This article was downloaded by: [UQ Library] On: 16 July 2015, At: 00:48 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Four new sesquiterpenes from the rhizomes of Curcuma phaeocaulis and their iNOS inhibitory activities a
a
a
a
b
Jiang-Hao Ma , Ying Wang , Yue Liu , Su-Yu Gao , Li-Qin Ding , c
a
ab
Feng Zhao , Li-Xia Chen & Feng Qiu a
Key Laboratory of Structure-Based Drug Design & Discovery, Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
Click for updates
b
Tianjin State Key Laboratory of Modern Chinese Medicine, School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China c
School of Pharmacy, Yantai University, Yantai 264005, China Published online: 05 Jun 2015.
To cite this article: Jiang-Hao Ma, Ying Wang, Yue Liu, Su-Yu Gao, Li-Qin Ding, Feng Zhao, Li-Xia Chen & Feng Qiu (2015) Four new sesquiterpenes from the rhizomes of Curcuma phaeocaulis and their iNOS inhibitory activities, Journal of Asian Natural Products Research, 17:5, 532-540, DOI: 10.1080/10286020.2015.1046449 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1046449
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [UQ Library] at 00:48 16 July 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 Vol. 17, No. 5, 532–540, http://dx.doi.org/10.1080/10286020.2015.1046449
Four new sesquiterpenes from the rhizomes of Curcuma phaeocaulis and their iNOS inhibitory activities Jiang-Hao Maa, Ying Wanga, Yue Liua, Su-Yu Gaoa, Li-Qin Dingb, Feng Zhaoc, Li-Xia Chena* and Feng Qiuab* a
Key Laboratory of Structure-Based Drug Design & Discovery, Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; bTianjin State Key Laboratory of Modern Chinese Medicine, School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; cSchool of Pharmacy, Yantai University, Yantai 264005, China
Downloaded by [UQ Library] at 00:48 16 July 2015
(Received 12 February 2015; final version received 27 April 2015) Dedicated to Professor Xin-Sheng Yao on the occasion of his 80th birthday.
Three new guaiane-type sesquiterpenes named phaeocaulisins K-M (1 – 3), and one germacrane-type sesquiterpenoid with new ring system of 1,5- and 1,8-ether groups named phagermadiol (4), were isolated from rhizomes of Curcuma phaeocaulis. Their structures were established based on extensive spectroscopic analysis. Compound 1, the first example of norsesquiterpene with tropone backbone, and compound 3 with a novel 1,2-dioxolane sesquiterpene alcohol were isolated from the genus Curcuma. All of the isolated compounds were tested for inhibitory activity against lipopolysaccharide-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compound 3 inhibited NO production with IC50 value of 6.05 ^ 0.43 mM. The plausible biosynthetic pathway for compounds 3 and 4 in C. phaeocaulis was also discussed. Keywords: Curcuma phaeocaulis; guaiane; germacrane; sesquiterpenes; nitric oxide
1. Introduction Curcuma phaeocaulis Valeton (Zingiberaceae), common name Rhizoma Curcumae (Ezhu in Chinese), is widely distributed in southern regions of China including Sichuan, Yunnan, Guangdong, and Fujian provinces. The rhizomes are an important crude drug frequently listed in prescriptions of traditional Chinese medicine for the treatment of Oketsu syndromes [1], which are caused by the obstruction of blood circulation, such as arthralgia, psychataxia, and dysmenorrhea. Previous studies indicated that the main bioactive constituents of Rhizoma Curcumae were diarylheptanoids [1,2] and sesquiterpenoids [3 – 6], which
possess anti-inflammatory [7,8], anti-tumor [9 – 11], antioxidant [12], vasorelaxant [13], hepatoprotective [14], and neuroprotective [15] activities. According to the Chinese pharmacopeia, three species of Rhizoma Curcumae (C. phaeocaulis Valeton, Curcuma kwangsiensis S.G. Lee et C.F. Liang, and Curcuma wenyujin Y.H. Chen et C. Ling) are officially approved as Chinese medicine [16]. Recently, our research group has been examining the constituents of C. kwangsiensis, C. phaeocaulis, and C. wenyujin as well as their inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) pro-
*Corresponding authors. Email: [email protected]; [email protected] q 2015 Taylor & Francis
Journal of Asian Natural Products Research 15
15 1
15
O
4
4
6
OH
14
H
HO
3
2
4 14
13
1
10
O O
O O
12
11
5
1
OH
8 3
5 14
H
O
1 8
HO
OH
10
10
3
533
12
7 5
11
OH 4
3
OH
13
Downloaded by [UQ Library] at 00:48 16 July 2015
Figure 1. Structures of compounds 1 –4 isolated from C. phaeocaulis.
germacrane-type sesquiterpenoid from the rhizomes of C. phaeocaulis (Figure 1). Herein, we describe the isolation and structural elucidation of these compounds and their in vitro anti-inflammatory evaluation on inhibitory activities against LPSinduced NO production in RAW 264.7 macrophages. Furthermore, the biogenetic pathway for compounds 3 and 4 is proposed (Scheme 1).
duction, and has hitherto reported the isolation and characterization of several new sesquiterpenoids and diarylheptanoids [1 – 6]. We found that sesquiterpenes were mainly isolated from C. wenyujin and C. phaeocaulis, while diarylheptanoids were the major compounds from C. kwangsiensis. Moreover, guaiane-type and germacrane-type sesquiterpenes were mainly isolated from C. wenyujin, while guaiane-type and eudesmane-type sesquiterpenes were the major compounds from C. phaeocaulis. Although obvious chemical differences were found among these three species, most of the isolated compounds exhibited remarkable inhibitory activities against NO production. As part of our ongoing research for biologically active sesquiterpenoids from the genus Curcuma, and in order to provide a potential explanation for usage of these three species as Chinese herbal medicine in the treatment of inflammatory diseases, materials in the remaining fractions were further fractionated using silica gel chromatography as well as HPLC to afford three new guaiane-type sesquiterpenes and one
OH
OH
H
OH
H
H
O photooxidation O2 HO
2. Results and discussion Compound 1 was obtained as colorless oil with the molecular formula C12H14O2 as deduced by HR-ESI-MS. The IR spectral data showed the presence of hydroxyl (3401 cm21), conjugated carbonyl group (1711 cm21), and double bonds (1622, 1553, 1519, 1451 cm21). The characteristic ultraviolet absorptions (lmax (log 1) ¼ 232 (4.13), 317 (3.51) nm, MeOH) suggested the presence of a tropone moiety, a structural feature that has rarely been identified in natural products [17]. In the 1H NMR spectrum (Table 1) of 1, three olefinic proton signals
H
HO
O
OOH
H
O epoxidation
rearrangement cyclization
O germacrone
HO
i
phaeocaulisin E
OH O O
O cyclization
germacrone-4,5-epoxide
OH
H 3
OH
O
cyclization
O O
OH 4
O OH
OH iii
ketonecarbonyl enolization O H2O hydroxylation O
OH 4-epimer of wenyujinin J
Scheme 1. Plausible biogenetic pathway for compounds 3 and 4.
