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Sesquiterpenoids from Chloranthus henryi and Their Antineuroinflammatory Activities by Li-Jun Wang a ), Juan Xiong a ), Shu-Ting Liu a ), Xin-Hua Liu b ), and Jin-Feng Hu* a ) a
) Department of Natural Products Chemistry, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, P. R. China (phone/fax: þ 86 21 51980172; e-mail: [email protected]) b ) Department of Pharmacology, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, P. R. China
Five new and seven known mono-sesquiterpenoids (1 – 5 and 6 – 12, resp.) together with five known lindenane-type disesquiterpenoids, 13 – 17, were isolated from the whole plant of Chloranthus henryi. Based on spectroscopic methods, the new structures were established to be (5S,6R,8S,10R)-6hydroxyeudesma-4(15),7(11)-diene-12,8-olide (1), 6a-hydroxyeudesma-4(15),7(11),8(9)-triene-12,8olide (2), 8,12-epoxy-1b-hydroxyeudesma-4(15),7,11-trien-6-one (3), 12-oxochloraniolide A (4), and (4a)-8-hydroxy-12-norcardina-6,8,10-trien-11-one (5), respectively. Among the isolates, compound 2, zederone epoxide (8), spicachlorantin G (13), chloramultilide A (14), shizukaol B (15), and spicachlorantin B (17) showed significant anti-neuroinflammatory effects by inhibiting nitric-oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells with relatively low cytotoxicity.
Introduction. – Chloranthus henryi Hemsl. (family Chloranthaceae) is a perennial herb that has been used as a folk medicine to promote blood circulation and to reduce swelling [1] [2]. Previous phytochemical investigations on this plant mainly resulted in the isolation of various sesquiterpenoids with broad biological features such as antitumor [3 – 6], hepatoprotective [6], antifungal [7], and tyrosinase inhibitory activities [8]. During our continuing search for novel bioactive sesquiterpenoids from plants [9 – 11], the chemical constituents of C. henryi were reinvestigated. In this work, three new eudesmanolides, 1 – 3, one new 8,9-secoguaiene, 4, and a new norcadinene, 5, along with twelve known mono- and disesquiterpenoids (Fig. 1) were identified. In addition, the anti-neuroinflammatory activities of the isolated compounds are discussed. Results and Discussion. – By our routine procedures [9] [10], five eudesmanolides, 1 – 3, 6, and 7, two secoguaienes, 4 and 10, three cadinenes, 5, 11, and 12, two germacrenes, 8 and 9, and five dimeric lindenane-type sesquiterpenes, 13 – 17 were isolated from the whole plant of C. henryi. By comparison of the spectroscopic data and physicochemical properties with those reported in the literature, the known compounds were identified as atractylenolide III (6) [12], chlomultin B (7) [13], zederone epoxide (8) [14] [15], (1S,4S,5S,10S)-1,10 : 4,5-diepoxygermacrone (9) [16], (7S,1(10)Z)-4,5secoguaia-1(10),11-diene-4,5-dione (10) [17], (4a,11b)-8,11-dihydroxycadina-6,8,10 2014 Verlag Helvetica Chimica Acta AG, Zrich
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Fig. 1. Structures of compounds 1 – 17
trien-12-oic acid g-lactone (11) and its 4-epimer 12 [4], spicachlorantin G (13) [18], chloramultilide A (14) [19], shizukaol B (15) [20], and spicachlorantins A and B (16 and 17, resp.) [21]. Structure Elucidation. The molecular formula of compound 1 was determined as C15H20O3 based on a pseudo-molecular ion peak at m/z 271.1324 ([M þ Na] þ ) in its positive-ion mode HR-ESI-MS, implying six degrees of unsaturation. Strong absorption bands (n ˜ max ) at 1738 and 1384 cm 1 in the IR spectrum, together with the UV maximum (lmax at 218 nm) indicated an a,b-unsaturated g-lactone moiety in the structure of 1. The 1H-NMR spectrum (Table 1) of 1 displayed signals of two Me groups as singlets (d(H) 0.90 (s, Me(14)) and 2.08 (2 br. s, Me(13)), two CHO groups (d(H) 4.79 (dd, J ¼ 11.9, 6.0, HC(8)) and 4.83 (br. dd, J ¼ 12.5, 2.2, HC(6)), and an exocylic C¼C bond (d(H) 4.76 and 5.10 (2 br. s, CH2(15)). The 13C- and DEPT-NMR spectra (Table 2) of 1 showed 15 signals attributed to two Me, five CH2 (one olefinic at 107.4), and three CH groups (two O-bearing at d(C) 68.0 and 76.5), and five quaternary Catoms (two olefinic (d(C) 121.7 and 146.1) and one ester C¼O C-atoms (d(C) 174.8)).
5.02 (dd, J ¼ 11.0, 3.5)
5.60 (s)
2.10 (br. s) 0.96 (s) 5.14 (br. s), 4.84 (br. s)
2.22 (d, J ¼ 3.5)
4.83 (br. dd, J ¼ 12.5, 2.2) 4.79 (dd, J ¼ 11.9, 6.0) 2.30 (dd, J ¼ 12.1, 6.0) 1.18 (dd, J ¼ 12.1, 11.9) 2.08 (br. s) 0.90 (s) 5.10 (br. s), 4.76 (br. s)
2.27 (d, J ¼ 2.2)
HC(6)
HC(8) HbC(9) HaC(9) HC(12) Me(13) Me(14) CH2(15) or Me(15) 1-OH 4-OH 6-OH 8-OH
) Overlapping signals within the same column. b ) Not detected.
2.41 (d, J ¼ 11.0)
2.06 (d, J ¼ 12.5)
HbC(1) HaC(1) HbC(2) HaC(2) HbC(3) HaC(3) HC(4) HC(5)
a
2 1.65 (ddd) a ) 1.64 (ddd) a ) 1.72 – 1.76 (m) 1.62 – 1.66 (m) a ) 2.41 (br. d, J ¼ 12.5) 2.01 (br. dd, J ¼ 13.0, 12.5)
1 1.60 (ddd) a ) 1.35 (ddd, J ¼ 13.5, 13.2, 5.0) 1.68 – 1.73 (m) a ) 1.64 – 1.68 (m) a ) 2.42 (ddd, J ¼ 13.5, 2.6, 2.5) 1.97 (ddd, J ¼ 13.5, 13.1, 5.3)
H-Atom
1.46 (d, J ¼ 5.6)
3.16 (d, J ¼ 17.2) 2.79 (d, J ¼ 17.2) 7.08 (br. s) 2.21 (d, J ¼ 1.1) 0.94 (s) 5.58 (br. s), 5.21 (d, J ¼ 1.3)
2.90 (s)
3.75 (ddd, J ¼ 11.7, 5.6, 4.0) 1.88 – 1.94 (m) 2.36 (ddd, J ¼ 13.3, 4.6, 2.4) 1.61 (br. dd, J ¼ 12.6, 4.4) 2.08 (ddd, J ¼ 14.1, 12.6, 4.9)
3
b
)
2.06 (br. s) 1.76 (br. s) 1.28 (s)
4.89 (br. s) 4.78 (br. s)
2.18 (dd, J ¼ 15.1, 8.5), 2.28 (dd, J ¼ 15.1, 5.6)
2.55 (q-like, J ¼ 6.0)
1.78 – 1.85 (m) a ) 1.78 – 1.85 (m) a ) 1.85 – 1.90 (m) a ) 1.85 – 1.90 (m) a )
3.18 (q-like, J ¼ 8.0)
4
Table 1. 1H-NMR (500 MHz, CDCl3 ) Data of 1 – 5. d in ppm, J in Hz.
11.4 (s)
2.62 (s) 2.19 (br. s) 1.09 (d, J ¼ 6.5)
6.67 (br. s)
2.55 (ddd) a ) 2.68 (ddd, J ¼ 15.9, 5.8, 2.5) 1.90 – 1.97 (m) 1.35 – 1.45 (m) 1.65 – 1.73 (m) 2.90 (ddd, J ¼ 15.9, 3.6, 1.8), 2.61 (dd, J ¼ 15.9, 10.6) a )
5
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Table 2. a
13
C-NMR (CDCl3 ) Data for 1 – 5. d in ppm, J in Hz.
C-Atom
1 )
2 b)
3 a)
4 a)
5 b)
C(1) C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(10) C(11) C(12) C(13) C(14) C(15)
40.9 23.2 37.2 146.1 58.9 68.0 160.8 76.5 47.2 36.8 121.7 174.8 9.4 17.9 107.4
39.3 23.5 37.1 145.3 57.7 64.9 147.5 146.7 118.2 39.1 123.7 171.1 9.5 20.2 108.1
78.4 32.5 35.7 139.6 56.2 192.2 119.2 164.5 36.2 45.3 119.5 139.2 9.1 12.8 113.9
50.4 24.6 38.1 82.1 48.2 22.4 144.6 165.91 c ) 112.0 145.2 140.3 165.94 c ) 10.1 23.7 25.6
127.0 26.7 30.7 28.8 39.8 137.1 121.1 158.9 117.1 144.8 206.2
a ) Recorded at 100 MHz. b ) Recorded at 125 MHz. c ) Distinguishable in the expanded spectrum.
