Seven pairs of new enantiomeric sesquiterpenoids from Curcuma phaeocaulis

Article abstract of DOI:10.1016/j.bioorg.2020.103820

Seven pairs of new enantiomeric sesquiterpenoids, (+)/(?)-phaeocauline A ? G [(+)/(?)-1–7], were isolated from the rhizomes of Curcuma phaeocaulis by chiral HPLC separation. Their structures, including absolute configurations, were determined by spectroscopic analyses and ECD data. The isolates were assessed for vasorelaxant, anti-platelet aggregative, and neuroprotective activities. Enantiomers (+)-1 and (?)-1 showed similar activity against abnormal platelet aggregation induced by arachidonic acid, while their C-4 epimers (+)-2 and (?)-2 were inactive, which indicated that those effects were stereoselective, but not enantioselective. Compounds (+)/(?)-3–5 exhibited vasorelaxant effects against KCl-induced contraction of rat aortic rings.

Full text of DOI:10.1016/j.bioorg.2020.103820

BioorganicChemistry99(2020)103820
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Seven pairs of new enantiomeric sesquiterpenoids from Curcuma phaeocaulis
Fei Liua,b,1, Jin-Feng Chena,b,1, Ming-Ming Qiaoa,b, Hao-Yu Zhaoa,b, Qin-Mei Zhoua,b, Li Guoa,b
,
⁎
⁎
Cheng Penga, , Liang Xionga,b,
a School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, People’s Republic of China
b Institute of Innovative Medicine Ingredients of Southwest Specialty Medicinal Materials, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, People’s
Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Seven pairs of new enantiomeric sesquiterpenoids, (+)/(−)-phaeocauline A − G [(+)/(−)-1–7], were isolated
from the rhizomes of Curcuma phaeocaulis by chiral HPLC separation. Their structures, including absolute
configurations, were determined by spectroscopic analyses and ECD data. The isolates were assessed for va-
sorelaxant, anti-platelet aggregative, and neuroprotective activities. Enantiomers (+)-1 and (−)-1 showed si-
milar activity against abnormal platelet aggregation induced by arachidonic acid, while their C-4 epimers (+)-2
and (−)-2 were inactive, which indicated that those effects were stereoselective, but not enantioselective.
Compounds (+)/(−)-3–5 exhibited vasorelaxant effects against KCl-induced contraction of rat aortic rings.
Curcuma phaeocaulis
Sesquiterpenoids
Enantiomers
Chiral separation
Vasorelaxant activity
Anti-platelet aggregative activity
1. Introduction
widely used to promote blood circulation and remove blood stasis in
China [15]. Recent studies have demonstrated that many of these ses-
Chirality is one of the most important properties of both macro-
scopic as well as microscopic objects in nature, and it is defined by the
presence of one or more asymmetric carbon atoms in an organic mo-
lecule. Enantiomers have the same planar chemical structure, but they
are mirror images of each other; such molecules exhibit similar physical
and chemical properties, except for their optical activities in opposite
directions [1]. As for their biological and or pharmaceutical activity,
some enantiomers exhibit the same activity, while others exhibit a large
difference or even opposite activity [2–4]. Therefore, it is very im-
portant to assess the biological activities of each enantiomer of a chiral
molecule. Furthermore, the increasing interest in exploring the phy-
siological activity and pharmacological action of enantiomers of chiral
drugs has resulted in an increased demand for enantioseparation. At
present, HPLC using the chiral stationary phase is one of the most ef-
fective methods to isolate and purify a small amount of a chiral sub-
stance [5].
quiterpenoids in C. phaeocaulis have anti-inflammatory [14,16], anti-
oxidative [17], cytotoxic [18], anti-platelet aggregative [19], and he-
patoprotective effects [20]. However, there are very limited studies on
the bioactivities of enantiomeric sesquiterpenoids from the genus Cur-
cuma. In this study, seven pairs of new enantiomeric sesquiterpenoids
[(+)/(−)-1–7] were obtained by chiral HPLC separation (Fig. 1). Ac-
cording to the traditional functions of C. phaeocaulis, their blood-acti-
vating activities, including vasorelaxant and anti-platelet aggregative
effects, were evaluated.
2. Experimental
2.1. General experimental procedures
Optical rotations were measured on an Anton Paar MCP 200 po-
larimeter (Anton Paar GmbH, Austria). ECD spectra were recorded
using an Applied Photophysics Chirascan and Chirascan-plus circular
dichroism spectrometer (Applied Photophysics Ltd., Leatherhead,
England). An Agilent Cary 600 FT-IR microscope instrument (Agilent
Technologies Inc., CA, USA) was used to measure IR spectra. NMR data
In recent years, sesquiterpenoids have become the focus of research
due to their diverse skeletal types and rich activities [6]. With the de-
velopment of research methods and techniques, many more en-
antiomers of sesquiterpenoids have been discovered [7–10]. Curcuma
phaeocaulis Val. (Peng Ezhu in Chinese) has been shown to contain a
large number of sesquiterpenoids [11–13], some of which are en-
antiomers [14]. Its rhizomes, known as Rhizoma curcumae, have been
were obtained by
a Bruker-AVIIIHD-600 spectrometer (Bruker
Corporation, MA, USA) or an Agilent-NMR-vnmrs 600 spectrometer
(Agilent Technologies Inc., CA, USA) with the solvent peaks used as
⁎ Corresponding authors at: School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, People’s Republic of China (L. Xiong).
E-mail addresses: pengcheng@cdutcm.edu.cn (C. Peng), xiling0505@126.com, xiling@cdutcm.edu.cn (L. Xiong).
1 Both authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.103820
Received 24 February 2020; Accepted 2 April 2020
Availableonline07April2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

F. Liu, et al.
BioorganicChemistry99(2020)103820
Fig. 1. Structures of compounds (+)/(−)-1–7.