OH
O
OH ii
84.4 153.4 136.1
140.5 188.8 141.5
150.9
4 5 6
7 8 9
10 11 12 13 14 15
ddd (17.4,8.0,4.8) ddd (17.4,9.3,4.8) ddd (10.5,4.8,2.9) ddd (10.5,9.3,8.0)
1.42, s 2.34, s
7.07, s
7.06, dd (11.5,3.0)
7.43, d (11.5)
2.82, 2.96, 2.19, 2.08,
dH (J in Hz)
146.3 74.7 29.4 29.3 20.6 25.2
155.0 188.1 140.9
45.2 150.7 130.9
30.8
148.2 34.8
dC
1.55, 1.57, 1.24, 2.26,
s s d (7.0) s
7.02, s
7.35, s
2.81, ddd (18.0,7.6,7.4) 2.92, ddd (18.0,7.4,7.2) 2.22– 2.24, m 1.58– 1.62, m 3.21, q (7.0)
dH (J in Hz)
2
73.9 86.3 23.5 28.5 22.9 30.5
156.9 106.3 45.0
80.5 47.8 122.7
39.5
50.8 23.3
dC
1.43, 1.33, 1.21, 1.19,
s s s s
1.71, d (14.0) 2.05, d (14.0)
2.75, dd (11.2,3.3) 5.76, d (3.3)
1.69– 1.70, m 1.67– 1.68, m 1.86, ddd (13.0,7.0,3.8) 1.66– 1.67, 2H, m
dH (J in Hz)
3
161.0 74.6 25.1 23.6 25.1 25.1
121.1 166.9 126.9
71.4 72.0 29.4
48.1
96.7 32.6
dC
1.33, 1.31, 1.28, 2.22,
s s s s
6.18, s
3.22, dd (13.8,6.3) 2.55, dd (12.6,6.3) 2.06, dd (13.8,12.6)
2.51, dd (13.2,8.3) 2.43– 2.46, m
2.18, 2H, t (8.3)
dH (J in Hz)
4
H NMR spectra measured at 600 MHz, 13C NMR spectra measured at 75 MHz; spectrum of compound 2 was obtained in CDCl3, and spectra of 1, 3, 4 were obtained in CD3OD.
a1
39.9
3
27.4 25.5
150.3 33.3
dC
1
H and 13C NMR spectral data for compounds 1 – 4.a
1
1 2
Position
Table 1.
Downloaded by [UQ Library] at 00:48 16 July 2015
534 J.-H. Ma et al.
Downloaded by [UQ Library] at 00:48 16 July 2015
Journal of Asian Natural Products Research were observed at dH 7.07 (1H, s), 7.06 (1H, dd, J ¼ 11.5, 3.0 Hz), and 7.43 (1H, d, J ¼ 11.5 Hz). In addition, the 1H NMR spectrum also revealed the presence of two quaternary methyl groups at dH 1.42 and 2.34 (each 3H, s). The 13C NMR spectrum exhibited 12 carbon signals composed of two methyls at dC 27.4 and 25.5, two methylenes at dC 33.3 and 39.9, one quaternary oxygenated carbon at dC 84.4, six olefinic carbons at dC 136.1, 140.5, 141.5, 150.3, 150.9, and 153.4, and a conjugated ketone carbonyl carbon at dC 188.8. These functionalities accounted for four out of the six degrees of unsaturation, and the remaining two degrees of unsaturation required compound 1 to be bicyclic. Detailed comparison of the aforementioned spectroscopic features with those of the known compound tropone implied that the hydroxyl group was located at C-4 in 1 [17]. This deduction was further supported by the HMBC correlations (Figure 2) of H3-14 (dH 1.42) with C-3 (dC 39.9), C-4 (dC 84.4), C-5 (dC 153.4), H2-3 (dH 2.19, 2.08) with C-1 (dC 150.3), C-4 (dC 84.4), C-2 (dC 33.3), together with H-6 (dH 7.43) with C1 (dC 150.3), C-4 (dC 84.4), C-8 (dC 188.8). The absolute configuration of 1 at the asymmetric C-4 could be established by comparison to the optical rotation value of tropone [17]. However, compound 1 was unstable and might isomerize. Several structural analogues with similar absorption wavelength were detected after NMR and HR-ESI-MS tests. Further isolation of tropone derivatives and a thorough investigation of the rearrangement mechanisms
535
are warranted. Compound 1 was given the trivial name phaeocaulisin K. Compound 2 was assigned the molecular formula C15H20O2 on the basis of HR-ESI-MS (m/z: 255.1358 [M þ Na]þ) and NMR data. The IR spectrum showed absorptions belonging to double bonds (1611, 1544, 1512, 1451 cm21) and conjugated carbonyl (1763 cm21) group, and the UV spectrum revealed a characteristic absorption maxima at 234 and 326 nm (MeOH, log 1 4.21 and 3.64, respectively) indicating the presence of a tropone chromophore [4]. The 1H and 13C NMR spectra of 2 were similar to those of phaeocaulisin D, which had been obtained previously from C. phaeocaulis by our research group [6]. However, a distinctive difference in their 13C NMR spectra was observed that the chemical shift of C-4 in 2 was at dC 45.2 rather than at dC 84.9 in phaeocaulisin D. Consideration of the chemical shifts suggested that the hydroxyl at C-4 of 2 had undergone deoxidation. This deduction was supported by the HMBC spectrum (Figure 2), which showed correlations from H3-14 (dH 1.24) to C-3 (dC 30.8), C-4 (dC 45.2), C-5 (dC 150.7) and from H-6 (dH 7.35) to C-1 (dC 148.2), C-11 (dC 74.7), C-5 (dC 150.7), C-7 (dC 155.0), C-4 (dC 45.2), C-8 (dC 188.1). On the same reason as 1, the absolute configurations of 2 could not be determined and it was named phaeocaulisin L. Compound 3 had molecular formula C15H24O5 based on HR-ESI-MS (m/z: 307.1517 [M þ Na]þ) and NMR data. The IR spectrum (3451, 1636, and 1117 cm 21) suggested presence of a
Figure 2. Selected HMBC correlations of compounds 1 –4.
Downloaded by [UQ Library] at 00:48 16 July 2015
536
J.-H. Ma et al.
hydroxyl group and double bond. The 1H NMR spectrum showed methyl signals at dH 1.21, 1.19, 1.33, and 1.43 (each 3H, s), one olefinic proton signal at dH 5.76 (1H, d, J ¼ 3.3 Hz). The 13C NMR data indicated 15 carbons, including two olefinic carbons at dC 122.7 and 156.9, one hemiketal carbon atom at dC 106.3, and three oxygenated carbons at dC 73.9, 80.5, and 86.3. The long-range correlations (Figure 2) from H3-12 (dH 1.43) and H3-13 (dH 1.33) to the oxygenated quaternary carbon C-11 (dC 86.3) and sp2 quaternary carbon C-7 (dC 156.9), and from H2-9 (dH 2.05, 1.71) to dioxygenated quaternary carbon C-8 (dC 106.3) indicated the presence of a 1,2dioxolane system with the guaiane skeleton. The remaining three degrees of unsaturation required compound 3 to be tricyclic. Guaiane moiety accounted for two out of three rings and the third ring was further confirmed by a 1,2-dioxolane ring between C-7 and C-8 [18]. The relative configuration of 3 was elucidated through NOESY experiments, which revealed correlations of H-1 with Me-14/Me-15, H-2a with Me-15, and H-2b with H-5 indicated that H-1, Me-14 and Me-15 were in the same orientation. No correlation between H-1 and H-5 indicated that H-1 and H-5 had a trans-configuration. The available spectral data were not enough to assign the stereochemistry of the quaternary chiral center C-8 (Figure 1). The 1,2dioxolane-3-ol of guaiane-type sesquiterpene is unprecedented in the genus of Curcuma. Thus, the isolation of compound 3 might show chemotaxonomic importance for C. phaeocaulis within the family. Compound 3 could be produced from phaeocaulisin E, a key biosynthetic precursor isolated from C. phaeocaulis by our research group, through the dye-mediated photooxidation [19] of olefinic carbons between C-7 and C-11 followed by attack of the conjugated ketone group at C-8 for the formation of a peroxo bridge between C-8 and C-11 (Scheme 1). It was given the trivial name phaeocaulisin M.
