Four new C10-polyacetylene glycosides from the rhizomes of Atractylodes lancea

Article abstract of DOI:10.1080/10286020.2016.1247811

An ongoing phytochemical investigation of the rhizomes of Atractylodes lancea resulted in the isolation of four new C₁₀-type polyacetylene glycosides (1?4). Their structures were elucidated on the basis of extensive spectroscopic data (UV, IR, 1D and 2D NMR, and HRESIMS). The absolute configurations of compounds 2?4 were determined by comparing the specific rotations of their aglycones. Notably, compounds 2 and 3 exhibited significant hepatoprotective activities against APAP-induced HepG2 cell injury at a concentration of 10?μM. Compounds 2 and 3 showed weak anti-inflammatory effects on LPS-induced NO production in microglia BV2 cells at a concentration of 10?μM.

Full text of DOI:10.1080/10286020.2016.1247811

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Four new C -polyacetylene glycosides from the
10
rhizomes of Atractylodes lancea
Kuo Xu, Zi-Ming Feng, Ya-Nan Yang, Jian-Shuang Jiang & Pei-Cheng Zhang
To cite this article: Kuo Xu, Zi-Ming Feng, Ya-Nan Yang, Jian-Shuang Jiang & Pei-Cheng Zhang
(
2016): Four new C -polyacetylene glycosides from the rhizomes of Atractylodes lancea,
10
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2016.1247811
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1247811
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Date: 28 October 2016, At: 07:46

Journal of asian natural Products research, 2016
ꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2016.1247811
ꢀ
Four new C -polyacetylene glycosides from the rhizomes of
10
Atractylodes lancea
Kuo Xu, Zi-Ming Feng, Ya-Nan Yang, Jian-Shuang Jiang and Pei-Cheng Zhang
sꢁꢆꢁꢇ Kꢇy lꢆbꢃꢅꢆꢁꢃꢅy ꢃꢈ Bꢄꢃꢆꢉꢁꢄvꢇ sꢊbꢋꢁꢆꢌꢉꢇ ꢆꢌꢂ fꢊꢌꢉꢁꢄꢃꢌ ꢃꢈ nꢆꢁꢊꢅꢆꢍ Mꢇꢂꢄꢉꢄꢌꢇꢋ, iꢌꢋꢁꢄꢁꢊꢁꢇ ꢃꢈ Mꢆꢁꢇꢅꢄꢆ Mꢇꢂꢄꢉꢆ,
Pꢇkꢄꢌg uꢌꢄꢃꢌ Mꢇꢂꢄꢉꢆꢍ cꢃꢍꢍꢇgꢇ ꢆꢌꢂ cꢀꢄꢌꢇꢋꢇ aꢉꢆꢂꢇmy ꢃꢈ Mꢇꢂꢄꢉꢆꢍ sꢉꢄꢇꢌꢉꢇꢋ, Bꢇꢄjꢄꢌg 100050, cꢀꢄꢌꢆ
ABSTRACT
ARTICLE HISTORY
rꢇꢉꢇꢄvꢇꢂ 30 Jꢊꢍy 2016
aꢉꢉꢇpꢁꢇꢂ 8 oꢉꢁꢃbꢇꢅ 2016
An ongoing phytochemical investigation of the rhizomes of
Atractylodes lancea resulted in the isolation of four new C -type
