A new patchoulane-type sesquiterpenoid glycoside from the roots of Croton crassifolius

Article abstract of DOI:10.1080/14786419.2016.1233413

A new patchoulane-type sesquiterpenoid glycoside (1), together with five known sesquiterpenoids (2–6), was isolated from the roots of Croton crassifolius. Their structures were elucidated on the basis of spectroscopic methods. This is the first report about the sesquiterpenoid glycoside from C. crassifolius. All the isolated compounds 1–6 were evaluated for their cytotoxic activities against human tumour cell lines HL-60 and A549, but none showed significant activity.

Full text of DOI:10.1080/14786419.2016.1233413

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new patchoulane-type sesquiterpenoid glycoside
from the roots of Croton crassifolius
Qing-Qing Yuan, Wei-Bin Song, Wen-Qiong Wang & Li-Jiang Xuan
To cite this article: Qing-Qing Yuan, Wei-Bin Song, Wen-Qiong Wang & Li-Jiang Xuan (2016): A
new patchoulane-type sesquiterpenoid glycoside from the roots of Croton crassifolius, Natural
Product Research, DOI: 10.1080/14786419.2016.1233413
To link to this article: http://dx.doi.org/10.1080/14786419.2016.1233413
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Date: 26 September 2016, At: 18:49

Natural Product research, 2016
ꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2016.1233413
ꢀ
A new patchoulane-type sesquiterpenoid glycoside from the
roots of Croton crassifolius
Qing-Qing Yuan , Wei-Bin Song , Wen-Qiong Wang and Li-Jiang Xuana,b
a,b
a,b
a,b
ꢅ
sꢁꢅꢁꢆ Kꢆy lꢅbꢃꢄꢅꢁꢃꢄy ꢃf dꢄꢇg rꢆꢈꢆꢅꢄꢉꢀ, sꢀꢅngꢀꢅi Inꢈꢁiꢁꢇꢁꢆ ꢃf Mꢅꢁꢆꢄiꢅ Mꢆꢂiꢉꢅ, cꢀinꢆꢈꢆ aꢉꢅꢂꢆmy ꢃf sꢉiꢆnꢉꢆꢈ,
b
sꢀꢅngꢀꢅi, P.r. cꢀinꢅ; cꢃꢊꢊꢆgꢆ ꢃf pꢀꢅꢄmꢅꢉy, univꢆꢄꢈiꢁy ꢃf cꢀinꢆꢈꢆ aꢉꢅꢂꢆmy ꢃf sꢉiꢆnꢉꢆꢈ, Bꢆijing, P.r. cꢀinꢅ
ABSTRACT
ARTICLE HISTORY
A new patchoulane-type sesquiterpenoid glycoside (1), together
with five known sesquiterpenoids (2–6), was isolated from the
roots of Croton crassifolius. Their structures were elucidated on
the basis of spectroscopic methods. This is the first report about
the sesquiterpenoid glycoside from C. crassifolius. All the isolated
compounds 1–6 were evaluated for their cytotoxic activities against
human tumour cell lines HL-60 and A549, but none showed significant
activity.
rꢆꢉꢆivꢆꢂ 19 Mꢅy 2016
aꢉꢉꢆpꢁꢆꢂ 12 aꢇgꢇꢈꢁ 2016
KEYWORDS
Croton crassifolius;
ꢈꢆꢈqꢇiꢁꢆꢄpꢆnꢃiꢂ gꢊyꢉꢃꢈiꢂꢆ;
ꢉyꢁꢃꢁꢃxiꢉ ꢅꢉꢁiviꢁy
1
. Introduction
The genus Croton belongs to the Euphorbiaceae family, comprising approximately 1300
species distributed throughout tropical and subtropical regions of the world (Salatino et al.
2007). The plants of Croton are generally known for containing diverse terpenoids with a
good range of biological activities (Kuo et al. 2007; Pan et al. 2014; Sun et al. 2014; Shahwar
et al. 2015). Croton crassifolius Geisel. is mainly distributed in southern China, Laos, Thailand,
and Vietnam (Qiu et al. 1996). The plant of C. crassifolius, which is known as ‘jiguxiang’ in
China, has been used for the treatment of cancer, stomachache, sore throat and rheumatism
(
Song 1999). Previous phytochemical investigations on C. crassifolius showed that the main
components were clerodane diterpenoids with cytotoxic (Boonyarathanakornkit et al. 1988;
Qiu et al. 2016), antiviral (Wang et al. 2012) and anti-angiogenic activities (Huang, Li et al.
2014; Wang et al. 2016). However, the sesquiterpenoids constituents of this plant species
are rarely reported (Zhang et al. 2014). As part of our ongoing search for new bioactive
CONTACT li-Jiꢅng Xꢇꢅn
ꢊjxꢇꢅn@ꢈimm.ꢅꢉ.ꢉn
sꢇppꢊꢆmꢆnꢁꢅꢊ ꢂꢅꢁꢅ fꢃꢄ ꢁꢀiꢈ ꢅꢄꢁiꢉꢊꢆ ꢉꢅn bꢆ ꢅꢉꢉꢆꢈꢈꢆꢂ ꢅꢁ ꢀꢁꢁp://ꢂx.ꢂꢃi.ꢃꢄg/10.1080/14786419.2016.1233413.
©
2016 Infꢃꢄmꢅ uK limiꢁꢆꢂ, ꢁꢄꢅꢂing ꢅꢈ tꢅyꢊꢃꢄ & Fꢄꢅnꢉiꢈ Gꢄꢃꢇp

