FITOTE-03078; No of Pages 6 Fitoterapia xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
Q42Q3 3 4 5
Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal
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F
journal homepage: www.elsevier.com/locate/fitote
Jin-Bo Zhang a, Ming-Li Liu a, Chang Li b, Yan Zhang c, Yi Dai a,⁎, Xin-Sheng Yao a,b a b c
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China State-Province Key Laboratory of Biomedicine-Pharmaceutics of China, Department of Pharmacology, Harbin Medical University, Harbin 150086, PR China
6
a r t i c l e
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a b s t r a c t
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Article history: Received 27 September 2014 Accepted in revised form 21 November 2014 Available online xxxx
20 21 22 23
Keywords: Nardosinane-type sesquiterpenoids Nardostachys chinensis Batal Myocardial injury
24
1. Introduction
25 26
Nardostachys chinensis Batal, belonging to the genus Nardostachys (Valerianaceae), is a perennial herbal plant distributed in the Himalayan mountain areas [1]. Its underground part is used as a traditional Chinese medicine for the treatment of stomachic and sedative effects [2]. Phytochemical studies on this plant led to the isolation of a series of sesquiterpenoids, including guaiane, aristolane and nardosinane-type compounds [3–6], most of which possessed various bioactivities, such as antinociceptive [5], antimalarial [6], and cytotoxic activities [7]. Our preliminary study on N. chinensis discovered an aristolane-chalcone derivative and a nor-aristolane sesquiterpenoid [8], as well as several new sesquiterpenes [9,10]. In this paper, we mainly reported the isolation and structural elucidation of four new nardosinanetype sesquiterpenoids nardosinanone F–I (1–2, 4–5), along with eight known ones (3, 6–12) (Fig. 1). As far as we know, nardosinane-type sesquiterpene is a rare class of sesquiterpenes, which has only been found in soft corals, liverworts and Nardostachys genus plants [3,11–14]. Compounds 1 and 2 are another two new nardosinane sesquiterpenes bearing a rare
P
8
Q5 14 15 16 17 18 19
33 34 35 36 37 38 39 40 41 42 43 44
R
R
O
31 32
N C
29 30
U
27 28
E
C
T
E
D
Four new nardosinane-type sesquiterpenoids nardosinanone F–I (1–2, 4–5), along with eight known sesquiterpenoids (3, 6–12) were isolated from the underground parts of Nardostachys chinensis Batal. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compounds 1 and 2 were new nardosinane sesquiterpenoids processing a rare 4,11-epoxy group in nature. In addition, compounds 1, 5–7, 11 and 12 showed protective effects on neonatal rat cardiomyocyte injury induced by hydrogen peroxide. © 2014 Published by Elsevier B.V.
⁎ Corresponding author. Tel.: +86 20 85220785; fax: +86 20 85221559. E-mail address: [email protected] (Y. Dai).
4,11-epoxy group in nature. Up to date, there is only one new nardosinane-type sesquiterpene with such structural feature reported from the underground parts of N. chinensis [3]. The plausible biogenetic pathway was also proposed. Moreover, compounds 1, 2, 4–7, 11 and 12 were evaluated for their protective effects on neonatal rat cardiomyocyte injury induced by hydrogen peroxide.
45 46
2. Experimental
52
2.1. General experimental procedures
53
Optical rotations were measured on a Jasco P-1020 polarimeter. HR-ESI-MS spectra were acquired using a Waters Synapt G2 mass spectrometer. The NMR spectra were measured with Bruker AV300 and 400 spectrometers in CDCl3. Diaion HP-20 (Mitsubishi-Chemical, Japan), silica gel (100– 200 mesh, 200–300 mesh, Qingdao Marine Chemical Ltd., China), octadecylsilanized (ODS) silica gel (50 μm; YMC Ltd., Japan) and Sephadex LH-20 (50 μm; Amersham Pharmacia Biotech, Sweden) were used for open column chromatography (CC). Preparative HPLC was performed on a Shimadzu LC6AD system equipped with a UV detector, using a Cosmosil C-18 column (20 × 250 mm, 5 μm; Nacalai Tesque Inc., Japan; detector set at 210 nm and 280 nm) with a flow rate of
54
http://dx.doi.org/10.1016/j.fitote.2014.11.020 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
47 48 49 50 51
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J.-B. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
2.2. Plant material
72 73
76
The plant material was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. and identified as N. chinensis by Dr. Qingcun Tian. A voucher specimen (20091020NC) was deposited at the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, China.