10
polyacetylene glycosides (1−4). Their structures were elucidated on
the basis of extensive spectroscopic data (UV, IR, 1D and 2D NMR,
and HRESIMS). The absolute configurations of compounds 2−4 were
determined by comparing the specific rotations of their aglycones.
Notably, compounds 2 and 3 exhibited significant hepatoprotective
activities against APAP-induced HepG2 cell injury at a concentration
of 10 μM. Compounds 2 and 3 showed weak anti-inflammatory
effects on LPS-induced NO production in microglia BV2 cells at a
concentration of 10 μM.
KEYWORDS
Atractylodes lancea;
pꢃꢍyꢆꢉꢇꢁyꢍꢇꢌꢇ gꢍyꢉꢃꢋꢄꢂꢇ;
ꢀꢇpꢆꢁꢃpꢅꢃꢁꢇꢉꢁꢄvꢇ ꢆꢉꢁꢄvꢄꢁy;
ꢆꢌꢁꢄ-ꢄꢌflꢆmmꢆꢁꢃꢅy ꢇffꢇꢉꢁ
1
. Introduction
As a kind of common traditional Chinese medicine, Atractylodes lancea was used for the
treatment of “strengthening spleen, removing cold, and improving eyesight” [1,2]. A literature
survey disclosed that A. lancea was rich in sesquiterpenoids, polyacetylenes, monoterpenes,
steroids, phenolic acid, and acylsucrose [3−10]. In our ongoing phytochemical investigations
on the water-soluble bioactive ingredients of the rhizomes of this plant [11], four new C -
10
type polyacetylene glycosides (1−4) were isolated using various column chromatographic
methods. eir chemical structures were elucidated on the basis of extensive spectroscopic
data. e stereochemistry of 1−4 was unambiguously established by the specific rotations,
whereas those of monosaccharide moieties were determined by GC af er the chiral derivati-
zation. All the isolated compounds were evaluated for the hepatoprotective activities against
N-acetyl-acetyl-p-aminophenol (APAP)-induced HepG2 cell injury and anti-inflammatory
effects on the lipopolysaccharide (LPS)-induced NO production in microglia BV2 cells.
2
. Results and discussion
Compound 1 had an enediyne chromophore that two olefinic bonds were conjugated on the
opposite sides of the diyne unit [12,13]. Its molecular formula of C H O was deduced by
2
1
28 11
CONTACT Pꢇꢄ-cꢀꢇꢌg Zꢀꢆꢌg
pꢉzꢀꢆꢌg@ꢄmm.ꢆꢉ.ꢉꢌ
©
2016 iꢌꢈꢃꢅmꢆ uK lꢄmꢄꢁꢇꢂ, ꢁꢅꢆꢂꢄꢌg ꢆꢋ tꢆyꢍꢃꢅ & fꢅꢆꢌꢉꢄꢋ Gꢅꢃꢊp

