ 176.4 (Figure S1). Therefore, we speculated that celosin H was a
product of methyl (C-23) oxidation in 1 (Pang et al. 2014). This speculation was confirmed
by the HMBC correlations between H-23 (δ 1.30) and C-2 (δ 70.4), C-3 (δ 89.7). In addition,
H
C
C
the NOESY correlation of H-24 (δ 1.36) and H-25 (δ 1.45) revealed that the β-configuration
H
H
of 24-CH and α-configuration of 23-CH , the correlations of 23-CH with H-2 (δ 4.63) and
3
3
3
H
H-3 (δ 4.15) revealed the α-configurations of H-2 and H-3, thus, the β-configurations of
H
2-OH and 3-OH were determined. Consequently, the structure of 1 was elucidated as 3-O-[β-
D-xylopyranosyl-(1 → 3)-β-D-glucuronopyranosyl]-2β-hydroxy-oleanolic acid-28-O-β-D-glu-
copyranoside, named celosin K.
2
5
Compound 2, white amorphous powder, m.p., 281 ~ 282 °C; ꢀ , +22.6 (c 0.25, MeOH),
D
and the Molish and Liebermann–Burchard reactions were positive. The IR spectrum showed
−
1
−1
the presence of hydroxyl group (3413 cm ) and carbonyl group (1728 cm ). The molecular
formula of 2 was determined as C H O from its positive-ion HR-ESI-MS (m/z 981.4667
4
7
74 20

4
X. LIU ET AL.
+
1
13
[M + Na] , calcd. 981.4671). The H and C NMR data of 2 indicated a pentacyclic triterpenoid
1
saponin containing one triterpene aglycone and three monosaccharides. The H NMR data
exhibited the characteristic signals for six tertiary methyl groups at δ 1.32 (H-24), 1.54 (H-25),
H
1.13 (H-26), 1.21 (H-27), 0.86 (H-29) and 0.84 (H-30), the corresponding carbon signals at δC
17.2, 14.9, 17.6, 26.2, 33.1 and 23.6, respectively. Two characteristic oxygenated methine
protons were also observed at δ 4.34 (1H, m) and 4.79 (1H, m), which correlated in the HSQC
H
spectrum with the carbon resonances at δ 82.7 and 70.9, respectively. The anomeric proton
C
signals at δ 5.26 (1H, d, J = 7.7 Hz, H-1′), 5.24 (1H, d, J = 7.6 Hz, H-1″) and 6.28 (1H, d, J = 8.1 Hz,
H
H-1″′) showed HSQC correlations with anomeric carbon signals at δ 105.7 (C-1′), 106.1 (C-1″)
C
and 95.7 (C-1″′). In addition, one trisubstituted olefinic proton at δ 5.38 (H-12) with two
H
typical olefinic carbon signals at δ 123.0 and 144.1, one oxydic-methine at δ 82.7 and one
C
C
carbony signal at δc 176.4, indicating the aglycone of 2 was a derivative of oleanolic acid.
The NMR data of 2 were very similar to those of 1, except for the absence of a methyl group
and appearance of –CH –OH group.
2
The presence of d-glucuronic acid, d-xylose and d-glucose in a 1:1:1 ratio was established
by acid hydrolysis followed by GC-FID analysis of the corresponding derivatives. Comparison
1
13
of the H and C NMR data of the oligosaccharide part with those of 1 indicated that 2
contained the same sugar units as 1. The linkage points of the sugar units to each other and
to the aglycone were determined by following HMBC correlations: δ 5.26 (H-1′) with δ 82.7
H
C
(
C-3), δ 5.24 (H-1″) with δ 86.3 (C-3′) and δ 6.28 (H-1″′) with δ 176.2 (C-28) (Figure S1).
H C H C
Therefore, we speculated that compound 2 was a product of methyl (C-23) oxidation in 1.
This speculation was confirmed by the HMBC correlations between H-23 (δ 3.64) and δ
H
C
8
2.7 (C-3), 42.9 (C-4), 47.5 (C-5) and 14.9 (C-24) (Figure S1). Hence, compound 2 was identified
as 3-O-[β-D-xylopyranosyl-(1 → 3)-β-D-glucuronopyranosyl]-2β, 23-dihydroxy-oleanolic acid-
8-O-β-D-glucopyranside, named celosin L.
