Article pubs.acs.org/jnp
Inhibition Potential of Cycloartane-Type Glycosides from the Roots of Cimicif uga dahurica against Soluble Epoxide Hydrolase Nguyen Phuong Thao,†,‡ Bui Thi Thuy Luyen,§ Ji Sun Lee,† Jang Hoon Kim,⊥ Nguyen Tien Dat,‡ and Young Ho Kim*,† †
College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Vietnam § Department of Pharmaceutical Industry, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam ⊥ Korea Atomic Energy Research Institute, Jeongeup, Jeollabuk-do 580-185, Republic of Korea ‡
S Supporting Information *
ABSTRACT: A phytochemical assay-guided fractionation of the 95% ethanol extract of Cimicif uga dahurica roots afforded 29 9,19cycloartane triterpenoid glycosides, including the new cimiricasides A−F (1−6). The structures of 1−6 were established using contemporary NMR methods and from the HRESIMS data, and the sugar moiety in each case was confirmed by acid hydrolysis and subsequent GC/MS analysis. Compounds 2, 4, 5, 7−9, 18, 25, and 29 showed soluble epoxide hydrolase inhibitory effects with IC50 values of 0.4 ± 0.1 to 24.0 ± 0.2 μM. The compounds were analyzed by enzyme kinetic studies to explore the binding mode between the ligand and receptor. Compounds 4 (mixed type), 8, 18, and 29 (noncompetitive type) bound to a preferred allosteric site, while compounds 2, 5, 7, 9, and 25 had competitive interactions at the active site. The binding mechanism of selected inhibitors was investigated using molecular docking and dynamics simulations. 25.0 μg/mL).12,13 This species is one of approximately 28 species in the genus Cimicif uga (now known as Actaea), which is found in tropical rainforests and dry forests in East Asia, Europe, and North America.14−16 In mainland China, Japan, and Korea, Cimicif uga species have been used to treat anxiety, colds and influenza, diarrhea, dyspepsia, headaches, hysteria, inflammation of the gums, problems with female sexual arousal, rheumatism, sore throats, toothache, and urticaria.14 In recent years, considerable efforts have been directed toward examining their bioactive constituents and biological effects. Studies of several Cimicif uga species have investigated potential therapeutic or protective activities against diabetes mellitus, hot flashes, HIV infection (replication in H9 lymphocytes), and osteoporosis, as well as antiprotozoal and other vasoactive properties.14,16−19 However, the sEH inhibitory effects from the components of C. dahurica have not been reported previously. From an active extract of C. dahurica, six new cycloartane-type glycosides, namely, cimiricasides A−F (1−6), and 23 known compounds were identified. Moreover, several structure- and ligand-based pharmacophore models, including different binding modes of the ligands, were suggested, thoroughly covering an active compound series for identification of the inhibition potential on sEH.
S
oluble epoxide hydrolase (sEH) catalyzes the hydrolysis of epoxyeicosatrienoic acids (EETs), lipid intermediates derived from arachidonic acid via the cytochrome P450 (CYP450) epoxygenase pathway. Notably, 5,6-, 8,9-, 11,12-, and 14,15-EET are the products of CYP450 epoxygenase and major anti-inflammatory arachidonic acid metabolites with various biological activities.1,2 Several pathways are associated with the degradation of EETs, but the major one is catalyzed by sEH. sEH converts the epoxide structure into the corresponding diol, decreasing its bioactivity and leading to its rapid metabolism. The inhibition potential of sEH is hypothesized to increase the cardiovascular properties of EETs, including inflammation, hypertension, cardiac hypertrophy, and atherosclerosis.3,4 Similarly, the cardioprotective properties of EETs are expected to be reduced when they are hydrolyzed to their corresponding dihydroxyeicosatrienoic acids (DHETs) by sEH.1,2,5−9 Although there is no known receptor for EETs, they block the nuclear translocation of NF-κB10 and downregulate iNOS and COX-2,11 all of which influence many downstream biological activities. sEH inhibitors have low solubility and relatively limited durations of action. Thus, there is a continuing need for novel compounds possessing sEH inhibitory effects. The purpose of this study was to identify new sEH inhibitors from a higher plant species. The 95% ethanol extract of Cimicif uga dahurica (Turcz.) Maxim. (Ranunculaceae) roots showed potential inhibitory activities on sEH (68.1 ± 1.2%, © 2017 American Chemical Society and American Society of Pharmacognosy
Received: February 24, 2017 Published: May 30, 2017 1867
DOI: 10.1021/acs.jnatprod.7b00166 J. Nat. Prod. 2017, 80, 1867−1875
Journal of Natural Products
Article
glycoside with one acetoxy group attached to the triterpenoid aglycone or monoglycoside moiety. The NMR spectra of 1 (Tables 1 and 2) and 24-epi-7,8didehydrocimigenol 3-O-β-D-xylopyranoside (15) 20 were closely comparable except for the presence of an additional acetylated group. The 1H and 13C NMR spectra of 1 showed one anomeric signal [δH 4.67 (d, J = 7.5 Hz, H-1′)/δC 104.6 (C-1′)]. The ROESY spectrum exhibited correlations associated with the H-3 proton with Me-29, H-1′ with H-3, and H1′ with Me-29, confirming the attachment of the monoglycoside moiety at C-3. The D-xylose moiety was suggested to be in the β-configuration by the large coupling constant of the anomeric proton (J1,2 = 7.5 Hz) in the 1H NMR spectrum (Table 1). Additionally, when combined with the key correlations observed in the ROESY spectrum of H-1′ (axial), H-2′ (equatorial), H-3′ (axial), and H-4′ (equatorial), it indicated