2.3. Extraction and isolation
references. HRESIMS data were acquired on a Waters Synapt G2 HDMS
instrument (Waters Corporation Milford, MA, USA). Column chroma-
tography was performed using silica gel (200–300 mesh; Yantai
Institute of Chemical Technology, Yantai, China) and Sephadex LH-20
(40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden). TLC
was performed using glass precoated silica gel GF254 plates (Qingdao
Marine Chemical Inc., Qingdao, China). HPLC separation was carried
out using an Agilent 1100 instrument with a Kromasil semi-preparative
The dry rhizomes of C. phaeocaulis (50 kg) were extracted with 95%
EtOH (3 × 50 L) under reflux three times (3 h each time). The EtOH
extract was evaporated under reduced pressure to yield a residue,
which was dispersed in H2O and partitioned with EtOAc. The EtOAc
soluble fraction (300 g) was separated on a silica gel (200–300 mesh)
column and eluted with a gradient of petroleum ether/EtOAc (5:1–0:1)
and EtOAc/MeOH (1:0–0:1) into 11 fractions (A–K).
C
18 column (250 × 10 mm2, 5 μm) for reversed-phase semi-preparative
HPLC and a Daicel Chiralpak AD-H column (250 × 4.6 mm2, 5 μm) for
normal-phase enantioseparation. Vasorelaxant activity assays were
conducted with a PL3508B6/C-V Panlab 8 Chamber Organ Bath System
(including stimulating electrodes, Panlab Eight-Chamber Organ Baths,
organ chambers, tissue hooks, Labchart Pro software). Anti-platelet
aggregation assays were measured on an SC-2000 Platelet ag-
gregometer (Beijing Success Technology Development Co., Ltd.,
Beijing, China). Healthy New Zealand rabbits of either sex weighing
2.2–2.5 kg were purchased from Dashuo Experimental Animal Co., Ltd
(Chengdu, China). PC12 cells were obtained from the Chinese Academy
of Sciences Cell Bank (Shanghai, China). Arachidonic acid (AA) and
adenosine diphosphate (ADP) were purchased from Dalian Meilun
Biotechnology Co., Ltd. Fetal bovine serum (FBS) was purchased from
Zhejiang Tianhang Biotechnology Co., Ltd, and dulbecco's modified
Eagle's medium (DMEM) was purchased from Hyclone (Logan, UT,
USA).
Fraction D (6 g) was applied to a Sephadex LH-20 column (CH2Cl2/
MeOH, 1:1) to afford four subfractions (D1–D4). Subfraction D2 (3.6 g)
was further separated by RP-MPLC with MeOH/H2O (20:80–0:100) to
afford eight fractions (D2-1 − D2-8). D2-2 (500 mg) was purified by a
Sephadex LH-20 column (petroleum ether/CH2Cl2/MeOH, 5:5:1) and a
silica gel column (CH2Cl2/ Me2CO, 50:1–0:1) successively to get D2-2-2-2
(15 mg). Compound 3 (2 mg, tR = 51.3 min) was obtained from D2-2-2-2
by RP semi-preparative HPLC (50% MeOH in H2O).
Fraction E (14 g) was subjected to Sephadex LH-20 column chro-
matography (petroleum ether/CH2Cl2/MeOH, 5:5:1) to obtain E2-3
(4.3 g), which was further separated by RP-MPLC with MeOH/H2O
(10:90–0:100) to afford 10 fractions (E2-3-1 − E2-3-10). E2-3-1 (303 mg)
was further purified by column chromatography on silica gel (CH2Cl2/
EtOAc, 50:1–1:1), followed by preparative TLC (CH2Cl2/ Me2CO, 9:1)
to yield E2-3-1-2-3 (23 mg) and E2-3-1-2-4 (28 mg). Compounds 4 (3 mg, tR
= 10.3 min) and 6 (2 mg, tR = 10.2 min) were obtained from E2-3-1-2-3
and E2-3-1-2-4 by HPLC (30% MeOH in H2O and 15% acetonitrile in
H2O), respectively. Separation of E2-3-10 (98 mg) by column chroma-
tography on silica gel (CH2Cl2/MeOH, 100:1–1:1) and HPLC (60%
MeOH in H2O) successively yielded compound 5 (2.5 mg, tR = 11.6
min).
2.2. Plant material
C. phaeocaulis was collected in Sanjiang Town, Chongzhou City,
Sichuan Province, China, in March 2018. The sample was identified by
Dr. Jihai Gao and deposited in the School of Pharmacy in Chengdu
University of TCM, Chengdu, China (voucher specimen: CP-20180303).
Fraction F (36 g) was separated into 19 fractions (F1 − F19) by RP-
MPLC with MeOH/H2O (10:90–0:100). Subfractions F5 (1.4 g), F11
(459 mg), and F15 (385 mg) were separated by Sephadex LH-20
2

F. Liu, et al.
BioorganicChemistry99(2020)103820
Table 1
Chiral separation of racemic mixtures 1–7.
Compound
Chiral phase
Mobile phase
Flow rate (mL/min)
Retention time (min)
Amount (mg)
Peak area ratio [(+)/(−)]
1
2
3
4
5
6
7
(+)-1
AD-H
n-hexane/ethanol, 25:1
n-hexane/ethanol, 10:1
n-hexane/ethanol, 50:1
n-hexane/ isopropyl alcohol, 10:1
n-hexane/ isopropyl alcohol, 10:1
n-hexane/ethanol, 10:1
n-hexane/ethanol, 10:1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
12.0
13.2
7.0
0.51
0.50
0.45
0.43
0.80
0.76
1.26
1.10
1.18
1.20
0.74
0.70
0.57
0.50
1.02:1
1.05:1
1.05:1
1.15:1
0.99:1
1.06:1
1.14:1
(−)-1
(+)-2
(−)-2
(+)-3
(−)-3
(+)-4
(−)-4
(+)-5
(−)-5
(+)-6
(−)-6
(+)-7
(−)-7
AD-H
5.4
AD-H
11.8
15.9
12.9
15.3
7.8
AD-H
AD-H
13.9
26.0
39.3
10.7
12.2
AD-H
AD-H
columns (petroleum ether/CH2Cl2/MeOH, 5:5:1) followed by silica gel
chromatography columns (CH2Cl2/ Me2CO, 20:1–0:1) to afford F5-6-1
(22 mg), F11-2-1 (11 mg), and F15-2-2 (14 mg), respectively. Then,
compounds 7 (1.3 mg, tR = 20.2 min), 1 (1.2 mg, tR = 47.8 min), and 2
(1 mg, tR = 53.7 min) were obtained from F5-6-1, F11-2-1, and F15-2-2 via
RP semi-preparative HPLC (50%, 55%, and 64% MeOH in H2O), re-
spectively. Finally, Racemic compounds 1–7 were submitted to chiral
separation on a Daicel Chiralpak AD-H column to afford their en-
antiomers (Table 1).