Compound 3 was obtained as a white amorphous powder, m.p., 281 ~ 283 °C; ꢀ , +20.3
2
2
5
D
(
c 0.18, MeOH), and the Molish and Liebermann–Burchard reactions were positive. The IR
−1
spectrum showed the presence of hydroxyl group (3395 cm ) and carbonyl group
(
−
1
1682 cm ) Its molecular formula, C H O , was deduced from HR-ESI-MS: m/z 1011.4771
48 76 21
+
[M + Na] (calcd., 1011.4777), indicating the presence of 11 unsaturation degrees. There were
1
3
4
8 carbon signals in C NMR spectrum, among which 30 carbon signals were assigned to
the aglycone, and 18 carbon signals to the sugar moiety. The NMR data of compound 3
showed the characteristic signals of a triterpenoid saponin. Six tertiary methyl groups at δH
1
.95 (H-24), 1.52 (H-25), 1.12 (H-26), 1.20 (H-27), 0.86 (H-29) and 0.84 (H-30), and the corre-
3
3
sponding six sp carbons sp carbons at δ 14.1, 16.8, 17.4, 26.1, 33.0 and 23.6 were observed,
C
as well as one olefinic proton at δ 5.38 (1H, t, J = 3.7 Hz) with two typical olefinic carbon
H
signals at δ 122.8 and 144.1. The presence of 2-OH group was indicated by the signal at δ
C
1
H
13
1
4.72 in the H NMR and 70.0 in the downfield region of C NMR. The H NMR spectrum
showed that three anomeric proton signals at δ 5.03 (1H, d, J = 7.8 Hz, H-1′), 5.15 (1H, d,
H
J = 7.9 Hz, H-1″) and 6.29 (1H, d, J = 8.1 Hz, H-1″′), and three corresponding anomeric carbon
signals at δ 105.1 (C-1′), 104.8 (C-2′), 95.7 (C-1″′) were observed using HSQC spectrum,
C
revealing that 3 contained three sugar units. Acid hydrolysis of 3 followed by GC-FID analysis
identified the sugar units as d-glucose. The β-configurations of d-glucose were determined
by the J1,2 values of 7.8, 7.9 and 8.1 Hz, respectively. The HMBC spectrum showed that the
cross peaks from H-1′ (δ 5.03) to C-3 (δc 86.3), from H-1″ (δ 5.15) to C-4′ (δc 80.7) and from
H
H
H-1″′ (δ 6.28) to C-28 (δc 176.4), which revealed the glycosylation positions and sugar
H

NATURAL PRODUCT RESEARCH
5
sequence of 3 (Figure S1). Compound 3 had the almost same aglycone structure as 1, except
for the absence of a methyl group and appearance of carboxylic group by comparing their
NMR data. Therefore, we speculated that compound 3 is a product of methyl (C-23) oxidation
in 1. This speculation was confirmed by the HMBC correlations between δ 4.64 (H-3) and
H
δc 180.6 (C-23), δ 1.95 (H-24) and δc 180.6 (C-23) (Figure S1). Hence, compound 3 was
H
identified as 3-O-[β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl]-2-hydroxyl-medicagenic
acid-28-O-β-D-glucopyranosyide, named celosin M.
3
. Experimental
.1. Materials and chemicals
The melting point was measured on a Yanaco micromelting point apparatus without cor-
3
1
13
2
rection. H (600 MHz), C (150 MHz) and D NMR spectra were obtained on a Bruker AVANCE
DRX-600 NMR spectrometer with tetramethylsilane (TMS) as an internal standard. HR-ESI-MS
was performed on a Waters Acquity UPLC system coupled to a Waters Micromass LCT Premier
time-of-flight mass spectrometer (Milford, MA, USA), equipped with electrospray interface.
Optical rotations were measured on a Rudolph Autopol VI90079 polarimeter. UV spectra
were recorded on a Beckman Coulter DU800 spectrophotometer. IR spectra were recorded
from KBr disc on the FT-IR (Bruker Tensor 27). Gas chromatography analysis was operated
on an HP-5892 II with FID detector, and an HP-20 M (Carbowx 20 M) capillary column
(
25 m × 0.32 mm × 0.3 μm) was used. Silica gel (100–200 mesh, and 200–300 mesh, Qindao
Haiyang Chemical Co. Ltd. Qingdao, China), Sephadex LH-20 (Amersham Pharmcia Biotech.