2
K. XU ET AL.
−
a negative HRESIMS adduct ion at m/z 501.1628 [M + COOH] . e IR spectrum showed
−
1
−1
−1
the presence of hydroxyl (3369 cm ), acetylenic (2205, 2129 cm ), and olefinic (1608 cm )
1
2,3
groups. From the H NMR data (Table 1), a trans-configured Δ olefinic bond at δ 6.09
H
8
,9
and 6.44 (J = 16.0 Hz), a cis-configured Δ olefinic bond at δ 5.73 and 6.27 (J = 11.0 Hz),
H
two methylenes at δ 4.15 (1H, overlap), 4.34 (1H, J = 4.5, 16.0 Hz), and 4.16 (1H, overlap),
H
as well as two anomeric protons at δ 4.14 (J = 7.5 Hz) and 4.85 (J = 3.0 Hz) were observed.
H
1
3
Correspondingly, the C NMR data (Table 1) exhibited four olefinic carbons at δ 107.1,
C
1
08.5, 144.8, and 149.0, two acetylenic bonds at δ 73.6, 78.0, 78.3, and 81.5, two oxygenated
C
primary carbons at δ 59.5 and 67.3, as well as a glucosyl moiety and an apiosyl moiety.
C
Further analyses of the HMBC data (Figure 2) disclosed that the glucosyl and apiosyl moi-
eties were substituted at C-1 and Glc-C-6′, respectively. eir β-ꢀ-forms were deduced by
3
3
the J (7.5 Hz) and J (3.0 Hz) values and GC af er acid hydrolysis. Consequently, the
1
′,2′
1′′,2′′
structure of compound 1 was elucidated as (2E,8Z)-deca-2,8-diene-4,6-diyne-1,10-diol-1-
O-β-ꢀ-apiofuranosyl-(1→6)-β-ꢀ-glucopyranoside (Figure 1).