Compound 4 showed IR absorptions attributed to hydroxyl groups (3421 cm21) and double bonds (1676, 1456 cm21). The molecular formula was determined to be C15H22O4 from its HR-ESI-MS and NMR data. The 1H NMR spectrum showed four methyl signals at dH 1.28, 1.31, 1.33, and 2.22 (each 3H, s), one olefinic proton at dH 6.18 (1H, s) and one oxygenated methine proton at dH 3.22 (1H, dd, J ¼ 13.8, 6.3 Hz). The 13C NMR spectrum revealed 15 carbon resonances, including four olefinic carbons at dC 161.0, 126.9, 166.9, 121.1, one doubly oxygenated quaternary carbon at dC 96.7 and three oxygenated carbons at dC 71.4, 72.0, and 74.6. The above NMR spectroscopic data were similar to wenyujinin J [20], a germacrane-type sesquiterpene possessing a 1,5-ether group isolated from C. wenyujin, except for the appearance of an oxygenated quaternary carbon and a hemiketal carbon instead of a ketone carbonyl carbon and an oxygenated tertiary carbon. The C-2 – C-3 and C-5 – C-6 moieties were indicated by the 1H – 1H COSY data (Figure 2). In the HMBC spectrum (Figure 2), correlations from Me15 to C-1/C-9/C-10 and Me-14 to C-3/C-4/ C-5 suggested the presence of Me-15– C10(C-1)– C-9 and Me-14 –C-4(C-3) – C-5 moieties. The HMBC correlations from Me-12 to C-7/C-11/C-13 and Me-13 to C7/C-11/C12 suggested the presence of an isopropanol moiety. The HMBC correlations from H2-2 to C-1/C-3/C-10, H-9 to C-1/C-7/C-8/C-15, H2-6 to C-1/C-5/C-7/ C-8/C-11, and H-5 to C-1/C-2/C-4/C-6/C7/C-10/C-14, together with the five degrees of unsaturation, indicated that two oxygen bridges connected C-1 – C-5 and C-1 – C-8. The NOESY correlations of H-5 with H-3a/H-6a and Me-14 with H6b/H-3b indicated that H-5 and Me-14 were not co-facial, and Me-14 and the oxygen bridge between C-1 and C-5 were randomly assigned as b-orientation, and H-5 and the oxygen bridge between C-1 and C-8 were a-oriented. This hypothesis
Downloaded by [UQ Library] at 00:48 16 July 2015
Journal of Asian Natural Products Research was further supported by a biosynthetic aspect and the molecular modeling. Compound 4 could be produced from germacrone, one of the constituents of C. phaeocaulis (Scheme 1). In the biogenetic process, epoxidation of germacrone [21] at C-4 –C-5 was followed by attack of C-1 to generate 4-epimer of wenyujinin J. Intermediate iii was presumed to be synthesized via the hydroxylation at C-11 and enolization of ketone carbonyl at C-8. Under enzyme catalysis of dehydrogenases, the intermediate iii may undergo cyclization to produce 4. Compound 4 was named phagermadiol. NO, produced from L-arginine by NO synthase, has various biological actions, e.g., as a defense and regulatory molecule for homeostatic equilibrium. However, in pathophysiologic conditions, such as inflammation, there is an increased production of NO by inducible NO synthase. Macrophages have been expected to be an origin of inflammation, because they contain various chemical mediators that may be responsible for several inflammatory stages. The inhibitory activity toward NO production, induced by LPS in murine macrophagederived RAW264.7 cells, was assayed [22]. In the present study, all isolated compounds were tested for their inhibitory effects on NO production induced by LPS in macrophages. Hydrocortisone (IC50 58.66 ^ 6.39 mM) was used as a positive control. The IC50 values indicated that compound 3 exhibited potent inhibitory activity against NO production (IC50 6.05 ^ 0.43 mM). Compound 2 exhibited moderate activity (IC50 54.27 ^ 4.23 mM), which was close to that of hydrocortisone, while compound 1 showed weak activity (IC50 82.47 ^ 6.67 mM) and compound 4 did not show inhibitory activity up to 100 mM concentration. Cell viability in the present experiment was determined by the 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide method to find whether
537
inhibition of NO production was due to cytotoxicity of test compounds (data not shown), and none of the compounds exhibited significant cytotoxicity at their effective concentration for the inhibition of NO production. 3. Experimental 3.1 General experimental procedures Optical rotations were measured with a Perkin-Elmer 241 polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were recorded on a Shimadzu UV 2201 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) and IR spectra were recorded on a Bruker IFS 55 spectrometer (Bruker Optics, Ettlingen, German) with KBr pellets. NMR experiments were performed on Bruker ARX-300 and AV600 spectrometers (Bruker Biospin, Fallanden, Switzerland) with tetramethylsilane as an internal standard. The chemical shifts are stated relative to TMS and expressed in d values (ppm), with coupling constants reported in Hz. HR-ESI-MS were obtained on an Agilent 6210 TOF mass spectrometer (Palo Alto, CA, USA). Silica gel GF254 prepared for TLC and silica gel (200 – 300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Sephadex LH-20 was a product of Pharmacia (Amersharm, Uppsala, Sweden). Octadecyl silica gel (10 mm) was purchased from Merck Chemical Company Ltd (Darmstadt, German). RP-HPLC separations were conducted using an LC-6AD liquid chromatograph with a YMC Pack ODS-A ˚, column (250 £ 20 mm, 5 mm, 120 A Kyoto, Japan) and SPD-10A VP UV/VIS detector (Shimadzu). All reagents were HPLC or analytical grade and were purchased from Tianjin Damao Chemical Company (Tianjin, China). Spots were detected on TLC plates under UV light or by heating after spraying with anisaldehyde-H2SO4 reagent.
538
J.-H. Ma et al.
Downloaded by [UQ Library] at 00:48 16 July 2015
3.2 Plant material Rhizomes of C. phaeocaulis were collected from Chengdou, Sichuan province, China, and identified by Professor Qishi Sun, Department of Pharmaceutical Botany, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (No. CP-20100715) has been deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University. 3.3 Extraction and isolation The rhizomes of C. phaeocaulis (10 kg) were cut into approximately 2 cm pieces and extracted with 95% EtOH (100 L £ 2 h £ 2). The resulting extract (0.6 kg) was concentrated in vacuo, suspended in H2O (3 l), and partitioned successively with cyclohexane, EtOAc, and n-BuOH (3 L £ 3). The EtOAc extract (105 g) was subjected to silica gel CC (10 £ 80 cm) eluted with cyclohexane/acetone (100:1, 40:1, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain six fractions (EA –EF). Fraction EA (23 g) was subjected to a silica gel column (6 £ 80 cm) and eluted with CH2Cl2/EtOAc (from 40:1 to 0:1) to produce seven fractions (EA1 – EA7). Fraction EA4 (180 mg) was chromatographed over Sephadex LH-20 (CH2Cl2/ MeOH, 1:1; 1.5 £ 30 cm) to give compounds 1 (3.5 mg) and 2 (23.2 mg). Fraction EB (10.6 g) was subjected to a silica gel column (6 £ 80 cm) and eluted with CH2Cl2/acetone (from 40:1 to 0:1) to yield EB1 – EB6. Separation of EB4 (1.4 g) on a reversed-phase C18 silica gel column (2.5 £ 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) yielded fractions EB4-1 to EB4-5. EB4-5 (80 mg) was purified by preparative TLC (CH 2 Cl 2/acetone, 3:1) to obtain 4 (11.5 mg). EB5 (2.3 g) was subjected to RP-C18 silica gel CC (2.5 £ 30 cm) eluted with MeOH/H2O (1:9– 8:2) to yield EB5-1 and EB5-2. EB5-2 (100 mg) was separated
by HPLC (50% MeOH/H2O, 6 ml/min) to afford compound 3 (28.8 mg, tR 39 min). 3.3.1
Phaeocaulisin K (1)
Colorless oil (MeOH); UV (MeOH) lmax (log 1) 232 (4.13), 317 (3.51) nm; IR (KBr) vmax 3401, 2927, 2855, 1711, 1666, 1622, 1553, 1519, 1451, 1384, 1255, 1190, 1095 cm 21; 1 H NMR (600 MHz, CD 3OD); and 13C NMR (75 MHz, CD3OD) spectral data, see Table 1; HRESI-MS: m/z 189.0913 [M – H]2 (calcd for C12H13O2, 189.0916). 3.3.2 Phaeocaulisin L (2) Colorless oil (MeOH); UV (MeOH) lmax (log 1) 234 (4.21), 326 (3.64) nm; IR (KBr) vmax 2959, 2933, 2871, 1763, 1647, 1611, 1544, 1512, 1451, 1422, 1376, 1291, 1191, 1174, 1126, 1064 cm 21; 1H NMR (600 MHz, CDCl 3); and 13C NMR (75 MHz, CDCl 3) spectral data, see Table 1; HR-ESI-MS: m/z 255.1358 [M þ Na] þ (calcd for C15H 20O 2Na, 255.1361). 3.3.3
Phaeocaulisin M (3)
Pale yellow oil; ½a25 D þ 83.3 (c 0.12, MeOH); UV (MeOH) lmax (log 1) 208 (3.78) nm; IR (KBr) vmax 3451, 1636, 1384, 1117 cm21; 1H NMR (600 MHz, CD 3OD); and 13 C NMR (75 MHz, CD3OD) spectral data, see Table 1; HRESI-MS: m/z 307.1517 [M þ Na]þ (calcd for C15H24O5Na, 307.1521). 3.3.4 Phagermadiol (4) Amorphous powder; ½a25 D – 36.7 (c 0.12, MeOH); UV (MeOH) lmax (log 1) 224 (3.86); IR (KBr) vmax 3421, 2979, 1676, 1456, 1383, 1313 cm 21; 1H NMR (600 MHz, CD 3OD); and 13C NMR (75 MHz, CD3OD) spectral data, see Table 1; HR-ESI-MS: m/z 289.1414 [M þ Na]þ (calcd for C15H22O4Na, 289.1416).