33.4 20.5 21.9 13
C-NMR
These data suggested that compound 1 was a derivative of atractylenolide III (6), a known eudesma-4(15),7(11)-diene-12,8-olide sesquiterpene, previously isolated from the perennial herb Atractylodes macrocephala [12]. Unlike 6, the tertiary OH group at C(8) was absent, and instead a secondary OH group emerged at C(6) in 1. The structure of 1 was determined by detailed 2D-NMR (COSY, HSQC, and HMBC) techniques. In the COSY NMR spectrum of 1, a spin system was observed between HC(5) (d(H) 2.06) and HC(6) (d(H) 4.83), and HC(6) and HOC(6) (d(H) 2.27 (d, J ¼ 2.2, D2Oexchangeable)), and a homoallylic long-range coupling between HC(6) and Me(13) (d(H) 2.08). In addition, HC(8) (d(H) 4.79) displayed a 3J correlation with C(12) (d(C) 174.8) in the HMBC spectrum (Fig. 2). The trans-fusion of the rings A and B, and the relative configuration of 1 were determined by analyses of the coupling constants (Table 1) and NOE correlations (Fig. 3). The large values of J(HC(5),HC(6)) (12.5 Hz) and J(HC(8), HaC(9)) (11.9 Hz) unambiguously indicated that HC(5), HC(6), HC(8), and HaC(9) were all in axial positions. Unambiguous NOE correlations Me(14) (d(H) 0.90)/HC(6), Me(14)/HaC(9) (d(H) 2.30) and HaC(9)/HC(8) revealed that Me(14), HC(6), and HC(8) are on the same side of the ring system. The absolute configuration at C(8) was established as (S) by the observation of a negative Cotton effect (De ¼ 35.6) at
Fig. 2. Observed key HMBCs of compounds 1 and 2
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Fig. 3. Observed key NOE correlations of compounds 1 and 3
227 nm, which was assigned to the pp* transition of the a,b-unsaturated g-lactone moiety containing an allylic O-substituent (HOC(6)) [22]. Thus, compound 1 was identified as (5S,6R,8S,10R)-6-hydroxyeudesma-4(15),7(11)-diene-12,8-olide. Compound 2 was assigned the molecular formula of C15H18O3 as deduced from HRESI-MS (m/z 269.1156 ([M þ Na] þ )), implying one more degree of unsaturation than compound 1. The 1H- and 13C-NMR data (Tables 1 and 2) of 2 were quite similar to those of 1. The only difference was that an additional trisubstituted C¼C bond (d(H) 5.60 (s, 1 H); d(C) 146.7 (Cq ) and 118.2 (CH)) was present in 2, and hence the signals (d(H) 4.79; d(C) 76.5 (CH)) for CH(8)O in 1 were absent in 2. Thus, compound 2 was assumed to be the 8,9-didehydro derivative of 1, which was confirmed by 2D-NMR experiments (Fig. 2). As for 1, the trans-A/B ring junction and the a-orientation of HOC(6) were ascertained by the large coupling constant (11.0 Hz) between HC(5) and HC(6), and the key NOE correlation Me(14) (d(H) 0.96)/HC(6) (d(H) 5.02). Therefore, compound 2 was identified as 6a-hydroxyeudesma-4(15),7(11),8(9)-triene12,8-olide. Compound 3 was found to have the same molecular formula as 2. The 1H- and 13 C-NMR spectra (Tables 1 and 2) displayed the signals of two Me groups (d(H) 0.94 (s, Me(14), d(C) 12.8 (C(14)); d(H) 2.21 (d, J ¼ 1.1, Me(13)), d(C) 9.1 (C(13)), of a CHO group (d(H) 3.75 (ddd, J ¼ 11.7, 5.6, 4.0, HC(1)); d(C) 78.4 (C(1)), of an exocyclic C¼C bond (d(H) 5.21 and 5.58 (d, J ¼ 1.3 and br. s, CH2(15)); d(C) 113.9 (C(15)), 139.6 (C(4)), and of a typical trisubstituted furan ring (d(H) 7.08 (br. s, HC(12)); d(C) 119.2 (C(7)), 164.5 (C(8)), 119.5 (C(11)), 139.2 (C(12)) [23]. These NMR data were very similar to those of chlomultin B (7), a known 8,12epoxyeudesmene derivative previously isolated from the whole plant of C. multistachys [13]. The only difference between these two compounds was that the C(3)¼C(4) bond in 7 was shifted to C(4)¼C(15) in 3, which was confirmed by 2D-NMR experiments. The relative configuration of 3 was then verified by NOESY NMR experiment. Clear NOE correlations HC(1)/HC(5), HC(1)/HaC(9), and Me(14)/HbC(9) were observed (Fig. 3). Thus, compound 3 was identified as 8,12-epoxy-1b-hydroxyeudesma4(15),7,11-trien-6-one. The 1H-NMR spectrum (Table 1) of compound 4 exhibited signals for one Me group (d(H) 1.28 (s, Me(15)), one vinylic Me group (d(H) 2.06 (br. s, Me(13)), and an isopropenyl group (d(H) 1.76 (br. s, Me(14)), 4.78 and 4.89 (br. s, CH2(9)). The 13 C-NMR spectrum (Table 2) exhibited 15 resonances attributed to three Me, four CH2 (one olefinic at d(C) 112.0), and two CH groups, and six quaternary C-atoms (one O-bearing (d(C) 82.1), three olefinic (d(C) 140.3, 144.6, and 145.2), and two ester C¼O
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C-atoms at (d(C) 165.91 and 165.94)). These data closely resembled those of chloraniolide A, a known 8,9-secoguaiene-type sesquiterpene lactone previously isolated from the roots of C. anhuiensis [24]. The only difference was that an additional ester C¼O group in 4 was derived from the hemiacetal C(12) in chloraniolide A. This was supported by the molecular formula (C15H20O4 ; m/z 287.1247 ([M þ Na] þ )) of 4, which has one more degree of unsaturation than that of chloraniolide A. Thus, compound 4 was elucidated as 12-oxochloraniolide A. Its relative configuration was secured by the observed NOE correlations HC(1)/HC(5) and HC(5)/Me(15), indicating that HC(1), HC(5), and Me(15) had the same orientation. Compound 5 had the molecular formula C14H18O2 as deduced from the HR-ESIMS, implying six degrees of unsaturation. In accordance with the molecular formula, only 14 C-atom signals were detected in the 13C-NMR spectrum of 5 (Table 2), including those of three Me, three CH2 , and two CH groups (one olefinic (d(C) 117.1)), and six quaternary C-atoms (five olefinc (d(C) 121.1, 127.0, 137.1, 144.8, 158.9) and one ketonic C¼O (d 206.2)). The 1H-NMR spectrum (Table 1) of 5 exhibited signals of one secondary Me (d(H) 1.09 (d, J ¼ 6.5, Me(15)), a vinylic Me (d(H) 2.19 (br. s, Me(14)), and one Ac Me group (d(H) 2.62 (s, Me(13)), and of an olefinic H-atom (d(H) 6.67 (br. s, HC(9))). These data evidenced general features similar to those of commipholinone, a known 12-norcadinane-type sesquiterpene with a benzene moiety in ring B previously isolated from the resinous exudates of Commiphora opobalsamum [25]. The C(2)¼O group in commipholinone was absent in 5. Thus, compound 5 was identified as 8-hydroxy-12-norcardina-6,8,10-trien-11-one, which was confirmed by 2D-NMR experiments. Particularly, a spin system of CH2CH2CH(Me)CH2 (CH2(2)/CH2(3)/ HC(4)(Me(15))/CH2(5) was detected in the COSY NMR spectrum. Similar to commipholinone, the pseudo-axial orientation of HC(4) (d(H) 1.65 – 1.73) was indicated by the large vicinal coupling constant (J ¼ 10.6 Hz) between HC(4) and HbC(5). Moreover, the optical rotation ([a] 25 D ¼ 244.0 (c ¼ 0.05, acetone)) of 5 was comparable with that of commipholinone ([a] 20 D ¼ 130.0 (c ¼ 0.05, acetone)) [25], suggesting that these two compounds (each bearing a single stereogenic center, i.e., C(4)) have the same absolute configuration. Biological Activity. Neuroinflammation has proven to be implicated in the pathogenesis of neurological disorders, such as Alzheimers disease (AD), Parkinsons disease (PD), and stroke [26] [27]. Inhibition of the synthesis or release of inflammatory mediators (e.g., nitric oxide (NO)) in overactivated microglia has been considered to be a potential therapeutic approach for neurodegenerative diseases [28]. In this study, all the isolates, 1 – 17, were evaluated for their inhibitory effects on NO production in lipopolysaccharide (LPS)-activated BV-2 cells. As shown in Table 3, compounds 2, 8, 13 – 15, and 17 decreased NO production with IC50 values ranging from 31.1 to 79.4 mm, while others were inactive (IC50 > 200 mm). Interestingly, the dimeric sesquiterpenes, 13 – 15 and 17, exhibited more potent anti-neuroinflammatory activities than the sesquiterpenes. Moreover, the bioactive compounds were found to have no statistically significant influence on the cell viability of BV-2 cells at 50 mm (Table 3), indicating that they exhibited an anti-neuroinflammatory effect without cytotoxicity. Conclusions. – In this study, a MeOH/AcOEt 1 : 1 extract of the whole plant of C. henryi was successively subjected to repeated column chromatography (CC) over silica
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Table 3. Inhibitory Effects on NO Production in LPS-stimulated BV-2 Cells Compound
IC50 a ) b ) [mm]
Cell viability b ) c ) [%]
2 8 13 14 15 17 l-NMMA d )
79.0 1.4 68.1 6.3 70.4 2.6 47.9 2.9 31.1 1.2 79.4 3.5 13.3 1.0
92.6 6.3 103.4 2.5 88.6 3.3 88.3 2.6 106.6 3.5 84.8 2.0 101.4 3.5
a ) IC50 Value of each compound was defined as the concentration [mm] of indicated compound that caused 50% inhibition of NO production in LPS-stimulated BV-2 cells. b ) The results are averages of three independent experiments, and the data were expressed as mean SD. c ) Cell viability after treatment with 50 mm of each compound was expressed as % of untreated control cells. d ) NG Monomethyl-l-arginine (l-NMMA), positive control.
