Eight new eudesmane- and eremophilane-type sesquiterpenoids from Atractylodes lancea

Article abstract of DOI:10.1016/j.fitote.2016.08.017

Phytochemical and pharmacological study on the rhizomes of Atractylodes lancea led to the identification of twenty-one compounds: six new eudesmane-type sesquiterpenoids (1–6), two new eremophilane-type sesquiterpenoids (7, 8), and thirteen known compound

Full text of DOI:10.1016/j.fitote.2016.08.017

Eight new eudesmane- and eremophilane-type sesquiterpenoids from Atracty-
lodes lancea
Kuo Xu, Zi-Ming Feng, Ya-Nan Yang, Jian-Shuang Jiang, Pei-Cheng
Zhang
PII:
DOI:
Reference:
S0367-326X(16)30244-1
doi: 10.1016/j.fitote.2016.08.017
FITOTE 3477
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
15 July 2016
22 August 2016
25 August 2016
Please cite this article as: Kuo Xu, Zi-Ming Feng, Ya-Nan Yang, Jian-Shuang Jiang,
Pei-Cheng Zhang, Eight new eudesmane- and eremophilane-type sesquiterpenoids from
Atractylodes lancea, Fitoterapia (2016), doi: 10.1016/j.fitote.2016.08.017
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ACCEPTED MANUSCRIPT
Eight new eudesmane- and eremophilane-type
sesquiterpenoids from Atractylodes lancea
Kuo Xu, Zi-Ming Feng, Ya-Nan Yang, Jian-Shuang Jiang, Pei-Cheng Zhang*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences,
Beijing 100050, People's Republic of China
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ABSTRACT
Phytochemical and pharmacological study on the rhizomes of Atractylodes lancea led to the
identification of twenty-one compounds: six new eudesmane-type sesquiterpenoids (1−6),
two new eremophilane-type sesquiterpenoids (7, 8), and thirteen known compounds (9−21).
These new compounds were elucidated using extensive spectroscopic analyses with
experimental and calculated electronic circular dichroism (ECD) for the configurational
assignments. Notably, this study was the first report on the isolation of two eremophilane-
type sesquiterpenoids (7, 8) from the genus Atractylodes. Compounds 5, 7, and 16 showed
potent hepatoprotective activities against N-acetyl-p-aminophenol (APAP)-induced HepG2
cell injury at a concentration of 10 µM (bicyclol as the positive drug).
Keywords:
Atractylodes lancea
eudesmane
eremophilane
sesquiterpenoids
hepatoprotective activity
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1. Introduction
Hepatic injury is the most common syndrome among all hepatic disorders.1 Medicinal
plants supply a rich source for screening live-protective ingredients. Atractylodes lancea
(Thunb.) DC., which is a perennial herb known as ''Cangzhu'', has been reputed in Traditional
Chinese Medicine for ''strengthening spleen, removing cold, and improving eyesight''.2
Eudesmane- and guaiane-type sesquiterpenoids and polyacetylenes are considered
characteristic phytochemicals.3−11 A literature survey disclosed that the extract and chemical
constituents from the rhizomes of A. lancea exhibit potent hepatoprotective effects.12 In our
search for hepatoprotective agents from A. lancea, six new eudesmane-type sesquiterpenoids
(1−6), two new eremophilane-type sesquiterpenoids (7, 8), and thirteen known compounds
(12−21) were isolated using various column chromatographic methods. The structures were
elucidated via 1D and 2D NMR spectroscopic analyses. The configurational assignments of
these new compounds were established using ECD (electronic circular dichroism), whereas
those of monosaccharide moieties were analysed by GC after the chiral derivatization. This
study was the first report on the isolation of eremophilane-type sesquiterpenoids from genus
Atractylodes. All isolated compounds were assayed for hepatoprotective activities against
APAP-induced HepG2 cell injury (bicyclol as the positive contrast). The information in this
paper will benefit subsequent phytochemical studies of genus Atractylodes.
2. Materials and methods
2.1. General experimental procedures
The specific rotations, UV, and ECD data were individually measured on JASCO P-2000,
JASCO V-650, and JASCO J-815 spectrometers (JASCO, Easton, MD, U.S.A.). IR spectra
were collected by a Nicolet 5700 instrument (Thermo Scientific, Waltham, MA, U.S.A.).
NMR spectra were run on a Bruker 500 Hz spectrometer (Bruker-Biospin, Billerica, MA,
U.S.A), and chemical shifts were given in δ (ppm) with DMSO-d6 peaks as the reference.
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HRESIMS data were collected using an Agilent 1100 series LC/MSD ESI/TOF instrument
(Agilent Technologies, Waldbronn, Germany). GC analyses were performed on an Agilent
7890A system. HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan), RP-18 (50 μm, YMC,
Kyoto, Japan) and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) were used
for chromatographic substrates. A Shimadzu LC-10AT system equipped with a SPD-10A
detector and an YMC-Pack ODS-A column (250 × 20 mm, 5 μm, Kyoto, Japan) was used for
reversed-phase preparative HPLC (P-HPLC). An Agilent 1260 series system equipped with
an Apollo C18 column (250 × 4.6 mm, 5 μm, Grace Davison) was used for HPLC analyses.
2.2 Plant materials
The rhizomes of A. lancea were collected at Huanggang City (Hubei Province, China) in
June 2014 and were identified by Prof. Lin 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).
2.3 Extraction and isolation
The dried rhizomes of A. lancea (100 kg) were extracted thrice with 80% EtOH (v/v)
under reflux condition for 2 h. The crude extract (25.6 kg) was suspended in 30 L distilled
H2O and separately partitioned with petroleum ether, EtOAc, and n-BuOH (three times each).
The n-BuOH fraction (1.2 kg) was chromatographed on an HP-20 column and eluted with a
step gradient of EtOH-H2O (v/v) to provide five fractions: A (H2O fraction, 824 g), B (15%
EtOH fraction, 88.6 g), C (30% EtOH fraction, 106.4 g), D (50% EtOH fraction, 53.3 g), and
E (95% EtOH fraction, 19.5 g). Fraction C (106.4 g) was chromatographed on an RP-18 and
eluted at a gradient of MeOH-H2O (0:100−100:0, v/v) to obtain fractions C1-C7 using HPLC
analyses. Fraction C1 (30.5 g) was chromatographed on an LH-20 column using distilled H2O
to obtain 123 subfractions (Fr. C1.1−Fr. C1.123). Subtractions Fr. C1.49−Fr. C1.56 were
further separated using P-HPLC, with 25% MeOH (v/v) to yield 11 (115 mg) and 14 (13 mg).
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Similarly, the purification of subfractions Fr. C1.57−Fr. C1.95 using P-HPLC produced 10
(20 mg), 15 (78 mg), 16 (13 mg), 17 (8 mg), 18 (55 mg), 20 (33 mg) and 21 (11 mg). Fraction
C2 (8.2 g) was chromatographed on an LH-20 column using distilled H2O to yield 30
subfractions (Fr. C2.1−Fr. C2.30). These subfractions were purified using P-HPLC with a
MeOH:H2O ratio of 30:70 (v/v). Subfraction Fr. C2.8 produced 19 (162 mg), and Fr.
C2.22−Fr. C2.27 gave 9 (4 mg). Fraction C3 (10.0 g) was eluted using distilled H2O on an
LH-20 column to yield 42 subfractions (Fr. C3.1−Fr. C3.42). Subfraction Fr. C3.4 was
purified using P-HPLC with 30% MeOH (v/v) to give 12 (75 mg). Fraction C4 (10.2 g) was
chromatographed on an LH-20 column using distilled H2O to yield 32 subfractions (Fr.
C4.1−Fr. C4.32). These subfractions were purified using P-HPLC with an MeOH:H2O ratio
of 30:70 (v/v). Fr. C4.2−Fr. C4.4 afforded 8 (6 mg). Fraction C6 (6.3 g) was chromatographed
using an LH-20 column with distilled H2O to produce 37 subfractions (Fr. C6.1−Fr. C6.37).
Then, Fr. C6.11−Fr. C6.14 were purified using P-HPLC with 35% MeOH (v/v) to produce 4
(19 mg), 5 (9 mg), 6 (20 mg), and 7 (17 mg). Fraction C7 (9.7 g) was separated using LH-20
with distilled H2O (subtractions Fr. C7.1−Fr. C7.35) and further purified using P-HPLC with
MeOH:H2O (40:60, v/v). Fr. C7.6−Fr. C7.7 yielded 2 (14 mg) and 3 (12 mg), Fr. C7.8−Fr.
C7.12 produced 1 (84 mg), and Fr. C7.29−Fr. C7.32 produced 13 (5 mg).