e UV data of compound 2 were characteristic of an enediyne chromophore [12,13].
It had a molecular formula of C H O , as established by an HRESIMS adduct ion at m/z
2
2
32 12
+
13
5
(
11.1805 [M + Na] and C NMR data. e IR spectrum proved the presence of hydroxyl
−1
−1
−1
3388 cm ), acetylenic (2259, 2130 cm ), and olefinic (1648 cm ) groups. e 1D NMR
2,3
data (Table 1) showed: a trans-configured Δ olefinic bond at δ 6.03 and 6.48 (J = 16.0 Hz);
H
an ethyl group at δ 0.95, 1.70 and δ 9.5, 28.2; two acetylenic bonds at δ 69.9, 73.3, 77.2,
H
C
C
and 81.9; two oxygenated carbons at δ 67.4 and 67.5; and two glucopyranosyl moieties.
C
With the exception of the sugar moieties, the above spectroscopic data were consistent
with those of (8R)-decene-4,6-diyne-1,8-diol [14]. Comprehensive analyses of the HMBC
spectrum (Figure 2) revealed the locations of the glucopyranosyl moieties at C-1 and C-8.
Enzymatic hydrolysis of 2 produced aglycone (2a) and glucose. e β-ꢀ-configuration of the
3
3
glucopyranosyl moieties was deduced by the J and J values and GC af er the chiral
1
′,2′
1′′,2′′
2
0
derivatization. e specific rotation of 2a {[ꢀ] −9.0 (c 0.39, CHCl )} was consistent with that
D
3
of (8R)-decene-4,6-diyne-1,8-diol {[ꢀ] −5.0 (c 0.70, CHCl )}. Accordingly, the structure of
D
3
compound 2 was defined as (2E, 8R)-decene-4,6-diyne-1,8-diol-O-di-β-ꢀ-glucopyranoside.
Compound 3 was also an enediyne derivative. e HRESIMS adduct ion at m/z 371.1343
−
[
1
M + COOH] provided a molecular formula of C H O . Careful comparisons of the
1
6
22
7
D NMR spectroscopic data (Table 1) of 2 and 3 revealed the absence of a glucopyranosyl
moiety in 3. A long-range correlation from the proton at δ 4.34 (Glc-1′-H) to the carbon
H
at δ 67.5 in the HMBC spectrum demonstrated that the β-ꢀ-glucopyranosyl moiety of 3
C
was substituted at C-8. e identical chemical shif s of C-8, C-9, and C-10 with those of 2
Figure 1. tꢀꢇ ꢉꢀꢇmꢄꢉꢆꢍ ꢋꢁꢅꢊꢉꢁꢊꢅꢇꢋ ꢃꢈ ꢉꢃmpꢃꢊꢌꢂꢋ 1–4.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
reflected an 8R configuration, which was evidenced by the specific rotation of its aglycone
2
0
{
[ꢀ] −8.6 (c 0.27, CHCl )}. Consequently, the structure of compound 3 was elucidated as
D
3
(
2E,8R)-decene-4,6-diyne-1,8-diol-8-O-β-ꢀ-glucopyranoside.
Compound 4 was obtained as a pink powder, and the UV spectrum indicated an enediyne
skeleton [12,13]. Its molecular formula of C H O corresponded to a negative adduct ion
1
6
22
7
−
at m/z 371.1354 [M + COOH] in the HRESIMS. In accordance to the detailed HSQC anal-
1
13
2,3
yses, the H and C NMR data (Table 1) showed a trans-configured Δ olefinic bond, two
acetylenic bonds, an ethyl group, a β-ꢀ-glucopyranosyl moiety, and two oxygenated carbons.