Downloaded by [UQ Library] at 00:48 16 July 2015
Journal of Asian Natural Products Research 3.4 NO Production bioassay The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 1 £ 105 cells/well, and stimulated with 1 mg/ml of LPS in the presence or absence of test compounds. After incubation at 378C for 24 h, 100 ml of cell-free supernatant was mixed with 100 ml of Griess reagent (mixture of equal volumes of reagent A and reagent B, A: 1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid, B: 0.1% (w/v) of N-(1naphthyl) ethylenediamine). Absorbance was measured in a microplate reader at 540 nm. Nitrite concentrations and the inhibitory rates were calculated by a calibration curve prepared with sodium nitrite standards [3,23]. Experiments were performed in triplicate, and data are expressed as the mean ^ SD of three independent experiments. Acknowledgements
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
We are grateful to Dr Paul Owusu Donkor for refining the language of our manuscript. [11]
Disclosure statement No potential conflict of interest was reported by the authors.
[12]
Funding This work was financially supported by grants in part from the National Natural Science Foundation of China (NSFC) [grant number 30973630], [grant number 81430095]; and the Medicinal Chemistry Subject Construction Project of Shenyang Pharmaceutical University [grant number 20130017].
References [1] J. Li, F. Zhao, M.Z. Li, L.X. Chen, and F. Qiu, J. Nat. Prod. 73, 1667 (2010). doi:10.1021/np100392m. [2] J. Li, C.R. Liao, J.Q. Wei, L.X. Chen, F. Zhao, and F. Qiu, Bioorg. Med. Chem.
[13]
[14]
[15] [16]
539
Lett. 21, 5363 (2011). doi:10.1016/j.bmcl. 2011.07.012. Y. Lou, F. Zhao, H. He, K.F. Peng, X.H. Zhou, L.X. Chen, F. Qiu, and J. Asian, Nat. Prod. Res. 11, 737 (2009). Y. Lou, F. Zhao, H. He, K.F. Peng, L.X. Chen, and F. Qiu, Chem. Biodivers. 7, 1245 (2010). doi:10.1002/cbdv. 200900160. Y. Liu, J.H. Ma, Y. Wang, P.O. Donker, Q. Li, S.Y. Gao, Y.G. Hou, Y. Xu, J.N. Cui, L.Q. Ding, F. Zhao, N. Kang, L.X. Chen, and F. Qiu, Eur. J. Org. Chem. 5540, 2014 (2014). Y. Liu, J.H. Ma, Q. Zhao, C.R. Liao, L.Q. Ding, L.X. Chen, F. Zhao, and F. Qiu, J. Nat. Prod. 76, 1150 (2013). doi:10.1021/np400202f. C. Tohda, N. Nakayama, F. Hatanaka, and K. Komatsu, Evid-Based Complement. Altern. 3, 255 (2006). doi:10.1093/ecam/ nel008. T. Masuda, A. Jitoe, J. Isobe, N. Nakatani, and S. Yonemori, Phytochemistry 32, 1557 (1993). doi:10.1016/0031-9422(93) 85179-U. X.P. Chen, L.X. Pei, Z.F. Zhong, J.J. Guo, Q.W. Zhang, and Y.T. Wang, Phytomedicine 18, 1238 (2011). doi:10.1016/j. phymed.2011.06.017. M.S. Ali, Y. Tezuka, S. Awale, A.H. Banskota, and S. Kadota, J. Nat. Prod. 64, 289 (2001). doi:10.1021/ np000496y. M. Li, Z. Zhang, D.L. Hill, H. Wang, and R. Zhang, Cancer Res. 67, 1988 (2007). doi:10.1158/0008-5472.CAN-06-3066. Q.F. Tao, Y. Xu, R.Y.Y. Lam, B. Schneider, H. Dou, P.S. Leung, S.Y. Shi, C.X. Zhou, L.X. Yang, R.P. Zhang, Y.C. Xiao, X. Wu, J. Sto¨ckigt, S. Zeng, C.H.K. Cheng, and Y. Zhao, J. Nat. Prod. 71, 12 (2008). doi:10.1021/ np070114p. H. Matsuda, T. Morikawa, K. Ninomiya, and M. Yoshikawa, Tetrahedron 57, 8443 (2001). doi:10.1016/S0040-4020(01) 00828-6. H. Matsuda, T. Morikawa, K. Ninomiya, and M. Yoshikawa, Bioorg. Med. Chem. 9, 909 (2001). doi:10.1016/S0968-0896 (00)00306-0. P. Dohare, P. Garg, U. Sharma, N.R. Jagannathan, and M. Ray, BMC Complement. Altern. Med. 8, 55 (2008). Pharmacopoeia Commission of People’s Republic of China, Pharmacopoeia of the People’s Republic of China, 2010 Chinese
540
[17] [18] [19]
Downloaded by [UQ Library] at 00:48 16 July 2015
[20]
J.-H. Ma et al. ed. (Chinese Medical Science and Technology Press, Beijing, 2010). M. Rohr, P. Naegeli, and J.J. Daly, Phytochemistry 18, 279 (1979). doi:10. 1016/0031-9422(79)80070-9. S.S. Sawant, D.T.A. Youssef, P.W. Sylvester, V. Wali, and K.A.E. Sayed, Nat. Prod. Commun. 2, 117 (2007). H.F. Wong and G.D. Brown, J. Chem. Res. 2002, 30 (2002). doi:10.3184/ 030823402103170376. G.P. Yin, L.C. Li, Q.Z. Zhang, Y.W. An, J.J. Zhu, Z.M. Wang, G.X. Chou, and
Z.T. Wang, J. Nat. Prod. 77, 2161 (2014). doi:10.1021/np400984c. [21] K. Masanori, U. Akira, U. Kaoru, and S. Sadao, Chem. Pharm. Bull. 35, 53 (1987). doi:10.1248/cpb.35.53. [22] H. Sumioka, L. Harinantenaina, K. Matsunami, H. Otsuka, M. Kawahata, and K. Yamaguchi, Phytochemistry 72, 2165 (2011). doi:10.1016/j.phytochem. 2011.08.004. [23] V.M. Dirsch, H. Stuppner, and A.M. Vollmar, Planta Med. 64, 423 (1998). doi:10.1055/s-2006-957473.