(5R,7R,10S)-Isopterocarpolone-11-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside
(1). White amorphous powder; []2D0 +7.3 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 241
(4.03) nm; ECD (MeOH) λmax (Δε) 210 (−2.96), 244 (+3.79), 327 (−0.51) nm; IR (KBr) νmax
:
1
3390, 2973, 2934, 2881, 1650, 1084, 1047 cm−1; H and 13C NMR data see Table 1;
HRESIMS m/z 553.2619 [M + Na]+ (calcd for C26H42O11Na, 553.2625).
(5R,7R,10S)-6''-O-acetylatractyloside I (2). White amorphous powder; []2D0 +18.6 (c
0.10, MeOH); UV (MeOH) λmax (log ε) 255 (3.93) nm; ECD (MeOH) λmax (Δε) 216 (−0.73),
258 (+2.19), 322 (−1.39) nm; IR (KBr) νmax: 3403, 2932, 1737, 1659, 1074 cm−1; 1H and 13C
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NMR data see Table 1; HRESIMS m/z 641.2781 [M + Na]+ (calcd for C29H46O14Na,
641.2785).
(5R,7R,10S)-6'-O-acetylatractyloside I (3). White amorphous powder; []2D0 −10.7 (c
0.08, MeOH); UV (MeOH) λmax (log ε) 254 (3.79) nm; ECD (MeOH) λmax (Δε) 260 (+1.33),
324 (−0.85) nm; IR (KBr) νmax: 3400, 2972, 2928, 1722, 1660, 1621, 1077, 1043 cm−1; 1H and
13C NMR data see Table 1; HRESIMS m/z 641.2775 [M + Na]+ (calcd for C29H46O14Na,
641.2785).
(5R,7R,10S)-3-Hydroxylisopterocarpolone-3-O-β-D-glucopyranoside
(4).
White
amorphous powder; []2D0 +7.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 255 (3.78) nm;
ECD (MeOH) λmax (Δε) 216 (−0.40), 257 (+2.00), 322 (−1.16) nm; IR (KBr) νmax: 3374, 2969,
2936, 1660, 1616, 1076 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 413.2186 [M
− H]− (calcd for C21H33O8, 413.2175).
(2S,7R,10S)-3-Hydroxylcarissone-11-O-β-D-glucopyranoside (5). White amorphous
powder; []2D0 −106.2 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 252 (3.95) nm; ECD
(MeOH) λmax (Δε) 252 (−15.06), 317 (+1.29) nm; IR (KBr) νmax: 3384, 2920, 1667, 1614,
1078, 1027 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 459.2238 [M + COOH]−
(calcd for C22H35O10, 459.2230).
(2R,7R,10S)-3-Hydroxylcarissone-11-O-β-D-glucopyranoside (6). White amorphous
powder; []2D0 +63.5 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 250 (4.00) nm; ECD (MeOH)
λmax (Δε) 251 (+9.43), 316 (−1.03) nm; IR (KBr) νmax: 3393, 2973, 2931, 1666, 1610, 1074,
1042 cm−1; 1H and 13C NMR data see Table 2; HRESIMS m/z 459.2237 [M + COOH]− (calcd
for C22H35O10, 459.2230).
(3S,4R,5R,7R)-3,11-Dihydroxy-11,12-dihydronootkatone-11-O-β-D-glucopyranoside
(7). White amorphous powder; []2D0 −75.0 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 241
(3.99) nm; ECD (MeOH) λmax (Δε) 247 (−3.14), 306 (+0.10) nm; IR (KBr) νmax: 3417, 2972,
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2878, 1665, 1075, 1033 cm−1; 1H and 13C NMR data see Table 3; HRESIMS m/z 459.2234 [M
+ COOH]− (calcd for C22H35O10, 459.2230).
(3S,4R,5S,7R)-3,4,11-Trihydroxy-11,12-dihydronootkatone-11-O-β-D-
glucopyranoside (8). White amorphous powder; []2D0 −34.1 (c 0.07, MeOH); UV (MeOH)
λmax (log ε) 241 (3.78) nm; ECD (MeOH) λmax (Δε) 249 (−3.24), 313 (+1.06) nm; IR (KBr)
νmax: 3381, 2973, 2937, 1670, 1078, 1034 cm−1; 1H and 13C NMR data see Table 3; HRESIMS
m/z 453.2104 [M + Na]+ (calcd for C21H34O9Na, 453.2101).
2.4 Acid hydrolysis of compound 1
Compound 1 (5 mg) was dissolved in 1 mol/L HCl-dioxane (1:1, 5 mL) and maintained at
60 °C for 6 h.13,14 After drying in vacuum, the residue was partitioned in H2O (5 mL) and
extracted thrice with EtOAc (5 mL). The aqueous solution was evaporated in vacuum to
obtain the monosaccharide residue. These monosaccharide residues were processed using a
reported method.15−17 The configurational assignments of apiose and glucose were established
by comparing the retention times of their chiral derivatives with those of standard substances,
which were prepared using the identical procedure (D-apiose 14.56 min, D-glucose 20.56 min).
2.5 Hepatoprotective activity assay
The standard MTT assay was used to assess the hepatoprotective activity. Human
hepatoma cells (HepG2) were cultured in a DMEM medium, which was supplemented with
10% (v/v) foetal calf serum (FCS) and penicillin (100 U/mL)-streptomycin (100 μg/mL)
solution, at 37 °C in a humidified atmosphere with 5% CO2. The growing cells were seeded in
96-well plates and incubated for 12 h. Then, these cells were treated with APAP (8 mM) and
various test samples (10 μM) and were further incubated for 48 h. Then, 100 μL of MTT
solution (0.5 mg/mL) was added to each well after the medium was removed, and the
solutions were incubated for an additional 4 h. The residuum was dissolved in 150 μL DMSO
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after emptying the culture medium, and the absorbance was quantified at 570 nm using a
microplate reader. Bicyclol was used as the positive contrast.
3. Results and discussion
Compound 1 was obtained as a white powder. An HRESIMS adduct ion at m/z 553.2619
[M + Na]+ corresponds to a molecular formula of C26H42O11, which suggests six degrees of
unsaturation. The IR absorptions indicate the presence of hydroxyl (3390 cm−1) and carbonyl
1
(1650 cm−1) groups. The H NMR data (Table 1) show an olefinic proton at δH 5.76, four
methyl protons at δH 0.76, 1.13, 1.17, and 1.87, and two anomeric protons at δH 4.30 and 4.79.
The 13C NMR data (Table 1) exhibit 26 carbons, eleven of which were attributed to a glucosyl
moiety (δC 97.1, 73.6, 76.9, 70.3, 75.2, and 68.1) and an apiosyl moiety (δC 109.3, 75.9, 78.8,
73.2, and 63.2). The remaining 15 carbons, which are attributable to an α,β-unsaturated
carbonyl moiety (δC 125.6, 164.2, and 198.1), an oxygenated tertiary carbon (δC 78.8), and
other aliphatic carbons, reveal a skeleton of eudesmane-type sesquiterpenoid. The NMR
spectroscopic data were similar to those of isopterocarpolone-11-O-β-D-glucopyranoside,
except for an additional apiosyl moiety.8 A key HMBC correlation (Figure 2) from the proton
at δH 4.79 to the carbon at δC 68.1 suggests that the apiosyl moiety was located at Glc-C-6'.
After the acid hydrolysis of 1, D-glucose was determined using GC analysis after the chiral
derivatization (20.57 min), whereas the β-configuration was deduced based on the coupling
constant (7.5 Hz) of the anomeric proton. The β-D-configuration of apiose was established by
13
the similar GC method (14.53 min) and C NMR data.18 Thus, compound 1 is elucidated as
isopterocarpolone-11-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside.
The
ROESY
correlations (Figure 3) of H-5 with H-1a, H-6a, H-7, and H-9b and that of H3-15 with H-1b,
H-6b, and H-9a indicate that the juncture of A- and B- rings is trans-configured, H-5 and
CH3-10 are axial, whereas C-7 hydroxyisopropyl is equatorial. According to Snatzke's rule for
a cyclohexenone unit,19,20 the negative (Δε = −0.51) Cotton effect (CE) at 327 nm (Figure 4),
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which is contributed by the n→π* electron transition of the α,β-unsaturated carbonyl moiety,
unambiguously favours a 5R,7R,10S configuration.
Compound 2 shows similar IR absorptions to 1, which can be assigned to hydroxyl (3403
cm−1) and carbonyl (1659, 1737 cm−1) groups. Its molecular formula C29H46O14 with seven
degrees of unsaturation was deduced based on an HRESIMS adduct ion at m/z 641.2781 [M +
Na]+. The preliminary inspections of the NMR data (Table 1) characterised the resonances as
a eudesmane-type sesquiterpenoid. Except for an additional acetyl group (δH 1.95; δC 20.6,
and 170.3), the 1D NMR data exhibit four methyl groups (δH 0.83, 1.12, 1.17, and 1.83), an
α,β-unsaturated carbonyl moiety (δC 145.0, 150.1, and 193.7), two β-D-glucopyranosyl
moieties, and an oxygenated tertiary carbon (δC 78.9), which are consistent with those of
atractyloside I.8 The HMBC spectrum demonstrates that the acetyl group is at Glc-C-6'' via a