gel, MCI, and Sephadex LH-20, and semi-preparative HPLC to furnish five eudesmanolides, 1 – 3, 6, and 7, two secoguaienes, 4 and 10, three cadinenes, 5, 11, and 12, two germacrenes, 8 and 9, and five lindenane-type sesquiterpene dimers, 13 – 17. Among them, (4a)-8-hydroxy-12-norcardina-6,8,10-trien-11-one (5) and (7S,1(10)Z)4,5-secoguaia-1(10),11-diene-4,5-dione (10) represent the first 12-norcadinane-type and the first 4,5-secoguaiene-type sesquiterpenes, respectively, from the Chloranthus genus. Additionally, the 1H- and 13C-NMR data of zederone epoxide (8) were completely assigned for the first time. Notably, this is the first report on antineuroinflammatory effect of the sesquiterpenes from the genus Chloranthus. The biological results (Table 3) showed that the anti-neuroinflammatory sesquiterpenes may lead to further investigation of their therapeutic potentials for treatment of neurodegenerative and aging-related diseases. The authors gratefully acknowledge Prof. Zhensheng Yao (Zhejiang Chinese Medical University) for the plant identification. This work was supported by NSFC grants (Nos. 81273401, 81202420), a STCSM grant (No. 11DZ1921203), grants from the Ph.D. Programs Foundation of Ministry of Education (MOE) of China (Nos. 20120071110049, 20120071120049), a MOST grant (No. 2011ZX09307-002-01), and the National Basic Research Program of China (973 Program, Grant No. 2013CB530700).
Experimental Part General. Column chromatography (CC): silica gel (SiO2 ; Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, P. R. China), MCI gel CHP20P (75 – 150 mm; Mitsubishi Chemical Industries, Tokyo, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, SE-Uppsala). TLC: Silica gel-precoated plates (GF254, 0.25 mm; Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, P. R. China), visualization with UV light (254 and/or 366 nm) and by spraying with 15% H2SO4/EtOH, followed by heating to 1208. Semi-prep. HPLC: Waters e2695 system with a Waters 2998 Photodiode Array Detector (PAD) and a Waters 2424 ELSD; a SunFire ODS column (5 mm, 250 10 mm). Optical rotations: JASCO P-1020 polarimeter. CD Spectra: JASCO-810 spectropolarimeter. UV Spectra: Shimadzu UV-2550. IR Spectra: Avatar 360 ESP FTIR spectrometer; ˜n in cm 1. NMR Spectra: Bruker Avance III 400 MHz spectrometer or a Bruker Avance DRX-500 spectrometer; d in ppm rel. to the residual solvent signals as internal standard, J in Hz. ESI-MS: Waters UPLC H Class-SQD or on an Agilent 1100 series mass spectrometer; in m/z. EI-MS:
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Agilent 5975c mass spectrometer; in m/z. HR-ESI-MS: Bruker Daltonics micrOTOF-QII mass spectrometer; in m/z. Plant Material. The whole plant of C. henryi was collected by one of the authors (S.-T. L.) in July 2011 from Jinggang Mountains, Jiangxi Province of P. R. China. The plant was identified by Prof. Zhensheng Yao (Zhejiang Chinese Medical University). A voucher specimen (No. 20110701) was deposited with the Herbarium of the Department of Natural Products Chemistry, School of Pharmacy at Fudan University. Extraction and Isolation. Dried whole plant of C. henryi (7.0 kg) was extracted with MeOH/AcOEt (50 : 50) at r.t. for three times (3 20 l). After filtration, the solvents were removed under vacuum to give a crude extract (475 g), which was suspended in H2O (2 l) and then partitioned successively with petroleum ether (PE; 3 1.5 l), AcOEt (3 1.5 l), and BuOH (3 1.5 l). The AcOEt extract (232.0 g) was subjected to CC (MCI gel (8 100 cm); MeOH/H2O 30 to 100%). The 70% MeOH fraction (35.0 g) was subjected to CC (7 70 cm; SiO2 , 100 – 200 mesh, 2.0 kg; PE/acetone gradient 4 : 1 to 0 : 1) to afford five fractions, Frs. A – E, monitored by TLC. Compound 15 (42.0 mg) was isolated from Fr. B obtained by CC (4 40 cm; SiO2 , 200 – 300 mesh, 200 g; PE/AcOEt 2 : 1) and further purified by gel permeation chromatography (GPC; 2 80 cm, Sephadex LH-20; MeOH). Fr. C was subjected to SiO2 CC (4 40 cm; 200 – 300 mesh, 190 g; PE/AcOEt 1 : 1 to 1 : 3) to give subfractions C1 – C3. Fr. C1 was submitted to CC SiO2 (40 2 cm, 200 – 300 mesh, 150 g; CH2Cl2/AcOEt 2 : 1) to furnish compound 16 (60.5 mg). Subfr. C2 was purified by semi-prep. HPLC (MeOH/H2O 35 : 65; 3 ml/min) to give compounds 14 (tR 47.0 min; 9.0 mg) and 17 (tR 44.5 min; 9.0 mg). Fr. D was subjected to CC (40 2 cm; SiO2 , 200 – 300 mesh, 150 g; CH2Cl2/MeOH 100 : 1) to afford compound 13 (9.1 mg). The 80% MeOH fraction (42.0 g) was purified by CC (7 70 cm; SiO2 , 100 – 200 mesh, 2.5 kg; PE/acetone 10 : 1 to 1 : 2) to yield six fractions, Frs. F – K. Fr. H was separated into three fractions, Frs. H1 – H3 by CC (4 40 cm; SiO2 , 200 – 300 mesh, 400 g; PE/ AcOEt 6 : 1 to 4 : 1). CC of Fr. H1 (2 40 cm; SiO2 , 200 – 300 mesh, 100 g; CH2Cl2/AcOEt 6 : 1) yielded compounds 2 (12.0 mg) and 8 (23.0 mg), while compound 9 (45.0 mg) was recrystallized from Fr. H2 in AcOEt. Compounds 7 (3.0 mg), 10 (2.0 mg), and a mixture of 11 and 12 (in total, 45.0 mg) were purified from Fr. I by repeated CC (SiO2 ; CH2Cl2/AcOEt 4 : 1, CH2Cl2/MeOH 60 : 1, and PE/AcOEt 4 : 1). Fr. J was separated by CC (4 40 cm; SiO2 , 200 – 300 mesh, 400 g; CH2Cl2/AcOEt 4 : 1 to 2 : 1) to give three subfractions, Frs. J1 – J3. Compound 6 (23.5 mg) was recrystallized from Fr. J2 in AcOEt, while compound 5 (6.2 mg) was isolated from Fr. J1 by CC (2 40 cm; SiO2 , 200 – 300 mesh, 130 g; PE/AcOEt 3 : 1). Fr. J3 was subjected to semi-prep. HPLC (MeOH/H2O 43 : 57; 3.0 ml/min) to give compounds 1 (tR 17 min; 6.0 mg), 3 (tR 15 min; 3.0 mg), and 4 (tR 23 min; 4.9 mg). (5S,6R,8S,10R)-6-Hydroxyeudesma-4(15),7(11)-dien-12,8-olide ( ¼ (4R,4aS,8aR)-4a,5,6,7,8,8a,9,9aOctahydro-4-hydroxy-3,8a-dimethyl-5-methylidenenaphtho[2,3-b]furan-2(4H)-one; 1). White amor4 phous powder. [a] 25 m, D ¼ 383.8 (c ¼ 0.08, MeOH). UV (MeOH): 218 (5.71). CD (c ¼ 8.06 10 MeOH): De227 35.6. IR (film): 3429 (br.), 2928, 2843, 1738, 1673, 1384, 1148, 1064, 1034. 