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Four new sesquiterpenes from the rhizomes of Curcuma phaeocaulis and their iNOS inhibitory activities a
a
a
a
b
Jiang-Hao Ma , Ying Wang , Yue Liu , Su-Yu Gao , Li-Qin Ding , c
a
ab
Feng Zhao , Li-Xia Chen & Feng Qiu a
Key Laboratory of Structure-Based Drug Design & Discovery, Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
Click for updates
b
Tianjin State Key Laboratory of Modern Chinese Medicine, School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China c
School of Pharmacy, Yantai University, Yantai 264005, China Published online: 05 Jun 2015.
To cite this article: Jiang-Hao Ma, Ying Wang, Yue Liu, Su-Yu Gao, Li-Qin Ding, Feng Zhao, Li-Xia Chen & Feng Qiu (2015) Four new sesquiterpenes from the rhizomes of Curcuma phaeocaulis and their iNOS inhibitory activities, Journal of Asian Natural Products Research, 17:5, 532-540, DOI: 10.1080/10286020.2015.1046449 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1046449
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [UQ Library] at 00:48 16 July 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 Vol. 17, No. 5, 532–540, http://dx.doi.org/10.1080/10286020.2015.1046449
Four new sesquiterpenes from the rhizomes of Curcuma phaeocaulis and their iNOS inhibitory activities Jiang-Hao Maa, Ying Wanga, Yue Liua, Su-Yu Gaoa, Li-Qin Dingb, Feng Zhaoc, Li-Xia Chena* and Feng Qiuab* a
Key Laboratory of Structure-Based Drug Design & Discovery, Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; bTianjin State Key Laboratory of Modern Chinese Medicine, School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; cSchool of Pharmacy, Yantai University, Yantai 264005, China
Downloaded by [UQ Library] at 00:48 16 July 2015
(Received 12 February 2015; final version received 27 April 2015) Dedicated to Professor Xin-Sheng Yao on the occasion of his 80th birthday.
Three new guaiane-type sesquiterpenes named phaeocaulisins K-M (1 – 3), and one germacrane-type sesquiterpenoid with new ring system of 1,5- and 1,8-ether groups named phagermadiol (4), were isolated from rhizomes of Curcuma phaeocaulis. Their structures were established based on extensive spectroscopic analysis. Compound 1, the first example of norsesquiterpene with tropone backbone, and compound 3 with a novel 1,2-dioxolane sesquiterpene alcohol were isolated from the genus Curcuma. All of the isolated compounds were tested for inhibitory activity against lipopolysaccharide-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compound 3 inhibited NO production with IC50 value of 6.05 ^ 0.43 mM. The plausible biosynthetic pathway for compounds 3 and 4 in C. phaeocaulis was also discussed. Keywords: Curcuma phaeocaulis; guaiane; germacrane; sesquiterpenes; nitric oxide
1. Introduction Curcuma phaeocaulis Valeton (Zingiberaceae), common name Rhizoma Curcumae (Ezhu in Chinese), is widely distributed in southern regions of China including Sichuan, Yunnan, Guangdong, and Fujian provinces. The rhizomes are an important crude drug frequently listed in prescriptions of traditional Chinese medicine for the treatment of Oketsu syndromes [1], which are caused by the obstruction of blood circulation, such as arthralgia, psychataxia, and dysmenorrhea. Previous studies indicated that the main bioactive constituents of Rhizoma Curcumae were diarylheptanoids [1,2] and sesquiterpenoids [3 – 6], which
possess anti-inflammatory [7,8], anti-tumor [9 – 11], antioxidant [12], vasorelaxant [13], hepatoprotective [14], and neuroprotective [15] activities. According to the Chinese pharmacopeia, three species of Rhizoma Curcumae (C. phaeocaulis Valeton, Curcuma kwangsiensis S.G. Lee et C.F. Liang, and Curcuma wenyujin Y.H. Chen et C. Ling) are officially approved as Chinese medicine [16]. Recently, our research group has been examining the constituents of C. kwangsiensis, C. phaeocaulis, and C. wenyujin as well as their inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) pro-
*Corresponding authors. Email: [email protected]; [email protected] q 2015 Taylor & Francis
Journal of Asian Natural Products Research 15
15 1
15
O
4
4
6
OH
14
H
HO
3
2
4 14
13
1
10
O O
O O
12
11
5
1
OH
8 3
5 14
H
O
1 8
HO
OH
10
10
3
533
12
7 5
11
OH 4
3
OH
13
Downloaded by [UQ Library] at 00:48 16 July 2015
Figure 1. Structures of compounds 1 –4 isolated from C. phaeocaulis.
germacrane-type sesquiterpenoid from the rhizomes of C. phaeocaulis (Figure 1). Herein, we describe the isolation and structural elucidation of these compounds and their in vitro anti-inflammatory evaluation on inhibitory activities against LPSinduced NO production in RAW 264.7 macrophages. Furthermore, the biogenetic pathway for compounds 3 and 4 is proposed (Scheme 1).
duction, and has hitherto reported the isolation and characterization of several new sesquiterpenoids and diarylheptanoids [1 – 6]. We found that sesquiterpenes were mainly isolated from C. wenyujin and C. phaeocaulis, while diarylheptanoids were the major compounds from C. kwangsiensis. Moreover, guaiane-type and germacrane-type sesquiterpenes were mainly isolated from C. wenyujin, while guaiane-type and eudesmane-type sesquiterpenes were the major compounds from C. phaeocaulis. Although obvious chemical differences were found among these three species, most of the isolated compounds exhibited remarkable inhibitory activities against NO production. As part of our ongoing research for biologically active sesquiterpenoids from the genus Curcuma, and in order to provide a potential explanation for usage of these three species as Chinese herbal medicine in the treatment of inflammatory diseases, materials in the remaining fractions were further fractionated using silica gel chromatography as well as HPLC to afford three new guaiane-type sesquiterpenes and one
OH
OH
H
OH
H
H
O photooxidation O2 HO
2. Results and discussion Compound 1 was obtained as colorless oil with the molecular formula C12H14O2 as deduced by HR-ESI-MS. The IR spectral data showed the presence of hydroxyl (3401 cm21), conjugated carbonyl group (1711 cm21), and double bonds (1622, 1553, 1519, 1451 cm21). The characteristic ultraviolet absorptions (lmax (log 1) ¼ 232 (4.13), 317 (3.51) nm, MeOH) suggested the presence of a tropone moiety, a structural feature that has rarely been identified in natural products [17]. In the 1H NMR spectrum (Table 1) of 1, three olefinic proton signals
H
HO
O
OOH
H
O epoxidation
rearrangement cyclization
O germacrone
HO
i
phaeocaulisin E
OH O O
O cyclization
germacrone-4,5-epoxide
OH
H 3
OH
O
cyclization
O O
OH 4
O OH
OH iii
ketonecarbonyl enolization O H2O hydroxylation O
OH 4-epimer of wenyujinin J
Scheme 1. Plausible biogenetic pathway for compounds 3 and 4.
