Diarylheptanoids from the rhizomes of curcuma kwangsiensis

Article abstract of DOI:10.1021/np100392m

Twelve new diarylheptanoids and six known compounds were isolated from rhizomes of Curcuma kwangsiensis. Structures of the new compounds were elucidated by spectroscopic and chemical methods as (3S)- and (3R)-1,7-bis(4-hydroxyphenyl)-(6E)-6-hepten-3-ol (1a and 1b), (3S)- and (3R)-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-(6E)-6-hepten-3-ol (2a and 2b), (3S)- and (3R)-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol (3a and 3b), (3R)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (4b), (3S)- and (3R)-3-acetoxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-(6E)-6-heptene (5a and 5b), (3S)- and (3R)-3-acetoxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl) heptanes (6a and 6b), and (E)-1,7-bis(4-hydroxyphenyl)-6-hepten-3-one (7). The absolute configurations were determined using the modified Mosher's method. Separation of the enantiomeric mixtures (1a and 1b, 2a and 2b, 3a and 3b, 4a and 4b, 5a and 5b, 6a and 6b) was achieved on a chiral column using acetonitrile-water mixtures as eluents. The S enantiomers exhibited negative specific optical rotations in MeOH, and the R enantiomers were positive. Inhibitory effects of the compounds on nitric oxide production in lipopolysaccaride-activated macrophages were evaluated.

Full text of DOI:10.1021/np100392m

J. Nat. Prod. 2010, 73, 1667–1671
1667
Diarylheptanoids from the Rhizomes of Curcuma kwangsiensis
Jun Li,† Feng Zhao,‡ Ming Zhi Li,† Li Xia Chen,† and Feng Qiu*,†
Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical UniVersity,
Shenyang 110016, People’s Republic of China, Key Laboratory of Structure-Based Drug Design & DiscoVery, Ministry of Education,
Shenyang Pharmaceutical UniVersity, Shenyang, 110016, People’s Republic of China, and School of Pharmacy, Yantai UniVersity,
No. 32 Road QingQuan, Laishan District, Yantai 264005, People’s Republic of China
ReceiVed June 11, 2010
Twelve new diarylheptanoids and six known compounds were isolated from rhizomes of Curcuma kwangsiensis. Structures
of the new compounds were elucidated by spectroscopic and chemical methods as (3S)- and (3R)-1,7-bis(4-
hydroxyphenyl)-(6E)-6-hepten-3-ol (1a and 1b), (3S)- and (3R)-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-(6E)-6-
hepten-3-ol (2a and 2b), (3S)- and (3R)-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol (3a and 3b), (3R)-
1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (4b), (3S)- and (3R)-3-acetoxy-1-(3,4-dihydroxyphenyl)-7-(4-
hydroxyphenyl)-(6E)-6-heptene (5a and 5b), (3S)- and (3R)-3-acetoxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxy-
phenyl)heptanes (6a and 6b), and (E)-1,7-bis(4-hydroxyphenyl)-6-hepten-3-one (7). The absolute configurations were
determined using the modified Mosher’s method. Separation of the enantiomeric mixtures (1a and 1b, 2a and 2b, 3a
and 3b, 4a and 4b, 5a and 5b, 6a and 6b) was achieved on a chiral column using acetonitrile-water mixtures as
eluents. The S enantiomers exhibited negative specific optical rotations in MeOH, and the R enantiomers were positive.
Inhibitory effects of the compounds on nitric oxide production in lipopolysaccaride-activated macrophages were evaluated.
Curcuma kwangsiensis S.G.Lee et C.F.Ling (Zingiberaceae) is
widely distributed in southwest regions of People’s Republic of
China including Guangxi, Sichuan, Guangdong, and Yunnan
Provinces. Its roots are one of the most important crude drugs,
frequently listed in prescriptions of traditional Chinese medicine
for the treatment of stomach trouble and “Oketsu”,1 various
syndromes caused by the obstruction of blood circulation, such as
arthralgia, psychataxia, and dysmenorhea.
Zingiberaceaeplantshavebeenreportedtoberichindiarylheptano-
ids,2,3 sesquiterpenoids,4,5 and monoterpenes,6 some of these
compounds possessing significant vasorelaxant,7 anticancer,8,9
antioxidant,2,10 anti-inflammatory,11 and heptoprotective activities12,13
and nitric oxide (NO) production inhibitory activities.14 During the
course of our studies on bioactive constituents from 70% EtOH
extracts of rhizomes of C. kwangsiensis, 12 new diarylheptanoids
(1a, 1b, 2a, 2b, 3a, 3b, 4b, 5a, 5b, 6a, 6b, and 7) were isolated
along with six known ones (4a and 8-12). Interestingly, by means
of the modified Mosher’s method, these diarylheptanoids with one
asymmetric carbon at the 3-position were found to exist as
enantiomeric mixtures (1a and 1b, 2a and 2b, 3a and 3b, 4a and
4b, 5a and 5b, 6a and 6b). The S enantiomer predominated in those
mixtures with different ratios of S and R enantiomers (2:1 for 1a
and 1b, 6:1 for 2a and 2b, 3:1 for 3a and 3b, 3:1 for 4a and 4b,
3:1 for 5a and 5b, and 3:2 for 6a and 6b). HPLC separation of the
six couples of diarylheptanoid isomers with one chiral center at
C-3 was achieved for the first time. This paper describes the
isolation and structural elucidation of these new diarylheptanoids
and reports the inhibitory effect of the compounds on NO production
in LPS-activated macrophages.