1H- and 13 C-NMR: see Tables 1 and 2, resp. ESI-MS: 271 ([M þ Na] þ ), 519 ([2M þ Na] þ ). HR-ESI-MS: 271.1324 (C15H20NaO þ3 ; calc. 271.1305). 6a-Hydroxyeudesma-4(15),7(11),8(9)-trien-12,8-olide ( ¼ (4R,4aS,8aS)-4a,5,6,7,8,8a-Hexahydro-4hydroxy-3,8a-dimethyl-5-methylidenenaphtho[2,3-b]furan-2(4H)-one; 2). White amorphous powder. [a] 25 D ¼ 133.5 (c ¼ 0.2, MeOH). UV (MeOH): 277 (5.60). IR (film): 3468 (br.), 2933, 2855, 1766, 1668, 1447, 1036, 888, 745. 1H- and 13C-NMR: see Tables 1 and 2, resp. EI-MS: 246 (79.8, M þ ), 231 (56.3, [M Me] þ ), 228 (100, [M H2O] þ ). HR-ESI-MS: 269.1156 (C15H18NaO þ3 ; calc. 269.1148). 8,12-Epoxy-1b-hydroxyeudesma-4(15),7,11-trien-6-one ( ¼ (4aS,8R,8aR)-5,6,7,8,8a,9-Hexahydro-8hydroxy-3,8a-dimethyl-5-methylidenenaphtho[2,3-b]furan-4(4aH)-one; 3). White amorphous powder. [a] 25 D ¼ 10.0 (c ¼ 0.3, CHCl3 ). UV (MeOH): 261 (5.13). IR (film): 3425 (br.), 2917, 2849, 1742, 1632, 1457, 1384, 1265, 1167, 1121, 1080, 1020, 756, 707. 1H- and 13C-NMR: see Tables 1 and 2, resp. ESI-MS: 269 ([M þ Na] þ ), 515 ([2M þ Na] þ ). HR-ESI-MS: 269.1144 (C15H18NaO þ3 ; calc. 269.1148). 12-Oxochloraniolide A ( ¼ 3-{[(1S,2R,5S)-2-Hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclopentyl]methyl}-4-methylfuran-2,5-dione; 4). White amorphous powder. [a] 25 D ¼ 88.8 (c ¼ 0.08, MeOH). UV (MeOH): 248 nm (5.20). IR (film): 3416 (br.), 2953, 2367, 2306, 1763, 1643, 1463, 1386, 1276, 1112, 920. 1H- and 13 C-NMR: see Tables 1 and 2, resp. ESI-MS: 287 ([M þ Na] þ ), 309 ([M þ HCOO] ). HR-ESI-MS: 287.1247 (C15H20NaO þ4 ; calc. 287.1254).
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(4a)-8-Hydroxy-12-norcardina-6,8,10-trien-11-one ( ¼ 1-[(7R)-5,6,7,8-Tetrahydro-2-hydroxy-4,7-dimethylnaphthalen-1-yl]ethanone; 5). White amorphous powder. [a] 25 D ¼ 244.0 (c ¼ 0.05, acetone). UV (MeOH): 257 (4.88). IR (film): 3390 (br.), 2950, 2924, 2869, 1740, 1622, 1600, 1568, 1455, 1411, 1359, 1300, 1239, 1222, 1148, 1048. 1H- and 13C-NMR: see Tables 1 and 2, resp. EI-MS: 218 (49.9, M þ ), 203 (100, [M Me] þ ). ESI-MS: 241 ([M þ Na] þ ), 459 ([2M þ Na] þ ). HR-ESI-MS: 241.1192 (C14H18NaO þ2 ; calc. 241.1199). Zederone Epoxide ( ¼ (6aR,7aS,9aS)-1a,6a,7a,8,9,9a-Hexahydro-1a,5,7a-trimethylbisoxireno[4,5 : 8,9]cyclodeca[1,2-b]furan-6(2H)-one; 8). White amorphous powder. [a] 25 D ¼ þ 38.3 (c ¼ 0.3, MeOH). 1H-NMR (500 MHz, CDCl3 ): 7.08 (br. s, HC(12)); 3.78 (s, HC(5)); 3.68 (d, J ¼ 17.0, HaC(9)); 2.93 (br. d, J ¼ 10.0, HC(1)); 2.82 (d, J ¼ 17.0, HbC(9)); 2.41 (br. d, J ¼ 11.0, HaC(3)); 2.21 (br. d, J ¼ 14.0, HaC(2)); 2.16 (s, Me(13)); 1.52 (m, HbC(2)); 1.47 (m, HbC(3)); 1.32 (s, Me(14)); 1.15 (s, Me(15)). 13C-NMR (125 MHz, CDCl3 ): 189.8 (C(6)); 156.1 (C(8)); 138.4 (C(12)); 123.4 (C(11)); 122.6 (C(7)); 69.0 (C(1)); 63.6 (C(4)); 63.2 (C(5)); 57.9 (C(10)); 39.5 (C(9)); 36.1 (C(3)); 23.8 (C(2)); 16.8 (C(14)); 15.3 (C(15)); 10.5 (C(13)). EI-MS: 262 (18.2, M þ ), 43 (100, C3H þ7 ). Measurement of NO Production and Cell Viability in LPS-Activated BV-2 Cells. The mouse microglia cell line BV-2 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and maintained in Dulbeccos modified Eagles medium containing 1800 mg/l NaHCO3 , supplemented with 10% fetal bovine serum, 100 U/ml penicilin, and 100 mg/ml streptomycin at 378 in a humidified atmosphere with 5% CO2 . The anti-neuroinflammatory activity in BV-2 cells was evaluated as described in [29] with modification. The NO production was quantified by nitrite accumulation in the culture medium using the Griess reaction kit (Beyotime Biotechnology, P. R. China) according to the manufacturers instructions. Briefly, BV-2 cells were pretreated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 mm) of indicated compounds for 4 h, and then stimulated with or without lipopolysaccharide (LPS; 1 mg/ml, SigmaAldrich) for 24 h. The isolated supernatants were mixed with an equal volume of Griess reagent. NaNO2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm with a microplate reader (M200, TECAN, Austria GmbH, Austria). Cell viability was measured using a MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide) colorimetric assay [30]. NG-monomethyl-l-arginine (l-NMMA; Beyotime, purity 99%), a well-known NO synthase inhibitor, served as a positive control. The IC50 values were determined by GriphPad Prism 5.
REFERENCES [1] Jiangsu New Medical College, Dictionary of the Traditional Chinese Medicine, Shanghai Science and Technology Press, Shanghai, 1986, p. 652. [2] X. W. Gan, L. Ma, Q. G., Chen, Q. Q. Chen, Q. Yu, L. H. Hu, Planta Med. 2009, 75, 1344. [3] B. Wu, S. He, Y.-J. Pan, Planta Med. 2006, 72, 1334. [4] B. Wu, S. He, X.-D. Wu, D.-K. Wu, Y.-J. Pan, Helv. Chim. Acta 2007, 90, 1586. [5] B. Wu, S. He, Y.-J. Pan, Tetrahedron Lett. 2007, 48, 453. [6] C.-J Li, D.-M. Zhang, Y.-M. Luo, S.-S. Yu, Y. Li, Y. Lu, Phytochemistry 2008, 69, 2867. [7] Y. M. Lee, J. S. Moon, B.-S. Yun, K. D. Park, G. J. Choi, J.-C. Kim, S.-H. Lee, S. U. Kim, J. Agric. Food Chem. 2009, 57, 5750. [8] B. Wu, S. He, X.-D. Wu, Y.-J. Pan, Chem. Biodiversity 2008, 5, 1298. [9] J. Xiong, S.-T. Liu, Y. Tang, W.-X. Wang, V.-B. Bui, Y. Zhao, H. Fan, G.-X. Yang, J.-F. Hu, Phytochem. Lett. 2013, 65, 586. [10] X.-W. Li, L. Weng, X. Gao, Y. Zhao, F. Pang, J.-H. Liu, H.-F. Zhang, J.-F. Hu, Bioorg. Med. Chem. Lett. 2011, 21, 366. [11] V.-B. Bui, S.-T. Liu, J.-J. Zhu, J. Xiong, Y. Zhao, G.-X. Yang, G. Xia, J.-F. Hu, Phytochem. Lett. 2012, 5, 685. [12] Q.-F. Zhang, S.-D. Luo, H.-Y. Wang, Chin. Chem. Lett. 1998, 9, 1097. [13] S. Zhang, Z.-S. Su, S.-P. Yang, J.-M. Yue, J. Asian Nat. Prod. Res. 2010, 12, 522. [14] H. Hikino, H.Takahashi, Y. Sakurai, T. Takemoto, N. S. Bhacca, Pharm. Soc. Jpn. 1966, 14, 550.
