Article pubs.acs.org/jnp
Sesquiterpenes from the Rhizomes of Curcuma heyneana Azis Saifudin,† Ken Tanaka,‡ Shigetoshi Kadota,† and Yasuhiro Tezuka*,† †
Division of Natural Product Chemistry, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan ‡ Division of Pharmacognosy, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan S Supporting Information *
ABSTRACT: Four new germacranes [heyneanones A−D (1− 4)], three new guaianes [4,10-epizedoarondiol (5), 15hydroxyprocurcumenol (6), 12-hydroxycurcumenol (7)], and two new spirolactones [curcumanolides C (8) and D (9)] were isolated from the rhizomes of Curcuma heyneana together with 13 known sesquiterpenes and two known labdane-type diterpenes. Among the isolated compounds, heyneanone A (1), heyneanone C (3), 4,10-epizedoarondiol (5), procurcumenol (16), aerugidiol (17), zerumin A (23), and (E)-15,16bisnorlabda-8(17),11-dien-13-one (24) inhibited protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 42.5, 35.2, 35.1, 45.6, 35.7, 10.4, and 14.7 μM, respectively.
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RESULTS AND DISCUSSION The powdered rhizomes of C. heyneana were extracted using MeOH, and the MeOH extract was separated by a combination of column chromatography and preparative TLC techniques to give nine new sesquiterpenes (1−9, Figure 1) together with 15 known compounds: gajutsulactone A (10),10 caulolactone B (11),11 zedoarondiol (12),12 isozedoarondiol (13),12 guaidiol A (14),13,14 epiguaidiol A (15),14 procurcumenol (16),15 aerugidiol (17),15 isocurcumenol (18),9 oxycurcumenol epoxide (19),9 a mixture of curcumanolides A and B (20, 21),16 cyperusol C (22),17 zerumin A (23),18 and (E)-15,16bisnorlabda-8(17),11-dien-13-one (24).19 The molecular formula, C15H24O4, of heyneanone A (1) was deduced from HRESIMS and 13C NMR data. Its IR spectrum had absorption bands at 3425 and 1715 cm−1, suggesting the presence of hydroxy and carbonyl functionalities. The 1H NMR spectrum of 1 (Table 1) had signals attributable to an olefinic proton (δH 5.72), two oxymethines (δH 4.47, 3.61), three vinylic methyls (δH 1.83, 6H; δH 1.74), a tertiary methyl (δH 1.26), and multiplets attributable to six protons. The 13C NMR spectrum (Table 1) showed 15 signals, including those of a carbonyl carbon (δC 204.1), two olefinic carbons (δC 142.2, 135.9, 134.9, 126.4), an oxygenated quaternary carbon (δC 87.1), two oxygenated methines (δC 81.1, 76.2), and three methylenes (δC 34.1, 36.3, 29.9). On the basis of analysis of COSY and HMQC spectra, the oxygenated methine (δH 4.47, δC 81.1) and two methylenes (δH 2.23, 1.83, δC 29.9; δH 1.95, 1.73, δC 36.3) were connected to the partial structure HO− C(1)H−C(2)H2−C(3)H2−, whereas the oxygenated methine (δH 3.61, δC 76.2) and methylene (δH 2.82, 2.57, δC 34.1) were connected to the partial structure HO−C(6)H−C(7)H2−
rotein tyrosine phosphatase 1B (PTP1B) is an enzyme found in important insulin-targeted organs such as the liver and muscle, and the inhibition or deletion of this enzyme improves insulin signaling and glucose circulation.1 An excess of this negative regulator is also linked to endoplasmic reticulum (ER) stress during obesity and aging.2 In addition, neuronal PTP1B is a significant negative modulator of leptin signaling,3 and deficiency of this enzyme in the brain affects production of leptin by adipocytes, glucose homeostasis, and insulin sensitivity.4 Therefore, modification and inhibition of this phosphatase will establish peripheral glucose homeostasis, increase energy expenditure, and reduce weight.4,5 Thus, the inhibition of this enzyme is a well-validated target for the treatment of type II diabetes and obesity.6 During our search for PTP1B inhibitors from Indonesian medicinal plants, we found that a methanol extract from the rhizomes of Curcuma heyneana had a potent PTP1B inhibitory activity with an IC50 value of 7.14 μg/mL.7 In Indonesia, a slurry prepared from fresh rhizomes of C. heyneana is used to treat skin diseases; its crude extract is used to treat fatigue, helminth infections, obesity, and rhumatism; and its pulverized form is used as a component in beauty treatments.8 Regarding the chemical constituents, sesquiterpenes of germacrane, guaiane, and humulane types as well as labdane-type diterpenes have been reported.9 However, information on these chemical constituents remains limited, and PTP1B inhibitory constituents have not been reported. Thus, we examined the constituents of this plant and isolated nine new sesquiterpenes (1−9), referred to as heyneanones A−D (1−4), 4,10epizedoarondiol (5), 15-hydroxyprocurcumenol (6), 12hydroxycurcumenol (7), and curcumanolides C (8) and D (9), in addition to 15 known compounds (10−24). Herein we describe their structures together with their PTP1B inhibitory activity. © 2013 American Chemical Society and American Society of Pharmacognosy
Received: October 5, 2012 Published: February 6, 2013 223
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Figure 1. Structures of compounds isolated from Curcuma heyneana.