OH
O
OH ii
84.4 153.4 136.1
140.5 188.8 141.5
150.9
4 5 6
7 8 9
10 11 12 13 14 15
ddd (17.4,8.0,4.8) ddd (17.4,9.3,4.8) ddd (10.5,4.8,2.9) ddd (10.5,9.3,8.0)
1.42, s 2.34, s
7.07, s
7.06, dd (11.5,3.0)
7.43, d (11.5)
2.82, 2.96, 2.19, 2.08,
dH (J in Hz)
146.3 74.7 29.4 29.3 20.6 25.2
155.0 188.1 140.9
45.2 150.7 130.9
30.8
148.2 34.8
dC
1.55, 1.57, 1.24, 2.26,
s s d (7.0) s
7.02, s
7.35, s
2.81, ddd (18.0,7.6,7.4) 2.92, ddd (18.0,7.4,7.2) 2.22– 2.24, m 1.58– 1.62, m 3.21, q (7.0)
dH (J in Hz)
2
73.9 86.3 23.5 28.5 22.9 30.5
156.9 106.3 45.0
80.5 47.8 122.7
39.5
50.8 23.3
dC
1.43, 1.33, 1.21, 1.19,
s s s s
1.71, d (14.0) 2.05, d (14.0)
2.75, dd (11.2,3.3) 5.76, d (3.3)
1.69– 1.70, m 1.67– 1.68, m 1.86, ddd (13.0,7.0,3.8) 1.66– 1.67, 2H, m
dH (J in Hz)
3
161.0 74.6 25.1 23.6 25.1 25.1
121.1 166.9 126.9
71.4 72.0 29.4
48.1
96.7 32.6
dC
1.33, 1.31, 1.28, 2.22,
s s s s
6.18, s
3.22, dd (13.8,6.3) 2.55, dd (12.6,6.3) 2.06, dd (13.8,12.6)
2.51, dd (13.2,8.3) 2.43– 2.46, m
2.18, 2H, t (8.3)
dH (J in Hz)
4
H NMR spectra measured at 600 MHz, 13C NMR spectra measured at 75 MHz; spectrum of compound 2 was obtained in CDCl3, and spectra of 1, 3, 4 were obtained in CD3OD.
a1
39.9
3
27.4 25.5
150.3 33.3
dC
1
H and 13C NMR spectral data for compounds 1 – 4.a
1
1 2
Position
Table 1.
Downloaded by [UQ Library] at 00:48 16 July 2015
534 J.-H. Ma et al.
Downloaded by [UQ Library] at 00:48 16 July 2015
Journal of Asian Natural Products Research were observed at dH 7.07 (1H, s), 7.06 (1H, dd, J ¼ 11.5, 3.0 Hz), and 7.43 (1H, d, J ¼ 11.5 Hz). In addition, the 1H NMR spectrum also revealed the presence of two quaternary methyl groups at dH 1.42 and 2.34 (each 3H, s). The 13C NMR spectrum exhibited 12 carbon signals composed of two methyls at dC 27.4 and 25.5, two methylenes at dC 33.3 and 39.9, one quaternary oxygenated carbon at dC 84.4, six olefinic carbons at dC 136.1, 140.5, 141.5, 150.3, 150.9, and 153.4, and a conjugated ketone carbonyl carbon at dC 188.8. These functionalities accounted for four out of the six degrees of unsaturation, and the remaining two degrees of unsaturation required compound 1 to be bicyclic. Detailed comparison of the aforementioned spectroscopic features with those of the known compound tropone implied that the hydroxyl group was located at C-4 in 1 [17]. This deduction was further supported by the HMBC correlations (Figure 2) of H3-14 (dH 1.42) with C-3 (dC 39.9), C-4 (dC 84.4), C-5 (dC 153.4), H2-3 (dH 2.19, 2.08) with C-1 (dC 150.3), C-4 (dC 84.4), C-2 (dC 33.3), together with H-6 (dH 7.43) with C1 (dC 150.3), C-4 (dC 84.4), C-8 (dC 188.8). The absolute configuration of 1 at the asymmetric C-4 could be established by comparison to the optical rotation value of tropone [17]. However, compound 1 was unstable and might isomerize. Several structural analogues with similar absorption wavelength were detected after NMR and HR-ESI-MS tests. Further isolation of tropone derivatives and a thorough investigation of the rearrangement mechanisms
535
are warranted. Compound 1 was given the trivial name phaeocaulisin K. Compound 2 was assigned the molecular formula C15H20O2 on the basis of HR-ESI-MS (m/z: 255.1358 [M þ Na]þ) and NMR data. The IR spectrum showed absorptions belonging to double bonds (1611, 1544, 1512, 1451 cm21) and conjugated carbonyl (1763 cm21) group, and the UV spectrum revealed a characteristic absorption maxima at 234 and 326 nm (MeOH, log 1 4.21 and 3.64, respectively) indicating the presence of a tropone chromophore [4]. The 1H and 13C NMR spectra of 2 were similar to those of phaeocaulisin D, which had been obtained previously from C. phaeocaulis by our research group [6]. However, a distinctive difference in their 13C NMR spectra was observed that the chemical shift of C-4 in 2 was at dC 45.2 rather than at dC 84.9 in phaeocaulisin D. Consideration of the chemical shifts suggested that the hydroxyl at C-4 of 2 had undergone deoxidation. This deduction was supported by the HMBC spectrum (Figure 2), which showed correlations from H3-14 (dH 1.24) to C-3 (dC 30.8), C-4 (dC 45.2), C-5 (dC 150.7) and from H-6 (dH 7.35) to C-1 (dC 148.2), C-11 (dC 74.7), C-5 (dC 150.7), C-7 (dC 155.0), C-4 (dC 45.2), C-8 (dC 188.1). On the same reason as 1, the absolute configurations of 2 could not be determined and it was named phaeocaulisin L. Compound 3 had molecular formula C15H24O5 based on HR-ESI-MS (m/z: 307.1517 [M þ Na]þ) and NMR data. The IR spectrum (3451, 1636, and 1117 cm 21) suggested presence of a
Figure 2. Selected HMBC correlations of compounds 1 –4.
Downloaded by [UQ Library] at 00:48 16 July 2015
536
J.-H. Ma et al.
hydroxyl group and double bond. The 1H NMR spectrum showed methyl signals at dH 1.21, 1.19, 1.33, and 1.43 (each 3H, s), one olefinic proton signal at dH 5.76 (1H, d, J ¼ 3.3 Hz). The 13C NMR data indicated 15 carbons, including two olefinic carbons at dC 122.7 and 156.9, one hemiketal carbon atom at dC 106.3, and three oxygenated carbons at dC 73.9, 80.5, and 86.3. The long-range correlations (Figure 2) from H3-12 (dH 1.43) and H3-13 (dH 1.33) to the oxygenated quaternary carbon C-11 (dC 86.3) and sp2 quaternary carbon C-7 (dC 156.9), and from H2-9 (dH 2.05, 1.71) to dioxygenated quaternary carbon C-8 (dC 106.3) indicated the presence of a 1,2dioxolane system with the guaiane skeleton. The remaining three degrees of unsaturation required compound 3 to be tricyclic. Guaiane moiety accounted for two out of three rings and the third ring was further confirmed by a 1,2-dioxolane ring between C-7 and C-8 [18]. The relative configuration of 3 was elucidated through NOESY experiments, which revealed correlations of H-1 with Me-14/Me-15, H-2a with Me-15, and H-2b with H-5 indicated that H-1, Me-14 and Me-15 were in the same orientation. No correlation between H-1 and H-5 indicated that H-1 and H-5 had a trans-configuration. The available spectral data were not enough to assign the stereochemistry of the quaternary chiral center C-8 (Figure 1). The 1,2dioxolane-3-ol of guaiane-type sesquiterpene is unprecedented in the genus of Curcuma. Thus, the isolation of compound 3 might show chemotaxonomic importance for C. phaeocaulis within the family. Compound 3 could be produced from phaeocaulisin E, a key biosynthetic precursor isolated from C. phaeocaulis by our research group, through the dye-mediated photooxidation [19] of olefinic carbons between C-7 and C-11 followed by attack of the conjugated ketone group at C-8 for the formation of a peroxo bridge between C-8 and C-11 (Scheme 1). It was given the trivial name phaeocaulisin M.