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[15] Y. Qu, F. M. Xu, S. Nakamura, H. Matsuda, Y. Pongpiriyadacha, L. J. Wu, M. Yoshikawa, J. Nat. Med. 2009, 63, 102. [16] K. Harimaya, J.-F. Gao, T. Ohkura, T. Kawamata, Y. Iitaka, Y.-T. Guo, S. Inayama, Chem. Pharm. Bull. 1991, 39, 843. [17] H.-L. Jiang, J. Chen, X.-J. Jin, J.-L. Yang, Y. Li, X.-J. Yao, Q.-X. Wu, Tetrahedron 2011, 67, 9193. [18] S.-Y. Kim, Y. Kashiwada, K. Kawazoe, K. Murakami, H.-D. Sun, S.-L. Li, Y. Takaishi, Chem. Pharm. Bull. 2011, 59, 1281. [19] S.-P. Yang, J.-M. Yue, Tetrahedron Lett. 2006, 47, 1129. [20] J. Kawabata, J. Mizutani, Phytochemistry 1992, 31, 1293. [21] S.-Y. Kim, Y. Kashiwada, K. Kawazoe, K. Murakami, H.-D. Sun, S.-L. Li, Y. Takaishi, Phytochem. Lett. 2009, 2, 110. [22] I. Uchida, K. Kuriyama, Tetrahedron Lett. 1974, 43, 3761. [23] T. Yuan, C.-R. Zhang, S.-P. Yang, S. Yin, W.-B Wu, L. Dong, J.-M. Yue, J. Nat. Prod. 2008, 71, 2021. [24] Y.-J. Xu, C.-P. Tang, M.-J. Tan, C.-Q. Ke, T. Wu, Y. Ye, Chem. Biodiversity 2010, 7, 151. [25] J.-L. Yang, Y.-P. Shi, Phytochemistry 2012, 76, 124. [26] T. Wyss-Coray, L. Mucke, Neuron 2002, 35, 419. [27] E. C. Hirsch, S. Hunot, Lancet Neurol. 2009, 8, 382. [28] X.-H. Liu, L.-L. Pan, Q.-H. Gong, Y.-Z. Zhu, Curr. Pharm. Biotechnol. 2010, 11, 895. [29] C. S. Kim, S. Y. Kim, E. Moon, M. K. Lee, K. R. Lee, Bioorg. Med. Chem. Lett. 2013, 23, 1771. [30] S.-B. Wu, Y. Wen, F. Pang, H.-F. Zhang, Z. Zhao, J.-F. Hu, Planta Med. 2010, 76, 82. Received August 23, 2013
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Sesquiterpenoids from Chloranthus henryi and Their Antineuroinflammatory Activities by Li-Jun Wang a ), Juan Xiong a ), Shu-Ting Liu a ), Xin-Hua Liu b ), and Jin-Feng Hu* a ) a
) Department of Natural Products Chemistry, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, P. R. China (phone/fax: þ 86 21 51980172; e-mail: [email protected]) b ) Department of Pharmacology, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, P. R. China
Five new and seven known mono-sesquiterpenoids (1 – 5 and 6 – 12, resp.) together with five known lindenane-type disesquiterpenoids, 13 – 17, were isolated from the whole plant of Chloranthus henryi. Based on spectroscopic methods, the new structures were established to be (5S,6R,8S,10R)-6hydroxyeudesma-4(15),7(11)-diene-12,8-olide (1), 6a-hydroxyeudesma-4(15),7(11),8(9)-triene-12,8olide (2), 8,12-epoxy-1b-hydroxyeudesma-4(15),7,11-trien-6-one (3), 12-oxochloraniolide A (4), and (4a)-8-hydroxy-12-norcardina-6,8,10-trien-11-one (5), respectively. Among the isolates, compound 2, zederone epoxide (8), spicachlorantin G (13), chloramultilide A (14), shizukaol B (15), and spicachlorantin B (17) showed significant anti-neuroinflammatory effects by inhibiting nitric-oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells with relatively low cytotoxicity.
Introduction. – Chloranthus henryi Hemsl. (family Chloranthaceae) is a perennial herb that has been used as a folk medicine to promote blood circulation and to reduce swelling [1] [2]. Previous phytochemical investigations on this plant mainly resulted in the isolation of various sesquiterpenoids with broad biological features such as antitumor [3 – 6], hepatoprotective [6], antifungal [7], and tyrosinase inhibitory activities [8]. During our continuing search for novel bioactive sesquiterpenoids from plants [9 – 11], the chemical constituents of C. henryi were reinvestigated. In this work, three new eudesmanolides, 1 – 3, one new 8,9-secoguaiene, 4, and a new norcadinene, 5, along with twelve known mono- and disesquiterpenoids (Fig. 1) were identified. In addition, the anti-neuroinflammatory activities of the isolated compounds are discussed. Results and Discussion. – By our routine procedures [9] [10], five eudesmanolides, 1 – 3, 6, and 7, two secoguaienes, 4 and 10, three cadinenes, 5, 11, and 12, two germacrenes, 8 and 9, and five dimeric lindenane-type sesquiterpenes, 13 – 17 were isolated from the whole plant of C. henryi. By comparison of the spectroscopic data and physicochemical properties with those reported in the literature, the known compounds were identified as atractylenolide III (6) [12], chlomultin B (7) [13], zederone epoxide (8) [14] [15], (1S,4S,5S,10S)-1,10 : 4,5-diepoxygermacrone (9) [16], (7S,1(10)Z)-4,5secoguaia-1(10),11-diene-4,5-dione (10) [17], (4a,11b)-8,11-dihydroxycadina-6,8,10 2014 Verlag Helvetica Chimica Acta AG, Zrich
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Fig. 1. Structures of compounds 1 – 17
trien-12-oic acid g-lactone (11) and its 4-epimer 12 [4], spicachlorantin G (13) [18], chloramultilide A (14) [19], shizukaol B (15) [20], and spicachlorantins A and B (16 and 17, resp.) [21]. Structure Elucidation. The molecular formula of compound 1 was determined as C15H20O3 based on a pseudo-molecular ion peak at m/z 271.1324 ([M þ Na] þ ) in its positive-ion mode HR-ESI-MS, implying six degrees of unsaturation. Strong absorption bands (n ˜ max ) at 1738 and 1384 cm 1 in the IR spectrum, together with the UV maximum (lmax at 218 nm) indicated an a,b-unsaturated g-lactone moiety in the structure of 1. The 1H-NMR spectrum (Table 1) of 1 displayed signals of two Me groups as singlets (d(H) 0.90 (s, Me(14)) and 2.08 (2 br. s, Me(13)), two CHO groups (d(H) 4.79 (dd, J ¼ 11.9, 6.0, HC(8)) and 4.83 (br. dd, J ¼ 12.5, 2.2, HC(6)), and an exocylic C¼C bond (d(H) 4.76 and 5.10 (2 br. s, CH2(15)). The 13C- and DEPT-NMR spectra (Table 2) of 1 showed 15 signals attributed to two Me, five CH2 (one olefinic at 107.4), and three CH groups (two O-bearing at d(C) 68.0 and 76.5), and five quaternary Catoms (two olefinic (d(C) 121.7 and 146.1) and one ester C¼O C-atoms (d(C) 174.8)).
5.02 (dd, J ¼ 11.0, 3.5)
5.60 (s)
2.10 (br. s) 0.96 (s) 5.14 (br. s), 4.84 (br. s)
2.22 (d, J ¼ 3.5)
4.83 (br. dd, J ¼ 12.5, 2.2) 4.79 (dd, J ¼ 11.9, 6.0) 2.30 (dd, J ¼ 12.1, 6.0) 1.18 (dd, J ¼ 12.1, 11.9) 2.08 (br. s) 0.90 (s) 5.10 (br. s), 4.76 (br. s)
2.27 (d, J ¼ 2.2)
HC(6)
HC(8) HbC(9) HaC(9) HC(12) Me(13) Me(14) CH2(15) or Me(15) 1-OH 4-OH 6-OH 8-OH
) Overlapping signals within the same column. b ) Not detected.