and H-6′), 6.66 (2H, d, J ) 8.7 Hz, H-3′′ and H-5′′), and δH 7.11
(2H, d, J ) 8.7 Hz, H-2′′ and H-6′′)] and trans-olefinic protons at
δH 5.98 (dt, J ) 16.0, 6.8 Hz, H-6) and δH 6.24 (d, J ) 16.0 Hz,
H-7). The 13C NMR spectrum revealed signals of two oxygenated
sp2 carbons (δ 156.5 and 157.8), and treatment of 1 with
trimethylsilyldiazomethane in MeOH gave a mixture of dimethyl
derivatives 1c and 1d. Thus, the presence of two 4-hydroxyphenyl
groups in 1 was evident. In addition, the 13C NMR spectrum showed
the presence of seven nonaromatic carbons in 1 [four methylenes
(δC 30.4, 32.3, 38.5, and 40.8), two olefinic (δC 128.3 and 131.2),
one hydroxymethine (δC 71.3)], suggesting a diarylheptanoid
structure. HMBC correlations from H-6 to C-1′′, H-7 to C-1′′, C-2′′,
and C-6′′, and H-2′′/H-6′′ to C-7 confirmed a double bond at C-6
Results and Discussion
Compound 1, a yellow oil, was obtained as an enantiomeric
mixture of 1a and 1b. The HRESIMS spectrum exhibited a unique
[M + NH4]+ ion at m/z 316.1897 corresponding to the molecular
formula C19H22O3 (calcd for C19H26NO3, 316.1907). The 1H NMR
spectrum of 1 revealed two para-substituted benzene rings [δH 6.64
(2H, d, J ) 8.5 Hz, H-3′ and H-5′), 6.97 (2H, d, J ) 8.5 Hz, H-2′
* Corresponding author. Tel: +86 24 23986463. Fax: +86 24 23993994.
E-mail: fengqiu2000@tom.com.
† Shenyang Pharmaceutical University.
‡ Yantai University.
10.1021/np100392m  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/29/2010

1668 Journal of Natural Products, 2010, Vol. 73, No. 10
Li et al.
Table 1. 1H and 13C NMR Data for Compounds 1-4 in CD3OD (δ in ppm, J in Hz)a
1
2
3
4
position
δH
δC
δH
δC
δH
δC
δH
δC
1a
1b
2
3
4
5a
5b
6
2.52 ddd (14.9, 9.6, 7.0)
2.64 ddd (14.9, 9.7, 5.7)
1.66 m
3.49 m
1.55 m
2.17 m
2.25 m
5.98 dt (16.0, 6.8)
6.24 d (16.0)
32.3 2.48 ddd, (14.8, 9.8, 5.5)
2.60 ddd, (14.8, 9.6, 6.8)
40.8 1.66 m
71.3 3.56 m
38.5 1.56 m
32.5 2.48 ddd, (14.8, 9.9, 6.2)
2.61 ddd, (14.8, 9.8, 6.2)
40.7 1.66 mb
71.4 3.52 m
32.5 2.52 ddd, (15.0, 9.1, 7.2)
2.63 ddd, (15.0, 8.8, 6.6)
40.7 1.72 m
71.9 3.60 m
38.4 1.63 m
32.6
40.7
71.4
38.2
30.4
38.5 1.47 mb
30.4 1.35 m
30.4 2.18 m
26.5 2.31 m
2.27 m
1.44 mb
128.3 6.00 dt (15.6, 7.3)
131.2 6.26 d (15.6)
134.6
130.5 6.62 d (1.9)
116.2
128.4 1.57 mb
131.1 2.52 t (7.5)
135.5
33.2 6.26 dt (16.3, 6.6)
36.2 6.39 d (16.3)
135.6
116.7 6.65 d (1.9)
146.2
128.0
131.5
135.4
116.7
146.2
144.3
116.4c
120.8
139.4
127.1
129.6
131.5
129.6
127.1
7
1′
2′
3′
4′
5′
6′
1′′
2′′
3′′
4′′
5′′
6′′
a
6.97 d (8.5)
6.64 d (8.5)
116.7 6.63 d (2.0)
146.2
144.3
116.4 6.67 d (8.1)
120.8 6.50 dd (8.1,2.0)
131.1
128.3 6.98 d (8.5)
116.4 6.69 d (8.5)
157.7
156.5
144.3
6.64 d (8.5)
6.97 d (8.5)
116.2 6.63 d (8.0)
130.5 6.49 dd (8.0,1.9)
131.1
128.3 7.13 d (8.6)
116.4 6.68 d (8.6)
157.8
116.4 6.68 d (8.6)
128.3 7.13 d (8.6)
1
116.4 6.66 d (7.6)
120.8 6.52 dd (7.6, 1.9)
135.0
7.11 d (8.7)
6.66 d (8.7)
130.4 7.32 mb
116.2 7.26 mb
156.4 7.16 m
116.2 7.26 mb
130.4 7.32 mb
6.66 d (8.7)
7.11 d (8.7)
116.4 6.69 d (8.5)
128.3 6.98 d (8.5)
The H NMR data of 1-3 were obtained at 600 MHz, while the H NMR data of 4 were obtained at 300 MHz. The 13C NMR data of 2 and 3 were
1
obtained at 150 MHz, while the 13C NMR data of 1 and 4 were obtained at 75 MHz. b The proton resonances are overlapped.
(δC 128.3) and C-7 (δC 131.2), attached to C-1′′. Placement of the
double bond was supported further by the splitting patterns of the
olefinic protons. The OH was placed at C-3, as determined by long-
range correlations from H-1 to C-3, C-1′, C-2′, and C-6′, H-2 to
C-1′, H-2′/H-6′ to C-1, and H-3 to C-1 in the HMBC spectrum.