long-range correlation (Figure 2) of H-6'' (δH 3.96 and 4.23) with C-7'' (δC 170.3). Therefore,
the structure of 2 was defined as 6''-O-acetylatractyloside I. The ROESY correlations (Figure
3) of H-5 with H-1a, H-6a, H-7, and H-9b and that of H3-15 with H-1b, H-6b, and H-9a
suggest that the axial configuration of H-5 and CH3-10, whereas C-7 hydroxyisopropyl was
equatorial. The ECD spectrum (Figure 4) of 2 show negative CE sign at 322 nm (Δε = −1.39),
which supports that the absolute configuration is 5R,7R,10S.19,20
Compounds 3 and 2 have the identical molecular formula of C29H46O14 and similar
13
spectroscopic data, which suggests that their structures are highly similar. In the C NMR
spectrum, the deshielded resonance of Glc-C-6' at δC 63.7 indicates that the acetyl group (δH
1.96; δC 20.7 and 170.1) in 3 was substituted at Glc-C-6' instead of Glc-C-6''. This assignment
was verified with a key HMBC cross-peak from the protons at δH 3.94 and 4.25 to the carbon
at δC 170.1. With the exception of this difference, other 1D NMR data of 3 and 2 were
matched. Thus, compound 3 was elucidated as (5R,7R,10S)-6'-O-acetylatractyloside I, based
on similar ROESY (Figure 3) and ECD data (Figure 4) to those of 2.
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The molecular formula C21H34O8 of compound 4 was established using an HRESIMS
quasi-molecular ion at m/z 413.2186 [M − H]−. Its IR spectrum exhibits the absorption
characteristic of hydroxyl (3374 cm−1) and carbonyl (1660 cm−1) groups. Detailed inspections
of the 1D NMR data (Table 2) with that of 2 reveal the absence of a 6''-O-acetyl-β-D-glucosyl
moiety in 4, which is supported by the resonance of C-11 at δC 70.6 in 4, instead of the
deshielded resonance at δC 78.9 in 2. In addition, their other NMR spectroscopic data are
highly similar. Consequently, compound 4 was elucidated as 3-hydroxylisopterocarpolone-3-
O-β-D-glucopyranoside. The consistent ROESY (Figure 3) and ECD data (Figure 4) with
those of 2 suggest that compound 4 has the 5R,7R,10S configuration.
Compounds 5 and 6 were isolated as white amorphous powders with the identical
molecular formula of C21H34O8, which corresponds to the [M + COOH]− adduct ions at m/z
459.2238 and 459.2237, respectively. The 1D NMR data (Table 2) of these two compounds
show an α,β-unsaturated carbonyl moiety (δC 124.4, 162.9, and 200.1 in 5; δC 125.6, 162.7,
and 200.1 in 6), four methyl groups [δH 1.13 (3H), 1.18 (6H), and 1.71 (3H) in 5; δH 1.18 (6H),
1.24 (3H), and 1.71 (3H) in 6], and a β-D-glucopyranosyl moiety. The identical molecular
formula and similar spectroscopic data imply that they are a pair of stereoisomers. In the
HMBC experiments, the long-range correlations (Figure 2) of H-1 with C-3 and C-5, H-14
with C-3, C-4, and C-5, H-15 with C-1, C-9, and C-10, and H-1' with C-11 indicate a
eudesmane-type sesquiterpenoid with a glucosyl unit at C-11. In association with HSQC and
1H-1H COSY (Figure 2) data, two spin systems [C(1)H2-C(2)H and C(6)H2-C(7)H-C(8)H2-
C(9)H2] were established, and a hydroxyl group was determined to locate at C-2. Thus, the
planar structure of 5 and 6 was identified as 3-hydroxylcarissone-11-O-β-D-glucopyranoside.
To designate their absolute configurations, ROESY and ECD analyses were performed. For 5,
the correlations (Figure 3) of H-2 with H-9a, H-7 with H-6a, and H3-15 with H-6b and H-9b
suggest that H-2 and H-7 are on the same side of the eudesmane-ring, whereas CH3-10 is on
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the opposite side. For 6, H3-15 correlates with H-2 and H-6b, and H-7 correlates with H-6a,
which suggests that H-2 and CH3-10 are on the same side of the eudesmane-ring and opposite
to H-7. Their ECD data (Figure 5) show opposite CE signs at approximately 315 nm (Δε =
+1.29 in 5; Δε = −1.03 in 6), which were derived from the n→π* electron transition of the
α,β-unsaturated carbonyl moiety. Based on Snatzke's rule for a cyclohexenone unit,19,20 the
positive CE of 5 enables the assignment of a 2S,7R,10S configuration, whereas the negative
CE of 6 favours a 2R,7R,10S configuration. Their configurational assignments are also
supported by ECD calculations (see supplementary data, S42), which was performed using an
MMFF94 force field and the time-dependent density functional theory (TDDFT) at the
B3LYP/6-31+G(d,p) level. The calculated ECD data of 5 and 6 (Figure 5) are consistent with
their experimental data.
Compound 7 has a molecular formula of C21H34O8 with five degrees of unsaturation, as
13
deduced by the [M + COOH]− adduct ion at m/z 459.2234 in the HRESIMS and C NMR
data. Its IR spectrum exhibit absorptions of hydroxyl (3417 cm−1) and carbonyl (1665 cm−1)
1
groups. The most obvious characteristic in the H NMR spectrum is that the four methyl
resonances distribute in a smaller range (δH 1.04−1.09) than those of 5 and 6. Fifteen carbon
resonances in the 13C NMR spectrum are attributed to be an eremophilane-type
sesquiterpenoid, except for a glucopyranosyl moiety.21 H-14 and H-15 have long-range
correlations with C-4 and C-5 in the HMBC spectrum, which indicates that CH3-4 and CH3-5
are substituted at the adjacent carbons. In combination with the HSQC data, the HMBC cross-
peaks of H-1 with C-3, H-3 with C-5 and C-14, and H-1' with C-11 suggest that a hydroxyl
group is at C-3 and that the β-D-glucopyranosyl moiety is connected at C-11. Therefore, 7 is
3
elucidated as 3,11-dihydroxy-11,12-dihydronootkatone-11-O-β-D-glucopyranoside. The J3,4
value (12.5 Hz) indicates a 3,4-trans configuration. The ROESY correlations (Figure 3) of H-
3 with H3-15 and H-4 with H-7 reveal that H-3 and CH3-5 are on the same side of the
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eremophilane-ring, whereas H-4 and H-7 are on the opposite side. The positive CE at 306 nm
(Figure 6) reflects the absolute configuration as 3S,4R,5R,7R.19,20 In addition, ECD
calculation is performed using an MMFF94 force field and the TDDFT method at the
B3LYP/6-31+G(d,p) level. The obtained theoretical data of the (3S,4R,5R,7R)-conformer is
consistent with the experimental data for compound 7. To the best of our knowledge, this
study was the first report on the isolation of eremophilane-type sesquiterpenoid from the
genus Atractylodes.
Compound 8 was shown to have a molecular formula of C21H34O9 using an HRESIMS
13
adduct ion at m/z 453.2104 [M + Na]+, which indicates five degrees of unsaturation. The C
NMR data (Table 3) reveal an α,β-unsaturated carbonyl moiety at δC 122.4, 171.9, and 198.0,
1
which is supported by an IR absorption at vmax 1670 cm−1. In the H NMR spectrum, an
olefinic proton at δH 5.78, an oxygenated proton at δH 4.20, four methyl protons at δH 0.98,
1.10, 1.11, and 1.25, a group of glucopyranosyl protons at δH 4.24, 2.88−3.13, 3.44, and 3.61,
and other aliphatic resonances at 1.01−2.53 were observed. The comparisons of its 1D data
(Table 3) with those of 7 indicate that 8 is also an eremophilane-type sesquiterpenoid
1
glucopyranoside. An assigned H-1H spin system [C(6)H2-C(7)H-C(8)H2-C(9)H2] (Figure 2)
and the HMBC correlations (Figure 2) from H-1 with C-3 and C-5, H-3 with C-5 and C-14,
H-14 with C-3, C-4, and C-5, H-15 with C-5, C-6, and C-10, and H-1' with C-11 determine
the locations of two hydroxyl groups (C-3 and C-4) and the β-D-glucopyranosyl unit (C-11).
Accordingly, 8 was identified as 3,4,11-trihydroxy-11,12-dihydronootkatone-11-O-β-D-
glucopyranoside. The unambiguous ROESY correlations (Figure 3) of H-3 with H3-15 and
H3-14 with H-7 suggest that H-3, OH-4, and CH3-5 are on the same side of the eremophilane-
ring, whereas CH3-4 and H-7 are on the opposite side. The experimental ECD data of 8 is
consistent with its calculated ECD data (Figure 6), which facilitates the assignment of a
3S,4R,5S,7R configuration for compound 8.19,20
12