Ltd. Shanghai, China) column chromatography, ODS (50 mesh, AA12550, YMC) and semiprep
high-performance liquid chromatography (HPLC) were used for separation. L-xylose, d-glu-
cose and d-glucuronic acid were purchased from J&K Scientific Co. Ltd (Beijing, China).
3
.2. Plant material
The seeds of C. cristata were purchased from Suqian city, Jiangsu Province, China, in
September 2013, and identified by Prof. Xiujia Zhou from Shanghai University of Traditional
Chinese Medicine (Shanghai, China). A voucher specimen (2013072105) was originally depos-
ited in the State Key Laboratory of New Drugs in Shanghai Institute of Pharmaceutical
Industry (Shanghai, China).
3
.3. Extraction and isolation
The air-dried seeds of C. cristata (5 kg) were powdered and extracted with 60% ethanol under
reflux for two times (each for 3 h). After filtration, combination and evaporation, the residues
were suspended in water, and partitioned successively with ethyl acetate and 1-butanol.
The 1-butanol soluble fraction (40.0 g) was subjected to a MCI gel (CHP 20P) column chro-
matograph eluted with MeOH/H O (0:100, 10:90, 20:80, 40:60, 60:40, 80:20 and 100:0), yield-
2
ing seven sub-fractions (Fr.A–Fr.G). The sub-fraction Fr.E (5.0 g) was subjected to reversed
phase silica gel column employing MeCN/H O (20:80–100:0), yielding three portions (Fr.
2
E1–Fr.E3). Fr.E2 was repeatedly chromatographed on a reverse-phase RP-C8 column eluting
with MeCN/H O (33:67) to yield six fractions (Fr.E2a–Fr.E2f). Purification of sub-fraction Fr.E2b
2

6
X. LIU ET AL.
by semi-prep HPLC eluting with MeOH/H O (45:55) yielded compound 1 (18 mg). Fr.E2d was
2
purified by semi-prep HPLC eluting with MeOH/H O (50:50) yielded compounds 2 (15 mg)
2
and 3 (10 mg).
3
.3.1. Celosin K (1)
2
5
White amorphous powder, m.p., 280 ~ 282 °C;
ꢀ
, +20.8 (c 0.25, MeOH). UV (MeOH), λ (log
D
max
−1
ε): 218.4 (4.50); IR ν 3418, 2936, 1735, 1632, 1462, 1371, 1258, 1159, 1070 cm ; HR-ESI-MS
max
+
1
m/z: [M + Na] 965.4713. H NMR (pyridine-d , 600 MHz): δ 2.15 (1H, d, J = 13.8 Hz, H-1), 1.07
6
H
(
1H, m, H-1), 4.63 (1H, m, H-2), 3.36 (1H, m, H-3), 0.91 (1H, d, J = 10.8 Hz, H-5), 1.53 (2H, m,
H-6), 1.64 (1H, m, H-7), 1.33 (1H, m, H-7), 1.58 (1H, m, H-9), 2.16 (1H, m, H-11), 1.94 (1H, m,
H-11), 5.40 (1H, t, J = 3.7 Hz, H-12), 2.33 (1H, t, J = 13.0 Hz, H-15), 1.17 (1H, d, J = 13.6 Hz, H-15),
2.06 (1H, m, H-16), 1.89 (1H, m, H-16), 3.16 (1H, dd, J = 13.6, 4.4 Hz, H-18), 1.74 (1H, m, H-19),
1.22 (1H, dd, J = 14.3, 3.6 Hz, H-19), 1.32 (1H, m, H-21), 1.05 (1H, m, H-21), 1.80 (1H, m, H-22),