2.41 (d, J ¼ 11.0)
2.06 (d, J ¼ 12.5)
HbC(1) HaC(1) HbC(2) HaC(2) HbC(3) HaC(3) HC(4) HC(5)
a
2 1.65 (ddd) a ) 1.64 (ddd) a ) 1.72 – 1.76 (m) 1.62 – 1.66 (m) a ) 2.41 (br. d, J ¼ 12.5) 2.01 (br. dd, J ¼ 13.0, 12.5)
1 1.60 (ddd) a ) 1.35 (ddd, J ¼ 13.5, 13.2, 5.0) 1.68 – 1.73 (m) a ) 1.64 – 1.68 (m) a ) 2.42 (ddd, J ¼ 13.5, 2.6, 2.5) 1.97 (ddd, J ¼ 13.5, 13.1, 5.3)
H-Atom
1.46 (d, J ¼ 5.6)
3.16 (d, J ¼ 17.2) 2.79 (d, J ¼ 17.2) 7.08 (br. s) 2.21 (d, J ¼ 1.1) 0.94 (s) 5.58 (br. s), 5.21 (d, J ¼ 1.3)
2.90 (s)
3.75 (ddd, J ¼ 11.7, 5.6, 4.0) 1.88 – 1.94 (m) 2.36 (ddd, J ¼ 13.3, 4.6, 2.4) 1.61 (br. dd, J ¼ 12.6, 4.4) 2.08 (ddd, J ¼ 14.1, 12.6, 4.9)
3
b
)
2.06 (br. s) 1.76 (br. s) 1.28 (s)
4.89 (br. s) 4.78 (br. s)
2.18 (dd, J ¼ 15.1, 8.5), 2.28 (dd, J ¼ 15.1, 5.6)
2.55 (q-like, J ¼ 6.0)
1.78 – 1.85 (m) a ) 1.78 – 1.85 (m) a ) 1.85 – 1.90 (m) a ) 1.85 – 1.90 (m) a )
3.18 (q-like, J ¼ 8.0)
4
Table 1. 1H-NMR (500 MHz, CDCl3 ) Data of 1 – 5. d in ppm, J in Hz.
11.4 (s)
2.62 (s) 2.19 (br. s) 1.09 (d, J ¼ 6.5)
6.67 (br. s)
2.55 (ddd) a ) 2.68 (ddd, J ¼ 15.9, 5.8, 2.5) 1.90 – 1.97 (m) 1.35 – 1.45 (m) 1.65 – 1.73 (m) 2.90 (ddd, J ¼ 15.9, 3.6, 1.8), 2.61 (dd, J ¼ 15.9, 10.6) a )
5
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Table 2. a
13
C-NMR (CDCl3 ) Data for 1 – 5. d in ppm, J in Hz.
C-Atom
1 )
2 b)
3 a)
4 a)
5 b)
C(1) C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(10) C(11) C(12) C(13) C(14) C(15)
40.9 23.2 37.2 146.1 58.9 68.0 160.8 76.5 47.2 36.8 121.7 174.8 9.4 17.9 107.4
39.3 23.5 37.1 145.3 57.7 64.9 147.5 146.7 118.2 39.1 123.7 171.1 9.5 20.2 108.1
78.4 32.5 35.7 139.6 56.2 192.2 119.2 164.5 36.2 45.3 119.5 139.2 9.1 12.8 113.9
50.4 24.6 38.1 82.1 48.2 22.4 144.6 165.91 c ) 112.0 145.2 140.3 165.94 c ) 10.1 23.7 25.6
127.0 26.7 30.7 28.8 39.8 137.1 121.1 158.9 117.1 144.8 206.2
a ) Recorded at 100 MHz. b ) Recorded at 125 MHz. c ) Distinguishable in the expanded spectrum.
33.4 20.5 21.9 13
C-NMR
These data suggested that compound 1 was a derivative of atractylenolide III (6), a known eudesma-4(15),7(11)-diene-12,8-olide sesquiterpene, previously isolated from the perennial herb Atractylodes macrocephala [12]. Unlike 6, the tertiary OH group at C(8) was absent, and instead a secondary OH group emerged at C(6) in 1. The structure of 1 was determined by detailed 2D-NMR (COSY, HSQC, and HMBC) techniques. In the COSY NMR spectrum of 1, a spin system was observed between HC(5) (d(H) 2.06) and HC(6) (d(H) 4.83), and HC(6) and HOC(6) (d(H) 2.27 (d, J ¼ 2.2, D2Oexchangeable)), and a homoallylic long-range coupling between HC(6) and Me(13) (d(H) 2.08). In addition, HC(8) (d(H) 4.79) displayed a 3J correlation with C(12) (d(C) 174.8) in the HMBC spectrum (Fig. 2). The trans-fusion of the rings A and B, and the relative configuration of 1 were determined by analyses of the coupling constants (Table 1) and NOE correlations (Fig. 3). The large values of J(HC(5),HC(6)) (12.5 Hz) and J(HC(8), HaC(9)) (11.9 Hz) unambiguously indicated that HC(5), HC(6), HC(8), and HaC(9) were all in axial positions. Unambiguous NOE correlations Me(14) (d(H) 0.90)/HC(6), Me(14)/HaC(9) (d(H) 2.30) and HaC(9)/HC(8) revealed that Me(14), HC(6), and HC(8) are on the same side of the ring system. The absolute configuration at C(8) was established as (S) by the observation of a negative Cotton effect (De ¼ 35.6) at
Fig. 2. Observed key HMBCs of compounds 1 and 2
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Fig. 3. Observed key NOE correlations of compounds 1 and 3
227 nm, which was assigned to the pp* transition of the a,b-unsaturated g-lactone moiety containing an allylic O-substituent (HOC(6)) [22]. Thus, compound 1 was identified as (5S,6R,8S,10R)-6-hydroxyeudesma-4(15),7(11)-diene-12,8-olide. Compound 2 was assigned the molecular formula of C15H18O3 as deduced from HRESI-MS (m/z 269.1156 ([M þ Na] þ )), implying one more degree of unsaturation than compound 1. The 1H- and 13C-NMR data (Tables 1 and 2) of 2 were quite similar to those of 1. The only difference was that an additional trisubstituted C¼C bond (d(H) 5.60 (s, 1 H); d(C) 146.7 (Cq ) and 118.2 (CH)) was present in 2, and hence the signals (d(H) 4.79; d(C) 76.5 (CH)) for CH(8)O in 1 were absent in 2. Thus, compound 2 was assumed to be the 8,9-didehydro derivative of 1, which was confirmed by 2D-NMR experiments (Fig. 2). As for 1, the trans-A/B ring junction and the a-orientation of HOC(6) were ascertained by the large coupling constant (11.0 Hz) between HC(5) and HC(6), and the key NOE correlation Me(14) (d(H) 0.96)/HC(6) (d(H) 5.02). Therefore, compound 2 was identified as 6a-hydroxyeudesma-4(15),7(11),8(9)-triene12,8-olide. Compound 3 was found to have the same molecular formula as 2. The 1H- and 13 C-NMR spectra (Tables 1 and 2) displayed the signals of two Me groups (d(H) 0.94 (s, Me(14), d(C) 12.8 (C(14)); d(H) 2.21 (d, J ¼ 1.1, Me(13)), d(C) 9.1 (C(13)), of a CHO group (d(H) 3.75 (ddd, J ¼ 11.7, 5.6, 4.0, HC(1)); d(C) 78.4 (C(1)), of an exocyclic C¼C bond (d(H) 5.21 and 5.58 (d, J ¼ 1.3 and br. s, CH2(15)); d(C) 113.9 (C(15)), 139.6 (C(4)), and of a typical trisubstituted furan ring (d(H) 7.08 (br. s, HC(12)); d(C) 119.2 (C(7)), 164.5 (C(8)), 119.5 (C(11)), 139.2 (C(12)) [23]. These NMR data were very similar to those of chlomultin B (7), a known 8,12epoxyeudesmene derivative previously isolated from the whole plant of C. multistachys [13]. The only difference between these two compounds was that the C(3)¼C(4) bond in 7 was shifted to C(4)¼C(15) in 3, which was confirmed by 2D-NMR experiments. The relative configuration of 3 was then verified by NOESY NMR experiment. Clear NOE correlations HC(1)/HC(5), HC(1)/HaC(9), and Me(14)/HbC(9) were observed (Fig. 3). Thus, compound 3 was identified as 8,12-epoxy-1b-hydroxyeudesma4(15),7,11-trien-6-one. The 1H-NMR spectrum (Table 1) of compound 4 exhibited signals for one Me group (d(H) 1.28 (s, Me(15)), one vinylic Me group (d(H) 2.06 (br. s, Me(13)), and an isopropenyl group (d(H) 1.76 (br. s, Me(14)), 4.78 and 4.89 (br. s, CH2(9)). The 13 C-NMR spectrum (Table 2) exhibited 15 resonances attributed to three Me, four CH2 (one olefinic at d(C) 112.0), and two CH groups, and six quaternary C-atoms (one O-bearing (d(C) 82.1), three olefinic (d(C) 140.3, 144.6, and 145.2), and two ester C¼O