that C-2′, C-3′, and C-4′ are α-, β-, and αconfigurations, respectively. These findings confirmed that the monoglycoside unit of 1 is a xylopyranoside moiety. Moreover, the obvious downfield shift of H-2′ [δH 5.39 (dd, J = 7.5, 8.0 Hz)] in 1, with an upfield shift of H-2′ [δH 3.96 (dd, J = 7.5, 8.0 Hz)] in 15, suggested that the acetylated moiety is esterified by the hydroxy group at C-2′, rather than at other positions of the triterpenoid aglycone moiety. Based on this, it was verified that the location of an acetyl moiety is attached at C-2′ in the xylopyranoside moiety of 1, further supported by the HMBC cross-peaks that were observed between δH 5.39 (H-2′) and δC 170.1 (-OAc, CO), and between δH 2.02 (-OAc, CH3) and 170.1 (-OAc, CO). Other 1H−1H COSY and HMBC correlations (Figure S37, Supporting Information) were used to identify unambiguously the structure of 1, and the sugar moiety was confirmed as a D-xylopyranoside by acid hydrolysis, GC/ MS analysis, and its positive specific rotation value ([α]25 D +34.2, c 0.15, H2O). The relative configuration of 1 was determined on the ROESY correlations and based on the generally comparable NMR data with 15,20 in which H-3, H-15, H-23, and H-24 were determined to have the α-, β-, β-, and βorientation, respectively. The side-chain signals associated with three proton resonances of H-24, H3-26, and H3-27 were used as the focal points for establishing the relative configuration. The H-24 proton exhibited ROESY cross-peaks with H-23, H326, and H3-27. Key ROESY cross-peaks were observed between H-23 and the neighboring H-22a, H-22b, and H-20 protons, and between H-15 and H3-18 (Supporting Information). Moreover, agreement of the 13C NMR chemical shifts at C-3 (δC 88.5), C-15 (δC 78.7), C-23 (δC 73.9), and C-24 (δC 84.1) in 1 with those of 15 [C-3 (δC 88.5), C-15 (δC 78.7), C-23 (δC 73.8), and C-24 (δC 84.3)] suggested that these two compounds have the same configurations at C-3, C-15, C-23, and C-24. However, it showed major differences from 7,8didehydrocimigenol 3-O-β-D-xylopyranoside (11) [C-3 (δC 88.5), C-15 (δC 78.4), C-23 (δC 71.4), and C-24 (δC 90.5)].20 These were consistent with those reported for the β-orientations at C-23 and C-24, but different from those published for an α-orientation at C-24.20,21 A noteworthy observation was that H-23 in 1 appeared as a ddd (J = 2.5, 4.2, 9.5 Hz), while H-23 in 11 appeared as a br d (J = 9.0 Hz). Moreover, H-24 in 1 showed an additional coupling of 4.2 Hz when compared with the H-24 (singlet) in 11. The additional coupling between H-23 and H-24 in 1 relative to 11 suggested that the dihedral angle between H-23 and H-24 in 1 is different from that in 11 (where the configurations at C-23 and C-24 are β- and α-orientations, respectively). The configuration was
■
RESULTS AND DISCUSSION Cimiricaside A (1) was found to be a white, amorphous powder, and the molecular formula C37H56O10 was determined by HRESIMS, with a protonated molecular ion peak at m/z 661.3953 [M + H]+ (calcd 661.3952) and a sodium adduct molecular ion peak at m/z 683.3773 [M + Na]+ (calcd 683.3771). A detailed assessment of the NMR data indicated that 1 is a cycloartane-type triterpenoid glycoside, a compound class known as major components of Cimicif uga species.14 The 1 H NMR spectrum of 1 exhibited the resonances of nonequivalent protons of a cyclopropyl methylene signal [δH 0.36 and 0.90 (each 1H, d, J = 4.2 Hz, H-19a/H-19b)], a secondary [δH 0.88 (d, J = 6.0 Hz, Me-21)], and six tertiary methyl groups [δH 1.07 (s, Me-18), 1.29 (s, Me-26), 0.97 (s, Me-27), 1.16 (s, Me-28), 1.17 (s, Me-29), and 0.87 (s, Me30)], an acetyl group [δH 2.02 (s, -OAc)], an olefinic methine proton [δH 5.95 (dd, J = 1.2, 7.4 Hz, H-7)], and an anomeric proton signal [δH 4.67 (d, J = 7.5 Hz, H-1′)] (Table 1). The above evidence, together with the diagnostic signals for two oxygen-bearing methine carbons [δC 73.9 (C-23) and 84.1 (C24)] and a ketal carbon [δC 112.5 (C-16)] in the 13C NMR spectrum, confirmed that 1 is a 9,19-cimigenol-type mono1868
DOI: 10.1021/acs.jnatprod.7b00166 J. Nat. Prod. 2017, 80, 1867−1875
1869
a
s s s s
m m m dd (4.2, 11.4) dd (4.2, 12.0) m m m dd (1.2, 7.4) m m m m br s m s d (4.2) d (4.2) m d (6.0) m ddd (2.5, 6.0, 13.0) ddd (2.5, 4.2, 9.5) d (4.2) s
4.67, 5.39, 4.02, 4.04, 3.55, 4.17,
d (7.5) dd (7.5, 8.0) br t (8.0) ddd (5.0, 8.0, 10.0) dd (10.0, 11.0) dd (5.0, 11.0)
2.02, s
0.97, 1.16, 1.17, 0.87,
1.32, 1.60, 1.83, 2.13, 3.25, 1.16, 1.51, 1.83, 5.95, 1.12, 2.08, 1.53, 1.68, 4.39, 1.36, 1.07, 0.36, 0.90, 1.62, 0.88, 0.98, 2.56, 4.50, 3.61, 1.29,
1
4.78, 3.97, 4.29, 4.17, 3.66, 4.29,
1.16, 1.34, 1.23, 0.97, 3.11,
1.29, 1.64, 1.90, 2.28, 3.42, 1.18, 1.52, 1.80, 6.03, 1.13, 2.11, 1.56, 1.70, 4.46, 1.37, 1.06, 0.41, 0.96, 1.67, 0.79, 0.92, 2.17, 4.56, 3.59, 1.20,
d (7.5) dd (7.5, 8.0) dd (8.0, 8.0) ddd (5.0, 8.0, 10.0) dd (10.0, 11.0)) dd (5.0, 11.0
s s s s s
m m m dd (4.2, 11.4) dd (4.2, 12.0) m m m dd (1.2, 7.4) m m m m s m s d (4.2) d (4.2) m d (6.0) m ddd (2.5, 6.0, 13.0) br d (9.0) s s
2
1.93, 1.86, 4.73, 3.92, 4.93, 4.12, 3.62, 4.26,
1.18, 1.33, 1.22, 0.95,
1.26, 1.66, 1.87, 2.25, 3.39, 1.17, 1.50, 1.79, 6.06, 1.14, 2.07, 1.56, 1.71, 4.45, 1.37, 1.06, 0.41, 0.95, 1.69, 0.80, 0.91, 2.19, 4.52, 4.24, 1.21,
s s d (7.5) dd (7.5, 8.0) br t (8.0) ddd (5.0, 8.0, 10.0) dd (10.0, 11.0) dd (5.0, 11.0)
s s s s
m m m m dd (4.2, 12.0) m m m dd (1.2, 7.4) m m m m s m s d (4.2) d (4.2) m d (6.0) m ddd (2.5, 6.0, 13.0) br d (9.0) s s
3
Assignments were made using the HMQC, HMBC, COSY, and ROESY spectra. bOverlapped signals.