e identical molecular formulas and highly similar spectroscopic data of 4 and 3 suggested
1
3
that they were a pair of optical isomers. e most obvious difference of their C NMR data
(
Table 1) was the chemical shif of C-8, which implied the opposite C-8 configurations. is
20
deduction was unambiguously confirmed by the specific rotation of 4a {[ꢀ] + 10.1 (c 0.04,
D
CHCl )}, which was produced from the enzymatic hydrolysis of 4. us, compound 4 was
3
elucidated as (2E,8S)-decene-4,6-diyne-1,8-diol-8-O-β-ꢀ-glucopyranoside.
All these new compounds were evaluated for hepatoprotective activities against APAP-
induced HepG2 cell injury at a concentration of 10 μM (Table 2). Compared with the
model group, compounds 2 and 3 showed significant hepatoprotective activities with the
cell survival ratios of 55.1% (p < 0.001) and 26.4% (p < 0.01), respectively (bicyclol with
3
2.7%, p < 0.01). Besides, all compounds were tested for the anti-inflammatory effects on
the LPS-induced NO production in the microglia BV2 cells at a concentration of 10 μM
1
13
Table 1. h nMr (500 Mhz) ꢆꢌꢂ c nMr (125 Mhz) ꢋpꢇꢉꢁꢅꢆꢍ ꢂꢆꢁꢆ ꢈꢃꢅ ꢉꢃmpꢃꢊꢌꢂꢋ 1–4 ꢄꢌ dMso-d .
6
1
2
3
4
No.
δH
4.34, ꢂꢂ (4.5, 16.0) 67.3
4.15, ꢃvꢇꢅꢍꢆp
δC
δH
δC
δH
4.06, bꢅꢋ
δC
δH
4.05, ꢂ (2.0)
δC
1
1
2
3
4
5
6
7
8
9
1
ꢆ
b
4.36, m
4.17, ꢂꢂ (4.5, 16.0)
67.4
–
60.8
60.8
–
6.54, ꢂꢁ (4.5, 16.0) 150.0 6.51, ꢂꢁ (4.5, 16.0) 149.8
6.44, ꢂꢁ (4.5, 16.0) 144.8 6.48, ꢂꢁ (4.5, 16.0) 145.5
6.09, ꢂ (16.0)
108.5
81.5
73.6
78.3
78.0
107.1
6.03, ꢂ (16.0)
–
108.1
77.2
73.3
69.9
81.9
67.5
28.2
9.5
5.83, ꢂ (16.0)
–
105.7 5.82, ꢂꢁ (2.0, 16.0) 105.8
–
–
–
–
77.5
72.9
70.0
81.6
67.5
28.2
9.5
–
–
–
77.5
73.0
69.2
82.4
69.3
27.6
9.0
–
–
–
–
–
–
–
5.73, ꢂ (11.0)
4.67, ꢁ (6.5)
1.70, m
0.95, ꢁ (7.5)
Gꢍꢉ
4.67, ꢁ (6.5)
1.70, m
0.95, ꢁ (7.5)
Gꢍꢉ
4.57, ꢁ (6.0)
1.70, m
0.94, ꢁ (7.5)
Gꢍꢉ
6.27, ꢂꢁ (6.5, 11.0) 149.0
0
4.16, ꢃvꢇꢅꢍꢆp
Gꢍꢉ
59.5
1
2
3
4
5
6
6
′
′
′
′
′
′ꢆ
′b
4.14, ꢂ (7.5)
2.98, ꢃvꢇꢅꢍꢆp
3.13, ꢁ (8.5)
2.99, ꢃvꢇꢅꢍꢆp
3.25, m
3.84, bꢅꢂ (11.5)
3.40, ꢂꢂ (7.0, 11.5)
apꢄ
102.2
73.4
76.4
70.2
75.6
67.7
–
4.34, ꢂ (7.5)
2.96, m
3.14, m
3.02, m
3.08, m
3.66, ꢃvꢇꢅꢍꢆp
3.41, ꢃvꢇꢅꢍꢆp
Gꢍꢉ
100.0
73.3
76.8
70.2
77.2
4.34, ꢂ (7.5)
2.95, m
3.15, m
3.00, m
3.09, m
100.0
73.3
76.8
70.2
77.2
4.24, ꢂ (7.5)
2.94, ꢁ (8.5)
3.11, m
3.06, m
3.08, m
101.5
73.5
76.7
69.9
77.0
61.0
–
61.2 3.66, ꢂꢂ (5.5, 11.5) 61.2 3.65, ꢂꢂ (5.5, 11.5)
–
3.41, ꢂꢂ (5.5, 11.5)
–
3.45, ꢂꢂ (5.5, 11.5)
1
2
3
4
4
5
6
6
′′
′′
′′
′′ꢆ
′′b
′′
′′ꢆ
′′b
4.85, ꢂ (3.0)
3.75, bꢅꢋ
109.3
75.8
78.8
73.2
–
63.0
–
4.14, ꢂ (7.5)
2.96, m
3.14, m
3.02, m
3.08, m
3.66, ꢃvꢇꢅꢍꢆp
3.41, ꢃvꢇꢅꢍꢆp
102.4
73.5
76.6
70.0
77.0
61.0
–
–
3.85, ꢂ (9.5)
3.58, ꢂ (9.5)
3.32, ꢃvꢇꢅꢍꢆp
–
–
–