77
2.3. Extraction and isolation
78
The air-dried underground parts (5 kg) of N. chinensis were chopped and refluxed twice with 60% EtOH for 2 h each time. The crude extract (850 g) was subjected to a macroporous resin HP-20 column chromatography (10 × 100 cm), eluted with water and aqueous ethanol in gradient (0:100; 10:90; 30:70; 50:50; 70:30; 95:5). The 70% EtOH–H2O eluate (171.7 g) was fractionated by silica gel CC (10 × 48 cm) eluted with CHCl3– MeOH (100:0 → 0:100) to afford twelve fractions (Fr. 1–12). Fr. 3 (CHCl3–MeOH 99:1 eluate, 53.4 g) was further chromatographed over silica gel (5.5 × 48 cm) using a gradient elution of petroleum ether–EtOAc to yield nine subfractions (Fr. 3.1–3.9). Fr. 3.8 (petroleum ether–EtOAc 10:1 eluate, 9.1 g) was then subjected to an opening ODS CC (3.5 × 18 cm) eluted with MeOH–H2O (50% → 100%) to afford ten fine-fractions (Fr. 3.8.1–3.8.10). Fr. 3.8.2 (60% MeOH–H2O eluate, 410.6 mg) was purified by preparative ODS HPLC with 45% MeOH–H2O to yield compound 1 (7.9 mg, tR = 18 min) and compound 3 (3.8 mg, tR = 16 min). Fr. 3.8.3 (60% MeOH–H2O eluate, 876.1 mg) was subjected to a Sephadex LH-20 CC (2 × 120 cm; CHCl3–MeOH 7:3) followed by preparative ODS HPLC with 55% MeOH–H2O to afford compounds 4 (36.9 mg, tR = 27 min) and 5 (73.1 mg, tR = 31 min). Fr. 5 (CHCl3–MeOH 99:1 eluate, 37.4 g) was chromatographed over silica gel (8 × 44 cm) using a gradient elution of petroleum ether–EtOAc to yield six subfractions (Fr. 5.1–5.6). Fr. 5.1 (petroleum ether–EtOAc 8:2 eluate, 6.3 g) was further
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103
C
E R
R
O
83 84
C
81 82
N
79 80
U
74 75
D
71
subjected to an opening ODS CC (3.5 × 18 cm) eluted with MeOH–H2O in gradient to give eight subfractions (Fr. 5.1.1– 5.1.8). Fr. 5.1.1 (40% MeOH–H2O eluate, 209 mg) was separated by preparative ODS HPLC with 45% MeOH–H2O to yield compound 7 (17.6 mg, tR = 30 min). Fr. 5.1.2 (50% MeOH– H2O eluate, 3.7 g) was further separated to an opening ODS CC (3.5 × 18 cm) eluted with 50% MeOH–H2O to give compound 9 (135 mg). Fr. 5.1.4 (60% MeOH–H2O eluate, 108 mg) was chromatographed on preparative ODS HPLC with 20% MeCN– H2O to afford compound 8 (10.7 mg, tR = 24 min), compound 11 (10.0 mg, tR = 45 min) and compound 12 (5.4 mg, tR = 49 min). Fr. 5.2 (petroleum ether–EtOAc 8:2 eluate, 3.1 g) was purified using Sephadex LH-20 CC (2 × 120 cm; CHCl3–MeOH 7:3) to afford compound 6 (2.9 g). Fr. 5.3 (petroleum ether– EtOAc 7:3 eluate, 10.6 g) was subjected to an opening ODS CC (3.5 × 30 cm) eluted with MeOH–H2O in gradient to give ten fractions (Fr. 5.3.1–5.3.10). Fr. 5.3.2 (eluted with 45% MeOH– H2O, 2.38 g) was chromatographed on Sephadex LH-20 (2 × 120 cm; 80% MeOH–H2O) to afford compound 10 (2.08 g) and Fr. 5.3.2.1 (291 mg), followed by preparative ODS HPLC with 40% MeOH–H2O to yield compound 2 (3.0 mg, tR = 20 min). Nardosinanone F (1): yellow, oil; [α]26 D −114.6 