1093, 1064, 952, 857, 786, 711, 653 cm−1
;
1H NMR (600 MHz, acet-
one‑d6) and 13C NMR (150 MHz, acetone‑d6) data, see Tables 2 and 3,
respectively; (+)-HRESIMS m/z 305.2098 [M+Na]+ (calcd for
C17H30O3Na, 305.2093).
(+)-Phaeocauline B and (−)-phaeocauline B [(+)-2 and (−)-2]:
Colorless oil; {[α]2D0 + 13.6 (c 0.06, MeOH); ECD (MeCN) λmax (Δε) 190
(+8.8), 208 (−6.0) nm; (+)-2};{[α]2D0 −15.8 (c 0.05, MeOH); ECD
(MeCN) λmax (Δε) 191 (−8.6), 208 (+8.6) nm; (−)-2}; UV (MeCN)
λmax (log ε) 193 (3.66) nm; IR νmax 3340, 3277, 2969, 2932, 1446,
(+)-Phaeocauline A and (−)-phaeocauline A [(+)-1 and (−)-1]:
Colorless oil; {[α]2D0 + 75.5 (c 0.08, MeOH); ECD (MeCN) λmax (Δε) 191
(−8.6) nm; (+)-1};{[α]D20 −78.2 (c 0.06, MeOH); ECD (MeCN) λmax
(Δε) 190 (+8.8) nm; (−)-1}; UV (MeCN) λmax (log ε) 193 (3.76) nm; IR
νmax 3362, 2971, 2932, 1670, 1452, 1378, 1299, 1219, 1183, 1134,
1405, 1374, 1280, 1228, 1181, 1140, 1094, 1062, 959, 858, 741 cm−1
;
1H NMR (600 MHz, acetone‑d6) and 13C NMR (150 MHz, acetone‑d6)
data, see Tables 2 and 3, respectively; (+)-HRESIMS m/z 305.2090 [M
+Na]+ (calcd for C17H30O3Na, 305.2093).
(+)-Phaeocauline C and (−)-phaeocauline C [(+)-3 and (−)-3]:
Table 2
1H NMR (600 MHz) data of compounds 1–7 (δ in ppm, J in Hz).
Position 1a
2a
3a
4a
5a
5b
6a
7a
1
2
2.01, m
2.27, ddd (10.7,
10.7, 5.6)
1.80, m
2.52, m
2.92, ddd (11.3, 8.0, 2.67, d (13.0)
8.0)
3.63, ddd (4.1, 3.4,
3.4)
1.73, m
1.57, m
1.74, m
1.59, m
1.72, m
1.84, m
1.84, m
1.99, m
1.89, m
2.07, m
1.66, m
2.33, m
1.66, m
1.46, ddd (13.0,
10.5, 8.2)
1.92, m
1.64, m
3
5
1.55, m
1.55, m
1.70, m
1.53, m
2.05, m
1.73, m
1.76, m
2.08, m
2.08, m
2.44, m
2.20, m
1.98, ddd (13.7, 13.7,
3.9)
1.84, dd (13.5, 8.2)
1.76, m
1.95, m
1.40, m
2.19, s
2.25, ddd (11.6, 2.5, 2.03, dd (10.7, 3.9)
2.5)
2.24, dd (11.3, 3.4) 2.40, m
2.26, dd (13.5,
1.9)
6
5.92, d (3.1)
6.05, d (3.9)
5.79, s
3.25, d (16.8)
2.33, d (16.8)
6.12, brs
6.81, brs
7
2.55, d (15.8)
2.36, d (15.8)
8
2.35, m
2.02, m
1.73, m
1.58, m
2.39, dd (15.7, 7.8)
1.99, m
1.64, m
1.64, m
1.82, m
1.57, m
1.85, m
2.52, m
1.97, m
2.48, m
1.93, m
2.82, dd (14.7, 7.5)
2.27, m
9
1.65, m
5.73, s
2.64, dd (13.0, 7.5) 5.79, brs
3.41, d (16.4)
2.24, d (16.4)
1.65, m
2.14, m
4.78, brs
4.67, brs
1.56, s
11
12
13
14
15
16
17
OH
1.26, s
1.28, s
0.94, d (6.8) 1.74, s
0.97, d (7.0) 1.79, s
1.27, s
1.29, s
1.25, s
4.70, m
7.21, s
2.13, brs
1.24, s
1.07, s
1.26, s
1.28, s
1.56, s
1.13, s
1.27, s
1.42, s
1.29, s
1.38, s
1.89, s
1.50, s
1.18, s
1.18, s
4.91, m
1.90, brs
3.36. q (7.0)
1.05, t (7.0)
3.50, s (OH-4)
3.37, q (7.0)
1.06, t (7.0)
3.15, s (OH-4)
2.58, s (OH-
4.45, s (OH-1)
3.30, s (OH-4)
3.16, s (OH-6)
3.76, d (4.1, OH-1)
3.17, s (OH-4)
1)
3.31, s (OH-11)
3.31, s (OH-11)
3.49, s (OH-
4)
3.35, s (OH-
11)
a
b
Data were measured in acetone‑d6.
Data were measured in pyridine‑d5.
3

F. Liu, et al.
BioorganicChemistry99(2020)103820
Table 3
acetone‑d6) data, see Tables 2 and 3, respectively; (+)-HRESIMS m/z
13C NMR (150 MHz) data of compounds 1–7 in acetone‑d6 (δ in ppm).
287.1263 [M+Na]+ (calcd for C15H20O4Na, 287.1259).