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In addition, thirteen known compounds (9−21) were isolated from the rhizomes of A.
lancea: (E)-isoconiferin (9),22 syringin (10),23 4-O-caffeoylquinic acid (11),24 (2E,8R)-decene-
4,6-diyne-1,8-diol-8-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (12),25 (2E,8E,12R)-
tetradecane-2,8-diene-4,6-diyne-1,12,14-triol-1-O-β-D-glucopyranoside (13).26 cichoriin (14).
27 scopoletin-7-O-β-D-glucopyranoside (15),28 scopoletin-7-O-β-D-xylopyranosyl-(1→6)-β-D-
glucopyranoside (16),29 scopoletin-7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside
(17),30
isoscopoletin-6-O-β-D-glucopyranoside
(18),31
phenylmethanol
7-O-β-D-
apiofuranosyl-(1→6)-β-D-glucopyranoside (19),32 (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-
carboline-3-carboxylic acid (20),33 and (7E)-sinapate-4-O-β-D-glucopyranoside (21).34
All compounds were assayed for hepatoprotective activities against APAP-induced
HepG2 cell injury. As shown in Table 4, compared with the model group, compounds 5, 7,
and 16 exhibited potent hepatoprotective activities with the cell survival rates of 34.6% (p <
0.01), 37.7% (p < 0.001), and 35.9% (p < 0.001) at a concentration of 10 µM (the positive
drug bicyclol with 32.7%, p < 0.01).
Conflict of interest
Author's declares that there are no conflicts of interest.
Acknowledgments
The authors thank Y. H. Wang, M. X. Jin, and X. Liu for processing NMR data. This
project was funded by the National Science and Technology Project of China
(2012ZX09301002-001003).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://///
13