Table 1. 1H and 13C NMR Data for Germacranes 1−4 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a
δH (J in Hz) 4.47, 2.23, 1.83, 1.95, 1.73,
t (6.8) m m dt (12.3, 6.2) m
3.61, dd (9.4, 1.6) 2.82, dd (13.8, 9.4) 2.57, br d (13.8)
5.72, br s
1.83, 1.83, 1.26, 1.74,
s s s s
2 δC 81.1, CH 29.9, CH2 36.3, CH2 87.1, C 76.2, CH 34.1, CH2 134.9, C 204.1, C 126.4, CH 142.2, C 135.9, C 22.8,a CH3 21.9a, CH3 21.6, CH3 19.7, CH3
3
δH (J in Hz) 4.32, 1.88, 1.69, 1.78, 1.75,
δC
t (6.2) m m m m
36.9, CH2 71.5, C 76.6, CH 31.1, CH2
3.65, dd (13.0, 1.8) 2.75, dd (13.0, 1.8) 2.31, t (13.0)
5.68, br s
1.64,a s 1.71,a s 1.26, s 1.69, s
δH (J in Hz)
75.4, CH 22.4, CH2
127.9, C 208.9, C 126.8, CH 141.1, C 136.1, C 21.7,a CH3 20.5,a CH3 23.0, CH3 17.0, CH3
4.46, 2.17, 1.93, 1.63, 1.58,
br m dt td dt
δC
d (7.1) (14.0, 3.6) (14.0, 3.6) (14.0, 3.6)
3.55, dd (13.2, 3.5) 2.88, t (13.2) 2.60, dd (13.2, 3.5)
5.71, br s
1.90,a s 2.17,a s 1.19, s 1.75, s
4 δH (J in Hz)
δC
73.2, CH 21.6, CH2
2.69, t (7.8)
206.3, C 43.2, CH2
27.9, CH2
2.84, t (7.8)
30.8, CH2
69.3, C 82.7, CH 31.4, CH2
7.09, br s
130.8, 198.7, 127.6, 144.1, 146.7, 23.7, 23.7, 26.0, 21.7,
C C CH C C CH3 CH3 CH3 CH3
7.27, d (9.7) 6.91, d (9.7)
1.55, 1.55, 2.28, 2.19,
s s s s
148.3, C 141.6, CH 188.0, C 154.5, 131.9, 131.9, 146.4, 74.1, 29.1, 29.1, 24.7, 30.1,
C CH CH C C CH3 CH3 CH3 CH3
May be interchanged in each column.
(Figure 2). In the HMBC spectrum, the protons of the vinylic methyl at δH 1.74 (H3-15) and olefinic H-9 (δH 5.72) correlated
with the quaternary olefinic C-10 (δC 142.2) and the oxygenated C-1 methine (δC 81.1). Therefore, the vinylic 224
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Figure 2. Selected COSY (bold lines) and HMBC (arrows) correlations for compounds 1 and 4−8.