6′
27 28 29 30 OCH3 COCH3 COCH3 1′ 2′ 3′ 4′ 5′
23 24 26
20 21 22
15 17 18 19
12
7 11
3 5 6
2
1
position
4.78, 3.96, 4.09, 4.17, 3.68, 4.30,
4.45, 4.38, 4.51, 1.60, 1.40, 1.26, 0.99,
1.31, 1.67, 1.90, 2.27, 3.42, 1.24, 1.47, 1.82, 5.80, 1.12, 2.10, 1.56, 1.69, 4.73, 1.76, 1.40, 0.44, 1.05, 2.09, 0.85, 1.78, 2.00,
d (7.5) dd (7.5, 8.0) br t (8.0) ddd (5.0, 8.0, 10.0) dd (10.0, 11.0) dd (5.0, 11.0)
s d (9.5) d (9.5) s s s s
m m m dd (4.2, 11.4) dd (4.2, 12.0) m m m dd (1.2, 7.4) m m m m s m s d (4.2) d (4.2) m d (6.0) dd (2.5, 6.0) ddd (2.5, 6.0, 13.0)
4
Table 1. 1H NMR Spectroscopic Data (600 MHz, Pyridine-d5) of Cimiricasides A−F (1−6), δH in ppm, J in Hza
4.79, 3.96, 4.29, 4.16, 3.68, 4.30,
1.51, 1.35, 1.26, 0.97,
1.30, 1.65, 1.88, 2.27, 3.42, 1.23, 1.59, 1.91, 5.93, 1.10, 2.10, 1.59, 1.91, 4.40, 1.76, 1.18, 0.44, 0.99, 1.72, 0.96, 1.75, 2.08, 4.41, 4.23, 1.46,
d (7.5) dd (7.5, 8.0) dd (8.0, 8.0) ddd (5.0, 8.0, 10.0) dd (10.0, 11.0) dd (5.0, 11.0)
s s s s
m m m br d (4.2) dd (4.2, 12.0) m m m dd (1.2, 7.4) m m m m s m s d (4.2) d (4.2) m d (6.0) m m m d (8.5) s
5
s s d (8.0) dd (8.0, 9.0) dd (3.4, 9.0) d (3.4) m
s s s s
3.53, dd (2.4, 11.4) 4.27, dd (5.4, 11.4)
2.04, 1.89, 4.77, 4.36, 3.96, 5.31, 4.28,
1.38, 1.35, 1.23, 0.94,
1.26, m 1.62, m 1.86, m 2.20, br d (4.2) 3.50, dd (4.2, 12.0) 1.21, m 1.62, m 1.96, m 5.90, dd (1.2, 7.4) 1.10, m 2.12, m 1.58, m 1.89, m 4.35, s 1.73, m 1.16, s 0.43, d (4.2) 0.98, d (4.2) 1.74, m 0.92, d (6.0) 1.74, m 1.99b 4.18, m 5.67, d (8.5) 1.42, s
6
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.7b00166 J. Nat. Prod. 2017, 80, 1867−1875
Journal of Natural Products
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Table 2. 13C NMR Spectroscopic Data (150 MHz, Pyridine-d5) of Cimiricasides A−F (1−6)a
a
position
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OCH3 COCH3 COCH3 COCH3 COCH3 1′ 2′ 3′ 4′ 5′ 6′
30.3, 29.7, 88.5, 40.2, 42.6, 21.9, 114.3, 148.2, 21.4, 28.2, 25.6, 34.0, 41.2, 50.9, 78.7, 112.5, 60.7, 21.4, 28.4, 23.5, 19.8, 29.5, 73.9, 84.1, 68.7, 30.8, 25.6, 18.6, 26.1, 14.2,
2 CH2 CH2 CH C CH CH2 CH C C C CH2 CH2 C C CH C CH CH3 CH2 CH CH3 CH CH CH C CH3 CH3 CH3 CH3 CH3
30.1, 29.4, 88.0, 40.2, 42.4, 21.5, 114.0, 147.7, 21.0, 28.1, 25.3, 33.8, 41.0, 50.3, 77.9, 112.0, 59.0, 21.4, 28.1, 23.7, 19.5, 37.7, 71.5, 88.0, 76.0, 19.1, 21.9, 18.2, 25.5, 14.1, 49.0,
3 CH2 CH2 CH C CH CH2 CH C C C CH2 CH2 C C CH C CH CH3 CH2 CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3 CH3
170.1, C 21.8, CH3 104.6, 75.7, 76.3, 71.4, 67.2,
CH CH CH CH CH2
107.3, 75.4, 78.4, 71.0, 66.9,
CH CH CH CH CH2
4
30.1, 29.3, 88.0, 40.1, 42.4, 21.5, 114.0, 147.7, 21.4, 27.9, 25.3, 33.8, 40.9, 50.2, 78.3, 112.5, 59.0, 21.9, 28.1, 23.6, 19.4, 37.5, 72.5, 86.5, 82.9, 21.2, 25.5, 18.2, 25.5, 14.1,
CH2 CH2 CH C CH CH2 CH C C C CH2 CH2 C C CH C CH CH3 CH2 CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3
170.4, 169.9, 21.9, 20.9, 107.3, 75.5, 80.5, 70.9, 66.9,
C C CH3 CH3 CH CH CH CH CH2
30.0, 29.3, 87.9, 40.1, 42.5, 21.5, 113.3, 148.6, 21.0, 28.2, 25.1, 31.9, 40.4, 53.1, 79.8, 115.2, 58.9, 19.7, 28.4, 27.7, 19.1, 39.8, 116.8, 88.6, 77.6, 83.6, 22.6, 19.7, 25.5, 14.0,
107.2, 75.3, 78.3, 70.9, 66.9,
5 CH2 CH2 CH C CH CH2 CH C C C CH2 CH2 C C CH C CH CH3 CH2 CH CH3 CH2 C CH C CH2 CH3 CH3 CH3 CH3
CH CH CH CH CH2
30.3, 29.5, 88.1, 40.3, 42.6, 21.1, 113.5, 148.9, 21.7, 28.3, 25.4, 33.7, 41.6, 49.8, 80.2, 102.5, 60.8, 22.6, 28.3, 27.6, 21.8, 32.2, 77.1, 80.4, 72.5, 27.1, 26.9, 18.1, 25.7, 14.3,
107.5, 75.5, 78.6, 71.2, 67.1,
6 CH2 CH2 CH C CH CH2 CH C C C CH2 CH2 C C CH C CH CH3 CH2 CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3
CH CH CH CH CH2
30.0, CH2 29.2, CH2 87.9, CH 40.1, C 42.4, CH 20.8, CH2 113.1, CH 148.7, C 21.5, C 28.1, C 25.1, CH2 33.5, CH2 41.2, C 49.7, C 79.7, CH 102.8, C 60.4, CH 22.3, CH3 28.0 CH2 26.7, CH 21.3, CH3 32.4, CH2 74.6, CH 81.1, CH 71.8, C 26.9, CH3 27.0, CH3 17.8, CH3 25.4, CH3 14.0, CH3 170.3, 170.2, 20.9, 20.6, 107.0, 73.8, 75.4, 72.8, 78.3, 62.9,
C C CH3 CH3 CH CH CH CH CH CH2
Assignments were confirmed from the DEPT, HMQC, HMBC, COSY, and ROESY spectra.