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References
[1] J.R. Mitchell, D.J. Jollow, W.Z. Potter, D.C. Davis, J.R. Gillette, B.B. Brodie,
Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism, J. Pharmacol. Exp.
Ther. 187 (1973) 185−194.
[2] Chinese Pharmacopoeia Commission, Pharmacopoeia of the People's Republic of China
(volumeⅠ). China Medical Science Press, Beijing, 2015, pp 161−162.
[3] K. Xu, J.S. Jiang, Z.M. Feng, Y.N. Yang, L. Li, C.X. Zang, P.C. Zhang, Bioactive
sesquiterpenoid and polyacetylene glycosides from Atractylodes lancea, J. Nat. Prod. 79
(2016) 1567−1575.
[4] M. Resch, A. Steigel, Z.L. Chen, R. Bauer, 5-Lipoxygenase and cyclooxygenase-1
inhibitory active compounds from Atractylodes lancea, J. Nat. Prod. 61 (1998) 347−350.
[5] H.X. Wang, C.M. Liu, Q. Liu, K. Gao, Three types of sesquiterpenes from rhizomes of
Atractylodes lancea, Phytochemistry 69 (2008) 2088−2094.
[6] M.S. Lehner, A. Steigel, R. Bauer, Diacetoxy-substituted polyacetylenes from
Atractylodes lancea, Phytochemistry 46 (1997) 1023−1028.
[7] Y. Nakai, T. Kido, K. Hashimoto, Y. Kase, I. Sakakibara, M. Higuchi, H. Sasaki, Effect
of the rhizomes of Atractylodes lancea and its constituents on the delay of gastric
emptying, J. Ethnopharmacol. 84 (2003) 51−55.
[8] J. Kitajima, A. Kamoshita, T. Ishikawa, A. Takano, T. Fukuda, S. Isoda, Y. Ida,
Glycosides of Atractylodes lancea, Chem. Pharm. Bull. 51 (2003) 673−678.
[9] K. Tanaka, A. Ina, Structure elucidation of acylsucrose derivatives from Atractylodes
lanceae rhizome and Atractylodes rhizome, Nat. Prod. Commun. 4 (2009) 1095−1098.
[10] S. Yahara, T. Higashi, K. Iwaki, T. Nohara, N. Marubayashi, I. Ueda, H. Kohda, K. Goto,
H. Izumi, M. Nuno, S. Katsuki, S. Isoda, M. Satake, Studies on the constituents of
Atractylodes lancea, Chem. Pharm. Bull. 37 (1989) 2995−3000.
14

