Antioxidant acetophenone glycosides from the roots of Euphorbia ebracteolata Hayata

Article abstract of DOI:10.1080/14786419.2017.1371160

A new acetophenone diglycoside, 2,4-dihydroxy-6-methoxy-3-methyl-acetophenone 4-O-gentiobioside (1), along with six know analogues (2–6), were isolated from the roots of Euphorbia ebracteolata. The structures of these isolates were determined by UV, HRESIMS, 1D and 2D NMR spectral analyses. In addition, all acetophenones showed moderate DPPH free radical scavenging capability, and no cytotoxicity against five human cancer cell lines.

Full text of DOI:10.1080/14786419.2017.1371160

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Antioxidant acetophenone glycosides from the
roots of Euphorbia ebracteolata Hayata
Anhua Wang, Xiaoxv Gao, Xiaokui Huo, Shanshan Huang, Lei Feng,
Chengpeng Sun, Baojing Zhang, Xiaochi Ma, Jingming Jia & Chao Wang
To cite this article: Anhua Wang, Xiaoxv Gao, Xiaokui Huo, Shanshan Huang, Lei Feng,
Chengpeng Sun, Baojing Zhang, Xiaochi Ma, Jingming Jia & Chao Wang (2017): Antioxidant
acetophenone glycosides from the roots of Euphorbia ebracteolata Hayata, Natural Product
Research, DOI: 10.1080/14786419.2017.1371160
To link to this article: http://dx.doi.org/10.1080/14786419.2017.1371160
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Date: 31 August 2017, At: 09:25

Natural Product research, 2017
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1371160
ꢀ
Antioxidant acetophenone glycosides from the roots of
Euphorbia ebracteolata Hayata
a,b
a,b
b
b
b
Anhua Wang , Xiaoxv Gao , Xiaokui Huo , Shanshan Huang , Lei Feng ,
b
b
b
a
b
Chengpeng Sun , Baojing Zhang , Xiaochi Ma , Jingming Jia and Chao Wang
ꢆ
sꢇꢀꢄꢄꢈ ꢄf tꢅꢆꢃiꢁiꢄnꢆꢈ cꢀinꢉꢂꢉ Mꢆꢁꢉꢅiꢆ Mꢉꢃiꢇꢆ, sꢀꢉnyꢆng Pꢀꢆꢅmꢆꢇꢉꢊꢁiꢇꢆꢈ univꢉꢅꢂiꢁy, sꢀꢉnyꢆng, Pꢉꢄpꢈꢉ’ꢂ
b
rꢉpꢊbꢈiꢇ ꢄf cꢀinꢆ; cꢄꢈꢈꢉgꢉ ꢄf Pꢀꢆꢅmꢆꢇy, aꢇꢆꢃꢉmy ꢄf Inꢁꢉgꢅꢆꢁivꢉ Mꢉꢃiꢇinꢉ, ꢆnꢃ dꢉpꢆꢅꢁmꢉnꢁ ꢄf Biꢄꢇꢀꢉmiꢂꢁꢅy
ꢆnꢃ Mꢄꢈꢉꢇꢊꢈꢆꢅ Biꢄꢈꢄgy, dꢆꢈiꢆn Mꢉꢃiꢇꢆꢈ univꢉꢅꢂiꢁy, dꢆꢈiꢆn, Pꢉꢄpꢈꢉ’ꢂ rꢉpꢊbꢈiꢇ ꢄf cꢀinꢆ
ABSTRACT
ARTICLE HISTORY
rꢉꢇꢉivꢉꢃ 3 Mꢆy 2017
aꢇꢇꢉpꢁꢉꢃ 1 aꢊgꢊꢂꢁ 2017
A new acetophenone diglycoside, 2,4-dihydroxy-6-methoxy-3-methyl-
acetophenone 4-O-gentiobioside (1), along with six know analogues
(
2–6), were isolated from the roots of Euphorbia ebracteolata. The
KEYWORDS
structures of these isolates were determined by UV, HRESIMS, 1D and
D NMR spectral analyses. In addition, all acetophenones showed
Euphorbia ebracteolata;
ꢆꢇꢉꢁꢄpꢀꢉnꢄnꢉ gꢈyꢇꢄꢂiꢃꢉ;
dPPh ꢂꢇꢆvꢉnging;
ꢆnꢁiꢄxiꢃꢆnꢁ ꢆꢇꢁiviꢁy;
ꢇyꢁꢄꢁꢄxiꢇiꢁy
2
moderate DPPH free radical scavenging capability, and no cytotoxicity
against five human cancer cell lines.
1
. Introduction
Euphorbia ebracteolata Hayata, an herbaceous plant in Euphorbiaceae family, was widely
distributed in the central and western regions of China (Zhang et al. 2011). For thousand
years or more, the roots of the plant were used to treat cancer (He et al. 2012; Yuan et al.
2016), lymphatic tuberculosis (Zhang 2016), ascites and some other diseases. In addition,
CONTACT cꢀꢆꢄ Wꢆng
wꢆꢇꢀ_ꢉꢃꢊ@ꢂinꢆ.ꢇꢄm; Jingming Jiꢆ
jiꢆjingming@163.ꢇꢄm
sꢊppꢈꢉmꢉnꢁꢆꢈ ꢃꢆꢁꢆ fꢄꢅ ꢁꢀiꢂ ꢆꢅꢁiꢇꢈꢉ ꢇꢆn bꢉ ꢆꢇꢇꢉꢂꢂꢉꢃ ꢆꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1292511.
©
2017 Infꢄꢅmꢆ uK limiꢁꢉꢃ, ꢁꢅꢆꢃing ꢆꢂ tꢆyꢈꢄꢅ & Fꢅꢆnꢇiꢂ Gꢅꢄꢊp