The absolute configuration of the chiral center at C-3 and the
enantiomeric ratio of 1a and 1b in 1 were determined by the
modified Mosher’s method.15,16 Thus, the dimethyl derivatives 1c
and 1d were converted to the corresponding (S)-MTPA esters 1e
and 1f and (R)-MTPA esters 1g and 1h, respectively. Analysis of
the 1H NMR spectra of the two Mosher ester mixtures established
the absolute configuration at C-3 of the enantiomeric diarylheptanoid
mixture as S and R in a ratio of 2:1. Therefore, the diarylheptanoid
1 was concluded to be a 2:1 mixture of (3S)- and (3R)-1,7-bis(4-
hydroxyphenyl)-(6E)-6-hepten-3-ol (1a and 1b).
) 8.5 Hz) and 6.98 (2H, d, J ) 8.5 Hz)]. However, in the 1H NMR
spectrum of 3, the trans-olefinic proton signals were replaced by
aliphatic protons [δH 1.57 (2H, m, H-6) and 2.52 (2H, t, J ) 7.5
Hz, H-7)]. The structure indicated was supported by 13C NMR,
COSY, and HSQC data (Table 1). The OH group was shown to be
at C-3 of the aliphatic chain by HMBC correlations of H-1 with
C-3, C-1′, C-2′, and C-6′, H-2 with C-1′, H-2′/H-6′ with C-1, and
H-3 with C-1. Compound 3 was therefore formulated as 1-(3,4-
dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-3-ol. The modified
Mosher’s method was also applied to determine the absolute
configuration at C-3 of 3, as well as the ratio of S and R
enantiomers. Compound 3 was elucidated as a 3:1 mixture of 3a
and 3b.
Compound 4 had the molecular formula C19H22O3. The NMR
data demonstrated that 4 and 1-(3,4-dihydroxyphenyl)-7-phenyl-
(6E)-6-hepten-3-ol3 should be the same compound. Determination
of the absolute configuration at C-3 of this diarylheptanoid using
the modified Mosher’s method found it to be a 3:1 mixture of (3S)-
and (3R)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (4a
and 4b).
Compound 2 was obtained as a mixture of enantiomers (2a and
2b). The molecular formula of 2 was determined to be C19H22O4
on the basis of HRESIMS (m/z 332.1845 [M + NH4]+) and NMR
data. The 1H NMR data of 2 were very similar to those of 1, with
the differences being that a set of para-substituted aromatic protons
were absent and were replaced by a set of 1,3,4-trisubstituted
aromatic protons [δH 6.63 (1H, d, J ) 8.0 Hz), 6.62 (1H, d, J )
1.9 Hz), and 6.49 (1H, dd, J ) 8.0, 1.9 Hz)]. The conclusion was
supported by the 13C NMR data. The COSY, HMQC, and HMBC
NMR experiments of 2 indicated that it was 1-(3,4-dihydroxyphe-
nyl)-7-(4-hydroxyphenyl)-(6E)-6-hepten-3-ol,17 which had been
obtained previously from Curcuma comosa Roxb. (Zingiberaceae);
however, the absolute configuration of this compound had not been
determined. In order to determine the absolute configuration at C-3,
the modified Mosher’s method was applied to the trimethyl
derivatives 2c and 2d, as a mixture prepared from 2. On the basis
Compound 5 was also obtained as a mixture of enantiomers (5a
and 5b), as a yellowish oil. The HRESIMS analysis (m/z 374.1957
[M + NH4]+) in combination with the 1H and 13C NMR data (Table
1
2) indicated the molecular formula to be C21H24O5. The H and
13C NMR data of 5 in the aromatic regions were very similar to
those of 2, suggesting that 5 also possessed a 1,3,4-trisubstituted
benzene ring and a 1,4-disubstituted benzene ring. However,
distinctive differences in the 13C NMR spectrum were observed
between these molecules. The chemical shifts of C-2 and C-4 of 5
were upfield 3.3 and 3.2 ppm, respectively, while that of C-3 was
downfield 3.8 ppm; in addition, an acetyl carbon signal at δC 21.3
and 173.1 was present in the 13C NMR spectrum of 5. Notably, the
HMBC correlations of a methyl proton at δH 2.00 with the ester
carbonyl carbon (δC 173.1) and C-3 (δC 75.2) and of H-3 (δH 4.91)
with the ester carbonyl carbon (δC 173.1) were observed. These
facts suggested that 5 was the C-3-acetylated derivative of 2.
1
of the differences of H NMR data between (S)-MTPA esters 2e
and 2f and (R)-MTPA esters 2g and 2h, compound 2 was
determined to be a 6:1 mixture of (3S)- and (3R)-1-(3,4-dihydroxy-
phenyl)-7-(4-hydroxyphenyl)-(6E)-6-hepten-3-ol (2a and 2b).