determined as β-oriented at C-23 and C-24 from the analysis of the spin coupling pattern. From all of this information, the structure of cimiricaside A (1) was established as 2′-O-acetyl24-epi-7,8-didehydrocimigenol 3-O-β-D-xylopyranoside. Cimiricaside B (2) was also obtained as a white, amorphous powder and gave the molecular formula C36H56O9, which was identified by the HRESIMS [M + H]+ protonated molecular ion peak at m/z 633.4005 (calcd 633.4002) and the sodium adduct molecular ion peak at m/z 655.3823 [M + Na]+ (calcd 655.3822). The 1H and 13C NMR spectra of 2 (Tables 1 and 2) were similar to those of 7,8-didehydrocimigenol 3-O-β-Dxylopyranoside (11),20 except for additional signals due to a methoxy group. Furthermore, the position of the attached methoxy group was supported by a downfield chemical shift of δC 76.0 (C-25) in the 13C NMR spectra (Table 2) and HMBC correlations of δH 3.11 (OMe) with C-25 (δC 76.0), and between δH 1.20 (s, H3-26) and 1.16 (s, H3-27) with C-25 (δC
76.0) (Figure S37, Supporting Information). From these data, it was concluded that there is a methoxy group attachment at C25 of 2. The relative configuration of 2 was deduced based on the ROESY correlations and other NMR data comparison with a previous report. The coupling pattern of H-3 (dd, J = 4.2, 12.0 Hz), which was similar to that of H-3 (dd, J = 4.2, 12.0 Hz) in 11,20 was used to determine that H-3 is α-oriented. Additionally, analysis of the relatively large coupling constants of H-23 [δH 4.56 (br d, J = 9.0 Hz)] and H-24 [δH 3.59 (s)], together with the 13C NMR data of 2 (Table 2) and those of reported 9,19-cycloartane-type glycosides,14,20,21 suggested that β- and α-orientations are evident at the C-23 and C-24 positions, respectively. Based on the above analysis, the same conformation and relative configuration of 11 was assigned for 2. Moreover, correlations between H-3/H-5, H-3/H3-29, and H-5/H3-29; between H3-28/H-17 and H-17/H-24; and between H3-18, H-15, and H-20 with H-23 were observed in 1870
DOI: 10.1021/acs.jnatprod.7b00166 J. Nat. Prod. 2017, 80, 1867−1875
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explained by the presence of a C-23−C-26 ether linkage in 4. This suggestion was confirmed by the HMBC cross-peaks observed between δH 4.38 (H-26a) and 4.51 (H-26b) with C23 (δC 116.8), C-25 (δC 77.6), and C-27 (δC 22.6) as well as cross-peaks between δH 1.60 (s, H3-27) with C-24 (δC 88.6), C25 (δC 77.6), and C-26 (δC 83.6), and between δH 1.78 (H22a), 2.00 (H-22b), and δH 4.45 (s, H-24) with C-23 (δC 116.8) (Figure 1). Additionally, the sugar moiety present in 4
the ROESY spectrum (Supporting Information), indicating the α-configurations of H-3 and H-24 and the β-configurations of H-15 and H-23. Therefore, the structure of 2 was determined as 25-O-methyl-7,8-didehydrocimigenol 3-O-β-D-xylopyranoside. The molecular formula of cimiricaside C (3) was determined as C39H58O11 from the protonated molecular ion peak [M + H]+ at m/z 703.4059 (calcd 703.4057) in the HRESIMS. In the 1 H NMR spectrum, signals were observed for two acetyl groups [δH 1.86 and 1.93 (each 3H, s)], an anomeric proton of the xylose moiety [δH 4.73 (d, J = 7.5 Hz, H-1′)], seven methyl groups [δH 1.06 (s, Me-18), 0.80 (d, J = 6.0 Hz, Me-21), 1.21 (s, Me-26), 1.18 (s, Me-27), 1.33 (s, Me-28), 1.22 (s, Me-29), and 0.95 (s, Me-30)], and proton signals at δH 0.41 and 0.95 (each 1H, d, J = 4.2 Hz, H-19a/H-19b) characteristic for the nonequivalent protons of a cyclopropyl methylene group. These observations suggested that 3 is also a triterpenoid glycoside with a cycloartane skeleton. Further analysis of the NMR spectroscopic data of 3 (Tables 1 and 2) and comparison with those of its analogue 24-epi-25-O-acetyl-7,8-didehydrocimigenol 3-O-β-D-xylopyranoside (14)21 indicated that these compounds have a similar aglycone, except for the presence of the signals associated with the sugar unit and an additional signal due to an acetyl group. In 14, an H-3′ resonance was observed at δH 4.05 (dd, J = 8.0, 8.0 Hz), while in 3 it was shifted downfield to δH 4.93 (br t, J = 8.0 Hz). Additionally, the C-3′ (δC 78.7) resonance in 14 was shifted downfield to δC 80.5 (C-3′) in 3, consistent with the presence of an acetyl group attached to the C-3′ of a xylose moiety. This inference was supported by the correlation between δH 4.93 (H-3′) and δC 169.9 (CO) in the HMBC spectrum (Figure S37, Supporting Information). The α-configuration for H-3 was determined in the same manner as for 2 on the basis of ROESY correlations between H-3 [δH 3.39 (dd, J = 4.2, 12.0 Hz)], H-5 (δH 1.17), and H3-29 (δH 1.22), whereas another correlation between H-15 (δH 4.45) and H3-18 (δH 1.06) confirmed the αconfiguration for the substituent OH-15. Based on the analysis of the proton coupling constants of H-23 [δH 4.52 (br d, J = 9.0 Hz)] and H-24 [δH 4.24 (s)] of 3 with those of known 9,19cycloartane-type glycosides,14,20,21 it was considered that β- and α-configurations should be assigned to C-23 and C-24, respectively. Therefore, the structure of 3 was determined as 25,3′-O-diacetyl 7,8-didehydrocimigenol 3-O-β-D-xylopyranoside. The sodium adduct molecular ion peak at m/z 655.3458 [M + Na]+ (calcd for C35H52NaO10, 655.3458) in the HRESIMS, together with the presence of 35 signals in its 13C NMR spectrum, supported a molecular formula of C35H52O10 for cimiricaside D (4, also obtained as a white, amorphous powder). According to the analysis of the 1D and 2D NMR spectroscopic data (Tables 1 and 2), 4 was found to be typical of a cycloartane triterpenoid monoglycoside and exhibited some similarities to those of 7,8-didehydrocimigenol 3-O-β-Dxylopyranoside (11),20 including peaks for a xylose moiety and resonances for a cyclopropane methylene group [δH 0.44 and 1.05 (each 1H, d, J = 4.2 Hz, H-19a/H-19b)], except for differences corresponding to the side-chain signals. The main differences in the 13C NMR spectra of both compounds 4 and 11 concerned the C-21 to C-27 resonances [δC 19.1 (C-21), 39.8 (C-22), 116.8 (C-23), 88.6 (C-24), 77.6 (C-25), 83.6 (C26), and 22.6 (C-27)]. The H-26 resonance was shifted downfield, from δH 1.21 (s) in 11 to δH 4.38 and 4.51 (each 1H, d, J = 9.5 Hz) in 4. The downfield shift of H-26 may be
Figure 1. Key HMBC (blue arrows), COSY (thick black bonds), and ROESY (red dotted double arrows) correlations for 4.