2
A. WANG ET AL.
Figure 1. aꢇꢉꢁꢄpꢀꢉnꢄnꢉ gꢈyꢇꢄꢂiꢃꢉꢂ (1−7) iꢂꢄꢈꢆꢁꢉꢃ fꢅꢄm ꢁꢀꢉ ꢅꢄꢄꢁꢂ ꢄf Euphorbia ebracteolata.
many types of structures mainly including terpenoids (Wang et al. 2010; Liu et al. 2014),
acetophenones (Wang & Ding 1999; Zhang et al. 2010; Delnavazi et al. 2015; Huang et al.
2017) and flavonoids were isolated from Euphorbia species. In the present study, a new
acetopenone glycoside and six known analogues were isolated from the roots of E. ebrac-
teolata and their structures were identified using UV, LC-MS, 1D NMR, 2D NMR (Figure 1).
Moreover, their antioxidant and cytotoxic effects were evaluated by DPPH scavenging exper-
iment (Zeng & Shi 2013) and against five human cancer cell lines, respectively.
2
. Results and discussion
Compound 1, obtained as a white amorphous powder, had the molecular formula C H O
32 14
2
2
+
established by the HRESIMS from its ion peak at m/z 543.1689 [M + Na] (calcd for C H O Na,
2
2 32 14
543.1792). The UV absorptions λmax 209.6, 287.5 nm of aromatic moiety was observed in the
UV spectrum. Analyses of the spectroscopic data of compound 1 suggested the structure
1
13
to be an acetophenone glycoside analogue (Huang et al. 2017). The H and C NMR spectra
showed a phloroglucinol unit (δ 162.8, 161.4 and 161.0) with C-substituents of an acetyl
C
(
δ 203.3 and 32.9; δ 2.58, s, 3H) and of a methyl (δ 7.4; δ 1.95, s, 3H), as well as O-substituents
C H C H
of a methoxyl (δ 56.0; δ 3.89, s, 3H) and of a disaccharide moiety (δ 103.7, 99.6, 69.1 and
C
H
C
6
0.9; δ 5.05, d, J = 7.5 Hz, 4.17, d, J = 7.5 Hz). The free phenolic hydroxyl was positioned on
H
C-2 based on the formation of intramolecular hydrogen bond at δ 13.93. The methyl and
H
the glucosyl linkage were ascribed to C-3 and C-4, respectively, as determined by the HMBC
correlations between the methyl (δ 1.95) with C-2, C-3 and C-4, and between H-1′ (δ 5.05)
H
H
with C-4 (δ 161.4). (Figure S1) The remaining methoxyl was consequently assigned on C-6.
C
The glucosyl moiety was elucidated to be β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside
on the basis of the diagnostic NMR data, and supported by the HMBC cross-peak of H-1″/C-6′
and acid hydrolysis experiments. Therefore, the structure of 1 was determined to be 2,4-dihy-
droxy-6-methoxy-3-methyl-acetophenone 4-O-gentiobioside.