Compound 3 was also obtained as a mixture of two enantiomers
(3a and 3b). The molecular formula, C19H24O4 ([M + NH4]+
334.2011, calcd for 334.2013), was determined by HRESIMS. The
1H NMR data of 3 showed aromatic signals similar to those of 2,
including 1,3,4-trisubstituted aromatic protons [δH 6.67 (1H, d, J
) 8.1 Hz), 6.63 (1H, d, J ) 2.0 Hz), and 6.50 (1H, dd, J ) 8.1,
2.0 Hz)] and 1,4-disubstituted aromatic protons [δH 6.69 (2H, d, J
In order to determine the absolute configuration of C-3 and the
ratio of S and R enantiomers, compound 5 was methylated to protect
the phenolic OH groups (5c and 5d) and then deacetylated with
K2CO3-MeOH to give the corresponding 3-OH derivatives (2c and
2d). Mosher acylation of the enantiomeric mixture (2c and 2d) with
both (R)- and (S)-MTPA chloride yielded the C-3 (S)- (2e and 2f)

Diarylheptanoids from Curcuma kwangsiensis
Journal of Natural Products, 2010, Vol. 73, No. 10 1669
Table 2. 1H and 13C NMR Data for Compounds 5, 6, and 7 in CD3OD (δ in ppm, J in Hz)a
5
6
7
position
δH
δC
δH
2.46 mb
1.78 m
4.84 m
1.58 mb
1.31 m
1.56 mb
2.50 mb
δC
δH
δC
1
2
3
4
5
6
7
2.47 m
1.82 m
4.91 m
1.71 m
2.16 m
32.2
37.4
75.2
35.3
30.2
32.3
37.3
75.4
35.1
25.8
2.79 br d (6.2)
2.77 br d (6.2)
30.2
45.7
212.9
43.7
2.59 t (7.4)
2.40 q (7.4)
5.99 dt (16.1, 7.1)
6.30 d (16.1)
28.3
5.98 dt (15.7, 7.3)
6.26 d (15.7)
127.6
131.5
134.6
116.6
146.3
144.5
116.5
120.7
130.9
128.3
116.4
157.8
116.4
128.3
21.3
32.7
35.9
126.9
131.7
133.4
130.4
116.3c
156.8
116.3c
130.4
130.8
128.3
116.4c
157.9
116.4c
128.3
1′
134.7
116.6
146.4
144.5
116.5
120.7
134.7
130.4
116.2
156.4
116.2
130.4
21.3
2′
6.60 d (2.0)
6.60 d (2.1)
6.66 d (7.9)
7.02 d (8.3)
6.70 d (8.3)
3′
4′
5′
6.65 d (8.1)
6.47 dd (8.1, 2.0)
6.70 d (8.3)
7.02 d (8.3)
6′
6.47 dd (7.9, 2.1)
1′′
2′′
3′′
4′′
5′′
6′′
3-OAc
7.15 d (8.7)
6.69 d (8.7)
6.97 d (8.2)
6.69 d (8.2)
7.17 d (8.3)
6.73 d (8.3)
6.69 d (8.7)
7.15 d (8.7)
2.00 s
6.69 d (8.2)
6.97 d (8.2)
2.00 s
6.73 d (8.3)
7.17 d (8.3)
173.1
173.1
a
1
The H NMR data of 5, 6, and 7 were obtained at 600 MHz. The 13C NMR data of 5 and 7 were obtained at 150 MHz, while the 13C NMR data of
6 were obtained at 75 MHz. b The proton resonances are overlapped. c Assignments may be exchanged.
Table 3. Inhibitory Effect of Compounds Isolated from
and (R)-R-methoxy-R-(trifluoromethyl) phenylacetyl (MTPA) esters
(2g and 2h). Consequently, compound 5 was elucidated as a 3:1
mixture of (3S)- and (3R)-3-acetoxy-1-(3,4-dihydroxyphenyl)-7-
(4-hydroxyphenyl)-(6E)-6-heptene (5a and 5b).
Curcuma kwangsiensis on NO Production Induced by LPS in
Macrophagesa
compound
IC50 ( SD (µM)
compound
IC50 ( SD (µM)
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
9.83 ( 0.79
16.11 ( 2.18
42.02 ( 1.88
20.56 ( 2.02
5.83 ( 0.52
9.66 ( 0.88
3.86 ( 0.65
7.34 ( 0.66
6.54 ( 0.58
15.63 ( 1.15
6a
6b
7
8
9
10
11
12
49.51 ( 3.32
28.49 ( 2.24
8.93 ( 0.81
6.98 ( 0.57
5.58 ( 0.47
10.62 ( 0.93
4.79 ( 0.42
2.69 ( 0.21
12.96 ( 1.16
40.64 ( 3.22
Compound 6 was also obtained as a mixture of two enantiomers
(6a and 6b). The molecular formula, C21H26O5, was determined by
1
HRESIMS. The H and 13C NMR data of 6 were very close to
those of 5, with the trans-olefinic resonances of a double bond [C-6
(δC 127.6) and C-7 (δC 131.5)] being replaced by saturated aliphatic
resonances. Compound 6 was identified as 3-acetoxy-1-(3,4-
dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane, a reduced derivative
of 5, on the basis of the 2D NMR (COSY, HSQC, and HMBC)
data. In order to determine the absolute configuration of C-3 by
the modified Mosher’s method, 6 was deacetylated with HCl-MeOH
to give the corresponding 3-deacetyl derivatives (3a and 3b) and
then converted to the corresponding S (3e and 3f) and R (3g and
3h) Mosher esters as performed for 3. Consequently, compound 6
was identified as a 3:2 mixture of (3S)- and (3R)-3-acetoxy-1-(3,4-
dihydroxyphenyl)-7-(4-hydroxyphenyl)heptanes (6a and 6b).
indomethacinb
hydrocortisoneb
a NO concentration of control group: 3.24
concentration of LPS-treated group: 33.46
control.