was determined to be a xylopyranoside moiety by the 13C NMR resonances, 1H NMR sugar spin−spin coupling patterns, and from the 1H−1H COSY, HSQC, and HMBC experiments conducted (Figure 1). The closely comparable NMR spectra for the sugar component of 4 (Tables 1 and 2) to those of 7− 29 and the previously reported 9,19-cycloartane-type glycoside derivatives,14,20,21 together with the acid hydrolysis experiments, suggested that the same D-xylopyranoside unit occurred in 4. In order to determine the relative configuration of 4, the ROESY spectrum was analyzed. ROESY correlations were observed for the proton network associated with the side-chain configuration and the presence of a hydroxy group at C-15, which further defined the relative configuration of 4. Key ROESY correlations between H3-28 (δH 1.40) with H-17 (δH 1.76) and H-17 (δH 1.76) with H-24 (δH 4.45) indicated that H-17, H-24, and Me-28 could be assigned as α-oriented. Other ROESY correlations between H3-18 with H-19b, H3-18/H-15, and H-19b/H-15, between H-3 with H-5, H-3 with H3-29, and H3-29 with H-5, indicated that H-3, H-15, and Me-29 are α-, β-, and α-oriented, respectively (Figure 1). Therefore, cimiricaside D (4) was assigned as 16β,23;16α,24;23,26-triepoxyl-cycloart7-en-3β,15α,25-triol 3-O-β-D-xylopyranoside. A molecular formula of C35H56O10 was established for cimiricaside E (5, obtained as a white, amorphous powder) based on the presence of 35 signals in its 13C NMR spectrum and the HRESIMS sodium adduct molecular ion peak at m/z 659.3775 [M + Na]+ (calcd 659.3771). Analysis of the 1H and 13 C NMR data of 5 (Tables 1 and 2) suggested that this compound shares several structural similarities with 24-epi-24O-hydroxy-7,8-didehydrohydroshengmanol 3-O-β-D-galactopyranoside,22 except for major differences in the resonances associated with the sugar unit. However, as observed in the NMR spectra, the sugar moiety resonances of 5 were the same as those in 2, confirming the presence of a xyloside moiety. By 1871
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diacetyl-7,8-didehydrohydroshengmanol 3-O-β-D-galactopyranoside. In addition to the above-mentioned new compounds (1−6), 23 known compounds (7−29, Figure S36, Supporting Information) were also isolated and identified, including cimiaceroside B (7),25 3β,15α,16α,24α-tetrahydroxy-25,26,27trinor-16,24-cyclo−cycloart-7-en-23-one 3-O-β-D-xylopyranoside (8),26 25-O-acetyl-7,8-didehydrocimigenol 3-O-β-D-xylopyranoside (9),20 25-O-acetylcimigenol 3-O-β-D-xylopyranoside (10),27 7,8-didehydrocimigenol 3-O-β-D-xylopyranoside (11),20 cimiside A (12),28 cimigenol 3-O-β-D-xylopyranoside (13),29 24-epi-25-O-acetyl-7,8-didehydrocimigenol 3-O-β-D-xylopyranoside (14),21 24-epi-7,8-didehydrocimigenol 3-O-β-D-xylopyranoside (15),20 3′-O-acetyl-24-epi-7,8-didehydrocimigenol 3-Oβ-D-xylopyranoside (16),21 (23R,24R)-16β,23;16α,24-diepoxy12β-acetoxy-cycloart-7-en-3β,15α,25-tetraol 3-O-β-D-xylopyranoside (17),28 (23R,24R)-16β,23;16α,24-diepoxy-cycloart-7en-3β,12β,15α,25-tetraol 3-O-β-D-xylopyranoside (18),28 7,8didehydro-25-anhydrocimigenol 3-O-β- D -xylopyranoside (19),30 25-anhydrocimigenol 3-O-β-D-xylopyranoside (20),30 cimiracemoside I (21),31 (26R)-cimicifugoside (22),32 3-O-β-Dxylopyranosyl-24S,25-dihydroxy-15-oxo-acta-(16R,23R)-16,23monoxoside (23),33 24-epi-24-O-acetyl-7,8-didehydrohydroshengmanol 3-O-β-D-xylopyranoside (24),20 24-O-acetylhydroshengmanol 3-O-β-D-xylopyranoside (25),34 7,8-didehydro-24-Oacetylhydroshengmanol 3-O-β-D-xylopyranoside (26),24 23-Oacetylshengmanol 3-O-β-D-xylopyranoside (27),27 23-O-acetyl7,8-didehydroshengmanol 3-O-β-D-xylopyranoside (28),35 and (23R,24R)-23-O-acetyl-3β,15,24β,25-tetrahydroxy-cycloart-7en-16-one 3-O-β-D-xylopyranoside (29).26 The inhibitory effects of the compounds isolated from C. dahurica were assessed against sEH in vitro. As shown in Table 3, the active compounds were subjected to enzymatic assays at
acid hydrolysis and subsequent GC/MS analysis, the sugar moiety was confirmed as D-xylose, which was attached to C-3 of the aglycone in 5. This position was confirmed by the correlation between δH 4.79 (d, J = 7.5 Hz, H-1′) and C-3 (δC 88.1) from the HMBC spectrum (Figure S37, Supporting Information). A key ROESY experiment showed correlations between H-15/H3-18, H-17/H3-21, and H3-18/H-20 but not between H-23 [δH 4.41 (m)] and δH 4.23 (d, J = 8.5 Hz, H-24). Moreover, the ROESY correlations of H-3 (δH 3.42) with H-5 (δH 1.23) and H3-29 (δH 1.26) indicated an α-configuration of H-3, whereas another observed correlation of δH 4.40 (H-15) with H3-18 (δH 1.18) was used to assign an α-configuration to OH-15. The correlation of the signal at δH 4.23 (H-24) with H17 (δH 1.76) and H3-21 (δH 0.96) indicated that H-24 has an αconfiguration. This finding also confirmed an β-configuration for the C-24 hydroxy group. Additionally, the large 1H NMR coupling constant of H-24 [δH 4.23 (d, J = 8.5 Hz)] also suggested the same configuration as those of several previously reported 9,19-cycloartane glycoside derivatives.21−23 Therefore, cimiricaside E (5) was determined as 24-epi-24-O-hydroxy-7,8didehydrohydroshengmanol 3-O-β-D-xylopyranoside. Cimiricaside F (6) was purified as a white, amorphous powder. A combination of HRESIMS (protonated molecular ion peak at m/z 751.4263 [M + H]+ (calcd 751.4263) and sodium adduct molecular ion peak at m/z 773.4083 [M + Na]+ (calcd 773.4088)) and its 13C NMR spectrum confirmed the molecular formula of C40H62O13. The NMR spectrum was analyzed after using 2D NMR methods, suggesting that 6 belongs to the same cycloartane triterpenoid glycoside type as cimiricaside E (5), but differed in the presence of two acetyl groups and from a variation between their sugar moieties. The sugar unit of 6 was confirmed as D-galactopyranoside by acid hydrolysis, GC/MS analysis, TLC comparison, and its positive specific rotation value. On comparing the NMR data of 6 with those of 24-epi-24-O-hydroxy-7,8-didehydrohydroshengmanol 3-O-β-D-galactopyranoside, their structures were found to be similar.22 The primary difference in the 1D NMR spectra of these compounds involved the C-4′ resonance of 6, which appeared upfield at δC 70.3/δH 4.61 in 24-epi-24-O-hydroxy7,8-didehydrohydroshengmanol 3-O-β-D-galactopyranoside (Tables 1 and 2). The position of an acetyl group in 6 was determined to be at the C-4′ position of