4
K. XU ET AL.
Figure 2. tꢀꢇ kꢇy hMBc ꢉꢃꢅꢅꢇꢍꢆꢁꢄꢃꢌꢋ ꢃꢈ ꢉꢃmpꢃꢊꢌꢂꢋ 1 ꢆꢌꢂ 2.
Table 2. hꢇpꢆꢁꢃpꢅꢃꢁꢇꢉꢁꢄvꢇ ꢆꢉꢁꢄvꢄꢁꢄꢇꢋ ꢆgꢆꢄꢌꢋꢁ aPaP-ꢄꢌꢂꢊꢉꢇꢂ hꢇpG2 ꢉꢇꢍꢍꢋ ꢄꢌjꢊꢅꢇ.
Cell survival ratios
(% of normal)d
(% of model)
Compounds
nꢃꢅmꢆꢍ
Mꢃꢂꢇꢍ
100.00 ± 5.94
39.87 ± 3.11
–
–
32.7
55.1
26.4
b
***
ꢆ
##
Bꢄꢉyꢉꢍꢃꢍ
52.87 ± 2.04
ꢉ
###
2
3
61.82 ± 2.70
50.39 ± 3.89
ꢉ
##
ꢆ
b
ꢉ
Bꢄꢉyꢉꢍꢃꢍ ꢆꢋ ꢁꢀꢇ pꢃꢋꢄꢁꢄvꢇ ꢂꢅꢊg; aPaP (8 mM)-ꢄꢌꢂꢊꢉꢇꢂ ꢉꢇꢍꢍꢋ ꢆꢋ ꢁꢀꢇ mꢃꢂꢇꢍ gꢅꢃꢊp; oꢁꢀꢇꢅ ꢉꢃmpꢃꢊꢌꢂꢋ wꢇꢅꢇ ꢁꢇꢋꢁꢇꢂ ꢆꢁ 10 μM;
ꢂ
rꢇꢋꢊꢍꢁꢋ ꢆꢅꢇ ꢇxpꢅꢇꢋꢋꢇꢂ ꢆꢋ ꢁꢀꢇ mꢇꢆꢌ ± sd (n = 3). ***p < 0.001 (vꢋ. ꢉꢃꢌꢁꢅꢃꢍ gꢅꢃꢊp); p < 0.001 p < 0.01 (vꢋ. mꢃꢂꢇꢍ gꢅꢃꢊp).
###
##
Table 3. iꢌꢀꢄbꢄꢁꢃꢅy ꢇffꢇꢉꢁꢋ ꢆgꢆꢄꢌꢋꢁ lPs-ꢄꢌꢂꢊꢉꢇꢂ no pꢅꢃꢂꢊꢉꢁꢄꢃꢌ ꢄꢌ BV2 ꢉꢇꢍꢍꢋ.
Compounds
Concentration (μM)
Inhibitory ratios (%)
2
3
10
10
10
20.83
11.27
72.50
ꢆ
cꢊꢅꢉꢊmꢄꢌ
ꢆcꢊꢅꢉꢊmꢄꢌ ꢆꢋ ꢁꢀꢇ pꢃꢋꢄꢁꢄvꢇ ꢂꢅꢊg.
(
Table 3). Compounds 2 and 3 exhibited weak anti-inflammatory effects with the inhibitory
ratios of 20.83 and 11.27%, respectively (curcumin with 72.50%).
3
. Experimental
3
.1. General experimental procedures
Optical rotations and UV spectra were measured on the JASCO P-2000, JASCO V-650
spectrophotometer, respectively (JASCO, Easton, MD, USA). IR spectra were conducted
on a Nicolet 5700 spectrometer (ermo Scientific, Waltham, MA, USA). NMR data were
recorded on a Bruker spectrometer (500 MHz) (Bruker-Biospin, Billerica, MA, USA).
Chemical shif s were given with the solvent peaks as the references (δ in ppm, J in Hz).
HRESIMS data were obtained by an Agilent 1100 series LC/MSD TOF (Agilent Technologies,
Waldbronn, Germany). GC experiments were conducted on an Agilent 7890A instrument.
Column chromatography was performed with RP-18 (50 μm, YMC, Kyoto, Japan), Diaion
HP-20 macroporous resin (Mitsubishi Chemical Corp., Tokyo, Japan), and Sephadex LH-20
(
Pharmacia Fine Chemicals, Uppsala, Sweden). A Shimadzu LC-10AT instrument equipped
with an YMC-Pack ODS-A column (250 × 20 mm, 5 μm, Japan) served as the reversed-phase
preparative HPLC. An Agilent 1260 series system with an Apollo C column (250 × 4.6 mm,
18
5
μm, Grace Davison) was used for HPLC analyses.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
3
.2. Plant materials