ACCEPTED MANUSCRIPT
[11] H. Kamauchi, K. Kinoshita, K. Takatori, T. Sugita, K. Takahashi, K. Koyama, New
sesquiterpenoids isolated from Atractylodes lancea fermented by marine fungus,
Tetrahedron 71 (2015) 1909−1914.
[12] Y. Kiso, M. Tohkin, H. Hikino, Antihepatotoxic principles of Atractylodes Rhizomes, J.
Nat. Prod. 46 (1983) 651−654.
[13] M.L. Gan, M.T. Liu, L.S. Gan, S. Lin, B. Liu, Y.L. Zhang, J.C. Zi, W.X. Song, J.G. Shi,
Dammarane glycosides from the root of Machilus yaoshansis, J. Nat. Prod. 75 (2012)
1373−1382.
[14] Y.W. An, Z.L. Zhan, J. Xie, Y.N. Yang, J.S. Jiang, Z.M. Feng, F. Ye, P.C. Zhang,
Bioactive octahydroxylated C21 steroids from the root bark of Lycium chinense, J. Nat.
Prod. 79 (2016) 1024−1034.
[15] S. Hara, H. Okabe, K. Mihashi, Gas-Liquid chromatographic separation of aldose
enantiomers as trimethylsilyl ethers of methyl 2-(polyhydroxyalkyl)-thiazolidine-4(R)-
carboxylates, Chem. Pharm. Bull. 35 (1987) 501−506.
[16] Z.M. Feng, S. Song, J. He, Y.N. Yang, J.S. Jiang, P.C. Zhang, Acyl glycosides with rare
β-D-apiofuranosyl-β-D-glucopyranosyl-β-D-apiofuranosyl from Erycibe hainanesis,
Carbohydr. Res. 380 (2013) 59−63.
[17] Y. Shen, Z.M. Feng, J.S. Jiang, Y.N. Yang, P.C. Zhang, Dibenzoyl and isoflavonoid
glycosides from Sophora flavescens: inhibition of the cytotoxic effect of D-
galactosamine on human hepatocyte HL-7702, J. Nat. Prod. 76 (2013) 2337−2345.
[18] J.R. Snyder, A.S. Serianni, DL-apiose substituted with stable isotopes: synthesis, N.M.R.-
spectral analysis, and furanose anomerization, Carbohydr. Res. 166 (1987) 85−99.
[19] G. Snatzke, Circulardichroismus-IX: Modifizierung der octantenregel für α,β-
ungesättigte ketone: transoide enone, Tetrahedron 21 (1965) 421−438.
15