the galactose moiety based on the downfield chemical shift of H-4′.20,22 This was supported further by a correlation observed in the HMBC spectrum between H-4′ (δH 5.31) and the carbonyl group at δC 170.2 (-OAc, CO) (Figure S37, Supporting Information). The β-configuration was assigned to the other acetyl group at the C-24 position, based on the diagnostic downfield chemical shift in comparison to the similar chemical shifts and multiplicities, ROESY correlations, and other NMR data of 6 and of 24-epi-24-O-hydroxy-7,8-didehydrohydroshengmanol 3O-β-D-galactopyranoside,22 which showed the same relative configuration. The similarity of the chemical shift and multiplicities at C-24 [δH 5.67 (d, J = 8.5 Hz)/δC 81.1] in 6 with a β-configuration [δH 4.10 (d, J = 8.4)/δC 80.5] and the major difference from an α-configuration [δH 4.82 (d, J = 2.0 Hz)/δC 80.2]24 supported a β-orientation for the configuration of C-24. This was consistent with other reports on the βconfiguration of C-24.20,22−24 Furthermore, this was supported by ROESY correlations of H3-28 (δH 1.35) with H-17 (δH 1.73), and of H-17 (δH 1.73) with H-24 (δH 5.67), indicating that H-17, H-24, and Me-28 are α-oriented. Based on these data, the structure of 6 was determined as 24-epi-24,4′-O-
Table 3. sEH Inhibitory Activities of Selected Compounds inhibition of compounds on sEHa compound 1 2 4 5 7 8 9 18 25 29 AUDAc
100 μM (%) 64.9 75.6 63.2 84.8 70.2 69.6 76.7 61.9 66.1 72.4
± ± ± ± ± ± ± ± ± ±
4.0 1.0 0.9 1.9 0.7 0.8 0.4 1.9 3.3 0.1
IC50 values (μM) 60.9 0.4 14.6 10.7 4.4 8.6 24.0 18.5 0.4 5.2 7.8
± ± ± ± ± ± ± ± ± ± ±
0.6 0.1 1.1 0.2 0.7 1.0 0.2 1.0 0.1 0.1 2.1 nM
binding mode, type NTb competitive mixed competitive competitive noncompetitive competitive noncompetitive competitive noncompetitive
a Compounds were tested in a set of experiments three times. bNT: Not tested. cAUDA (25.0 nM) was used as a positive control.
concentrations of 0.31−100 μM to determine IC50 values. Compounds 2, 4, 5, 7−9, 18, 25, and 29 gave inhibition rates of >60% (at 100 μM) compared to the control, 12-(3-adamantan1-yl-ureido)-dodecanoic acid (AUDA). These results were considered very promising. In particular, compounds 2 and 25, which demonstrated the most potent inhibition, also showed very low IC50 values (0.4 μM). Interestingly, the cycloartane glycoside derivatives 11 and 24, with only minor differences found in 2 and 25, respectively, were devoid of any sEH inhibitory effects. Compounds 4, 5, 7−9, 18, and 29 exhibited potent inhibitory effects, with IC50 values of 8.6−24.0 μM. In 1872
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position and participated in hydrogen bonding. The majority shared common residues (Asp335, Tyr343, Tyr383, Tyr466, Met469, Met503, and Gln505) for hydrogen bonding. Detailed information on the cycloartane glycoside derivatives tested is provided in Table 4 (Figure S39, Supporting Information). Molecular dynamics simulation, as an alternative approach, has been used extensively to explore atomic-level dynamic information regarding flap motion. All molecular dynamic studies were performed using Gromacs (version 5.1.2). The total and potential energies were from approximately −8.0 × e+5 to −9.8 × e+5 kJ/mol, respectively. The root-mean-square deviation (RMSD) of the protein was determined to examine the stability of the trajectory for the receptor−ligand complex. A comparison of the RMSD and subsequent conformational changes indicated binding mobility in the groups of inhibitors. The complexes of receptors and ligands 2 and 25 had an average RMSD of approximately 0.3 nm after 5 ns. The RMSD graphic showed that their simulation operated stably, without enzyme denaturation, over 20 ns (Figure S40C, Supporting Information). The conformation stabilized gradually, at ∼5 ps, with slight changes in the backbone structure. The root mean-square fluctuations (RMSFs) of receptor residues indicated the local mobility of the receptor. The results were created by plotting the RMSFs for the protein residues. Residues of the receptor with compound 2 showed flexibility with an RMSF < 0.35 nm, and the residues of the receptor with 25 also had an RMSF < 0.4 nm (Figure S40D, Supporting Information). Through this plot, the catalytic triad of the protein showed similar movements. In particular, Asp496 and His524 showed dramatic flexibility of 0.239 (2)/0.188 (25) and 0.149/0.158 nm, respectively. In contrast, Asp335 showed little fluidity of 0.0637 (2) and 0.0532 (25). Two ligands had full hydrogen bonds, with one- to three- (the former) or five- (the latter) hydrogen bonds for inhibitory activity during the 20,000 ps period (Figures S40E−F, Supporting Information). The ligands and key amino acids of the receptor and their distances involved in hydrogen bonding with ligands 2 and 25 were determined in a molecular simulation. The receptor complex with ligand 2 confirmed that this was sometimes located within 0.3 nm of a key amino acid (Tyr466), but not Asp335 (2.80, 3.01), in the catalytic triad. The other complex revealed that Gln384 (2.76, 2.63, 3.02) was a key amino acid in the hydrogen bond interaction with 25 because it consistently had a 0.35 nm distance. Inhibitor 2, although a competitive inhibitor, only formed a hydrogen bond with Asp335 (2.80 and 3.01) (catalytic triad), and this made an approach at a 0.35 nm distance with Asp335 and His523. The latter did not interact with the catalytic triad (Figure S41, Supporting Information). Finally, 11 frames were acquired at 2 ns intervals over the entire simulation time. Through the overlapped frames, two ligands (2 and 25) showed harmonic movements without collisions in the flexible sEH (Figure S42, Supporting Information). Of these ligands, 2 showed circular movement in the z-axis in the first docked state, whereas 25 moved around parts of the active site and the right pocket and then moved on the axis of the abscissa. Additionally, noncompetitive inhibitors (8 and 29) were bonded into the full right and left pocket, respectively. They showed dependent movement at their nearby loop. Through these results, they may not directly block substrate binding activity. However, their binding with the flexible loop was suggested to indirectly affect necessary enzyme mobility and the catalytic triad in the catalytic reaction (Figure S43, Supporting Information).