1.71 (1H, m, H-22), 1.30 (3H, s, H-23), 1.36 (3H, s, H-24), 1.45 (3H, s, H-25), 1.11 (3H, s, H-26),
1.26 (3H, s, H-27), 0.88 (3H, s, H-29), 0.85 (3H, s, H-30), 5.02 (1H, d, J = 7.1 Hz, H-1′), 4.06 (1H,
t, J = 8.4 Hz, H-2′), 4.33 (1H, m, H-3′), 4.43 (1H, d, J = 3.9 Hz, H-4′), 4.61 (1H, d, J = 9.6 Hz, H-5′),
.31 (1H, d, J = 7.4 Hz, H-1″), 4.01 (1H, m, H-2″), 4.12 (1H, m, H-3″), 4.32 (1H, m, H-4″), 4.31 (1H,
m, H-5″), 3.68 (1H, t, J = 10.2 Hz, H-5″), 6.29 (1H, J = 8.1 Hz, H-1″′), 4.17 (1H, d, J = 8.5 Hz, H-2″′),
5
4
2
.25 (1H, d, J = 8.9 Hz, H-3″′), 4.32 (1H, m, H-4″′), 3.99 (1H, m, H-5″), 4.42 (1H, dd, J = 12.0,
.5 Hz, H-6″′), 4.36 (1H, dd, J = 12.0, 4.4 Hz, H-6″′); 13C NMR (pyridine-d , 150 MHz): δ 44.2
6
C
(
3
(
1
C-1), 70.4 (C-2), 89.7 (C-3), 39.0 (C-4), 56.0 (C-5), 18.3 (C-6), 33.1 (C-7), 40.0 (C-8), 48.4 (C-9),
7.0 (C-10), 23.4 (C-11), 123.0 (C-12), 144.0 (C-13), 42.2 (C-14), 28.1 (C-15), 23.9 (C-16), 46.9
C-17), 41.7 (C-18), 46.1 (C-19), 30.7 (C-20), 34.0 (C-21), 32.5 (C-22), 29.5 (C-23), 18.5 (C-24),
6.5 (C-25), 17.5 (C-26), 26.1 (C-27), 176.4 (C-28), 33.1 (C-29), 23.6 (C-30), 106.1 (C-1′), 74.2
(C-2′), 86.1 (C-3′), 71.4 (C-4′), 77.1 (C-5′), 172.4 (C-6′), 106.4 (C-1″), 75.2 (C-2″), 78.0 (C-3″), 70.8
(C-4″), 67.3 (C-5″), 95.7 (C-1″′), 74.1 (C-2″′), 78.8 (C-3″′), 71.3 (C-4″′), 79.2 (C-5″′), 62.2 (C-6″′)
(Table S1 and S2).
3
.3.2. Celosin L (2)
2
5
White amorphous powder, m.p., 281 ~ 282 °C;
ꢀ
, +22.6 (c 0.25, MeOH). UV (MeOH), λ (log
D
max
−1
ε): 218.4 (4.46); IR ν 3413, 2926, 1728, 1680, 1432, 1205, 1141, 1074, 1034 cm ; HR-ESI-MS
max
+
1
m/z: [M + Na] 981.4667. H NMR (pyridine-d , 600 MHz): δ 2.18 (1H, d, J = 13.8 Hz, H-1), 1.14
6
H
(
1H, m, H-1), 4.79 (1H, m, H-2), 4.34 (1H, m, H-3), 1.78 (1H, m, H-5), 1.46 (2H, m, H-6), 1.64 (1H,
m, H-7), 1.33 (1H, m, H-7), 1.69 (1H, m, H-9), 2.07 (1H, m, H-11), 1.95 (1H, m, H-11), 5.38 (1H, t,
J = 3.7 Hz, H-12), 2.31 (1H, t, J = 13.1 Hz, H-15), 1.09 (1H, m, H-15), 2.02 (1H, m, H-16), 1.91 (1H,
m, H-16), 3.14 (1H, dd, J = 13.7, 4.0 Hz, H-18), 1.71 (1H, m, H-19), 1.19 (1H, m, H-19), 1.30 (1H,
m, H-21), 1.04 (1H, d, J = 11.2 Hz, H-21), 1.80 (1H, m, H-22), 1.70 (1H, m, H-22), 4.32 (1H, m,
H-23), 3.65 (1H, m, H-23), 1.32 (3H, s, H-24), 1.54 (3H, s, H-25), 1.13 (3H, s, H-26), 1.21 (3H, s,
H-27), 0.86 (3H, s, H-29), 0.84 (3H, s, H-30), 5.26 (1H, d, J = 7.7 Hz, H-1′), 4.08 (1H, t, J = 8.6 Hz,
H-2′), 4.18 (1H, m, H-3′), 4.42 (1H, d, J = 3.9 Hz, H-4′), 4.52 (1H, d, J = 9.6 Hz, H-5′), 5.24 (1H, d,
J = 7.4 Hz, H-1″), 4.00 (1H, m, H-2″), 4.14 (1H, m, H-3″), 4.31 (1H, m, H-4″), 4.32 (1H, m, H-5″),
3
.68 (1H, m, H-5″), 6.28 (1H, J = 8.1 Hz, H-1″′), 4.16 (1H, m, H-2″′), 4.25 (1H, d, J = 8.9 Hz, H-3″′),