2
Q.ꢀQ. YꢁAꢂ ET AL.
Figure 1. cꢀꢆmiꢉꢅꢊ ꢈꢁꢄꢇꢉꢁꢇꢄꢆꢈ ꢃf ꢉꢃmpꢃꢇnꢂꢈ 1–6.
constituents from traditional plants with medicinal interest, we carried out the investigation
of the roots of C. crassifolius. The present paper deals with the isolation and structural elu-
cidation of a new patchoulane-type sesquiterpenoid glycoside (1), together with five known
sesquiterpenoids (2–6) (Figure 1). The cytotoxic activities of 1–6 were evaluated against
human tumour cell lines HL-60 and A549.
2
. Results and discussion
The air-dried and powdered roots of C. crassifolius were extracted with 70% acetone and
partitioned with petroleum and CHCl . The water-soluble and CHCl fraction were subjected
3
3
to MCI resin, HW-40F, reversed-phase C18, and repeated silica gel column chromatography
CC), followed by Sephadex LH20, to afford a new sesquiterpenoid glycoside (1), together
(
with five known sesquiterpenoids (2–6). Their structures were elucidated on the basis of
spectroscopic methods including HRESIMS, 1D and 2D ꢂMR spectra and comparison with
the data reported in the literature.
Compound 1 was obtained as a white amorphous powder and determined to have a
+
molecular formula of C H O by HRESIMS ([M + ꢂa] m/z 435.1988, Calcd 435.1989) and
2
1
32
8
13
1
C ꢂMR data. The H ꢂMR spectrum (Table S1) displayed three methyl groups at δ 0.76 (s,
H
H-12), 0.96 (s, H-13), and 0.99 (d, J = 6.3 Hz, H-14), and a glucose moiety at δ 2.91 (dd, J = 8.3,
H
8
.3 Hz, H-2′), 3.00 (dd, J = 9.1, 9.1 Hz, H-4′), 3.04–3.13 (overlapped, H-3′, 5′), 3.39 (dd, J = 11.6,
1
3
6.1 Hz, H-6′b), 3.64 (1H, dd, J = 11.6, 2.1 Hz, H-6′a), and 4.10 (d, J = 7.8 Hz, H-1′). The C ꢂMR
spectrum (Table S1) showed 21 carbon signals including 3 methyl groups, 5 methylenes, 8
methines and 5 quaternary carbons, which were extremely similar to those of cyperenoic
acid (2) (Boonyarathanakornkit et al. 1988). The main difference between 1 and 2 was the
presence of one glucose moiety (δ 105.5, 77.3, 77.0, 74.3, 70.7 and 61.7) in 1. The HMBC
C
correlations from H-1′ (δ 4.10) to C-9 (δ 105.1) indicated that the glucose moiety was
H
C
attached to C-9 (Figure S1). The sugar was determined as d-glucose by acid hydrolysis and
comparison with authentic sample on TLC (Huang, Lu et al. 2014). Meanwhile, the large
coupling constant (7.8 Hz) of the anomeric proton at δ 4.10 (H-1′) revealed that the glucose
H
was in the β-configuration. The relative stereochemistry of 1 was established by the ROESY