e rhizomes of Atractylodes lancea were collected in Huanggang City, Hubei Province of
China, in June 2014 and authenticated by associate professor L. Ma. A voucher specimen
(
ID-s-2596) was deposited in the herbarium at the Department of Medicinal Plants, Institute
of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050, China.
3
.3. Extraction and isolation

e rhizomes of A. lancea (100 kg) were extracted thrice with 80% EtOH for 2 h at 85 °C.

e extract (25.6 kg) was suspended in H O (30 L) and partitioned with petroleum ether,
2
EtOAc, and n-BuOH (three times each), respectively. e n-BuOH fraction (1.2 kg) was
chromatographed on an HP-20 column with a step gradient of EtOH-H O (v/v) to yield five
2
fractions: A (H O, 824 g), B (15% EtOH, 88.6 g), C (30% EtOH, 106.4 g), D (50% EtOH,
2
5
3.3 g), and E (95% EtOH, 19.5 g). Fraction C was chromatographed on an RP-18 and
eluted at a gradient of MeOH-H O (0:100−100:0, v/v) to obtain fractions C1–C10 using
2
HPLC analyses. Fraction C3 (10.0 g) was chromatographed on an LH-20 column using
H O to yield 42 subfractions (Fr. C3.1−Fr. C3.42). ese subfractions were purified by the
2
reversed-phase preparative HPLC with a MeOH:H O ratio of 30:70 (v/v). Subfraction Fr.
2
C3.4 afforded 2 (24.3 mg), d Fr. C3.5−Fr. C3.6 gave 3 (79.2 mg). Fraction C4 (10.2 g) was
chromatographed on an LH-20 column using H O to obtain 32 subfractions (Fr. C4.1−Fr.
2
C4.32). e purification of subfractions Fr. C4.10−Fr. C4.11 using the reversed-phase pre-
parative HPLC produced 4 (21.2 mg) and Fr. C4.12−Fr. C4.13 yielded 1 (4.9 mg).
3
.3.1. Compound 1
2
0
Brown amorphous powder,[ꢀ]D − 26.8 (c 0.07, MeOH: H O = 1:4); UV (MeOH) λ (log ε)
2
max
2
2
18 (4.29), 262 (3.78), 277 (4.02), 294 (4.18), 313 (4.11) nm; IR (KBr) ν 3369, 2882, 2205,
max
−1 1
13
129, 1608, 1049 cm ; H and C NMR spectral data, see Table 1; HRESIMS: m/z 501.1628
−
[
M + COOH] (calcd for C H O ,501.1608).
2
2
29 13
3
.3.2. Compound 2
2
0
Brown amorphous powder,[ꢀ]D − 92.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 214 (4.42),
2
1
41 (3.53), 254 (3.88), 268 (4.02), 284 (3.91) nm; IR (KBr) νmax: 3388, 2930, 2259, 2130,
−
1 1
13
648, 1078, 1044 cm ; H and C NMR spectral data see Table 1; HRESIMS: m/z 511.1805
+
[
M + Na] (calcd for C H O Na, 511.1791).
2
2
32 12
3
.3.3. Compound 3
2
0
Brown amorphous powder, [ꢀ] − 119.1 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 214
D
(
4.45), 240 (3.72), 253 (3.88), 267 (4.00), 283 (3.92) nm; IR (KBr) νmax: 3361, 2971, 2934,
−1
1
13
2
881, 2231, 2136, 1678, 1079 cm ; H and C NMR spectral data, see Table 1; HRESIMS:
−
m/z 371.1343 [M + COOH] (calcd for C H O 371.1342).
1
7
23
9
3
.3.4. Compound 4
2
0
Pink amorphous powder,[ꢀ] + 37.8 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 213 (4.64),
D
2
40 (3.83), 253 (4.00), 268 (4.14), 283 (4.03) nm; IR (KBr) νmax: 3360, 2928, 2882, 2360,

6
K. XU ET AL.
−
1
1
13
2
3
230, 1655, 1081, 1025 cm ; H and C NMR spectral data see Table 1; HRESIMS: m/z
−
71.1354 [M + COOH] (calcd for C H O , 371.1342).
1
7
23
9
3
.5. Determination of the absolute configurations of sugars
Compound 1 (3 mg) was hydrolyzed with 1 mol/L HCl-dioxane (v/v = 1:1, 5 ml) at 60 °C
for 6 h [15]. Compounds 2−4 (each 5 mg) were individually hydrolyzed with snailase
(
w/w = 1:2, 3 ml H O as the solvent) at 37 °C for 24 h. e obtained hydrolysates were
2
extracted with EtOAc thrice (3 ml) to yield EtOAc extracts and monosaccharide residues
af er evaporating the solvents. e EtOAc extracts of 2−4 was purified by the reversed-
phase preparative HPLC with 20% CH CN to yield the corresponding aglycones (2a−4a).
3

e monosaccharide residues were processed based on the reported method [16−18]. e
absolute configurations of glucose and apiose were determined by comparing the retention
times of their trimethylsilyl-ꢁ-cysteine derivatives with those of authentic sugars prepared
by a similar procedure: ꢀ-glucose at 20.56 min and ꢀ-apiose at 14.56 min.
3
.6. Biological activity assay
e hepatoprotective activity was tested on HepG2 cells (bicyclol as the positive contrast),

whereas the anti-inflammatory activity was evaluated in microglial BV2 cell (curcumin as
the positive drug). e detailed processes were based on the reported method [11].
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work described in this paper was supported by the National Science and Technology Project of
China [grant number 2012ZX09301002-001003].
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