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C-atoms at (d(C) 165.91 and 165.94)). These data closely resembled those of chloraniolide A, a known 8,9-secoguaiene-type sesquiterpene lactone previously isolated from the roots of C. anhuiensis [24]. The only difference was that an additional ester C¼O group in 4 was derived from the hemiacetal C(12) in chloraniolide A. This was supported by the molecular formula (C15H20O4 ; m/z 287.1247 ([M þ Na] þ )) of 4, which has one more degree of unsaturation than that of chloraniolide A. Thus, compound 4 was elucidated as 12-oxochloraniolide A. Its relative configuration was secured by the observed NOE correlations HC(1)/HC(5) and HC(5)/Me(15), indicating that HC(1), HC(5), and Me(15) had the same orientation. Compound 5 had the molecular formula C14H18O2 as deduced from the HR-ESIMS, implying six degrees of unsaturation. In accordance with the molecular formula, only 14 C-atom signals were detected in the 13C-NMR spectrum of 5 (Table 2), including those of three Me, three CH2 , and two CH groups (one olefinic (d(C) 117.1)), and six quaternary C-atoms (five olefinc (d(C) 121.1, 127.0, 137.1, 144.8, 158.9) and one ketonic C¼O (d 206.2)). The 1H-NMR spectrum (Table 1) of 5 exhibited signals of one secondary Me (d(H) 1.09 (d, J ¼ 6.5, Me(15)), a vinylic Me (d(H) 2.19 (br. s, Me(14)), and one Ac Me group (d(H) 2.62 (s, Me(13)), and of an olefinic H-atom (d(H) 6.67 (br. s, HC(9))). These data evidenced general features similar to those of commipholinone, a known 12-norcadinane-type sesquiterpene with a benzene moiety in ring B previously isolated from the resinous exudates of Commiphora opobalsamum [25]. The C(2)¼O group in commipholinone was absent in 5. Thus, compound 5 was identified as 8-hydroxy-12-norcardina-6,8,10-trien-11-one, which was confirmed by 2D-NMR experiments. Particularly, a spin system of CH2CH2CH(Me)CH2 (CH2(2)/CH2(3)/ HC(4)(Me(15))/CH2(5) was detected in the COSY NMR spectrum. Similar to commipholinone, the pseudo-axial orientation of HC(4) (d(H) 1.65 – 1.73) was indicated by the large vicinal coupling constant (J ¼ 10.6 Hz) between HC(4) and HbC(5). Moreover, the optical rotation ([a] 25 D ¼ 244.0 (c ¼ 0.05, acetone)) of 5 was comparable with that of commipholinone ([a] 20 D ¼ 130.0 (c ¼ 0.05, acetone)) [25], suggesting that these two compounds (each bearing a single stereogenic center, i.e., C(4)) have the same absolute configuration. Biological Activity. Neuroinflammation has proven to be implicated in the pathogenesis of neurological disorders, such as Alzheimers disease (AD), Parkinsons disease (PD), and stroke [26] [27]. Inhibition of the synthesis or release of inflammatory mediators (e.g., nitric oxide (NO)) in overactivated microglia has been considered to be a potential therapeutic approach for neurodegenerative diseases [28]. In this study, all the isolates, 1 – 17, were evaluated for their inhibitory effects on NO production in lipopolysaccharide (LPS)-activated BV-2 cells. As shown in Table 3, compounds 2, 8, 13 – 15, and 17 decreased NO production with IC50 values ranging from 31.1 to 79.4 mm, while others were inactive (IC50 > 200 mm). Interestingly, the dimeric sesquiterpenes, 13 – 15 and 17, exhibited more potent anti-neuroinflammatory activities than the sesquiterpenes. Moreover, the bioactive compounds were found to have no statistically significant influence on the cell viability of BV-2 cells at 50 mm (Table 3), indicating that they exhibited an anti-neuroinflammatory effect without cytotoxicity. Conclusions. – In this study, a MeOH/AcOEt 1 : 1 extract of the whole plant of C. henryi was successively subjected to repeated column chromatography (CC) over silica
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Table 3. Inhibitory Effects on NO Production in LPS-stimulated BV-2 Cells Compound
IC50 a ) b ) [mm]
Cell viability b ) c ) [%]
2 8 13 14 15 17 l-NMMA d )
79.0 1.4 68.1 6.3 70.4 2.6 47.9 2.9 31.1 1.2 79.4 3.5 13.3 1.0
92.6 6.3 103.4 2.5 88.6 3.3 88.3 2.6 106.6 3.5 84.8 2.0 101.4 3.5
a ) IC50 Value of each compound was defined as the concentration [mm] of indicated compound that caused 50% inhibition of NO production in LPS-stimulated BV-2 cells. b ) The results are averages of three independent experiments, and the data were expressed as mean SD. c ) Cell viability after treatment with 50 mm of each compound was expressed as % of untreated control cells. d ) NG Monomethyl-l-arginine (l-NMMA), positive control.
gel, MCI, and Sephadex LH-20, and semi-preparative HPLC to furnish five eudesmanolides, 1 – 3, 6, and 7, two secoguaienes, 4 and 10, three cadinenes, 5, 11, and 12, two germacrenes, 8 and 9, and five lindenane-type sesquiterpene dimers, 13 – 17. Among them, (4a)-8-hydroxy-12-norcardina-6,8,10-trien-11-one (5) and (7S,1(10)Z)4,5-secoguaia-1(10),11-diene-4,5-dione (10) represent the first 12-norcadinane-type and the first 4,5-secoguaiene-type sesquiterpenes, respectively, from the Chloranthus genus. Additionally, the 1H- and 13C-NMR data of zederone epoxide (8) were completely assigned for the first time. Notably, this is the first report on antineuroinflammatory effect of the sesquiterpenes from the genus Chloranthus. The biological results (Table 3) showed that the anti-neuroinflammatory sesquiterpenes may lead to further investigation of their therapeutic potentials for treatment of neurodegenerative and aging-related diseases. The authors gratefully acknowledge Prof. Zhensheng Yao (Zhejiang Chinese Medical University) for the plant identification. This work was supported by NSFC grants (Nos. 81273401, 81202420), a STCSM grant (No. 11DZ1921203), grants from the Ph.D. Programs Foundation of Ministry of Education (MOE) of China (Nos. 20120071110049, 20120071120049), a MOST grant (No. 2011ZX09307-002-01), and the National Basic Research Program of China (973 Program, Grant No. 2013CB530700).
Experimental Part General. Column chromatography (CC): silica gel (SiO2 ; Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, P. R. China), MCI gel CHP20P (75 – 150 mm; Mitsubishi Chemical Industries, Tokyo, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, SE-Uppsala). TLC: Silica gel-precoated plates (GF254, 0.25 mm; Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, P. R. China), visualization with UV light (254 and/or 366 nm) and by spraying with 15% H2SO4/EtOH, followed by heating to 1208. Semi-prep. HPLC: Waters e2695 system with a Waters 2998 Photodiode Array Detector (PAD) and a Waters 2424 ELSD; a SunFire ODS column (5 mm, 250 10 mm). Optical rotations: JASCO P-1020 polarimeter. CD Spectra: JASCO-810 spectropolarimeter. UV Spectra: Shimadzu UV-2550. IR Spectra: Avatar 360 ESP FTIR spectrometer; ˜n in cm 1. NMR Spectra: Bruker Avance III 400 MHz spectrometer or a Bruker Avance DRX-500 spectrometer; d in ppm rel. to the residual solvent signals as internal standard, J in Hz. ESI-MS: Waters UPLC H Class-SQD or on an Agilent 1100 series mass spectrometer; in m/z. EI-MS:
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Agilent 5975c mass spectrometer; in m/z. HR-ESI-MS: Bruker Daltonics micrOTOF-QII mass spectrometer; in m/z. Plant Material. The whole plant of C. henryi was collected by one of the authors (S.-T. L.) in July 2011 from Jinggang Mountains, Jiangxi Province of P. R. China. The plant was identified by Prof. Zhensheng Yao (Zhejiang Chinese Medical University). A voucher specimen (No. 20110701) was deposited with the Herbarium of the Department of Natural Products Chemistry, School of Pharmacy at Fudan University. Extraction and Isolation. Dried whole plant of C. henryi (7.0 kg) was extracted with MeOH/AcOEt (50 : 50) at r.t. for three times (3 20 l). After filtration, the solvents were removed under vacuum to give a crude extract (475 g), which was suspended in H2O (2 l) and then partitioned successively with petroleum ether (PE; 3 1.5 l), AcOEt (3 1.5 l), and BuOH (3 1.5 l). The AcOEt extract (232.0 g) was subjected to CC (MCI gel (8 100 cm); MeOH/H2O 30 to 100%). The 70% MeOH fraction (35.0 g) was subjected to CC (7 70 cm; SiO2 , 100 – 200 mesh, 2.0 kg; PE/acetone gradient 4 : 1 to 0 : 1) to afford five fractions, Frs. A – E, monitored by TLC. Compound 15 (42.0 mg) was isolated from Fr. B obtained by CC (4 40 cm; SiO2 , 200 – 300 mesh, 200 g; PE/AcOEt 2 : 1) and further purified by gel permeation chromatography (GPC; 2 80 cm, Sephadex LH-20; MeOH). Fr. C was subjected to SiO2 CC (4 40 cm; 200 – 300 mesh, 190 g; PE/AcOEt 1 : 1 to 1 : 3) to give subfractions C1 – C3. Fr. C1 was submitted to CC SiO2 (40 2 cm, 200 – 300 mesh, 150 g; CH2Cl2/AcOEt 2 : 1) to furnish compound 16 (60.5 mg). Subfr. C2 was purified by semi-prep. HPLC (MeOH/H2O 35 : 65; 3 ml/min) to give compounds 14 (tR 47.0 min; 9.0 mg) and 17 (tR 44.5 min; 9.0 mg). Fr. D was subjected to CC (40 2 cm; SiO2 , 200 – 300 mesh, 150 g; CH2Cl2/MeOH 100 : 1) to afford compound 13 (9.1 mg). The 80% MeOH fraction (42.0 g) was purified by CC (7 70 cm; SiO2 , 100 – 200 mesh, 2.5 kg; PE/acetone 10 : 1 to 1 : 2) to yield six fractions, Frs. F – K. Fr. H was separated into three fractions, Frs. H1 – H3 by CC (4 40 cm; SiO2 , 200 – 300 mesh, 400 g; PE/ AcOEt 6 : 1 to 4 : 1). CC of Fr. H1 (2 40 cm; SiO2 , 200 – 300 mesh, 100 g; CH2Cl2/AcOEt 6 : 1) yielded compounds 2 (12.0 mg) and 8 (23.0 mg), while compound 9 (45.0 mg) was recrystallized from Fr. H2 in AcOEt. Compounds 7 (3.0 mg), 10 (2.0 mg), and a mixture of 11 and 12 (in total, 45.0 mg) were purified from Fr. I by repeated CC (SiO2 ; CH2Cl2/AcOEt 4 : 1, CH2Cl2/MeOH 60 : 1, and PE/AcOEt 4 : 1). Fr. J was separated by CC (4 40 cm; SiO2 , 200 – 300 mesh, 400 g; CH2Cl2/AcOEt 4 : 1 to 2 : 1) to give three subfractions, Frs. J1 – J3. Compound 6 (23.5 mg) was recrystallized from Fr. J2 in AcOEt, while compound 5 (6.2 mg) was isolated from Fr. J1 by CC (2 40 cm; SiO2 , 200 – 300 mesh, 130 g; PE/AcOEt 3 : 1). Fr. J3 was subjected to semi-prep. HPLC (MeOH/H2O 43 : 57; 3.0 ml/min) to give compounds 1 (tR 17 min; 6.0 mg), 3 (tR 15 min; 3.0 mg), and 4 (tR 23 min; 4.9 mg). (5S,6R,8S,10R)-6-Hydroxyeudesma-4(15),7(11)-dien-12,8-olide ( ¼ (4R,4aS,8aR)-4a,5,6,7,8,8a,9,9aOctahydro-4-hydroxy-3,8a-dimethyl-5-methylidenenaphtho[2,3-b]furan-2(4H)-one; 1). White amor4 phous powder. [a] 25 m, D ¼ 383.8 (c ¼ 0.08, MeOH). UV (MeOH): 218 (5.71). CD (c ¼ 8.06 10 MeOH): De227 35.6. IR (film): 3429 (br.), 2928, 2843, 1738, 1673, 1384, 1148, 1064, 1034. 1H- and 13 C-NMR: see Tables 1 and 2, resp. ESI-MS: 271 ([M þ Na] þ ), 519 ([2M þ Na] þ ). HR-ESI-MS: 271.1324 (C15H20NaO þ3 ; calc. 271.1305). 6a-Hydroxyeudesma-4(15),7(11),8(9)-trien-12,8-olide ( ¼ (4R,4aS,8aS)-4a,5,6,7,8,8a-Hexahydro-4hydroxy-3,8a-dimethyl-5-methylidenenaphtho[2,3-b]furan-2(4H)-one; 2). White amorphous powder. [a] 25 D ¼ 133.5 (c ¼ 0.2, MeOH). UV (MeOH): 277 (5.60). IR (film): 3468 (br.), 2933, 2855, 1766, 1668, 1447, 1036, 888, 745. 1H- and 13C-NMR: see Tables 1 and 2, resp. EI-MS: 246 (79.8, M þ ), 231 (56.3, [M Me] þ ), 228 (100, [M H2O] þ ). HR-ESI-MS: 269.1156 (C15H18NaO þ3 ; calc. 269.1148). 8,12-Epoxy-1b-hydroxyeudesma-4(15),7,11-trien-6-one ( ¼ (4aS,8R,8aR)-5,6,7,8,8a,9-Hexahydro-8hydroxy-3,8a-dimethyl-5-methylidenenaphtho[2,3-b]furan-4(4aH)-one; 3). White amorphous powder. [a] 25 D ¼ 10.0 (c ¼ 0.3, CHCl3 ). UV (MeOH): 261 (5.13). IR (film): 3425 (br.), 2917, 2849, 1742, 1632, 1457, 1384, 1265, 1167, 1121, 1080, 1020, 756, 707. 1H- and 13C-NMR: see Tables 1 and 2, resp. ESI-MS: 269 ([M þ Na] þ ), 515 ([2M þ Na] þ ). HR-ESI-MS: 269.1144 (C15H18NaO þ3 ; calc. 269.1148). 12-Oxochloraniolide A ( ¼ 3-{[(1S,2R,5S)-2-Hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclopentyl]methyl}-4-methylfuran-2,5-dione; 4). White amorphous powder. [a] 25 D ¼ 88.8 (c ¼ 0.08, MeOH). UV (MeOH): 248 nm (5.20). IR (film): 3416 (br.), 2953, 2367, 2306, 1763, 1643, 1463, 1386, 1276, 1112, 920. 1H- and 13 C-NMR: see Tables 1 and 2, resp. ESI-MS: 287 ([M þ Na] þ ), 309 ([M þ HCOO] ). HR-ESI-MS: 287.1247 (C15H20NaO þ4 ; calc. 287.1254).
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(4a)-8-Hydroxy-12-norcardina-6,8,10-trien-11-one ( ¼ 1-[(7R)-5,6,7,8-Tetrahydro-2-hydroxy-4,7-dimethylnaphthalen-1-yl]ethanone; 5). White amorphous powder. [a] 25 D ¼ 244.0 (c ¼ 0.05, acetone). UV (MeOH): 257 (4.88). IR (film): 3390 (br.), 2950, 2924, 2869, 1740, 1622, 1600, 1568, 1455, 1411, 1359, 1300, 1239, 1222, 1148, 1048. 1H- and 13C-NMR: see Tables 1 and 2, resp. EI-MS: 218 (49.9, M þ ), 203 (100, [M Me] þ ). ESI-MS: 241 ([M þ Na] þ ), 459 ([2M þ Na] þ ). HR-ESI-MS: 241.1192 (C14H18NaO þ2 ; calc. 241.1199). Zederone Epoxide ( ¼ (6aR,7aS,9aS)-1a,6a,7a,8,9,9a-Hexahydro-1a,5,7a-trimethylbisoxireno[4,5 : 8,9]cyclodeca[1,2-b]furan-6(2H)-one; 8). White amorphous powder. [a] 25 D ¼ þ 38.3 (c ¼ 0.3, MeOH). 1H-NMR (500 MHz, CDCl3 ): 7.08 (br. s, HC(12)); 3.78 (s, HC(5)); 3.68 (d, J ¼ 17.0, HaC(9)); 2.93 (br. d, J ¼ 10.0, HC(1)); 2.82 (d, J ¼ 17.0, HbC(9)); 2.41 (br. d, J ¼ 11.0, HaC(3)); 2.21 (br. d, J ¼ 14.0, HaC(2)); 2.16 (s, Me(13)); 1.52 (m, HbC(2)); 1.47 (m, HbC(3)); 1.32 (s, Me(14)); 1.15 (s, Me(15)). 13C-NMR (125 MHz, CDCl3 ): 189.8 (C(6)); 156.1 (C(8)); 138.4 (C(12)); 123.4 (C(11)); 122.6 (C(7)); 69.0 (C(1)); 63.6 (C(4)); 63.2 (C(5)); 57.9 (C(10)); 39.5 (C(9)); 36.1 (C(3)); 23.8 (C(2)); 16.8 (C(14)); 15.3 (C(15)); 10.5 (C(13)). EI-MS: 262 (18.2, M þ ), 43 (100, C3H þ7 ). Measurement of NO Production and Cell Viability in LPS-Activated BV-2 Cells. The mouse microglia cell line BV-2 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and maintained in Dulbeccos modified Eagles medium containing 1800 mg/l NaHCO3 , supplemented with 10% fetal bovine serum, 100 U/ml penicilin, and 100 mg/ml streptomycin at 378 in a humidified atmosphere with 5% CO2 . The anti-neuroinflammatory activity in BV-2 cells was evaluated as described in [29] with modification. The NO production was quantified by nitrite accumulation in the culture medium using the Griess reaction kit (Beyotime Biotechnology, P. R. China) according to the manufacturers instructions. Briefly, BV-2 cells were pretreated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 mm) of indicated compounds for 4 h, and then stimulated with or without lipopolysaccharide (LPS; 1 mg/ml, SigmaAldrich) for 24 h. The isolated supernatants were mixed with an equal volume of Griess reagent. NaNO2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm with a microplate reader (M200, TECAN, Austria GmbH, Austria). Cell viability was measured using a MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide) colorimetric assay [30]. NG-monomethyl-l-arginine (l-NMMA; Beyotime, purity 99%), a well-known NO synthase inhibitor, served as a positive control. The IC50 values were determined by GriphPad Prism 5.
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