Position
1
2
3
4
5
6
7
2.4. Vasorelaxant activity assay
1
48.7
22.5
41.3
79.9
50.7
123.0
150.4
23.6
37.4
79.3
73.2
29.3
29.4
22.8
18.9
56.2
16.7
50.1
23.6
40.9
80.3
50.6
122.9
150.9
24.1
37.5
79.7
73.2
29.2
29.2
26.4
19.0
55.8
16.7
81.0
30.0
40.9
77.4
153.6
122.1
87.0
36.9
32.1
85.3
34.6
18.2
18.2
29.8
21.2
82.9
34.3
30.3
70.6
69.6
24.4
133.8
195.8
129.8
148.4
139.2
21.7
22.4
15.8
20.0
48.9
25.9
40.5
80.1
56.0
122.6
151.6
29.7
38.6
155.9
73.3
29.2
29.2
27.4
106.5
47.4
73.2
26.1
34.7
71.5
60.4
196.8
120.9
167.8
34.0
42.1
119.6
139.6
9.3
2
25.5
The vasorelaxant activity of the isolates against KCl-induced con-
tractions of rat aorta rings was measured as described previously [21].
The experimental details are provided in the Supplementary Data.
3
35.4
4
145.5
48.2
5
6
74.2
7
52.6
2.5. Anti-platelet aggregative activity assay
8
197.1
127.4
160.6
111.2
9
10
11
12
13
14
15
16
17
The anti-platelet aggregative effects of the isolates were evaluated
by suppressing ADP- or AA-induced platelet aggregation in rabbit pla-
telets [22]. The experimental details are provided in the Supplementary
Data.
30.5
25.9
22.0
2.6. Neuroprotective activity assay
The protection of the isolates on glutamate-induced cytotoxicity in
PC12 cells was studied using the MTT method [23]. The experimental
details are provided in the Supplementary Data.
Colorless oil; {[α]2D0 + 26.6 (c 0.03, MeOH); ECD (MeCN) λmax (Δε) 197
(−61.2), 221 (+12.6) nm; (+)-3};{[α]2D0 −23.0 (c 0.04, MeOH); ECD
(MeCN) λmax (Δε) 197 (+55.4), 221 (−11.5) nm; (−)-3}; UV (MeCN)
λmax (log ε) 185 (4.03) nm; IR νmax 3483, 3439, 1699, 1465, 1448,
1363, 1342, 1298, 1221, 1184, 1144, 1114, 1067, 1041, 1020, 963,
3. Results and discussion
906, 867, 848, 830, 758, 675 cm−1
;
1H NMR (600 MHz, acetone‑d6)
Compound 1 was assigned with the molecular formula C17H30O3 by
an ion peak at m/z 305.2098 [M+Na]+ in the HRESIMS. The 1H NMR
data (Table 2) of 1 showed signals of four tertiary methyl groups, one
ethoxy group, one olefinic proton, and several aliphatic methylenes and
methines. A total of 17 carbon signals, corresponding to five methyl
groups, five methylenes, three methines (one olefinic), and four qua-
ternary carbons (three oxygenated and one olefinic), were revealed by
its 13C NMR spectrum (Table 3) and DEPT analysis. These data in-
dicated that compound 1 was a guaiane sesquiterpenoid similar to
4α,10β,11-trihydroxy-1,5-trans-guaiane-6,7-ene [24], with the excep-
tion of the replacement of a hydroxy group with an ethoxy group in 1
[δH 3.36 (2H, q, J = 7.0 Hz), 1.05 (3H, t, J = 7.0 Hz); δC 56.2 (t), 16.7
(q)]. Three oxygenated quaternary carbons were confirmed to be C-4
(δC 79.9), C-10 (δC 79.3), and C-11 (δC 73.2) according to the HMBC
correlations (Fig. 2) of H3-14 with C-3, C-4, and C-5; of H3-12 with C-7,
C-11, and C-13; and of H3-15 with C-1, C-9, and C-10, while the ethoxy
group was located at C-10 based on the HMBC correlation (Fig. 2) from
H2-16 to C-10. The NOE correlations of H-1 with H3-14 and H-16 and of
H-5 with OH-4 and H3-15 (Fig. 3) indicated that compound 1 has the
same relative configuration as that of 4α,10β,11-trihydroxy-1,5-trans-
guaiane-6,7-ene. Consequently, the structure of 1 was identified as
(1β,5α)-10β-ethoxyguaia-6(7)-en-4α,11-diol.
and 13C NMR (150 MHz, acetone‑d6) data, see Tables 2 and 3, respec-
tively; (+)-HRESIMS m/z 275.1629 [M+Na]+ (calcd for C15H24O3Na,
275.1623).
(+)-Phaeocauline D and (−)-phaeocauline D [(+)-4 and (−)-4]:
Colorless oil; {[α]2D0 + 89.2 (c 0.04, MeOH); ECD (MeCN) λmax (Δε) 238
(+8.4), 270 (−5.8), 342 (−2.5) nm; (+)-4}; {[α]2D0 −78.6 (c 0.06,
MeOH); ECD (MeCN) λmax (Δε) 238 (−8.6), 270 (+5.9), 342 (+2.5)
nm; (−)-4}; UV (MeCN) λmax (log ε) 263 (3.12), 241 (3.09) nm; IR νmax
3348, 2979, 2943, 1649, 1590, 1432, 1416, 1372, 1302, 1288, 1274,
1221, 1176, 1149, 1109, 1083, 1064, 1030, 1000, 970, 944, 909, 882,
852, 793, 707, 650 cm−1; 1H NMR (600 MHz, acetone‑d6) and 13C NMR
(150 MHz, acetone‑d6) data, see Tables
2 and 3, respectively;
(+)-HRESIMS m/z 271.1311 [M+Na]+ (calcd for C15H20O3Na,
271.1310).
(+)-Phaeocauline E and (−)-phaeocauline E [(+)-5 and (−)-5]:
Colorless oil; {[α]2D0 + 28.7 (c 0.03, MeOH); ECD (MeCN) λmax (Δε) 191
(−39.1), 208 (+4.6), 218 (−3.6) nm; (+)-5}; {[α]2D0 −26.6 (c 0.03,
MeOH); ECD (MeCN) λmax (Δε) 192 (+39.1), 208 (−3.8), 218 (+3.7)
nm; (−)-5}; UV (MeCN) λmax (log ε) 196 (4.01) nm; IR νmax 3393,
3183, 2918, 2848, 1644, 1468, 1419, 1371, 877, 816, 720, 644 cm−1
;
1H NMR (600 MHz, acetone‑d6 or pyridine‑d5) and 13C NMR (150 MHz,
acetone‑d6) and data, see Tables 2 and 3, respectively; (+)-HRESIMS
m/z 259.1679 [M+Na]+ (calcd for C15H24O2Na, 259.1674).