.33 (1H, m, H-4″′), 3.99 (1H, m, H-5″′), 4.43 (1H, dd, J = 9.4, 2.8 Hz, H-6″′), 4.37 (1H, m, H-6″′);
C NMR (pyridine-d , 150 MHz): δ 44.3 (C-1), 70.9 (C-2), 82.7 (C-3), 42.9 (C-4), 47.5 (C-5), 17.9
4
13
6
C
(C-6), 32.8 (C-7), 40.0 (C-8), 48.5 (C-9), 36.9 (C-10), 23.9 (C-11), 123.0 (C-12), 144.1 (C-13), 42.2
(C-14), 28.2 (C-15), 23.3 (C-16), 47.0 (C-17), 41.7 (C-18), 46.1 (C-19), 30.7 (C-20), 33.9 (C-21),

NATURAL PRODUCT RESEARCH
7
32.5 (C-22), 64.7 (C-23), 14.9 (C-24), 17.2 (C-25), 17.6 (C-26), 26.2 (C-27), 176.4 (C-28), 33.1
(C-29), 23.6 (C-30), 105.7 (C-1′), 74.2 (C-2′), 86.3 (C-3′), 71.4 (C-4′), 77.1 (C-5′), 172.5 (C-6′), 106.1
(C-1″), 75.2 (C-2″), 78.0 (C-3″), 71.0 (C-4″), 67.3 (C-5″), 95.7 (C-1″′), 74.1 (C-2″′), 78.8 (C-3″′), 71.1
(C-4″′), 79.2 (C-5″′), 62.2 (C-6″′) (Table S1 and S2).
3
.3.2. Celosin M (3)
2
5
White amorphous powder, m.p., 281 ~ 283 °C;
ꢀ
, +20.3 (c 0.18, MeOH). UV (MeOH), λ (log
D
max
−1
ε): 218.4 (4.33); IR ν
3395, 2945, 1682, 1444, 1204, 1137, 1072 cm ; HR-ESI-MS m/z:
max
1
+
[
M + Na] 1011.4777. H NMR (pyridine-d , 600 MHz): δ 2.27 (1H, m, H-1), 1.27 (1H, m, H-1),
6
H
4.72 (1H, m, H-2), 4.64 (1H, d, J = 3.7 Hz, H-3), 1.73 (1H, m, H-5), 1.62 (2H, m, H-6), 1.62 (1H, m,
H-7), 1.35 (1H, m, H-7), 1.73 (1H, m, H-9), 2.12 (1H, m, H-11), 1.98 (1H, m, H-11), 5.38 (1H, t,
J = 3.7 Hz, H-12), 2.31 (1H, m, H-15), 1.09 (1H, m, H-15), 2.06 (1H, m, H-16), 1.89 (1H, m, H-16),
3
1
1
.16 (1H, dd, J = 13.7, 4.8 Hz, H-18), 1.71 (1H, m, H-19), 1.21 (1H, m, H-19), 1.30 (1H, m, H-21),
.04 (1H, m, H-21), 1.80 (1H, m, H-22), 1.69 (1H, m, H-22), 1.95 (3H, s, H-24), 1.52 (3H, s, H-25),
.12 (3H, s, H-26), 1.20 (3H, s, H-27), 0.86 (3H, s, H-29), 0.84 (3H, s, H-30), 5.03 (1H, d, J = 7.8 Hz,
H-1′), 3.90 (1H, t, J = 8.6 Hz, H-2′), 3.84 (1H, m, H-3′), 4.26 (1H, m, H-4′), 4.15 (1H, m, H-5′), 4.44
1H, m, H-6′), 4.39 (1H, m, H-6′), 5.15 (1H, d, J = 7.9 Hz, H-1″), 4.03 (1H, m, H-2″), 4.16 (1H, m,
(
H-3″), 4.32 (1H, m, H-4″), 4.15 (1H, m, H-5″), 4.44 (1H, m, H-6″), 4.35 (1H, m, H-6″), 6.29 (1H, d,
J = 8.1 Hz, H-1″′), 4.16 (1H, m, H-2″′), 4.25 (1H, d, J = 8.9 Hz, H-3″′), 4.33 (1H, m, H-4″′), 3.94 (1H,
13
m, H-5″), 4.47 (1H, m, H-6″′), 4.23 (1H, m, H-6″′); C NMR (pyridine-d , 150 MHz): δ 44.2 (C-1),
6
C
7
(
4
(
0.0 (C-2), 86.0 (C-3), 52.8 (C-4), 48.7 (C-5), 21.1 (C-6), 32.9 (C-7), 40.3 (C-8), 48.7 (C-9), 36.8
C-10), 23.9 (C-11), 122.8 (C-12), 144.1 (C-13), 42.2 (C-14), 28.1 (C-15), 23.3 (C-16), 46.9 (C-17),
1.7 (C-18), 46.1 (C-19), 30.7 (C-20), 33.9 (C-21), 32.5 (C-22), 180.6 (C-23), 14.1 (C-24), 16.8