Figure 3. NOE’s observed in difference NOE spectra (arrow with edge at one end) and ROESY correlations (arrows with edge at both ends) for compounds 1−5, 8, and 9.
confirmed by 2D NMR spectra, showing the same correlations as those for 1. The relative configurations of 2 and 3 were also established by analyzing NOEs observed in difference NOE spectra and ROESY spectra (Figure 3). Thus, heyneanones B (2) and C (3) were determined to be a C-4 epimer, 1β,4α,5βtrihydroxy-7(11),9-germacradien-8-one, and a C-1 epimer, 1α,4β,5β-trihydroxy-7(11),9-germacradien-8-one, respectively. Heyneanone D (4) was isolated as a colorless oil with the molecular formula C15H20O3. Its 1H NMR spectrum exhibited signals attributable to two vinyl methyls (δH 2.28, 2.19), two aliphatic methyls (δH 1.55, 6H), three olefinic protons (δH 7.27, 7.09, 6.91), and two methylenes (δH 2.84, 2.69). The 13C NMR spectrum of 4 showed 15 signals, including those of two carbonyls (δC 206.3, 188.0), two methylenes (δC 43.2, 30.8), four methyls (δC 30.1; δC 29.1, 2C; δC 24.7), an oxygenated quaternary carbon (δC 74.1), and six olefinic carbons (δC 154.5; δC 148.3; δC 146.4; δC 141.6; δC 131.9, 2C). These signals were analyzed by COSY, HMQC, HMBC, and difference NOE spectra (Figures 2, 3), resulting in the structure (4Z,7Z,9Z)-11hydroxy-4,7,9-germacratriene-1,6-dione. 4,10-Epizedoarondiol (5) was obtained as a brown oil with the molecular formula C15H24O3, and its IR spectrum showed absorptions attributable to hydroxy (3411 cm−1) and carbonyl (1669 cm−1) groups. The 1H and 13C NMR spectra of 5 (Table
methyl (C-15), olefinic methine (C-9), and oxygenated methine (C-1) were connected to the olefinic quaternary C10. The methyl protons at δH 1.26 (H3-14) correlated with the carbons of a methylene at δC 36.3 (C-3), an oxygenated methine at δC 76.2 (C-5), and an oxygenated quaternary carbon at δC 87.1 (C-4), indicating that the methyl (C-14), methylene (C-3), and oxygenated methine (C-5) should be connected to the oxygenated quaternary carbon (C-4). In contrast, the methylene protons at δH 2.82 and 2.57 (H2-6) correlated with the olefinic quaternary carbon at δC 134.9 (C-7) and the carbonyl carbon at δC 204.1 (C-8), and the vinylic methyl protons at δH 1.83 (H3-12, H3-13) correlated with the olefinic quaternary carbons at δC 134.9 (C-7) and 135.9 (C-11). On the basis of these data, 1 was concluded to be a germacrane-type sesquiterpene. The relative configuration of 1 was established by analysis of NOEs detected in difference NOE experiments using the Dreiding stereomodel (Figure 3). Thus, heyneanone A was concluded to be 1β,4β,5β-trihydroxy-7(11),9-germacradien-8-one (1). Heyneanones B (2) and C (3) were shown to have the same molecular formula, C15H24O4, as 1 on the basis of analyses of the MS and NMR data. The 1H and 13C NMR spectra of 2 and 3 were similar and resembled those of 1 (Table 1). Thus, 2 and 3 were considered to be stereoisomers of 1, and this was 225
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Table 2. 1H and 13C NMR Data for Guaianes 5−7 5a δH
position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a,b,c
1.88, 2.11, 1.88, 2.24, 1.72,
m m td (9.4, 7.2) m m
2.45, t (13.1) 3.19, d (13.1) 2.18, m
3.05, d (12.3) 2.87, d (12.3)
1.73, s 2.04, s 1.40,d s 1.41,d s
6b δC 55.0, CH 40.3, CH2 22.1, CH2 79.4, C 50.6, CH 28.9, CH2 136.8, C 203.1, C 58.7, CH2 71.2, 138.4, 22.0, 22.9, 30.4, 22.9,
C C CH3 CH3 CH3 CH3
2.45, 1.63, 1.58, 1.87, 1.79,
δH
δC
q (9.8) m m m m
47.7, CH 26.0, CH2
1.87, m 2.64, br d (13.9) 2.19, br t (13.9)
40.0, CH2 79.9, C 53.8, CH 28.6, CH2 136.1, C 199.4, C 126.3, CH
6.14, br s
1.75, 1.79, 1.25, 4.26, 4.21,
7c
s s s d (15.6) d (15.6)
155.4, 137.4, 22.4, 21.3, 24.3, 64.6,
C C CH3 CH3 CH3 CH2
1.94, 1.83, 1.51, 1.88, 1.60, 1.83,
δH
δC
m m m m m m
52.3, CH 28.7, CH2
2.73, d (15.6) 2.25, d (15.6)
32.3, CH2 41.4, CH 86.8, C 37.1, CH2
5.71, br s
125.8, C 102.6, C 127.1, CH
3.95, 1.83, 1.00, 1.63,
140.2, 140.9, 65.8, 14.7, 12.2, 21.0,
s (2H) br s d (6.6) s
C C CH2 CH3 CH3 CH3
Measured in pyridine-d5, CDCl3, and methanol-d4, respectively. dMay be interchanged.