The HRESIMS data of 2 indicated that it possesses the same mole-
cular formula as 1. Comparison of the 1H, 13C, and 2D NMR data of 2
and 1 suggested that the two compounds have the same planar struc-
ture. Compound 2 was identified as the C-4 epimer of compound 1
(+)-Phaeocauline F and (−)-phaeocauline F [(+)-6 and (−)-6]:
Colorless oil; {[α]2D0 + 47.1 (c 0.03, MeOH); ECD (MeCN) λmax (Δε) 213
(+9.3), 239 (−18.3) nm; (+)-6}; {[α]2D0 −42.5 (c 0.04, MeOH); ECD
(MeCN) λmax (Δε) 213 (−9.3), 239 (+18.9) nm; (−)-6}; UV (MeCN)
λ
max (log ε) 234 (3.37), 190 (3.49) nm; IR νmax 3344, 2952, 2829, 1657,
1438, 1401, 1380, 1344, 1285, 1245, 1150, 1118, 1096, 1074, 1002,
976, 877, 866, 851, 828 cm−1; 1H NMR (600 MHz, acetone‑d6) and 13
C
NMR (150 MHz, acetone‑d6) data, see Tables 2 and 3, respectively;
(+)-HRESIMS m/z 215.1050 [M+Na]+ (calcd for C12H16O2Na,
215.1048).
(+)-Phaeocauline G and (−)-phaeocauline G [(+)-7 and (−)-7]:
Colorless oil; {[α]2D0 + 64.5 (c 0.05, MeOH); ECD (MeCN) λmax (Δε) 274
(+0.9), 312 (−2.5) nm; (+)-7}; {[α]2D0 −59.5 (c 0.05, MeOH); ECD
(MeCN) λmax (Δε) 273 (−0.9), 312 (+2.5) nm; (−)-7}; UV (MeCN)
λ
max (log ε) 205 (3.28), 269 (2.50) nm; IR νmax 3391, 3183, 2920, 2853,
1643, 1562, 1460, 1422, 1252, 1200, 1113, 1064, 1029, 962, 810, 763,
Fig. 2. Key 1H−1H COSY and HMBC correlations of 1 and 3–7.
720 cm−1
;
1H NMR (600 MHz, acetone‑d6) and 13C NMR (150 MHz,
4

F. Liu, et al.
BioorganicChemistry99(2020)103820
Fig. 3. Key NOE correlations of 1, 2, 4, 6, and 7 and ROESY correlations of 3.
according to the NOE correlations (Fig. 3) of H-1 with OH-4 and H-16
and of H-5 with H3-14 and H3-15. Thus, compound 2 was determined to
be (1α,5β)-10α-ethoxyguaia-6(7)-en-4α,11-diol.
data, and the empirical pyridine-induced deshielding effect. The large
coupling constant between H-1 and H-5 (J = 11.3 Hz) showed the
presence of the trans-guaiane skeleton [28]. The NOE correlation of H3-
14 with H-5 revealed the anti-orientation of OH-4 and H-5 (Fig. 3). To
further verify the relative configuration using the empirical pyridine-
induced deshielding effect [29,30], the 1H NMR data of 5 were col-
lected in acetone‑d6 and pyridine‑d5, respectively. The downfield shift
(Δδ + 0.40) was observed for H-1 in pyridine‑d5, which confirmed that
OH-4 and H-1 are cofacial. Therefore, the structure of 5 was defined as
(1α,5β)-guaia-6(7),10(15)-dien-4α,11-diol.
Compound 3 was obtained as a colorless oil. Its molecular formula
was elucidated as C15H24O3 by the positive HRESIMS data (m/z
275.1629 [M+Na]+; calcd. for C15H24O3Na, 275.1623). The 1H NMR
data (Table 2) of 3 displayed signals for two tertiary methyl groups (δH
1.29 and 1.42), two secondary methyl groups [δH 0.94 (d, J = 6.8 Hz)
and 0.97 (d, J = 7.0 Hz)], and one olefinic proton at 5.79 (s). A total of
15 carbon signals (Table 3) attributed to 4 × CH3, 4 × CH2, 2 × CH
(one olefinic methine), and 5 × C (one olefinic carbon and four oxy-
genated carbons) were revealed in the 13C NMR and DEPT spectra. The
above data indicated that 3 is very similar to a known 7,10-epoxy
guaiane sesquiterpenoid, pubinernoid B [25], with the exception of an
additional hydroxy group at C-1 (δC 81.0) in 3. This assignment was
confirmed by 1H−1H COSY correlations (Fig. 2) and the HMBC corre-
lations of H3-15 with C-1, C-9, and C-10; of OH-1 with C-1 and C-2; and
of OH-4 with C-3, C-4, and C-14 (Fig. 2). The relative configuration was
established by analysis of ROESY data. Correlations of H3-14 with H3-
13 and of OH-1 with OH-4 (Fig. 3) demonstrated that H3-14, isopropyl-
7, and 7-O-10 bridge were cofacial, whereas OH-1 and OH-4 occupied
the opposite face. Thus, the structure of compound 3 was determined as
7β,10β-epoxyguaia-5(6)-en-1α,4α-diol.