ꢂATꢁRAL PRODꢁCT RESEARCH
3
spectrum. The key ROESY correlations of Me-14 with H-9, and H-10 with Me-12 indicated
that Me-14 and H-9 were cofacial and randomly assigned as α-oriented, while H-10 and
Me-12 were on the opposite side and assigned to be β-oriented (Figure S1). Based on the
above analysis, the structure of compound 1 was determined to be a new sesquiterpenoid
glycoside, and it was named cyperenoic acid-9-O-β-d-glucopyranoside.
The known compounds cyperenoic acid (2), 4β,10α-aromadendranediol (3) (Hisakatsu
et al. 1989), aromadendrane-4α,8α,10α-triol (4) (Maatooq 2002), aromadendrene diol (5)
(Martínez et al. 1986) and 4β,9β-dihydroxyaromadendrene (6) (Andrés & Antonio 1989), were
characterised by comparison of physical and spectroscopic data with reported literature
values. Compounds 1, 3–6 were isolated from C. crassifolius for the first time.
The six isolated compounds 1–6 were evaluated for their cytotoxic activities against two
human tumour cell lines HL-60 and A549 using MTT and SRB assays. However, none showed
significant activity (cellular proliferation inhibition rate < 50% at 20 μM), and the results were
presented in Table S2.
3
. Experimental
3
.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR spectrum was recorded
on a Perkin-Elmer 577 spectrometer using KBr discs. One-dimensional and two-dimensional
1
ꢂMR experiments were performed at room temperature using a Bruker Ascend 500 MHz ( H)
13
and 126 MHz ( C) spectrometer. HRESIMS analyses were carried out on an Agilent 6224 TOF
mass spectrometer with an ESI interface. Silica gel (200–300 mesh, Qingdao Haiyang Chemical
Co Ltd), C reversed-phase (RP-18) silica gel (20–45 μm; Fuji Silysia Chemical Ltd), Toyopearl
18
HW-40F (32–63 μm; Tosoh) and Sephadex LH-20 gel (Amersham Biosciences) were used for
CC. Pre-coated silica gel GF254 plates (Qingdao Haiyang Chemical Co.Ltd) were used for TLC.
All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co, Ltd).
3
.2. Plant material
The dried roots of C. crassifolius were collected from Guangxi Province, People’s Republic of
China in December 2011, and authenticated by Prof. He-Ming Yang. A voucher specimen
(SIMMXLJ-89) was deposited at the Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, People’s Republic of China.
3
.3. Extraction and isolation
Air-dried and pulverised roots of C. crassifolius (5.0 kg) were soaked in 70% acetone (3 × 25 L;
4 h each time) at room temperature. After removing the solvent under reduced pressure
2
at 40 °C, the combined extracts were concentrated to afford the crude extract, which was
suspended in water and partitioned successively with petroleum ether and CHCl . The
3
water-soluble fraction (371 g) was subjected to MCI gel CC (MeOH–H O, 0:100 to 100:0) to
2
afford fractions C1–C6. Fraction C5 was separated using aToyopearl HW-40F column (MeOH–
H O, 0:100 to 70:30) to afford subfractions C51–C54. Subfraction C52 was further purified
2
through repeated ODS C (MeOH–H O, 0:100 to 20:80) and Sephadex LH-20 (MeOH) columns
18
2
to afford compound 1 (10 mg). Fraction C4 was separated using an ODS C18 (MeOH–H O,
2