Compound 6 had 1H and 13C NMR spectra similar to those of procurcumenol (16),15 isolated from the same extract. However, 1H and 13C NMR spectra indicated the disappearance of one of the two methyl groups in 16 and the presence of signals attributable to a hydoxymethylene group (Table 2). The hydoxymethylene group was determined to be C-15 on the basis of HMBC correlations of the hydroxymethylene protons (δH 4.26, 4.21) with the olefinic carbons at δC 126.3 (C-9) and 155.4 (C-10) and of the olefinic proton (δH 6.14) with the hydroxymethylene carbon (δC 64.6) (Figure 2). Thus, compound 6 was concluded to be 15-hydroxyprocurcumenol. The 1H and 13C NMR spectra of compound 7 exhibited signals attributable to two tertiary methyls (δH 1.63, δC 21.0; δH 1.83, δC 14.7), a secondary methyl (δH 1.00, δC 12.2), three aliphatic methylenes (δH 1.83, 1.51, δC 28.7; δH 1.88, 1.60, δC 32.3), an oxygenated methylene (δH 3.95, 2H, δC 65.8), two aliphatic methines (δH 1.94, δC 52.3; δH 1.83, δC 41.4), an olefinic methine (δH 5.71, δC 127.1), two oxygenated quaternary carbons (δC 86.8, 102.6), and three olefinic quaternary carbons (δC 140.9, 140.2, 125.8) (Table 2). These signals were similar to those of curcumenol,9 which was previously isolated from the same plant; however, they showed the presence of signals attributable to an oxymethylene (δH 3.95, 2H, δC 65.8) and the lack of signals for one of the two methyl groups. In addition, the 13C NMR data analyzed by the HMBC spectrum (Figure 2) revealed a high-field shift (1.0 ppm) of C-6 caused by a γ-gauche effect.20 Thus, 7 was considered to be 12-hydroxycurcumenol, which was confirmed by the ROESY correlation between the hydroxymethylene protons (δH 3.95) and H-6 at δH 2.25. Curcumanolides C (8) and D (9) had the same molecular formula, C15H22O3, in the HRESIMS and similar 1H and 13C NMR spectra, suggesting that they were stereoisomers. Their IR spectra exhibited absorbtions of hydroxy (8, 3380 cm−1; 9, 3389 cm−1) and lactone carbonyl groups (8, 1736 cm−1; 9, 1741 cm−1). The 1H NMR spectra of 8 and 9 exhibited signals attributable to four methyls, an exo-olefinic group, two
2) exhibited signals for two aliphatic and two vinylic methyls, four methylenes, and two methines, together with those of a carbonyl carbon (δC 203.1), a tetrasubstituted olefinic group (δC 138.4, 136.8), and two oxygenated quaternary carbons (δC 79.4, 71.2). On the basis of analysis of COSY and HMQC spectra, three methylenes (δH 2.24, 1.72, δC 22.1; δH 2.11, 1.88, δC 40.3; δH 3.19, 2.18, δC 28.9) and two methines (δH 1.88, δC 55.0; δH 2.45, δC 50.6) were connected to the partial structure −C(3)H2−C(2)H2−C(1)H−C(5)H−C(6)H2− (Figure 2). In the HMBC spectrum of 5, the methyl protons at δH 1.40 (H314) correlated with the oxygenated