NATURAL PRODUCT RESEARCH
3
According to the previously reports, compounds 2–7 were identified as acetophenone
glycosides 2,4-dihydroxy-6-methoxy-acetophenone 4-O-β-D-xylopyranosyl-(1→6)-β-D-
glucopyranoside (2) (Huang et al. 2017), 2,4-dihydroxy-6-methoxy-3-methyl-acetophenone
4
6
-O-β-D-xylopyranosyl-(1→6) -β-D-glucopyranoside (3) (Wang & Ding 1999), 2,4-dihydroxy-
-methoxy- acetophenone 4-O-a-L-arabinopyranosyl(1→6)-β-D-glucopyranoside (4) (Huang
et al. 2017), 2,4-dihydroxy-6-methoxy-3-methyl-acetophenone 4-O-α-L-arabinopyranosyl
1→6)-β-D-glucopyranoside (5) (Wang & Ding 1999), 2,4-dihydroxy-6-methoxy-acetophenone
-O-β-D-glucopyranoside (6) (Prasad 1999), and 2,4-hydroxy-6-methoxy-3-methyl-acetophe-
(
4
none 4-O-β-D-glucopyranoside (7) (Huang et al. 2017), respectively.
The anti-oxidant effects of acetophenone glycosides (1–7) have been evaluated using
DPPH scavenging assay. All of the isolated compounds displayed anti-oxidant effects with
the IC values ranging from 22.20–36.50 μg/mL (Table 1). Additionally, all of the compounds
50
displayed no cytotoxic activity against five huaman cancer cell lines (MCF-7, A549, Hep-3B,
U118 and U87) using MTT assay.
3
. Experimental
3
.1. General experimental procedures
UV spectra were measured by a JASCO V-650 UV–vis spectrophotometer in methanol.
HRESIMS data were obtained using an Agilent 1100 series LC/MSD ion trap mass spectrom-
eter using direction injection. NMR spectra were run in DMSO-d on a Varian Mercury NMR
6
spectrometer. Analytical HPLC data were collected on an UltiMate 3000 instrument (Thermo
Scientific dionex). Preparative HPLC was given by an Saipuruisi MH-LC 52 instrument with
an Elite UV2300 detector and a SHIMADZU C18 column (250 × 20 mm, 5 μm). Column chro-
matographic separations were carried out on silica gel H-60 (Qingdao Marine Chemical
Group Corporation, Qingdao, China), Macroporous resin D101 (Zhengzhou Qinshi technol-
ogy Co. Ltd., Zhengzhou, China), Acetonitrile and Methanol were used of chromatographic
grade purchasing from Sigma-Aldrich in American. All other solvents were used of chemical
grade (Kermel Chemical Co. Ltd., Tianjin, China).
3
.2. Plant material
The roots of E. ebracteolata were collected in the Bozhou city of Anhui province and identified
by Prof Qing-ShanYang, Anhui University of Chinese Medicine. The specimen was deposited
in College of Pharmacy, Dalian Medical University (D-2014–05).
Table 1. tꢀꢉ ꢆnꢁiꢄxiꢃꢆnꢁ ꢆꢇꢁiviꢁy ꢆgꢆinꢂꢁ dPPh ꢄf ꢁꢀꢉ iꢂꢄꢈꢆꢁꢉꢃ ꢇꢄmpꢄꢊnꢃꢂ 1−7.
Samples
IC50 (μg/mL)
cꢄmpꢄꢊnꢃ 1
cꢄmpꢄꢊnꢃ 2
cꢄmpꢄꢊnꢃ 3
cꢄmpꢄꢊnꢃ 4
cꢄmpꢄꢊnꢃ 5
cꢄmpꢄꢊnꢃ 6
cꢄmpꢄꢊnꢃ 7
Viꢁꢆmin c
34.62 ± 0.5
36.20 ± 1.6
35.63 ± 1.4
32.33 ± 0.7
33.17 ± 0.5
23.23 ± 1.8
22.50 ± 0.8
1.57 ± 0.02
Nꢄꢁꢉ: dꢆꢁꢆ wꢉꢅꢉ pꢅꢉꢂꢉnꢁꢉꢃ ꢆꢂ mꢉꢆnꢂ ± sd ꢄf ꢁꢀꢅꢉꢉ pꢆꢅꢆꢈꢈꢉꢈ mꢉꢆꢂꢊꢅꢉmꢉnꢁꢂ.