4
Q.ꢀQ. YꢁAꢂ ET AL.
40:60 to 100:0) to afford subfractions C41–C42. Subfraction C42 was further purified through
Sephadex LH-20 (MeOH) column to afford compound 4 (12 mg). The CHCl (420 g) fraction
3
was loaded onto a silica gel CC and eluted with a gradient of petroleum ether/ethyl acetate
(
25:1, 15:1, 10:1, 5:1, 2:11:1 and 0:1) to give fractions B1–B10. Fraction B5 was subjected to
a silica gel column using a petroleum ether/acetone (50:1, 20:1, 10:1, 5:1, 2:11:1 and 0:1)
gradient system, finally obtaining six fractions B51–B56. Compound 2 (55 mg) was crystal-
lised from fraction B53. Fraction B9 was further purified through repeated silica gel and
Sephadex LH-20 (MeOH) columns to afford compound 3 (30 mg). Fraction B7 was subjected
to silica gel CC eluted with a gradient of petroleum ether/ethyl acetate (4:1, 3:1, 2:1 and 1:1)
to obtain fractions B71–B75. Compound 5 (8 mg) and 6 (8 mg) were yielded from B73 by
repeated silica gel and Sephadex LH-20 (MeOH) columns.
2
0
Cyperenoic acid-9-O-β-d-glucopyranoside (1): white powder; [ꢀ] −51.7 (c 0.10, MeOH);
D
−
1
+
IR (KBr): 3453, 1643 cm ; HR-ESI-MS: m/z 435.1988 [M + ꢂa] (Calcd for 435.1989, C H O ꢂa);
21
32
8
1
H ꢂMR (500 MHz, DMSO-d ): δ 1.49 (1H, dd, J = 13.2, 7.5 Hz, H-2a), 1.64 (1H, dt, J = 13.2,
6
9.8 Hz, H-2b), 2.51–2.61 (1H, overlapped, H-3a), 2.62–2.70 (1H, m, H-3b), 2.10–2.18 (1H, m,
H-6a), 2.51–2.61 (1H, overlapped, H-6b), 1.91 (1H, dt, J = 6.6, 3.2 Hz, H-7), 2.03 (1H, ddd,
J = 13.4, 7.1, 3.8 Hz, H-8a), 1.73 (1H, ddd, J = 13.4, 10.4, 2.6 Hz, H-8b), 3.04–3.13 (3H, over-
lapped, H-9, 3′, 5′), 1.97 (1H, dd, J = 10.1, 6.3 Hz, H-10), 0.76 (3H, s, H-12), 0.96 (3H, s, H-13),
0.99 (3H, d, J = 6.3 Hz, H-14), 4.10 (1H, d, J = 7.8 Hz, H-1′), 2.91 (1H, t, J = 8.3 Hz, H-2′), 3.00 (1H,
d, J = 9.1 Hz, H-4′), 3.64 (1H, dd, J = 11.6, 2.1 Hz, H-6′a), 3.39 (1H, dd, J = 11.6, 6.1 Hz, H-6′b).
13
C ꢂMR (126 MHz, DMSO-d ): δ 66.5 (C-1), 25.5 (C-2), 36.7 (C-3), 124.6 (C-4), 166.2 (C-5), 30.9
6
(C-6), 47.4 (C-7), 35.5 (C-8), 80.9 (C-9), 42.3 (C-10), 40.9 (C-11), 25.3 (C-12), 19.2 (C-13), 13.5
(C-14), 166.0 (C-15), 105.1 (C-1′), 73.9 (C-2′), 76.6 (C-3′), 70.2 (C-4′), 76.9 (C-5′), 61.3 (C-6′).
3
.4. Acid hydrolysis of 1
Compound 1 (2 mg) was hydrolysed with 5% HCl (3 mL) at 100 °C for 2 h. The mixture was
neutralised with 10% ꢂa CO and extracted with CH Cl (3 × 5 mL). The sugar was confirmed
2
3
2
2
to be d-glucose through comparison of the TLC (n-BuOH/EtOAc/pyridine/H O, 6:1:5:4) with
2
2
0
an authentic sample of glucose (R : 0.4) and by its optical activity ([ꢀ] +45.7, c 0.04, H O).
f
D
2
3
.5. Cytotoxic activity
The cytotoxic activities against the cancer cell lines HL-60 and A549 were determined by the
MTT (Mosmann 1983) and SRB (Vichai & Kirtikara 2006) assays, respectively. Briefly, 100 μL
−1
of each cell line (5 × 104 cells mL ) was seeded in 96-well microplates and incubated at
7 °C for 24 h. Then, the test compound (20 μM) was added and the microplates were further
3
incubated for 72 h. Compounds that inhibited 50% of the growth of the cancer cells were
evaluated again at six concentrations; each concentration of the compound was tested in
two parallel wells. Cisplatin was used as a positive control.
4
. Conclusions
One new patchoulane-type sesquiterpenoid glycoside, named cyperenoic acid-9-O-β-d-
glucopyranoside (1), together with five known sesquiterpenoids (2–6), was isolated from
the roots of C. crassifolius. All the isolated compounds showed no significant cytotoxic activity

ꢂATꢁRAL PRODꢁCT RESEARCH
5
against human tumour cell lines HL-60 and A549. This is the first report about the sesquit-
erpenoid glycoside from C. crassifolius.
Disclosure statement
ꢂo potential conflict interest was reported by the authors.
Funding
This research was supported by the ꢂational Science & Technology Major Project ‘Key ꢂew Drug
Creation and Manufacturing Program’[grant number SIMM1601ZZ-03].
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