(
0.21 µM. NO
(
2.13 µM. b Positive
In addition to 12 new diarylheptanoids (1a, 1b, 2a, 2b, 3a, 3b,
4b, 5a, 5b, 6a, 6b, and 7), six known ones, (3S)-1-(3,4-dihydroxy-
phenyl)-7-phenyl-(6E)-6-hepten-3-ol (4a),3 1-(4-hydroxyphenyl)-
7-phenyl-(6E)-6-hepten-3-one (8),3 1,7-bis(4-hydroxyphenyl)-3-
heptanone(9),181-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-
3-heptanone (10),19 1,7-bis(4-hydroxyphenyl)hepta-4E-6E-dien-3-
one (11),8 and 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-
(4E)-4-hepten-3-one (12),20 were isolated and identified by
comparison of their spectroscopic data with those reported in the
literature.
Separation of the enantiomeric mixtures (1a and 1b, 2a and 2b,
3a and 3b, 4a and 4b, 5a and 5b, 6a and 6b) was achieved by
reversed-phase chiral HPLC using a Chiralpak AD-RH column (150
mm × 4.6 mm, 5 µm) with acetonitrile-water mixtures as eluents.
The specific optical rotations of the isolated S enantiomers (1a,
2a, 3a, 4a, 5a, and 6a) were negative, and the R enantiomers were
opposite.
All compounds isolated were examined for their inhibitory effects
on NO production induced by LPS in macrophages (Table 3). Cell
viability was determined by the MTT method to find whether
inhibition of NO production was due to cytotoxicity of the test
compounds. As shown in Table 3, indomethacin (IC50 12.96 ( 1.16)
and hydrocortisone (IC50 40.64 ( 3.22) were used as positive
controls.21-23 All of the isolated diarylheptanoid derivatives
significantly suppressed NO production. Compounds 1a, 3a, 3b,
4a, 4b, 5a, 7, 8, 9, 11, and 12 showed strong inhibition of NO
Compound 7 was obtained as a yellowish oil. The molecular
1
formula of 7 was determined as C19H20O3 by HRESIMS. The H
NMR spectrum of 7 showed the presence of two para-substituted
benzene rings [δH 6.70 (2H, d, J ) 8.3 Hz, H-3′ and H-5′), 7.02
(2H, d, J ) 8.3 Hz, H-2′ and H-6′), 6.73 (2H, d, J ) 8.3 Hz, H-3′′
and H-5′′), and 7.17 (2H, d, J ) 8.3 Hz, H-2′′ and H-6′′)] and two
trans-olefinic protons at δH 5.99 (dt, J ) 16.1, 7.1 Hz) and 6.30
(1H, d, J ) 16.1 Hz). The 13C NMR spectrum displayed 19 signals
consistent with four methylene (δC 28.3, 30.2, 43.7, and 45.7), two
olefinic (δC 126.9 and 131.7), one carbonyl (δC 212.9), and 12
aromatic ring carbons and suggesting a diarylheptanoid structure.
The 1H NMR spectrum exhibited some similarities between 7 and
1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one,3 except that the
H-4′′ signal of aromatic ring was absent in 7, being replaced by an
OH group. Furthermore, the 13C NMR spectrum showed a
significant downfield shift for C-4′′ (δC 157.9) compared to that of
the known compound. This deduction was supported by HSQC and
HMBC spectra. The attachment of 4-hydroxyphenyl moieties at
C-1 and C-7, respectively, was confirmed by the HMBC correlations
of H-1 with C-1′, C-2′, and C-6′, H-2 with C-1′, H-2′/H-6′ with
C-1, H-7 with C-1′′, C-2′′, and C-6′′, H-6 with C-1′′, and H-2′′/
H-6′′ with C-7. Therefore, compound 7 was identified as (E)-1,7-
bis(4-hydroxyphenyl)-6-hepten-3-one.

1670 Journal of Natural Products, 2010, Vol. 73, No. 10
Li et al.
UV-vis spectrometric detector and by a Chiralpak AD-RH column
[150 mm × 4.6 mm, 0.5 mL/min, detection at 220 nm, eluted with a
mixture of A (acetonitrile) and B (water)]. Compound 1 (5.0 mg) was
chromatographed, using acetonitrile-water (50:50), to provide com-
pounds 1a (3.1 mg, tR 14.2 min) and 1b (1.5 mg, tR 10.3 min);
compound 2 (4.0 mg), to provide compounds 2a (3.0 mg, tR 17.4 min)
and 2b (0.5 mg, tR 14.0 min), A:B ) 4:6; compound 3 (3.0 mg), to
provide compounds 3a (2.0 mg, tR 20.0 min) and 3b (0.7 mg, tR 17.1
min), A:B ) 3:7; compound 4 (2.5 mg), to provide compounds 4a
(1.5 mg, tR 25.7 min) and 4b (0.6 mg, tR 23.5 min), A:B ) 45:55;
compound 5 (5.5 mg), to provide compounds 5a (4.0 mg, tR 8.0 min)
and 5b (1.2 mg, tR 14.0 min), A:B ) 1:1; compound 6 (2.3 mg), to
provide compounds 6a (1.1 mg, tR 126.9 min) and 6b (0.8 mg, tR 117.2
min), A:B ) 3:7.
(3S)-/(3R)-1,7-Bis(4-hydroxyphenyl)-(6E)-6-hepten-3-ol (1a and
1b): yellowish oil; [R]25D -3.5 (c 0.1, MeOH, 1a), [R]25D +3.5 (c 0.1,
MeOH, 1b); UV (MeOH) λmax (log ε) 226 (4.05), 280 (3.47) nm; IR
νmax (KBr) 3375, 2933, 2856, 1610, 1515, 1448, 1365, 1238, 1113,
1023, 820 cm-1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD,
75 MHz), Table 1; HRESIMS m/z 316.1897 [M + NH4]+ (calcd for
C19H26NO3, 316.1907).
production induced by LPS in macrophages with IC50 values of
9.83, 5.83, 9.66, 3.86, 7.34, 6.54, 8.93, 6.98, 5.58, 4.79, and 2.69
µM, respectively. Compounds 1b, 5b, and 10 exhibited moderate
activities, which were close to that of indomethacin.