Compound 4 showed IR absorptions attributed to hydroxyl groups (3421 cm21) and double bonds (1676, 1456 cm21). The molecular formula was determined to be C15H22O4 from its HR-ESI-MS and NMR data. The 1H NMR spectrum showed four methyl signals at dH 1.28, 1.31, 1.33, and 2.22 (each 3H, s), one olefinic proton at dH 6.18 (1H, s) and one oxygenated methine proton at dH 3.22 (1H, dd, J ¼ 13.8, 6.3 Hz). The 13C NMR spectrum revealed 15 carbon resonances, including four olefinic carbons at dC 161.0, 126.9, 166.9, 121.1, one doubly oxygenated quaternary carbon at dC 96.7 and three oxygenated carbons at dC 71.4, 72.0, and 74.6. The above NMR spectroscopic data were similar to wenyujinin J [20], a germacrane-type sesquiterpene possessing a 1,5-ether group isolated from C. wenyujin, except for the appearance of an oxygenated quaternary carbon and a hemiketal carbon instead of a ketone carbonyl carbon and an oxygenated tertiary carbon. The C-2 – C-3 and C-5 – C-6 moieties were indicated by the 1H – 1H COSY data (Figure 2). In the HMBC spectrum (Figure 2), correlations from Me15 to C-1/C-9/C-10 and Me-14 to C-3/C-4/ C-5 suggested the presence of Me-15– C10(C-1)– C-9 and Me-14 –C-4(C-3) – C-5 moieties. The HMBC correlations from Me-12 to C-7/C-11/C-13 and Me-13 to C7/C-11/C12 suggested the presence of an isopropanol moiety. The HMBC correlations from H2-2 to C-1/C-3/C-10, H-9 to C-1/C-7/C-8/C-15, H2-6 to C-1/C-5/C-7/ C-8/C-11, and H-5 to C-1/C-2/C-4/C-6/C7/C-10/C-14, together with the five degrees of unsaturation, indicated that two oxygen bridges connected C-1 – C-5 and C-1 – C-8. The NOESY correlations of H-5 with H-3a/H-6a and Me-14 with H6b/H-3b indicated that H-5 and Me-14 were not co-facial, and Me-14 and the oxygen bridge between C-1 and C-5 were randomly assigned as b-orientation, and H-5 and the oxygen bridge between C-1 and C-8 were a-oriented. This hypothesis
Downloaded by [UQ Library] at 00:48 16 July 2015
Journal of Asian Natural Products Research was further supported by a biosynthetic aspect and the molecular modeling. Compound 4 could be produced from germacrone, one of the constituents of C. phaeocaulis (Scheme 1). In the biogenetic process, epoxidation of germacrone [21] at C-4 –C-5 was followed by attack of C-1 to generate 4-epimer of wenyujinin J. Intermediate iii was presumed to be synthesized via the hydroxylation at C-11 and enolization of ketone carbonyl at C-8. Under enzyme catalysis of dehydrogenases, the intermediate iii may undergo cyclization to produce 4. Compound 4 was named phagermadiol. NO, produced from L-arginine by NO synthase, has various biological actions, e.g., as a defense and regulatory molecule for homeostatic equilibrium. However, in pathophysiologic conditions, such as inflammation, there is an increased production of NO by inducible NO synthase. Macrophages have been expected to be an origin of inflammation, because they contain various chemical mediators that may be responsible for several inflammatory stages. The inhibitory activity toward NO production, induced by LPS in murine macrophagederived RAW264.7 cells, was assayed [22]. In the present study, all isolated compounds were tested for their inhibitory effects on NO production induced by LPS in macrophages. Hydrocortisone (IC50 58.66 ^ 6.39 mM) was used as a positive control. The IC50 values indicated that compound 3 exhibited potent inhibitory activity against NO production (IC50 6.05 ^ 0.43 mM). Compound 2 exhibited moderate activity (IC50 54.27 ^ 4.23 mM), which was close to that of hydrocortisone, while compound 1 showed weak activity (IC50 82.47 ^ 6.67 mM) and compound 4 did not show inhibitory activity up to 100 mM concentration. Cell viability in the present experiment was determined by the 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide method to find whether
537
inhibition of NO production was due to cytotoxicity of test compounds (data not shown), and none of the compounds exhibited significant cytotoxicity at their effective concentration for the inhibition of NO production. 3. Experimental 3.1 General experimental procedures Optical rotations were measured with a Perkin-Elmer 241 polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were recorded on a Shimadzu UV 2201 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) and IR spectra were recorded on a Bruker IFS 55 spectrometer (Bruker Optics, Ettlingen, German) with KBr pellets. NMR experiments were performed on Bruker ARX-300 and AV600 spectrometers (Bruker Biospin, Fallanden, Switzerland) with tetramethylsilane as an internal standard. The chemical shifts are stated relative to TMS and expressed in d values (ppm), with coupling constants reported in Hz. HR-ESI-MS were obtained on an Agilent 6210 TOF mass spectrometer (Palo Alto, CA, USA). Silica gel GF254 prepared for TLC and silica gel (200 – 300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Sephadex LH-20 was a product of Pharmacia (Amersharm, Uppsala, Sweden). Octadecyl silica gel (10 mm) was purchased from Merck Chemical Company Ltd (Darmstadt, German). RP-HPLC separations were conducted using an LC-6AD liquid chromatograph with a YMC Pack ODS-A ˚, column (250 £ 20 mm, 5 mm, 120 A Kyoto, Japan) and SPD-10A VP UV/VIS detector (Shimadzu). All reagents were HPLC or analytical grade and were purchased from Tianjin Damao Chemical Company (Tianjin, China). Spots were detected on TLC plates under UV light or by heating after spraying with anisaldehyde-H2SO4 reagent.
538
J.-H. Ma et al.
Downloaded by [UQ Library] at 00:48 16 July 2015
3.2 Plant material Rhizomes of C. phaeocaulis were collected from Chengdou, Sichuan province, China, and identified by Professor Qishi Sun, Department of Pharmaceutical Botany, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (No. CP-20100715) has been deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University. 3.3 Extraction and isolation The rhizomes of C. phaeocaulis (10 kg) were cut into approximately 2 cm pieces and extracted with 95% EtOH (100 L £ 2 h £ 2). The resulting extract (0.6 kg) was concentrated in vacuo, suspended in H2O (3 l), and partitioned successively with cyclohexane, EtOAc, and n-BuOH (3 L £ 3). The EtOAc extract (105 g) was subjected to silica gel CC (10 £ 80 cm) eluted with cyclohexane/acetone (100:1, 40:1, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain six fractions (EA –EF). Fraction EA (23 g) was subjected to a silica gel column (6 £ 80 cm) and eluted with CH2Cl2/EtOAc (from 40:1 to 0:1) to produce seven fractions (EA1 – EA7). Fraction EA4 (180 mg) was chromatographed over Sephadex LH-20 (CH2Cl2/ MeOH, 1:1; 1.5 £ 30 cm) to give compounds 1 (3.5 mg) and 2 (23.2 mg). Fraction EB (10.6 g) was subjected to a silica gel column (6 £ 80 cm) and eluted with CH2Cl2/acetone (from 40:1 to 0:1) to yield EB1 – EB6. Separation of EB4 (1.4 g) on a reversed-phase C18 silica gel column (2.5 £ 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) yielded fractions EB4-1 to EB4-5. EB4-5 (80 mg) was purified by preparative TLC (CH 2 Cl 2/acetone, 3:1) to obtain 4 (11.5 mg). EB5 (2.3 g) was subjected to RP-C18 silica gel CC (2.5 £ 30 cm) eluted with MeOH/H2O (1:9– 8:2) to yield EB5-1 and EB5-2. EB5-2 (100 mg) was separated
by HPLC (50% MeOH/H2O, 6 ml/min) to afford compound 3 (28.8 mg, tR 39 min). 3.3.1
Phaeocaulisin K (1)
Colorless oil (MeOH); UV (MeOH) lmax (log 1) 232 (4.13), 317 (3.51) nm; IR (KBr) vmax 3401, 2927, 2855, 1711, 1666, 1622, 1553, 1519, 1451, 1384, 1255, 1190, 1095 cm 21; 1 H NMR (600 MHz, CD 3OD); and 13C NMR (75 MHz, CD3OD) spectral data, see Table 1; HRESI-MS: m/z 189.0913 [M – H]2 (calcd for C12H13O2, 189.0916). 3.3.2 Phaeocaulisin L (2) Colorless oil (MeOH); UV (MeOH) lmax (log 1) 234 (4.21), 326 (3.64) nm; IR (KBr) vmax 2959, 2933, 2871, 1763, 1647, 1611, 1544, 1512, 1451, 1422, 1376, 1291, 1191, 1174, 1126, 1064 cm 21; 1H NMR (600 MHz, CDCl 3); and 13C NMR (75 MHz, CDCl 3) spectral data, see Table 1; HR-ESI-MS: m/z 255.1358 [M þ Na] þ (calcd for C15H 20O 2Na, 255.1361). 3.3.3
Phaeocaulisin M (3)
Pale yellow oil; ½a25 D þ 83.3 (c 0.12, MeOH); UV (MeOH) lmax (log 1) 208 (3.78) nm; IR (KBr) vmax 3451, 1636, 1384, 1117 cm21; 1H NMR (600 MHz, CD 3OD); and 13 C NMR (75 MHz, CD3OD) spectral data, see Table 1; HRESI-MS: m/z 307.1517 [M þ Na]þ (calcd for C15H24O5Na, 307.1521). 3.3.4 Phagermadiol (4) Amorphous powder; ½a25 D – 36.7 (c 0.12, MeOH); UV (MeOH) lmax (log 1) 224 (3.86); IR (KBr) vmax 3421, 2979, 1676, 1456, 1383, 1313 cm 21; 1H NMR (600 MHz, CD 3OD); and 13C NMR (75 MHz, CD3OD) spectral data, see Table 1; HR-ESI-MS: m/z 289.1414 [M þ Na]þ (calcd for C15H22O4Na, 289.1416).