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 221 (4.02) nm, 258 (4.02) nm, 323 (4.02) nm; IR (KBr) νmax 3381, 2985, 2937, 1713 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 253.1200 [M + Na]+ (calcd for C15H18O2Na 253.1204). Nardosinanone G (2): yellow, oil; [α]26 D −73.4 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 206 (4.12) nm, 256 (3.05) nm; IR (KBr) νmax 3385, 2986, 2933, 1710 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 247.1335 [M + H]+ (calcd for C15H19O3 247.1334). Nardosinanone H (4): yellow, oil; [α]26 D −343.0 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 229 (4.03) nm, 265 (3.09) nm; IR (KBr) νmax 2989, 1667 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 231.1389 [M + H]+ (calcd for C15H19O2 231.1385). Nardosinanone I (5): yellow, oil; [α]26 D −253.8 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 247 (3.01) nm; IR (KBr) νmax 2985, 1662 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 233.1536 [M + H]+ (calcd for C15H21O2 233.1542).
T
69 70
8.0 mL/min. Thin-layer chromatography (TLC) was performed on silica gel GF254 plates (Yantai Chemical Inst., China), and spots were visualized by spraying with concentrated sulfuric acid–vanillin solution followed by heating.
E
67 68
P
Fig. 1. Chemical structures of compounds 1–12.
R O
O
F
2
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
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J.-B. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx t1:1 Q1 t1:2
3
Table 1 1 H NMR and 13C NMR data for compounds 1 and 2 (400/100 MHz, in CDCl3) and 4 and 5 (300/75 MHz, in CDCl3).
t1:3
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
t1:4 t1:5
1 2
126.0 124.2
6.92 (d, 5.7) 6.23 (dd, 9.4, 5.7)
127.3 197.0
6.44 (s)
128.9 199.4
6.67 (s)
136.9 26.0
t1:6
3
135.7
5.92 (d, 9.4)
42.3
2.32 (m)
26.4
t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15
4 5 6 7 8 9 10 11 12
80.2 49.0 57.5 145.1 131.4 187.6 137.7 79.0 27.3
2.32 (m)
32.3 40.8 52.2 148.3 130.8 187.9 140.4 57.4 55.5
6.90 (dd, 6.2, 2.8) 2.26 (m) 2.16 (m) 1.42 (m) 1.55 (m) 1.99 (m)
t1:16 t1:17 t1:18
13 14 15
31.9 20.6 24.3
4
45.8
2.44 (d, 17.5) 2.76 (d, 17.5)
0.87 (s)
84.7 52.0 57.1 146.6 130.5 189.0 154.6 81.3 27.9
0.94 (s)
34.8 42.0 55.0 150.9 128.7 186.8 157.0 141.1 118.7
1.44 (s) 0.97 (s) 1.36 (s)
32.2 20.5 24.9
1.43 (s) 1.27 (s) 1.35 (s)
18.7 19.9 15.4
154 155
E
152 153 Q9
R
150 151 Q8
3. Results and discussion
157
The extract was subjected to column chromatography to yield four new nardosinane-type sesquiterpenoids nardosinanone F–I (1–2, 4–5), as well as eight known compounds, including nardonoxide (3) [3], nardosinonediol (6), kanshone E (7), isonardosmone (8) [17], kanshone A (9) [18], desoxo-narchinolA (10) [7], narchinol B (11) [19], and narchinol A (12) [20]. Nardosinanone F (1) was obtained as a yellow oil with [α]26 D −114.6 (c 0.50, MeOH). HR-ESI-MS of 1 displayed the pseudomolecular ion peak at m/z 253.1200 [M + Na]+ (calcd for C15H18O2Na, 253.1204) corresponding to the molecular formula C15H18O2 with seven degrees of unsaturation. The 13C