quaternary carbon at δC 79.4 (C-4) and the methylene carbon at δC 22.1 (C-3), whereas methine H-5 correlated with the oxygenated quaternary carbon at δC 79.4 (C-4) and the olefinic carbon at δC 136.8 (C-7). In addition, the methylene proton at δH 3.19 (H-6) exhibited HMBC correlations with the carbons at δC 136.8 (C-7), 138.4 (C-11), and 203.1 (C-8), whereas the methylene proton at δH 2.87 (H-9) exhibited correlations with the carbons at δC 136.8 (C-7), 203.1 (C-8), 71.2 (C-10), and 55.0 (C-1). Moreover, the methyl protons at δH 1.41 (H3 -15) exhibited HMBC correlations with the carbons at δC 58.7 (C-9), 71.2 (C-10), and 55.0 (C-1), whereas both the methyl protons at δH 1.73 (H3-12) and 2.04 (H3-13) exhibited correlations with the carbons at δC 136.8 (C-7) and 138.4 (C-11). On the basis of these and other HMBC correlations (Figure 2), 5 was concluded to be a guaiane-type sesquiterpene. The relative configuration of 5 was established by analysis of NOEs detected in difference NOE experiments using the Dreiding stereomodel (Figure 3). The large coupling constants (J = 13.1 Hz) of H-5 with H-1 and H-6 at δH 2.18 indicated their trans relationship. Difference NOE experiments irradiating H3-14 and H-5 enhanced the intensity of H-6 at δH 3.19, whereas irradiation of the latter proton enhanced that of H-5. Therefore, H3-14, H5, and H-6 at δH 3.19 should have a cis relationship. In contrast, irradiation of H3-15 enhanced the intensities of H-1. Thus, 4,10-epizedoarondiol (5) was determined to be (1α,4α,5β,10β)-4,5-dihydroxy-7(11)-guaien-8-one. 226
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methines, two methylenes, and an olefinic proton (Table 3). The 13C NMR spectra of 8 and 9 exhibited 15 carbon signals
Table 4. PTP1B and TCPTP Inhibitory Activities of Isolated Compoundsa PTP1B
Table 3. 1H and 13C NMR Data for Curcumanolides 8 and 9 compound 8
9
position
δH
δC
1
2.85, t (9.8)
54.6, CH
2
2.05, m
28.7, CH2
3
1.95, m 2.13, m
27.2, CH2
1.64, m 4 5 6 7 8 9 10 11 12 13 14 15 a
2.25, dq (9.8, 7.3) 6.84, s
4.91, br s 4.71, br s 1.49, 1.54, 0.82, 1.67,
s s d (6.8) s
41.9, CH 96.4, 147.2, 141.1, 172.1, 140.8, 115.1,
C CH C C C CH2
68.9, C 30.8,a CH3 28.8,a CH3 12.4, CH3 22.5, CH3
δH 2.75, dd (11.5, 7.6) 2.05, dtd (8.6, 7.6, 2.8) 1.92, m 2.22, dtd (14.3, 7.6, 2.8) 1.35, dtd (14.3, 7.6, 4.9) 2.37, quintet (7.6) 6.96, s
4.92, br s 4.65, br s 1.49, 1.54, 1.03, 1.69,
s s d (7.3) s
1 3 5 16 17 23 24 RK-682 ulsolic acid
δC 52.9, CH 28.5, CH2
28.4, CH2
TCPTP
inhibition (%)b at 25 μg/mL
IC50 (μM)
IC50 (μM)
± ± ± ± ± ± ± ± ±
42.5 35.2 35.1 45.6 35.7 10.4 14.7 5.62 2.75
62.0 36.7 15.7 26.8 45.9 7.4 9.3 5.10 2.40
77.5 86.2 86.0 69.3 87.5 96.7 88.8 99.1 96.7
2.1 4.7 2.1 4.6 3.0 0.8 0.5 0.5 0.7
a
Compounds 2, 4, 6−15, and 18−22 exhibited weak (