ACCEPTED MANUSCRIPT
[20] J.K. Gawronski, Circular dichroism and stereochemistry of chiral conjugated
cyclohexenones, Tetrahedron 38 (1982) 3−26.
[21] G. Savona, F. Piozzi, M.C.D.L. Torre, O. Servettaz, B. Rodriguez, A valencane
sesquiterpenoid from Teucrium carolipaui, Phytochemistry 26 (1987) 571−572.
[22] Y.N. Sun, W. Li, X.T. Yan, S.Y. Yang, Y.H. Kim, Chemical constituents from the stems
of Acanthopanax divaricatus var. albeofructus, Biochem. Syst. Ecol. 57 (2014) 164−168.
[23] Z. Yuan, B.Y. Zhou, Z.C. Zhang, T.Z.L. Men, X. Li, Glycosides from Glehnia littoralis,
J. Shenyang Pharm. Univ. 19 (2002) 183−185.
[24] Y.X. Wang, E. Wang, J. Shang, H. Wang, Caffeoylquinic acid derivatives from the seeds
of Vernonia anthelmintica, China J. Chin. Mater. Med. 37 (2012) 1590−1592.
[25] J. Kitajima, A. Kamoshita, T. Ishikawa, A. Takano, T. Fukuda, S. Isoda, Y. Ida,
Glycosides of Atractylodes ovata, Chem. Pharm. Bull. 51 (2003) 1106−1108.
[26] J. He, Y. Shen, J.S. Jiang, Y.N. Yang, Z.M. Feng, P.C. Zhang, S.P. Yuan, Q. Hou, New
polyacetylene glucosides from the florets of Carthamus tinctorius and their weak anti-
inflammatory activities, Carbohydr. Res. 346 (2011) 1903−1908.
[27] M. Gawronska-Grzywacz, T. Krzaczek, Flavonoids and coumarins from Hieracium
pilosella L. (Asteraceae), Acta Soc. Bot. Pol. 78 (2009) 189−195.
[28] Y. Chen, X. Feng, X.D. Jia, Y.F. Dong, J.Y. Liang, M. Wang, H. Sun, Chemical
constituents from the flower bud of Lonicera macranthoides Hand. –Mazz, Chin. Tradit.
Herb. Drugs, 39 (2008) 823−825.
[29] M.L. Gan, C.G. Zhu, Y.L. Zhang, J.C. Zi, W.X. Song, Y.C. Yang, J.G. Shi, Constituents
from a water-soluble portion of ethanolic extract of Iodes cirrhosa, China J. Chin. Mater.
Med. 35 (2010) 456−467.
[30] H. Liu, J.Z. Shen, H.X Zhang, Chemical Constituents from Stems of Clausena excavata,
J. Chin. Med. Mater. 37 (2014) 2012−2015.
16

ACCEPTED MANUSCRIPT
[31] W.S. Feng, J.C. Wang, Y.H. He, X.K. Zheng, K. Song, Y.L. Zhang, M. Li, W. Zhao,
Chemical constituents from the flower buds of Magnolia biondii Pamp, Chin. Pharm. J.
50 (2015) 2103−2106.
[32] G.G. Deng, Z.J. Cui, X.W. Yang, Chemical constituents from polarity part in roots of
Angelica dahurica var. formosana cv. Chuanbaizhi, China J. Chin. Mater. Med. 40 (2015)
3805−3810.
[33] H.J. Song, Y.X. Liu, Y.X. Liu, Y.Q. Huang, Y.Q Li, Q.M. Wang, Design, synthesis, anti-
TMV, fungicidal, and insecticidal activity evaluation of 1,2,3,4-tetrahydro-β-carboline-3-
carboxylic acid derivatives based on virus inhibitors of plant sources, Bioorg. Med.
Chem. Lett. 24 (2014) 5228–5233.
[34] Y. Xu, J.Y. Liang, Z.M. Zou, Studies on chemical constituents of rhizomes of Smilax
china, China J. Chin. Mater. Med. 33 (2008) 2497−2499.
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Fig. 1. The structures of compounds 1–21.
Fig. 2. Key HMBC and 1H-1H COSY correlations of compounds 1, 2, 4, 5, and 8.
Fig. 3. Key ROESY correlations of compounds 1−8.
18

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Fig. 4. Experimental ECD spectra of compounds 1−4.
Fig. 5. Experimental and calculated ECD spectra of compounds 5 and 6.
Fig. 6. Experimental and calculated ECD spectra of compounds 7 and 8.
19

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Table 1
1H NMR (500 MHz) and 13C NMR (125 MHz) data (δ in ppm, J in Hz) for compounds 1−3 in DMSO-d6
1
2
3
Position
Position
Position
δH
δC
δH
δC
δH
δC
1a
1b
2
2.25, d (15.5)
2.03, d (15.5)
53.9 1a
2.28, d (16.0)
2.19, d (16.0)
53.8 1a
2.27, d (16.0)
2.18, d (16.0)
53.8
1b
2
1b
2
198.1
193.7
193.3
144.2
150.4
46.2
3
5.76, s
125.6
164.2
46.9
3
145.0
150.1
46.4
3
4
4
4
5
2.35, d (11.5)
2.22, d (14.0)
0.99, d (14.0)
1.54, m
5
2.41, d (12.0)
2.20, m
1.00, m
1.52, m
1.55, m
1.23, m
1.45, m
1.32, m
5
2.47, d (12.0)
2.21, m
0.98, m
1.51, m
1.58, m
1.23, m
1.45, m
1.31, m
6a
6b
7
23.1 6a
6b
23.7 6a
6b
23.9
47.5
7
47.2
7
47.3
21.4
8a
8b
9a
9b
10
11
12
13
14
15
Glc-1'
2'
1.57, m
21.7 8a
8b
21.6 8a
8b
1.24, m
1.45, m
39.2 9a
9b
39.0 9a
9b
38.8
1.35, m
37.1 10
78.8 11
22.7 12
25.0 13
21.6 14
16.6 15
97.1 Glc-1'
73.6 2'
76.9 3'
70.3 4'
75.2 5'
68.1 6'a
6'b
36.7 10
78.9 11
22.7 12
25.0 13
14.6 14
16.6 15
103.0 Glc-1'
74.3 2'
76.5 3'
69.9 4'
77.2 5'
61.0 6'a
6'b
36.6
78.6
22.7
25.0
14.6
16.4
102.0
74.2
76.1
70.2
74.0
63.7
1.13, s
1.12, s
1.13, s
1.17, s
1.17, s
1.17, s
1.87, s
1.83, s
1.81, s
0.76, s
0.83, s
0.83, s
4.30, d (7.5)
2.91, t (8.5)
3.14, t (8.5)
2.96, t (9.0)
3.22, m
4.51, d (7.5)
3.11, overlap
3.16, t (8.5)
3.08, overlap
3.01, overlap
3.59, brd (11.5)
3.41, m
4.60, d (7.5)
3.13, overlap
3.19, overlap
3.04, overlap
3.25, overlap
4.25, brd (11.5)
3.94, m
3'
4'
5'
6'a
6'b
Api-1''
2''
3.80, brd (11.0)
3.36, overlap
4.79, d (3.0)
3.68, d (3.0)
109.3 Glc-1''
75.9 2''
78.8 3''
73.2 4''
5''
4.35, d (7.5)
2.93, t (8.5)
3.16, t (8.5)
3.02, overlap
3.32, overlap
4.23, brd (11.5)
3.96, m
97.0 7'
73.6 8'
76.6 Glc-1''
70.2 2''
73.3 3''
64.2 4''
5''
170.1
20.7
97.1
73.7
77.1
70.2
76.6
61.3
1.96, s
3''
4.30, d (7.5)
2.90, t (8.5)
3.14, overlap
3.04, overlap
3.05, overlap
3.62, m
4''a
4''b
5''
3.83, d (9.5)
3.56, d (9.5)
3.31, d (11.0)
63.2 6''a
6''b
7''
170.3 6''a
20.6 6''b
8''
1.95, s
3.38, m
20