164 165 166 167
O
N C
162 163
U
160 161
R
156
158 159
D
Primary culture of neonatal rat cardiomyocytes was prepared from 1 to 3-day-old Wistar rats by trypsin according to the method described in Lu's report [15,16]. Firstly, 1 μM of different compounds was used to evaluate protective effects on neonatal rat cardiomyocytes from H2O2 injury (100 μM), and salvianolic acid B was used as the positive control. Then the effective compounds were further studied using the dose dependent effect with different concentrations (0.01 μM, 0.1 μM, 1 μM).
1 2 3 15
4
O
O
O 9
10 5
8 6
O
O
O 1H-1H
7
11
14
O
17.7 22.3 16.6
12
2.82 (d, 4.8) 2.68 (d, 4.8) 1.07 (s) 1.01 (s) 0.93 (d, 6.6)
E
147
4.98 (br s) 4.96 (br s) 1.48 (s) 1.21 (s) 0.99 (d, 6.2)
2.14 (d, 6.3) 6.88 (dd, 10.1, 6.3) 6.28 (d, 10.1)
NMR and DEPT-135 spectra revealed 15 carbon signals, attributable to four methyl, six methine (five olefinic), and five quaternary carbons (one carbonyl, two oxygenated, and one olefinic). In the 1H–1H COSY spectrum (Fig. 2), two sets of spin coupling systems suggested the presence of the following structural units: C-1–C-2–C-3 and C-6–C-7–C-8. Moreover, the key HMBC correlations of H3-14/C-4, C-5, C-6 and C-10; H315/C-3 and C-4; H-1/C-9; H-7/C-9; and H3-12/C-6 and C-11 permitted the establishment of a nardosinane-type skeleton bearing the carbonyl group at C-9. The chemical shifts of C-4 (δC 80.2) and C-11 (δC 79.0) indicated that each of C-4 and C-11 owned a hydroxyl group. Taking into account the molecular formula, which showed two oxygens in the structure, the gross structure of 1 was deduced as a novel nardosinane sesquiterpene bearing a rare 4,11-epoxy group. In the NOESY spectrum, the correlations from H3-14 to H-6/H3-15 demonstrated that H3-14, H3-15 and H-6 are located in the same orientation (Fig. 3). The nardosinane sesquiterpene is a rather rare type of sesquiterpenes in nature; they are considered to derive from an aristolane precursor. (+)-Aristolone occurs in liverworts and sponges, while the (−)-aristolone is characteristic of terrestrial plants and soft corals [9,21]. Compound 1 had the same rotation sign (−114.6) as (−)-aristolone, and demonstrated that they belonged to the same enantiomeric series. Thus, the absolute configuration of 1 was assigned as 4S, 5R, 6R. Nardosinanone G (2) was obtained as a yellow oil with [α]26 D −73.4 (c 0.50, MeOH). It possessed the molecular formula of
C
2.4. Culture of neonatal rat cardiomyocytes and the assay for cell viability
3.25 (d, 6.5) 6.88 (dd, 10.1, 6.5) 6.30 (d, 10.1)
P
3.07 (d, 6.2) 6.87 (dd, 10.0, 6.2) 6.33 (d, 10.0)
T
2.93 (d, 6.3) 6.81 (dd, 10.1, 6.3) 6.31 (d, 10.1)
145 146
148 149
5
F
2
R O O
1
O
O
COSY
HMBC
13
1
2
4
5
Fig. 2. Key HMBC and 1H–1H COSY correlations of compounds 1–2 and 4–5.