4
A. WANG ET AL.
3
.3. Extraction and isolation
The roots of E. ebracteolata (20 kg) were crushed and extracted by 80% aqueous EtOH under
Reflux (1.5 h × 3). The EtOH extracts (1300 g) were fractionally extracted with petroleum
ether, ethyl acetate, n-BuOH successively (10L × 3 times). The n-BuOH extract (330 g) were
separated by macroporous resin to get three EtOH/water fractions (30% EtOH fraction, 60%
EtOH fraction, 90% EtOH fraction). The 30% EtOH fraction (12 g) was dealed with a silica gel
column by elution with CH Cl –MeOH (20:1-1:1) in sequence to give fractions 1–102. Fraction
2
2
2
3 (143 mg) was separated in CH CN-0.03% TFA in water (15:85; 8 ml/min; 210, 250 nm) to
3
get compounds 6 (rt. 55 min) and 7 (rt. 59 min). Fraction 59 was isolated firstly by a fully
automatic preparative HPLC with CH CN-water (10%-30%, 60 min; 10 ml/min; 210, 250 nm),
3
and the sub-fractions were purified by preparative HPLC in CH CN-water (0.03% TFA, 15:85;
3
8
3
ml/min; 210, 250 nm) to afford compounds 1 (rt. 34 min; 5 mg), 2 (rt. 38 min; 11 mg), 3 (rt.
7 min; 6 mg), 4 (rt. 43 min; 10 mg) and 5 (rt. 41 min; 12 mg).
3
(
.3.1. 2,4-dihydroxy-6-methoxy-3-methyl-acetophenone 4-O-gentiobioside
Compound 1)
A white amorphous powder; [ꢀ] –15.3 (c 0.1 CH OH). UV(CH CN) λ 209.6 and 287.5 nm.
2
5
D
3
3
max
1
H NMR (600 MHz, DMSO-d ), δ 13.93 (1H, s, 2-OH), 6.35 (1H, s, H-5), 5.05 (1H, d, J = 7.5 Hz,
6
H
H-1′), 4.17 (1H, d, J = 7.5 Hz, H-1″), 4.02 (1H, dd, J = 11.5, 1.5 Hz, H-6′), 3.89 (3H, s, 6-OCH3),
.70 (1H, m, H-5′), 3.64 (1H, brd, J = 11.5 Hz, H-6″), 3.60 (1H, dd, J = 11.5, 7.0 Hz, H-6′), 3.42
1H, brd, J = 11.5 Hz, H-6″), 3.31 (1H, m, H-2′), 3.31 (1H, m, H-4′), 3.31 (1H, m, H-5″), 3.10 (1H,
m, H-3″), 3.03 (1H, m, H-4″), 3.03 (1H, m, H-3′), 2.93 (1H, t, J = 8.5 Hz, H-2″), 2.58 (3H, s, 8-CH ),
3
(
3
1
3
1
.95 (3H, s, 3-CH ). C NMR (150 MHz, DMSO-d ), δ 203.3 (C-7), 162.8 (C-2), 161.4 (C-4), 161.0
3
6
C
(C-6), 105.7(C-1), 105.1 (C-3), 103.7 (C-1″), 99.6 (C-1′), 90.1 (C-5), 73.0 (C-2′), 76.8 (C-3′), 69.9
(
(
C-4′), 76.8 (C-3″), 76.4 (C-5″), 75.8 (C-5′), 73.5 (C-2″), 69.9 (C-4″), 69.1 (C-6′), 60.9 (C-6″), 56.0
+
6-OCH ), 32.9 (C-8), 7.4 (3-CH ). HRESIMS: m/z 543.1689 [M + Na] (calcd for C H O Na,
3
3
22 32 14
543.1792).
3
.4. Hydrolysis of compound 1
Five milligram powder of compound 1 was dissolved in DMSO (The least amount) and par-
ticipated in a hydrolysis reaction with 10% H SO in the 85 °C water bath over 2 h. Next, the
2
4
mixture was neutralised by the saturated sodium bicarbonate solution. The reaction mixture
was extracted by EtOAc, and the water-layer was evaporated in vacuo by a rotatory evapo-
rator, the residue was dissolved by DMSO in order to dispose inorganic salt. Subsequently,
The DMSO fraction including saccharide was dried by a lyophilizer and next analysed by
LC-MS with comparing to standard substance d-glucose (chromatography column: Luna
NH , 5um, 4.6 mm × 25 cm) (Shi et al. 2009).
2
3
.5. DPPH scavenging assay
The free radical scavenging rates of these samples were detected by DPPH assay as the
previous and acknowledged method, which was widely used in the literature for more than
20 years (Apak et al. 2016; William et al. 2017; Zhao et al. 2017). Meanwhile, vitamin C was
used as a positive control.

NATURAL PRODUCT RESEARCH
5
3
.6. Cytotoxicity assay
Compounds 1−7 were tested for cytotoxicity against MCF-7, A549, Hep-3B, U118 and U87
cell lines by means of the MTT method as described (Wang et al. 2014).
4
. Conclusions
The present paper have obtained 7 acetophenone glycosides from the roots of E. ebracteo-
lata, including one new compound. On the basis of the structure analysis, the anti-oxidant
effects of these isolated compounds have been evaluated in vitro using DPPH scavenging
bioassay. Compared with the positive control vitamin C, all of the acetophenone glycosides
displayed moderate DPPH scavenging effects.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China [grant number 81473423],
[grant number 81503201], [grant number 81622047]; the Program for Innovative Research Team of the
Ministry of Education and Program for Liaoning Innovative Research Team in University.
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