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Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) data (δ in ppm, J in Hz) for compounds 4−6 in DMSO-d6
4
5
6
Position
δH
δC
δH
δC
δH
δC
1a
1b
2
2.32, d (16.5)
2.18, d (16.5)
53.7
1.87, dd (6.0, 13.0)
1.61, d (13.0)
43.8
68.0
1.91, dd (5.5, 12.5)
1.51, d (12.5)
46.0
68.2
193.8
144.9
149.9
46.3
4.06, dd (6.0, 13.0)
4.16, dd (5.5, 14.0)
3
200.1
124.4
162.9
28.7
200.1
125.6
162.7
28.3
4
5
2.45, d (11.5)
1.98, m
1.03, m
1.31, m
1.56, m
1.24, m
1.46, m
1.30, m
6a
6b
7
24.2
2.75, d (12.5)
2.01, t (12.5)
1.41, m
2.83, d (14.5)
1.85, overlap
1.48, m
48.8
21.3
50.2
22.1
48.4
21.9
8a
8b
9a
9b
10
11
12
13
14
15
Glc-1'
2'
1.83, m
1.87, m
1.73, m
1.67, m
39.1
1.75, m
37.7
1.62, m
42.3
1.56, m
1.26, m
36.7
70.6
26.7
27.7
14.7
16.4
102.8
74.3
76.5
69.9
77.2
61.0
37.3
78.0
22.6
24.9
11.3
27.2
97.2
73.8
77.2
70.4
76.6
61.3
36.8
78.1
22.4
24.9
11.0
22.7
97.2
73.8
77.1
70.3
76.5
61.3
1.07, s
1.18, s
1.18, s
1.08, s
1.18, s
1.18, s
1.87, s
1.71, s
1.71, s
0.82, s
1.13, s
1.24, s
4.53, d (7.5)
3.11, overlap
3.17, t (8.5)
3.09, overlap
3.01, overlap
4.30, d (7.5)
2.90, t (8.5)
3.14, t (8.5)
3.01, t (8.5)
3.04, m
4.30, d (7.5)
2.90, t (8.5)
3.14, t (8.5)
3.02, t (8.5)
3.04, m
3'
4'
5'
6'a
6'b
3.59, brd (11.5)
3.41, m
3.61, dd (2.0, 11.5)
3.37, m
3.61, dd (2.0, 11.5)
3.38, m
21

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Table 3
1H NMR (500 MHz) and 13C NMR (125 MHz) data (δ in ppm, J in Hz) for compounds 7 and 8 in DMSO-
d6
7
8
Position
δH
δC
δH
δC
1
5.76, s
122.8
198.8
73.6
42.6
41.4
34.8
5.78, s
122.4
198.0
76.7
78.7
45.9
34.0
2
3
3.81, d (12.5)
4.20, s
4
1.94, dq (6.5, 12.5)
5
6a
6b
7
1.87, d (13.0)
1.13, t (13.0)
1.38, m
2.21, brd (14.0)
1.01, brd (14.0)
1.49, m
40.6
25.4
42.3
24.2
8a
8b
9a
9b
10
11
12
13
14
15
Glc-1'
2'
1.65, m
1.77, m
1.65, m
1.64, m
2.44, m
28.2
2.53, m
28.3
2.18, m
2.29, m
175.4
77.9
22.4
24.5
11.8
20.2
97.1
73.8
77.0
70.3
76.6
61.2
171.9
78.3
22.7
24.3
17.1
23.9
97.0
73.7
77.0
70.4
76.6
61.2
1.08, s
1.10, s
1.09, s
1.11, s
1.04, d (6.5)
1.06, s
0.98, s
1.25, s
4.25, d (7.5)
2.86, t (8.5)
3.12, t (8.5)
2.98, t (8.5)
3.03, m
4.24, d (7.5)
2.88, t (8.5)
3.13, t (8.5)
3.01, overlap
3.04, overlap
3.61, brd (11.0)
3.44, overlap
3'
4'
5'
6'a
6'b
3.61, brd (11.5)
3.37, overlap
22

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Table 4
Hepatoprotective activities against APAP-induced HepG2 cells injure
Cell Survival Rates
Compounds
(% of normal)
100.00 ± 5.94
39.87 ± 3.11***
52.87 ± 2.04##
53.67 ± 1.11##
54.90 ± 0.66###
43.75 ± 3.69#
54.16 ± 0.61###
52.85 ± 1.72##
52.81 ± 6.35#
(% of model)
Normal
—
Model
—
Bicyclol
32.7
5
34.6
7
37.7
14
16
17
18
9.8
35.9
32.6
32.5
a) Bicyclol as the positive drug; b) APAP (8 mM)-induced cells as the model group; c) Other compounds were
tested at 10 μM; d) Results are expressed as the mean ± SD (n = 3); ***p < 0.001 (vs control group), ###p <
0.001, ##p < 0.01, #p < 0.05 (vs model group).
23

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GRAPHIC ABSTRACT
24