C-25), 17.4 (C-26), 26.1 (C-27), 176.4 (C-28), 33.0 (C-29), 23.6 (C-30), 105.1 (C-1′), 74.6 (C-2′),
7
7
6.4 (C-3′), 80.7 (C-4′), 76.5 (C-5′), 62.2 (C-6′), 104.8 (C-1″), 74.7 (C-2″), 79.2 (C-3″), 71.0 (C-4″),
8.1 (C-5″), 61.9 (C-6″), 95.7 (C-1″′), 74.1 (C-2″′), 78.8 (C-3″′), 71.1 (C-4″′), 78.3 (C-5″′), 62.2 (C-6″′)
(
Table S1 and S2).
3
.4. Acid hydrolysis and sugar analysis of compounds 1–3
The acid hydrolysis and sugar analysis of the isolated three new compounds were performed
as previously described in the literature (Zong et al. 2015). Each compound (3 mg) was
dissolved in 2 M trifluoroacetic acid for 2 h at 120 °C. The reaction mixture was extracted
with dichloromethane, and the supernatant was evaporated to dryness under N flow. The
2
residue was dissolved in 0.2 mL pyridine containing L-cysteine methyl ester hydrochloride
(
10 mg/mL) and reacted at 70 °C for 1 h. This reaction was evaporated under N flow, after
2
which 0.2 mL trimethysilyimidazole was added. The mixture was heated at 70 °C for another
h, and then partitioned between n-hexane and water. The organic layer was analysed by
1
GC-FID. Temperature conditions were as follows: injector temperature at 280 °C, the initial
oven temperature was 160 °C for 1 min, then linearly increased to 280 °C and held for 5 min.
The standard sugar samples were subjected to the same reaction and GC-FID conditions.
The sugar unit of compounds 1–3 were identified by comparision with authentic samples:
L-xylose (t = 10.67 min), D-glucose (t = 13.84 min), D-glucuronic acid methyl ester
R
R
(
tR = 15.08 min), respectively.

8
X. LIU ET AL.
4
. Conclusion
Phytochemical investigation of Semen C. cristata allowed the isolation and characterisation
of three new oleanane-type saponins. Previous investigation revealed the existence of trit-
erpenoid saponin in C. cristata, this results could have a significant chemotaxonomical mean-
ing in the further studies for classification of the Celosia species. Moreover, the biological
activities of these new compounds should be evaluated in the next work.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work is funded by National Natural Science Foundation of China [grant number 41306133], [grant
number 81202104]; the Key Research and Development Plan of Shandong Province [grant number
2015GSF115015]; partly supported by Research project of Shanghai Municipal Health and Family
Planning Commission [grant number 201540027]; Shanghai Fengxian District Science and Technology
Project [grant number 20131101], [grant number 20141001].
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