Compound 6 has the molecular formula of C12H16O2 with five in-
dices of hydrogen deficiency, as indicated by the HRESIMS and 13C
NMR data. The 1H NMR data (Table 2) of 6 showed proton signals at-
tributed to an allylic methyl group (δH 1.90), a terminal double bond
(δH 4.67 and 4.78), and a conjugated trisubstituted double bond (δH
5.79). The 13C NMR and DEPT data (Table 3) revealed the presence of
12 carbons, including one methyl, five methylenes (one olefinic), two
methines (one olefinic), two olefinic quaternary carbons, one carbonyl
carbon, and one oxygen-bearing quaternary carbon. The above func-
tionalities accounted for three out of the five indices of hydrogen de-
ficiency, and the remaining two indices suggested 6 to be bicyclic. Fi-
nally, compound 6 was assigned to be a degraded cadinene by the
1H−1H COSY correlations of H-1/H2-2/H2-3 and the HMBC correla-
tions of H3-15 with C-1, C-9, and C-10; of H-2 with C-1, C-3, C-4, C-6,
and C-10; of H-11 with C-3, C-4, and C-5; of H-5 with C-1, C-3, C-4, C-6,
and C-7; of H-7 with C-1, C-5, C-6, C-8, and C-9; and of H-9 with C-1
and C-7 (Fig. 2). The NOE correlations of H-1 with H-2a (δH 2.07) and
of OH-6 with H-2b (δH 1.66) suggested that the two six-membered rings
are trans-fused (Fig. 3). Thus, the structure of 6 was elucidated as (1α)-
6β-hydroxy-12,13,14-trinorcadinane-4(11),9(10)-dien-8-one.
The molecular formula (C15H20O3) of compound 4 was determined
from its [M+Na]+ ion peak at m/z 271.1311 (calcd. 271.1310) in its
HRESIMS. The 1H, 13C, and DEPT NMR data (Tables 2 and 3) of 4
suggested that it is also a guaiane type sesquiterpene similar to 1-
epiaerugidiol [26]. The presence of the 4,5-epoxide ring in 4 was de-
termined by the molecular formula (C15H20O3), the up-field shift of C-4
(δC 70.6), and the down-field shift of C-5 (δC 69.6), as well as the ab-
sence of the proton signal of H-5. The planar structure of 4 is supported
by 1H−1H COSY and HMBC data, especially the correlations from H3-
14 to C-3, C-4, and C-5; from H3-12 to C-7, C-11, and C-13; from H3-15
to C-1, C-9, and C-10; and from H-6 to C-1, C-4, and C-8 (Fig. 2). In the
1D NOE experiment of 4, H3-14 and OH-1 were enhanced when H-6a
(δH 3.25) was irradiated (Fig. 3), which indicated that OH-1, H-6a (δH
3.25), and H3-14 are cofacial. Thus, the structure of compound 4 was
established as 1β-hydroxy-4α,5α-epoxyguaia-7(11),9(10)-dien-8-one.
The HRESIMS, 1H and 13C NMR data (Tables 2 and 3) of compound
5 showed that it is an isomer of 4β,12-dihydroxyguaian-6,10-diene, a
known guaiane-type sesquiterpene isolated from the rhizomes of Alisma
orientale [27]. Detailed analysis of the 1H−1H COSY, HSQC, and HMBC
data confirmed that they have the same planar structure. The relative
configuration of 5 was elucidated by 1H−1H coupling constants, NOE
The molecular formula of compound 7 was assigned as C15H20O4,
which is the same as that of an eudesmane-type sesquiterpene, curco-
lonol [31]. A comparison of the 1H and 13C NMR data of 7 (Tables 2 and
3) and curcolonol suggested that the two compounds are structurally
quite similar. Compound 7 was identified as the C-5 epimer of curco-
lonol, according to the 1H−1H COSY and HMBC data (Fig. 2) combined
with the NOE correlations from H3-15 to OH-1, H-5, and H-9b (δH
2.24); from H3-14 to H-5; and from H-9a (δH 3.41) to OH-4 (Fig. 3).
Thus, compound 7 was identified to be (5β)-1β,4α-dihydroxy-8,12-
epoxyeudsma-7(8),11(12)-dien-6-one.
Interestingly, although compounds 1–7 all contain multiple chiral
centers, their specific optical rotations are close to zero and no cotton
effects were observed in their ECD spectra. It suggested that they may
be isolated as racemic mixtures. Subsequently, compounds 1–7 were
5

F. Liu, et al.
BioorganicChemistry99(2020)103820
Fig. 4. The experimental and calculated ECD spectra of 1–7. The TDDFT calculations of ECD spectra of 1–7 were conducted at the CAM-B3LYP/DGDZVP level.
subjected to chiral HPLC separation on a chiral column, yielding (+)-1/
(−)-1, (+)-2/(−)-2, (+)-3/(−)-3, (+)-4/(−)-4, (+)-5/(−)-5,
(+)-6/(−)-6, and (+)-7/(−)-7, respectively (Table 1). Each pair of
enantiomers showed opposite ECD curves and specific optical rotations.
Since the relative configurations of 1–7 have been determined as
mentioned above, ECD calculations were performed to define the ab-
solute configurations [32–34] (Supplementary Data, Experimental
Section). Consequently, the seven pairs of enantiomers were
6

F. Liu, et al.
BioorganicChemistry99(2020)103820
Fig. 5. Vasorelaxant activities of 3–5 against KCl-induced contraction of rat aorta rings. The data are presented as the means
S.E.M., n = 5. (One-way ANOVA
followed by Dunnett’s post hoc test, *p < 0.05 and **p < 0.01 vs. control group).
determined to be (+)-(1S,4R,5R,10S)-10-ethoxyguaia-6(7)-en-4,11-
diol [(+)-phaeocauline A, (+)-1], (−)-(1R,4S,5S,10R)-10-ethox-
yguaia-6(7)-en-4,11-diol [(−)-phaeocauline A, (−)-1], (+)-
(1R,4R,5S,10R)-10-ethoxyguaia-6(7)-en-4,11-diol [(+)-phaeocauline
B, (+)-2], (−)-(1S,4S,5R,10S)-10-ethoxyguaia-6(7)-en-4,11-diol [(−)-
phaeocauline B, (−)-2], (+)-(1S,4R,7S,10S)-7,10-epoxyguaia-5(6)-en-
1,4-diol [(+)-phaeocauline C, (+)-3], (−)-(1R,4S,7R,10R)-7,10-epox-
yguaia-5(6)-en-1,4-diol [(−)-phaeocauline C, (−)-3], (+)-(1S,4R,5S)-
1-hydroxy-4,5-epoxyguaia-7(11),9(10)-dien-8-one [(+)-phaeocauline
D, (+)-4], (−)-(1R,4S,5R)-1-hydroxy-4,5-epoxyguaia-7(11),9(10)-
dien-8-one [(−)-phaeocauline D, (−)-4], (+)-(1R,4R,5S)-guaia-
6(7),10(15)-dien-4,11-diol [(+)-phaeocauline E, (+)-5], (−)-
(1S,4S,5R)-guaia-6(7),10(15)-dien-4,11-diol [(−)-phaeocauline E, (−)-
5], (+)-(1R,6S)-6-hydroxy-12,13,14-trinorcadinane-4(11),9(10)-dien-
8-one [(+)-phaeocauline F, (+)-6], (−)-(1S,6R)-6-hydroxy-12,13,14-
trinorcadinane-4(11),9(10)-dien-8-one [(−)-phaeocauline F, (−)-6],
(+)-(1R,4R,5S,10R)-1,4-dihydroxy-8,12-epoxyeudsma-7(8),11(12)-
dien-6-one [(+)-phaeocauline G, (+)-7], and (−)-(1S,4S,5R,10S)-1,4-
dihydroxy-8,12-epoxyeudsma-7(8),11(12)-dien-6-one [(−)-phaeocau-
line G, (−)-7] by comparison of the calculated ECD spectra with the
experimental ECD spectra (Fig. 4).