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
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O
F
4
210
P
D
E
T
C
208 209
E
206 207
configuration determined by NOESY data, an absolute configuration of 4S, 5R, 6R was assigned to 2. Nardosinanone H (4) was obtained as a yellow oil with [α]26 D −343.0 (c 0.50, MeOH). Its molecular formula C15H18O2 was deduced by HR-ESI-MS, with seven degrees of unsaturation. The 13C NMR and DEPT-135 spectra revealed 15 carbons, including three methyl, two methylene (one olefinic), five methine (three olefinic), and five quaternary carbons (two carbonyl and two olefinic). The remaining two degrees of unsaturation required 4 to be a bicyclic sesquiterpene containing two terminal double bonds. The 1H–1H COSY showed two structural units: C-3–C-4–C-15 and C-6–C-7–C-8 (Fig. 2). Additionally, the HMBC correlations of H3-14/C-4, C-5, C-6 and C-10; H-1/C-3 and C-9; H-4/C-2; H-7/C-9; and H3-13/C-6, C-11 and C-12 permitted the establishment of the planar structure of 4 (Fig. 1). The NOESY correlations from H3-14 to
O
O O deoxidation epoxidation O
O
7
[O]
O
OH
O
8
[-H2O] OH
[O]
O
[-H] O
OH
O
O
[-H2O]
O
OH
9
10
1 [H] [-H2O]
O
cleavage
OH
OH
epoxidation
[-H2O] [O]
O
O
O
O
4
5
O
O
R
204 205
R
202 203
O
200 201
C
198 199
C15H18O3, which was determined by HREIMS at m/z 247.1335 [M + H]+ (calcd for C15H19O3 247.1334), with seven degrees of unsaturation. 1D- and 2D-NMR data analyses demonstrated that 2 was a nardosinane skeleton bearing two carbonyl groups at C-2 and C-9 (Fig. 2). The comparative analysis of 1H- and 13C NMR data (Table 1) of 1 and 2 revealed that two olefinic carbons at δC 124.2, 135.7 in 1 were replaced by one carbonyl carbon at δC 197.0 and one methylene carbon at δC 45.8 in 2. This assumption was further confirmed by the HMBC correlations H3-15/C-3 and H-3/C-2. NOESY correlations from H3-14 to H-6 and H3-15 indicated that 2 had the same relative configuration as 1. In the CD spectrum of 2, the negative Cotton effects at 343 nm (Fig. S2-8, Supplementary data) confirmed the 6R configuration in 2 according to octane rule of cyclohexanone [22] (the exciton coupling of n–π* transition in α, β-unsaturated ketone moiety of cyclohexenone). Considering the relative
N
196 197
U
195
R O
Fig. 3. Key NOESY correlations of compounds 1, 4 and 5.
nardosinonediol (6)
3
2
[O] O
O O
HO
[-H] OH
OH 11
12 Fig. 4. Possible biosynthetic pathway of compounds 1–12.
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
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J.-B. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
B
120
120
0.01µM 0.1µM 1µM
80
*
*
*
*
*
*
*
60
40
viability (%)
100 80
* *
* * * *
*
*
*
*
*
* *
* *
*
0
sa
lv
ia
no
H
co nt ro l
F
0
R O O
20
ro l 2 lic O2 co a m cid p co ou B m nd po -1 co u m nd p co ou 2 m nd p co ou 4 m nd po -5 co u m nd co po -6 m un dp co oun 7 m d po -11 un d12
20
co
*
60 40
nt
viability (%)
100
sa H 2O lv ia 2 no lic ac id B co m po un d1 co m po un d5 co m po un d6 co m po un dco 7 m po un d11 co m po un d12
A
5
260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287
Conflict of interest
288
D
289
The authors declare no conflict of interest.