turn, six compounds (2, 5, 7, 8, 25, and 29) showed the most potent sEH inhibitory effects, with IC50 values that were below 10 μM (Table 3). Nine cycloartane glycoside derivatives, 2, 4, 5, 7−9, 18, 25, and 29, were used to investigate the binding mechanisms in the presence of inhibitors (0.09−50.0 μM) at various substrate concentrations (1.25−20.0 μM). As a result (Table 3), compounds 8, 18, and 29 were found to be noncompetitive inhibitors, with intercepts on the negative x-axis, that were shown to intercept with an allosteric site.36 The data for 4 showed several lines that crossed to the left of the vertical axis and above the horizontal axis; hence, increasing inhibitor concentrations resulted in a decrease in Vmax and an increase in Km,36 indicating that mixed-type inhibition occurred (interaction with the free enzyme or the enzyme substrate complex at an allosteric site). The analysis of enzyme kinetics confirmed competitive binding between compounds 2, 5, 7, 9, 25, and the sEH receptor and ligands. Compounds 2, 5, 7, 9, and 25 are competitive inhibitors that crossed at a point on the vertical axis and at different major points on the horizontal axis, with an intercept on the positive y-axis (Figure S38, Supporting Information). Binding kinetics are emerging as key factors for predicting inhibitor efficacy. The structures determined of the purified cycloartane glycoside derivatives were docked, based on a kinetic study. To simulate the binding interaction between sEH and the inhibitors, molecular docking was simulated using AutoDock (version 4.2). The docking scores supported the importance of the domain for the bioactivity of the compounds. All compounds evaluated were docked favorably in the active site, as well as in the right pockets next to the active site. Their calculated AutoDock scores ranged from −8.70 to −10.55 kcal/ mol (Table 4). According to these results, the binding site of Table 4. Hydrogen Bonds and AutoDock Score between Receptor and Selected Inhibitors a
inhibitor
hydrogen bonds (Å)
2 4
Asp335 (2.80, 3.01) and Tyr466 (3.00) Tyr343 (2.87), Met469 (3.01), Met503 (2.93), and Gln505 (3.19) Asp335 (2.57, 2.73), Trp336 (2.72), Met369 (3.23, 3.26), Asn378 (2.68), and Tyr466 (3.08) Asp335 (2.59), Ser374 (3.09), and Tyr383 (2.52) Met339 (3.16), Asn378 (2.43, 2.90), and Gln 502 (2.67) Asp509 (3.14), Tyr343 (2.96), Met469 (3.00), Met503 (2.88), Gln505 (2.81), and His 506 (3.22) Tyr343 (2.69), Met469 (2.91), Gln502 (2.72), Met503 (2.87), and Gln505 (2.96) Tyr383 (3.26) and Gln384 (2.76, 2.63, 3.02) Trp525 (2.96)
5 7 8 9 18 25 29 a
AutoDock score (kcal/mol) −9.72 −9.85 −8.76 −9.82 −8.82 −10.55 −10.29 −8.70 −9.99
Amino acid sequence number of receptor.
the competitive inhibitors 5, 7, 9, and 25 was located in a portion of the active site and right pocket, forming a hydrogen bond. In contrast, the other inhibitors (2, 4, 8, 18, and 29) fit only into the right pocket, near the active site. Overall, the cycloartane-type glycosides showed similar docking results (2, 5, and 7), and formed hydrogen bonds with Asp335. Additionally, compounds 2, 4, 5, 7−9, 18, 25, and 29 showed similar results because a sugar unit was present at the same 1873
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Cimiricaside A (1). white, amorphous powder; [α]24 D −69.4 (c 0.11, MeOH); IR (KBr) νmax 3409, 2965, 1743, 1649, 1374, 1304, 1237, 1164, 1043, 973, 841 cm−1; 1H (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz) data, see Tables 1 and 2; HRESIMS m/z 661.3953 [M + H]+ (calcd for C37H57O10, 661.3952) and 683.3773 [M + Na]+ (calcd for C37H56NaO10, 683.3771). Cimiricaside B (2). white, amorphous powder; [α]24 D −43.1 (c 0.10, MeOH); IR (KBr) νmax 3465, 2946, 1648, 1456, 1373, 1258, 1159, 1077, 1023, 970, 838 cm−1; 1H (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz) data, see Tables 1 and 2; HRESIMS m/z 633.4005 [M + H]+ (calcd for C36H57O9, 633.4002) and 655.3823 [M + Na]+ (calcd for C36H56NaO9, 655.3822). Cimiricaside C (3). white, amorphous powder; [α]24 D −19.5 (c 0.10, MeOH); IR (KBr) νmax 3439, 2957, 1735, 1655, 1458, 1366, 1249, 1081, 1045, 972, 836 cm−1; 1H (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz) data, see Tables 1 and 2; HRESIMS m/z 703.4059 [M + H]+ (calcd for C39H59O11, 703.4057). Cimiricaside D (4). white, amorphous powder; [α]24 D −39.9 (c 0.10, MeOH); IR (KBr) νmax 3409, 2937, 1731, 1646, 1453, 1380, 1111, 1043, 977, 850 cm−1; 1H (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz) data, see Tables 1 and 2; HRESIMS m/z 655.3458 [M + Na]+ (calcd for C35H52NaO10, 655.3458). Cimiricaside E (5). white, amorphous powder; [α]24 D −31.8 (c 0.11, MeOH); IR (KBr) νmax 3370, 2964, 1638, 1455, 1381, 1156, 1073, 1043, 972 cm−1; 1H (pyridine-d5, 600 MHz) and 13C NMR (pyridined5, 150 MHz) data, see Tables 1 and 2; HRESIMS m/z 659.3775 [M + Na]+ (calcd for C35H56NaO10, 659.3771). Cimiricaside F (6). white, amorphous powder; [α]24 D −89.7 (c 0.12, MeOH); IR (KBr) νmax 3420, 2937, 1737, 1649, 1456, 1373, 1240, 1168, 1071, 1044, 990, 825 cm−1; 1H (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz) data, see Tables 1 and 2; HRESIMS m/ z 751.4263 [M + H]+ (calcd for C40H63O13, 751.4263) and 773.4083 [M + Na]+ (calcd for C40H62NaO13, 773.4088). Determination of the Absolute Configuration of Sugar Components of 1−6. This was conducted according to published protocols with modifications, as detailed in a previous paper36 (Supporting Information). sEH Effect Assay. sEH inhibition assays on the extracts and the isolated compounds were performed as described previously by Thao et al.36 Briefly, 50 μL of sEH (31.6 ng/mL) and 20 μL of various concentrations of the compounds dissolved in MeOH were added in a 96-well plate containing 80 μL of 25 mM Bis-Tris-HCl buffer containing 0.1 mg/mL BSA (pH = 7.0), mixed with 50 μL of 40 μM 3phenyl-cyano(6-methoxy-2-naphthalenyl)methyl ester-2-oxiraneacetic acid (PHOME). After starting the enzyme reaction at 37 °C, products by hydrolysis of the substrate were monitored at excitation and emission of 330 and 465 nm during 1 h.