at C-10 are closely related to this activity. This study not only enriched
the enantiomeric sesquiterpenoids in the genus Curcuma, but also partly
revealed the material basis of Curcuma phaeocaulis for activating blood
circulation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
Support was received from the National Natural Science Foundation
of China (Grant No. 81903777), the Postdoctoral Science Foundation of
China (Grant No. 2019M653362), the Youth Science and Technology
Innovation Research Team Program of Sichuan (Grant No.
2017TD0001), the Scientific Research Innovation Team Construction in
Sichuan Provincial University) (Grant No. 18TD0017), and the “Xing lin
Scholar” Plan of Chengdu University of Traditional Chinese Medicine
(Grant Nos. YXRC2018005 and BSH2018009).
Since the rhizomes of C. phaeocaulis are a traditional Chinese
medicine for promoting blood circulation and removing stasis, the
isolates were investigated for anti-platelet aggregative and vasorelaxant
activities. Enantiomers (+)-1 and (−)-1 showed similar activity
against abnormal platelet aggregation induced by arachidonic acid
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103820.
(AA) with inhibition rates of 27.78
4.36% and 31.63
7.10%,
respectively, while their C-4 epimers (+)-2 and (−)-2 were inactive at
References
0.1 mM (the inhibition of aspirin was 72.89
7.65% at 0.1 mM).
Compounds (+)/(−)-3–5 exhibited vasorelaxant effects against KCl-
[1] L. Huang, L.X. Dai, The Chemistry and Biology of Chiral Drug, Chemical Industry
Press, Beijing, 2002, pp. 2–4.
induced contraction of rat aortic rings with maximal vasorelaxation
[2] A.T. Rufino, M. Ribeiro, F. Judas, L. Salgueiro, M.C. Lopes, C. Cavaleiro,
A.F. Mendes, J. Nat. Prod. 77 (2014) 264–269, https://doi.org/10.1021/
np400828x.
(Emax
)
of 35.51
3.65%/38.96
3.26%, 39.42
4.63%/
40.93
5.68%, and 47.71
4.35%/45.64
6.85%, respectively
(Fig. 5), and there were no significant differences in the activities of
each pair of the enantiomers. In addition, the isolates were investigated
for their neuroprotective activities based on the results of the previous
neuroprotective studies of similar sesquiterpenoids from C. phaeocaulis
[14,32].14,29 However, they did not show significant activity at 50 μM.
[3] L.A. Nguyen, H. He, C. Pham-Huy, Int. J. Biomed. Sci. 2 (2006) 85–100.
[4] R.W. Fitch, B.B. Snider, Q. Zhou, B.M. Foxman, A.A. Pandya, J.L. Yakel, T.T. Olson,
N. Al-Muhtasib, Y. Xiao, K.D. Welch, K.E. Panter, J. Nat. Prod. 81 (2018)
1029–1035, https://doi.org/10.1021/acs.jnatprod.8b00062.
[5] Y. Wang, Y. Zhou, C. Ma, B. Yang, R. Feng, Y. Zhang, J. Fu, W. Chen, Y. Sun, J. Ma,
Q. Zhang, Y. Deng, Y. Zhang, W. He, Acta Pharm. Sin. B 2 (2012) 527–533, https://
doi.org/10.1016/j.apsb.2012.06.006.
[6] G.H. Ma, K.X. Chen, L.Q. Zhang, Y.M. Li, Med. Chem. Res. 28 (2019) 1339–1358,
https://doi.org/10.1007/s00044-019-02385-7.
[7] L.L. Xu, H.L. Chen, P. Hai, Y. Gao, C.D. Xie, X.L. Yang, I. Abe, Org. Lett. 21 (2019)
1078–1081, https://doi.org/10.1021/acs.orglett.8b04123.
4. Conclusion
[8] M.M. Qiao, F. Liu, Y. Liu, L. Guo, Q.M. Zhou, C. Peng, L. Xiong, Bioorg. Chem. 92
(2019) 103275, https://doi.org/10.1016/j.bioorg.2019.103275.
[9] R. Zhang, X. Feng, G. Su, Z. Mu, H. Zhang, Y. Zhao, S. Jiao, L. Cao, S. Chen, P. Tu,
X. Chai, J. Nat. Prod. 81 (2018) 1711–1720, https://doi.org/10.1021/acs.jnatprod.
7b01071.
In summary, seven pairs of new enantiomeric sesquiterpenoids,
[(+)/(−)-1–7], were obtained from the rhizomes of Curcuma phaeo-
caulis by chiral HPLC separation, and their structures were determined
by comprehensive spectroscopic analyses and ECD data. (+)/(−)-1
showed moderate activity against abnormal platelet aggregation in-
duced by arachidonic acid (AA), while (+)/(−)-3–5 exhibited vasor-
elaxant effects against KCl-induced contraction of rat aortic rings. The
difference in anti-platelet aggregative activity between (+)/(−)-1 and
(+)/(−)-2 indicated that those effects were stereoselective, but not
enantioselective. Moreover, a comparison of the vasorelaxant effects of
compounds (+)/(−)-2 and (+)/(−)-5 suggested that the substituents
[10] C.Y. Zhang, L. Luo, J. Xia, Y.N. Song, L.J. Zhang, M. Zhang, K. Rahman, Y. Ye,
H. Zhang, J.Y. Zhu, Nat. Prod. Res. 32 (2018) 2893–2899, https://doi.org/10.1080/
14786419.2017.1389937.