Downloaded by [UQ Library] at 00:48 16 July 2015
Journal of Asian Natural Products Research 3.4 NO Production bioassay The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 1 £ 105 cells/well, and stimulated with 1 mg/ml of LPS in the presence or absence of test compounds. After incubation at 378C for 24 h, 100 ml of cell-free supernatant was mixed with 100 ml of Griess reagent (mixture of equal volumes of reagent A and reagent B, A: 1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid, B: 0.1% (w/v) of N-(1naphthyl) ethylenediamine). Absorbance was measured in a microplate reader at 540 nm. Nitrite concentrations and the inhibitory rates were calculated by a calibration curve prepared with sodium nitrite standards [3,23]. Experiments were performed in triplicate, and data are expressed as the mean ^ SD of three independent experiments. Acknowledgements
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
We are grateful to Dr Paul Owusu Donkor for refining the language of our manuscript. [11]
Disclosure statement No potential conflict of interest was reported by the authors.
[12]
Funding This work was financially supported by grants in part from the National Natural Science Foundation of China (NSFC) [grant number 30973630], [grant number 81430095]; and the Medicinal Chemistry Subject Construction Project of Shenyang Pharmaceutical University [grant number 20130017].
References [1] J. Li, F. Zhao, M.Z. Li, L.X. Chen, and F. Qiu, J. Nat. Prod. 73, 1667 (2010). doi:10.1021/np100392m. [2] J. Li, C.R. Liao, J.Q. Wei, L.X. Chen, F. Zhao, and F. Qiu, Bioorg. Med. Chem.
[13]
[14]
[15] [16]
539
Lett. 21, 5363 (2011). doi:10.1016/j.bmcl. 2011.07.012. Y. Lou, F. Zhao, H. He, K.F. Peng, X.H. Zhou, L.X. Chen, F. Qiu, and J. Asian, Nat. Prod. Res. 11, 737 (2009). Y. Lou, F. Zhao, H. He, K.F. Peng, L.X. Chen, and F. Qiu, Chem. Biodivers. 7, 1245 (2010). doi:10.1002/cbdv. 200900160. Y. Liu, J.H. Ma, Y. Wang, P.O. Donker, Q. Li, S.Y. Gao, Y.G. Hou, Y. Xu, J.N. Cui, L.Q. Ding, F. Zhao, N. Kang, L.X. Chen, and F. Qiu, Eur. J. Org. Chem. 5540, 2014 (2014). Y. Liu, J.H. Ma, Q. Zhao, C.R. Liao, L.Q. Ding, L.X. Chen, F. Zhao, and F. Qiu, J. Nat. Prod. 76, 1150 (2013). doi:10.1021/np400202f. C. Tohda, N. Nakayama, F. Hatanaka, and K. Komatsu, Evid-Based Complement. Altern. 3, 255 (2006). doi:10.1093/ecam/ nel008. T. Masuda, A. Jitoe, J. Isobe, N. Nakatani, and S. Yonemori, Phytochemistry 32, 1557 (1993). doi:10.1016/0031-9422(93) 85179-U. X.P. Chen, L.X. Pei, Z.F. Zhong, J.J. Guo, Q.W. Zhang, and Y.T. Wang, Phytomedicine 18, 1238 (2011). doi:10.1016/j. phymed.2011.06.017. M.S. Ali, Y. Tezuka, S. Awale, A.H. Banskota, and S. Kadota, J. Nat. Prod. 64, 289 (2001). doi:10.1021/ np000496y. M. Li, Z. Zhang, D.L. Hill, H. Wang, and R. Zhang, Cancer Res. 67, 1988 (2007). doi:10.1158/0008-5472.CAN-06-3066. Q.F. Tao, Y. Xu, R.Y.Y. Lam, B. Schneider, H. Dou, P.S. Leung, S.Y. Shi, C.X. Zhou, L.X. Yang, R.P. Zhang, Y.C. Xiao, X. Wu, J. Sto¨ckigt, S. Zeng, C.H.K. Cheng, and Y. Zhao, J. Nat. Prod. 71, 12 (2008). doi:10.1021/ np070114p. H. Matsuda, T. Morikawa, K. Ninomiya, and M. Yoshikawa, Tetrahedron 57, 8443 (2001). doi:10.1016/S0040-4020(01) 00828-6. H. Matsuda, T. Morikawa, K. Ninomiya, and M. Yoshikawa, Bioorg. Med. Chem. 9, 909 (2001). doi:10.1016/S0968-0896 (00)00306-0. P. Dohare, P. Garg, U. Sharma, N.R. Jagannathan, and M. Ray, BMC Complement. Altern. Med. 8, 55 (2008). Pharmacopoeia Commission of People’s Republic of China, Pharmacopoeia of the People’s Republic of China, 2010 Chinese
540
[17] [18] [19]
Downloaded by [UQ Library] at 00:48 16 July 2015
[20]
J.-H. Ma et al. ed. (Chinese Medical Science and Technology Press, Beijing, 2010). M. Rohr, P. Naegeli, and J.J. Daly, Phytochemistry 18, 279 (1979). doi:10. 1016/0031-9422(79)80070-9. S.S. Sawant, D.T.A. Youssef, P.W. Sylvester, V. Wali, and K.A.E. Sayed, Nat. Prod. Commun. 2, 117 (2007). H.F. Wong and G.D. Brown, J. Chem. Res. 2002, 30 (2002). doi:10.3184/ 030823402103170376. G.P. Yin, L.C. Li, Q.Z. Zhang, Y.W. An, J.J. Zhu, Z.M. Wang, G.X. Chou, and
Z.T. Wang, J. Nat. Prod. 77, 2161 (2014). doi:10.1021/np400984c. [21] K. Masanori, U. Akira, U. Kaoru, and S. Sadao, Chem. Pharm. Bull. 35, 53 (1987). doi:10.1248/cpb.35.53. [22] H. Sumioka, L. Harinantenaina, K. Matsunami, H. Otsuka, M. Kawahata, and K. Yamaguchi, Phytochemistry 72, 2165 (2011). doi:10.1016/j.phytochem. 2011.08.004. [23] V.M. Dirsch, H. Stuppner, and A.M. Vollmar, Planta Med. 64, 423 (1998). doi:10.1055/s-2006-957473.