The diarylheptanoids with a carbonyl group at C-3 exhibited
conspicuous inhibitory activity (e.g., 7, 8, 9, 10, 11, 12). For each
pair of enantiomers, the inhibitory effect of the S enantiomer was
not significantly different from that of the corresponding R
enantiomer. Introduction of OH or OCH3 groups to the phenyl
groups and the presence of double bonds on the heptyl chain were
not important for the activity.
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a Perkin-Elmer 241 polarimeter. UV spectra were recorded on a
Shimadzu UV 2201 spectrophotometer. IR spectra were conducted on
a Bruker IFS 55 spectrometer. NMR experiments were performed on
Bruker ARX-300 and AV-600 spectrometers. The chemical shifts are
stated relative to TMS and expressed in δ values (ppm), with coupling
constants reported in Hz. HRESIMS were obtained on a Bruker APEX-
II mass spectrometer, and ESIMS were recorded on an Agilent 1100-
LC/MSD TrapSL mass spectrometer. Silica gel GF254 prepared for TLC
and silica gel (200-300 mesh) for column chromatography (CC) were
obtained from Qingdao Marine Chemical Factory (Qingdao, People’s
Republic of China). Sephadex LH-20 was a product of Pharmacia.
Octadecyl silica gel was purchased from Merck Chemical Company
Ltd. RP-HPLC separations were conducted using a Waters 600 series
pumping system equipped with a Waters 490 UV detector and
performed with a C18 column (250 mm × 20 mm, 10 µm; GL Science
Inc.). Separation of enantiomers was carried out on a Chiralpak AD-
RH column (150 mm × 4.6 mm, 5 µm; Daicel Chemical Industries,
Ltd.) using a Shimadzu LC-6A liquid chromatography instrument
equipped with a Shimadzu SPD-6AV UV-vis detector. All reagents
were HPLC or analytical grade and were purchased from Tianjin Damao
Chemical Company. Spots were detected on TLC plates under UV light
or by heating after spraying with anisaldehyde-H2SO4 reagent.
Plant Material. Rhizomes of Curcuma kwangsiensis were collected
from Guangxi Province, China, and identified by Professor Qishi Sun,
of the School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University. A voucher specimen (AG-20061015) has
been deposited in the herbarium of the Department of Natural Products
Chemistry, Shenyang Pharmaceutical University.
Extraction and Isolation. The rhizomes of C. kwangsiensis (10 kg)
were cut into approximately 2 cm pieces and extracted with 70% EtOH
(100 L) for 2 h. The resulting EtOH extract was concentrated in vacuo,
suspended in H2O, and partitioned successively with cyclohexane,
EtOAc, and n-BuOH. The EtOAc extract (65 g) was subjected to silica
gel CC with CHCl3-MeOH (100:1 to 0:100) to obtain nine fractions
(A-I), which were combined according to TLC analysis. Fraction C
(6 g) was chromatographed on a Sephadex LH-20 column with
CHCl3-MeOH (1:1) to give three subfractions (FC.1-FC.3). Fraction
C.3 (1.2 g) was purified by ODS open column chromatography
[MeOH-H2O (1:9 to 8:2)] and resolution of fraction C.34 by RP-HPLC
with MeOH-H2O (3:2) to afford compound 7 (23.2 mg, tR 78.4 min)
and compound 11 (20.5 mg, tR 86.3 min). Fraction F (12.5 g), eluted
with CHCl3-MeOH (1:9), was chromatographed over silica gel, eluting
with a gradient of increasing MeOH (1:9 to 8:2) in CHCl3, to give five
subfractions (FF.1-FF.5). Fraction F.3 (2.4 g) was purified by
preparative TLC [CHCl3-MeOH (1:9)] to obtain 1 (56.3 mg, Rf )
0.32) and 8 (15.1 mg, Rf ) 0.43), and subfraction F.4 (540 mg) was
separated by RP-HPLC with MeOH-H2O (1:1) to afford compound 4
(18.2 mg, tR 83.2 min), compound 5 (28.4 mg, tR 70.4 min), and
compound 6 (21.6 mg, tR 93.5 min). Fraction G (8.6 g) was subjected
to CC on Sephadex LH-20 [CHCl3-MeOH (1:1)] to give four
subfractions (FG.1-FG.4). Fraction G.2 (640 mg) was purified by RP-
HPLC [MeOH-H2O (11:9)] to obtain 12 (11.4 mg, tR 36.9 min), 10
(21.2 mg, tR 48.3 min), and 9 (19.2 mg, tR 60.1 min). Fraction G.4
(320 mg) was purified by RP-HPLC [MeOH-H2O (9:10)] to obtain
compounds 2 (23.2 mg, tR 50.2 min) and 3 (12.7 mg, tR 55.9 min).
Separation of Enantiomeric Mixtures. In order to afford sufficient
quantities of enantiopure compounds for biological assay, the isolated
enantiomeric mixtures were separated using a Shimadzu LC-6A liquid
chromatography instrument equipped with a Shimadzu SPD-6AV
(3S)-/(3R)-1-(3,4-Dihydroxyphenyl)-7-(4-hydroxyphenyl)-(6E)-6-
hepten-3-ol (2a and 2b): yellowish oil; [R]25 -3.7 (c 0.1, MeOH,
D
2a), [R]25 +3.7 (c 0.05, MeOH, 2b); UV (MeOH) λmax (log ε) 223
D
(3.85), 278 (3.16) nm; IR νmax (KBr) 3372, 2940, 1712, 1609, 1513,
1446, 1368, 1242, 1172, 1021, 967, 811 cm-1; 1H NMR (CD3OD, 600
MHz) and 13C NMR (CD3OD, 150 MHz), Table 1; HRESIMS m/z
332.1845 [M + NH4]+ (calcd for C19H26NO4, 332.1856).