[11] J.H. Ma, F. Zhao, Y. Wang, Y. Liu, S.Y. Gao, L.Q. Ding, L.X. Chen, F. Qiu, Org.
Biomol. Chem. 13 (2015) 8349–8358, https://doi.org/10.1039/c5ob00964b.
[12] J.H. Ma, F. Zhao, Y. Wang, Y. Liu, S.Y. Gao, L.Q. Ding, L.X. Chen, F. Qiu,
Phytochem. Lett. 14 (2015) 221–225, https://doi.org/10.1016/j.phytol.2015.10.
017.
[13] Y. Liu, J.H. Ma, Q. Zhao, C.R. Liao, L.Q. Ding, L.X. Chen, F. Zhao, F. Qiu, J. Nat.
7

F. Liu, et al.
BioorganicChemistry99(2020)103820
Prod. 76 (2013) 1150–1156, https://doi.org/10.1039/c5ob00964b.
[25] S.X. Huang, J. Yang, W.L. Xiao, Y.L. Zhu, R.T. Li, L.M. Li, J.X. Pu, X. Li, S.H. Li,
H.D. Sun, Helv. Chim. Acta 89 (2006) 1169–1175, https://doi.org/10.1002/hlca.
200690114.
[14] G. Xia, D. Sun, J. Ma, Y. Liu, F. Zhao, P.O. Donkor, L. Ding, L. Chen, F. Qiu, Sci. Rep.
7 (2017) 43576, https://doi.org/10.1038/srep43576.
[15] National Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of
China (II); Chinese Medical Science and Technology Press: Beijing, 2015, pp.
274−275.
[26] H.J. Jang, J.H. Kim, H.M. Oh, M.S. Kim, J.H. Jo, K. Jung, S. Lee, Y.H. Kim, W.S. Lee,
S.W. Lee, M.C. Rho, Chem. Pharm. Bull. 64 (2016) 1062–1066, https://doi.org/10.
1248/cpb.c16-00086.
[16] S. Oh, A.R. Han, H.R. Park, E.J. Jang, H.K. Kim, M.G. Jeong, H. Song, G.H. Park,
E.K. Seo, E.S. Hwang, Chem. Biodivers. 11 (2014) 1034–1041, https://doi.org/10.
1016/j.bioorg.2014.11.002.
[27] H.G. Jin, Q. Jin, A.R. Kim, H. Choi, J.H. Lee, Y.S. Kim, D.G. Lee, E.R. Woo, Arch.
Pharm. Res. 35 (2012) 1919–1926, https://doi.org/10.1007/s12272-012-1108-5.
[28] E. Tsankova, V. Enev, Tetrahedron 43 (1987) 4425–4432, https://doi.org/10.1016/
S0040-4020(01)90318-7.
[17] Y.F. Hao, C.L. Lu, D.J. Li, L. Zhu, J.G. Jiang, J.H. Piao, Food Funct. 5 (2014)
1369–1373, https://doi.org/10.1039/c3fo60687b.
[29] A. Madaio, V. Piccialli, D. Sica, G.J. Corriero, J. Nat. Prod. 52 (1989) 952–961,
https://doi.org/10.1021/np50065a007.
[18] X. Chen, L. Pei, Z. Zhong, J. Guo, Q. Zhang, Y. Wang, Phytomedicine 18 (2011)
1238–1243, https://doi.org/10.1016/j.phymed.2011.06.017.
[30] A. Migliuolo, G. Notaro, V. Piccialli, D.J. Sica, J. Nat. Prod. 53 (1990) 1414–1424,
https://doi.org/10.1021/np50072a003.
[19] C. Mao, H. Xie, T.J. Lu, Chin. Med. Mater. 23 (2000) 212–213.
[20] H. Matsuda, T. Morikawa, K. Ninomiya, M. Yoshikawa, Bioorg. Med. Chem. 9
(2001) 909–916, https://doi.org/10.1016/s0968-0896(00)00306-0.
[21] Y. Liu, F. Liu, M.M. Qiao, L. Guo, M.H. Chen, C. Peng, L. Xiong, Org. Lett. 21 (2019)
1197–1201, https://doi.org/10.1021/acs.orglett.9b00149.
[31] W.J. Syu, C.C. Shen, M.J. Don, J.C. Ou, G.H. Lee, C.M. Sun, J. Nat. Prod. 61 (1998)
1531–1534, https://doi.org/10.1021/np980269k.
[32] Q.M. Zhou, M.H. Chen, X.H. Li, C. Peng, D.S. Lin, X.N. Li, Y. He, L. Xiong, J. Nat.
Prod. 81 (2018) 1919–1927, https://doi.org/10.1021/acs.jnatprod.7b00690.
[33] F. Liu, J.F. Chen, Y. Wang, L. Guo, Q.M. Zhou, C. Peng, L. Xiong, Biomed.
Pharmacother. 118 (2019) 109273, https://doi.org/10.1016/j.biopha.2019.
109273.
[22] J.J. Chen, T.H. Tsai, H.R. Liao, L.C. Chen, Y.H. Kuo, P.J. Sung, C.L. Chen, C.S. Wei,
Molecules 21 (2016) 1385, https://doi.org/10.3390/molecules21101385.
[23] D. Yu, Y. Duan, Y. Bao, C. Wei, L. An, J. Ethnopharmacol. 98 (2005) 89–94, https://
doi.org/10.1016/j.jep.2004.12.027.
[34] J. Zhu, X. Lu, X. Fan, R. Wu, H. Diao, R. Yu, H. Xu, J. Zi, Chin. Chem. Lett. 29 (2018)
1261–1263, https://doi.org/10.1016/j.cclet.2017.11.042.
[24] Z.Y. Jiang, X.M. Zhang, J. Zhou, F.X. Zhang, J.J. Chen, Y. Lü, L. Wu, Q.T. Zheng,
Chem. Pharm. Bull. 55 (2007) 905–907, https://doi.org/10.1248/cpb.55.905.
8