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation, IR, NMR, ESI, HRESIMS, and GC/MS data collection, and TLC and MPLC were carried out in a manner similar to procedures described in a previous paper.36 Plant Material. The roots (3.5 years old) of Cimicif uga dahurica were purchased from a herbal company, Naemome Dah, Ulsan, Korea, in February 2016 (35°32′08.3″ N 129°19′10.1″ E). Y.H.K. identified this sample. A voucher specimen representing this collection has been deposited at the Herbarium of the College of Pharmacy, Chungnam National University, Daejeon, Korea, under accession number CNU16003. Extraction and Isolation. The dried roots of C. dahurica (2.5 kg) were cut into pieces and extracted with 95% aqueous EtOH (3 × 5.0 L) under ultrasonic agitation at 90 Hz and 40 °C. The ethanol solution was removed of solvent under a vacuum and was filtered through a Büchner funnel to produce a dried brown extract (65.3 g). Since the EtOH extract significantly reduced sEH inhibitory activity (with 68.1 ± 1.23% at a concentration of 25.0 μg/mL), it was suspended in distilled water and successively partitioned with n-hexane and CH2Cl2 to afford n-hexane (9.6 g, A), CH2Cl2 (15.2 g, B), and water fractions (40.5 g, W). The water fraction (W) showed potent sEH inhibitory activity (with 70.1 ± 1.21% at the same concentration), which was greater than that in the presence of the ethanol extract. Thus, this extract of C. dahurica roots was chosen for subsequent studies. The H2O fraction was separated using a Diaion HP-20 column and was eluted with a gradient solvent mixture of MeOH-H2O (25:75, 50:50, 65:35, 75:25, to pure MeOH, stepwise) to yield four fractions (W-1 to W-4), based on TLC analysis. Fraction W-2 (2.1 g) was subjected to passage over a silica gel column (with a solvent mixture of CHCl3-MeOH-H2O, 6.5:1:0.1) and then an open YMC RP-C18 silica gel column (65 → 100%, H2O-MeOH) to afford compounds 1 (15.2 mg) and 9 (11.7 mg). When the same steps were repeated as above, compounds 6 (7.5 mg), 19 (18.2 mg), 20 (10.1 mg), 21 (5.0 mg), and 27 (3.0 mg) were also obtained by purifying subfraction W-2 on YMC RP-C18 silica gel, followed by passage over a Sephadex LH-20 column using mixtures of MeOH−acetone−H2O (8:1:1). Next, fraction W-3 (1.7 g) was subjected to YMC RP-C18 silica gel CC and was eluted with a solvent mixture of H2O−MeOH (5:1, 3:1, 1:1, and 100% MeOH) to afford four subfractions (W-3.1 to W-3.4). Further purification of subfraction W-3.1 (0.42 g) via silica gel column eluted with CH2Cl2−MeOH (7:1), yielded a compound mixture (18.3 mg) that was further purified by preparative TLC (acetone−H2O, 1:2) to yield compounds 14 (9.5 mg) and 16 (5.6 mg). In a similar process to that describe above, subfraction W-3.3 (0.07 g) was subjected to YMC RP-C18 silica gel CC (MeOH−H2O, 1:1) and then was further purified by silica gel CC using CH2Cl2−acetone (1:2) to yield compounds 2 (5.1 mg), 3 (10.2 mg), and 10 (12.7 mg). Next, subfraction W-3.4 (0.11 g) yielded compounds 11 (15.4 mg), 13 (3.8 mg), and 28 (22.5 mg) after fractionation via repeated silica gel CC with EtOAc−MeOHH2O (14:1:0.5) and then was separated over C18 reversed-phase silica gel using MeOH−H2O (42:58). The fractionation of subfraction W-4 (1.3 g) was separated via silica gel CC and eluted repeatedly with CHCl3−EtOAc (8:1, 4:1, and 2:1) to yield five subfractions (W-4.1 to W-4.5), which were separated by passage over a Sephadex LH-20 column and then applied to repeated silica gel CC (LiChroprep RP-C18 column) to yield 5 (5.3 mg), 15 (10.3 mg), 17 (20.5 mg), and a mixture that was further fractionated via silica gel CC and was eluted successively with CH2Cl2−EtOAc− H2O (5:1:0.1) and CHCl3−acetone (1:1) to yield compounds 18 (5.1 mg), 22 (9.8 mg), and 24 (6.5 mg). Finally, subfraction W-4.5 (0.65 g) was fractionated over silica gel and eluted successively with EtOAc− MeOH−H2O (7:1:0.08) and further purified via C18 reversed-phase silica gel (MeOH−H2O, 35:65) to afford compounds 4 (12.5 mg), 7 (4.5 mg), 8 (15.5 mg), 12 (8.2 mg), 23 (3.5 mg), and a compound mixture. This mixture was then repeatedly purified using a C18 reversed-phase silica gel CC (acetone−H2O, 20:80) to yield compounds 25 (9.5 mg), 26 (11.2 mg), and 29 (7.5 mg).
Enzyme activity (%) = [S40 − S0/C40 − C0] × 100 where C40 and S40 are the fluorescence of control and inhibitor after 40 min, and S0 and C0 are the fluorescence of inhibitor and control at 0 min. In this testing, AUDA (IC50 = 7.8 ± 2.1 nM) was used as a positive control. sEH Kinetic Assay. This assay was determined under steady-state conditions as described using the protocol of Thao et al.36 Molecular Docking Simulation. This part of the work was conducted according to published protocols,36 with modifications (Supporting Information). Molecular Dynamics Simulation. This part of the work was conducted according to published protocols,36 with modifications (Supporting Information). Statistical Analysis. Statistical analysis was perform as previously described.36
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00166. 1874
DOI: 10.1021/acs.jnatprod.7b00166 J. Nat. Prod. 2017, 80, 1867−1875
Journal of Natural Products
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Article
1 H, 13C, HSQC, HMBC, 1H−1H COSY, and ROESY spectra of six cimiricasides A−F (1−6) (Figures S1 to S35). Structures of the known compounds 10−17, 19− 24, and 26−28 (Figure S36). Selected key HMBC and COSY correlations for compounds 1−3, 5, and 6 (Figure S37). Kinetic, molecular docking, and molecular dynamics studies for selected compounds (Figures S38 to S43). (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-42-821-5933. Fax: +82-42-823-6566. E-mail: yhk@ cnu.ac.kr. ORCID
Young Ho Kim: 0000-0002-5212-7543 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the Priority Research Center Program (2009−0093815) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology, Republic of Korea.
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