(3S)-/(3R)-1-(3,4-Dihydroxyphenyl)-7-(4-hydroxyphenyl)heptan-
3-ol (3a and 3b): yellowish oil; [R]25D -2.9 (c 0.1, MeOH, 3a), [R]25
D
+2.9 (c 0.05, MeOH, 3b); UV (MeOH) λmax (log ε) 219 (3.94), 282
(3.32) nm; IR νmax (KBr) 3375, 2933, 2856, 1610, 1515, 1448, 1365,
1
1281, 1238, 1113, 957, 820 cm-1; H NMR (CD3OD, 600 MHz) and
13C NMR (CD3OD, 150 MHz), Table 1; HRESIMS m/z 334.2011 [M
+ NH4]+ (calcd for C19H28NO4, 334.2013).
(3R)-1-(3,4-Dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (4b):
yellowish oil; [R]25 +5.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε)
D
201 (4.15), 283 (3.64) nm; IR νmax (KBr) 3382, 2940, 2860, 1687, 1613,
1514, 1451, 1379, 1144, 1026, 830 cm-1; 1H NMR (CD3OD, 300 MHz)
and 13C NMR (CD3OD, 75 MHz), Table 1; HRESIMS m/z 316.1899
[M + NH4]+ (calcd for C19H26NO3, 316.1907).
(3S)-/(3R)-3-Acetoxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphe-
nyl)-(6E)-6-heptene (5a and 5b): yellowish oil; [R]25 -5.7 (c 0.1,
D
MeOH, 5a), [R]25D +5.7 (c 0.1, MeOH, 5b); UV (MeOH) λmax (log ε)
208 (4.26), 262 (3.04) nm; IR νmax (KBr) 3397, 2938, 1709, 1654, 1611,
1
1516, 1445, 1379, 1263, 1200, 1028, 819 cm-1; H NMR (CD3OD,
600 MHz) and 13C NMR (CD3OD, 150 MHz), Table 2; HRESIMS
m/z 374.1957 [M + NH4]+ (calcd for C21H28NO5, 374.1962).
(3S)-/(3R)-3-Acetoxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphe-
nyl)heptanes (6a and 6b): yellowish oil; [R]25 -5.3 (c 0.1, MeOH,
D
6a), [R]25 +5.3 (c 0.05, MeOH, 6b); UV (MeOH) λmax (log ε) 209
D
(4.17), 262 (3.12) nm; IR νmax (KBr) 3397, 2939, 2846, 1733, 1613,
1517, 1459, 1374, 1242, 1116, 1026, 959, 820 cm-1; 1H NMR (CD3OD,
600 MHz) and 13C NMR (CD3OD, 75 MHz), Table 2; HRESIMS m/z
376.2127 [M + NH4]+ (calcd for C21H30NO5, 376.2118).
(E)-1,7-Bis(4-hydroxyphenyl)-6-hepten-3-one (7): yellowish oil;
UV (MeOH) λmax (log ε) 201 (4.86), 262 (3.05) nm; IR νmax (KBr)
3362, 2939, 1701, 1611, 1514, 1447, 1372, 1234, 1031, 830 cm-1; 1H
NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), Table
2; HRESIMS m/z 314.1740 [M + NH4]+ (calcd for C19H24NO3,
314.1751).
Methylation of 1, 2, 3, and 5. An enantiomeric mixture of 1 (1a
and 1b) (2 mg) and trimethylsilyldiazomethane (2.0 M solution in
n-hexane) (0.5 mL) in MeOH (0.5 mL) was stirred at room temperature
overnight. After the excess trimethylsilyldiazomethane was decomposed
with AcOH, the reaction mixture was evaporated to dryness. The
resulting residue was purified by preparative TLC (cyclohexane-acetone,
4:1, Rf ) 0.46), yielding a mixture of 1c and 1d (1.8 mg). Similarly,
methylation of 2 (2a and 2b) (2.2 mg), 3 (3a and 3b) (2.2 mg), and 5
(5a and 5b) (3.6 mg) yielded a mixture of 2c and 2d (2.0 mg), 3c and
3d (1.8 mg), and 5c and 5d (3.4 mg), respectively.
Compounds 1c and 1d: yellowish oil; 1H NMR (300 MHz, CDCl3)
δ 7.26 (2H, d, J ) 8.5 Hz, H-2′′, 6′′), 7.12 (2H, d, J ) 8.4 Hz, H-2′,
6′), 6.84 (2H, d, J ) 8.5 Hz, H-3′′, 5′′), 6.82 (2H, d, J ) 8.4 Hz, H-3′,

Diarylheptanoids from Curcuma kwangsiensis
Journal of Natural Products, 2010, Vol. 73, No. 10 1671
5′), 6.35 (1H, d, J ) 15.8 Hz, H-7), 6.07 (1H, dt, J ) 15.8, 7.3 Hz,
H-6), 3.80 (3H, s, OMe), 3.78 (3H, s, OMe), 3.70 (1H, m, H-3), 2.67
(2H, m, H-1), 2.31 (2H, m, H-5), 1.77 (2H, m, H-2), 1.65 (2H, m,
H-4).
Griess reagent (0.1% N-[1-naphthyl]ethylenediamine and 1% sulfanil-
amide in 5% H3PO4). Cytotoxicity was determined by the MTT
colorimetric assay, after 24 h incubation with test compounds. The
-
concentration of NO2 was calculated by a working line from 0, 1, 2,
Compounds 2c and 2d: yellowish oil; 1H NMR (300 MHz, CDCl3)
δ 7.26 (2H, d, J ) 8.8 Hz, H-2′′, 6′′), 6.84 (2H, d, J ) 8.8 Hz, H-3′′,
5′′), 6.78 (1H, d, J ) 8.7 Hz, H-5′), 6.74 (1H, dd, J ) 8.7, 2.0 Hz,
H-6′), 6.73 (1H, d, J ) 2.0 Hz, H-2′), 6.36 (1H, d, J ) 16.2 Hz, H-7),
6.07 (1H, dt, J ) 16.2, 7.0 Hz, H-6), 3.86 (3H, s, OMe), 3.85 (3H, s,
OMe), 3.80 (3H, s, OMe), 3.71 (1H, m, H-3), 2.69 (2H, m, H-1), 2.32
(2H, m, H-5), 1.79 (2H, m, H-2), 1.65 (2H, m, H-4).
5, 10, 20, 50, and 100 µM sodium nitrite solutions, and the inhibitory
-
rate on NO production induced by LPS was calculated by the NO2
levels as follows:
-
-
[NO2
]
- [NO2 ]
LPS LPS+sample
Inhibitory rate (%) ) 100 ×
-
-
[NO2
]
- [NO2 ]
LPS untreated
Compounds 3c and 3d: yellowish oil; 1H NMR (300 MHz, CDCl3)
δ 7.08 (2H, d, J ) 8.4 Hz, H-2′′, 6′′), 6.82 (2H, d, J ) 8.4 Hz, H-3′′,
5′′), 6.78 (1H, d, J ) 7.7 Hz, H-5′), 6.73 (1H, dd, J ) 7.7, 1.8 Hz,
H-6′), 6.72 (1H, d, J ) 1.8 Hz, H-2′), 3.87 (3H, s, OMe), 3.86 (3H, s,
OMe), 3.78 (3H, s, OMe), 3.62 (1H, m, H-3), 2.69 (2H, m, H-1), 2.32
(2H, m, H-5), 1.79 (2H, m, H-2), 1.65 (2H, m, H-4).
Experiments were performed in triplicate, and data are expressed as
the mean ( SD of three independent experiments.
Acknowledgment. This research was supported by the Program for
Liaoning Excellent Talents in University (LR201037).
Compounds 5c and 5d: yellowish oil; 1H NMR (300 MHz, CDCl3)
δ 7.26 (2H, d, J ) 8.6 Hz, H-2′′, 6′′), 6.83 (2H, d, J ) 8.6 Hz, H-3′′,
5′′), 6.78 (1H, d, J ) 8.9 Hz, H-5′), 6.71 (1H, dd, J ) 8.9, 1.8 Hz,
H-6′), 6.70 (1H, d, J ) 2.0 Hz, H-2′), 6.32 (1H, d, J ) 16.0 Hz, H-7),
6.03 (1H, dt, J ) 16.0, 6.7 Hz, H-6), 5.00 (1H, m, H-3), 3.86 (3H, s,
OMe), 3.85 (3H, s, OMe), 3.80 (3H, s, OMe), 2.57 (2H, m, H-1), 2.21
(2H, m, H-5), 2.05 (3H, s, 3-OAc), 1.87 (2H, m, H-2), 1.74 (2H, m,
H-4).
Hydrolysis of Enantiomeric Mixtures of 5c, 5d, and 6 (6a and
6b). To a solution of a mixture of 5c and 5d (3.0 mg) in MeOH (1.5
mL) was added anhydrous K2CO3 (10 mg), and the mixture was stirred
at 30 °C until deacetylation was complete (9 h). Then the reaction
mixture was evaporated to dryness. The residue was purified by
preparative TLC (cyclohexane-acetone, 4:1, Rf ) 0.42) and yielded a
mixture of 2c and 2d (2.4 mg).
Supporting Information Available: HRESIMS and 1D and 2D
NMR spectra of 1-7 and their derivatives. This material is available
free of charge via the Internet at http://pubs.acs.org.
References and Notes
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NO Production Bioassay. Mouse monocyte-macrophage RAW
264.7 cells (ATCC TIB-71) were purchased from the Chinese Academy
of Science. RPMI 1640 medium, penicillin, streptomycin, and fetal
bovine serum were purchased from Invitrogen (New York). Li-
popolysaccharide (LPS), dimethylsufoxide (DMSO), 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), indomethacin, and
hydrocortisone were obtained from Sigma Co. (St. Louis, MO). RAW
264.7 cells were suspended in RPMI 1640 medium supplemented with
penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% heat-
inactivated fetal bovine serum. The cells were harvested with trypsin
and diluted to a suspension in fresh medium. The cells were seeded in
96-well plates with 1 × 105 cells/well and allowed to adhere for 2 h at
37 °C in 5% CO2 in air. Then, the cells were treated with 1 µg/mL of
LPS for 24 h with or without various concentrations of test compounds.
DMSO was used as a solvent for the test compounds, which were
applied at a final concentration of 0.2% (v/v) in cell culture supernatants.
NO production was determined by measuring the accumulation of nitrite
in the culture supernatant using Griess reagent.24 Briefly, 100 µL of
the supernatant from incubates was mixed with an equal volume of
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NP100392M