290
This research was financially supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” of China (Grant No. 2011ZX09201201-28), the National Program on Key Basic Research Project of China (973 Program Grant No. 2012CB518606), and the Program of Introducing Talents of Discipline to Universities (B13038).
291 292
Appendix A. Supplementary data
298
The HRESI-MS, 1D NMR, and 2D NMR for compounds 1–2 and 4–5 are available in Supplementary data. Supplementary data to this article can be found online at http://dx.doi.org/10. 1016/j.fitote.2014.11.020.
299 300
References
303
E
Acknowledgments
T
C
E
258 259
R
256 257
R
254 255
O
252 253
N C
250 251
H3-15/H-6 and from H3-13 to H-4 demonstrated the relative configuration as shown in Fig. 3. Taking the biosynthesis of natural products into account, aristolane sesquiterpenes (the putative biosynthetic precursor of nardosinane) isolated from this plant bear an R-configuration methyl group at C-4 [5,6,8,23]. Thus, the absolute configuration of 4 was determined as 4R, 5R, 6S. Nardosinanone I (5), obtained as a yellow oil with [α]26 D −253.8 (c 0.50, MeOH), was assigned with the molecular formula C15H20O2 according to its HR-ESI-MS at m/z 233.1536 [M + H]+ (calcd for C15H21O2, 233.1542), indicating six degrees of unsaturation. The 1H and 13C NMR data of 5 were very similar to those of kanshone A (9) [18], except that one oxygenated quaternary carbon (δC 57.4, C-11) and one methylene (δC 55.5; C-12) in 5 replaced one hydroxy group and one methyl in 9, indicating a three-membered epoxide ring at C-11 and C-12. Meanwhile, 2D-NMR correlations suggested the deduction and established the planar structure of 5 as shown in Fig. 2. The NOESY correlations from H3-14 to H3-15/H-6 and from H3-13 to H-4 indicated the relative configuration. 5 had the same rotation sign (−253.8) as 1; it could be concluded that the absolute configuration of 5 was assigned as 4R, 5R, 6R, 11S. In a previous phytochemistry investigation, an epoxy dienone (38 in reference) had been synthesized [24], however the NMR spectral data were not available. In the present study, we carried out detailed NMR data analysis and confirmed its structure as nardosinanone I. The plausible biogenetic pathway for 1–12 was proposed as shown in Fig. 4. The aristolane-type sesquiterpene precursor, nardosinonediol (6), would be transformed into 1–12 through a series of biochemical reactions including oxidation, dehydration, and cleavage as well as epoxidation reactions. Eight compounds (1, 2, 4–7, 11, 12) were evaluated for their protective effects on neonatal rat cardiomyocyte injury induced by hydrogen peroxide (H2O2) with salvianolic acid B as the positive control. Compounds 1, 5, 6, 7, 11 and 12 had protective effects on H2O2 induced myocardial injury (p b 0.05, vs H2O2 group) (Fig. 5). Compounds 1, 5, 11 and 12 can dosedependently protect neonatal rat cardiomyocytes from H2O2 injury from 0.01 to 1 μM (p b 0.05, vs H2O2 group) (Fig. 5).
U
248 249
P
Fig. 5. Effect of compounds 1, 2, 4–7, 11, and 12 on neonatal rat cardiomyocyte injury induced by H2O2.
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O
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Contents lists available at ScienceDirect
Fitoterapia
Q42Q3 3 4 5
Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal
R O O
1Q2
F
journal homepage: www.elsevier.com/locate/fitote
Jin-Bo Zhang a, Ming-Li Liu a, Chang Li b, Yan Zhang c, Yi Dai a,⁎, Xin-Sheng Yao a,b a b c
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China State-Province Key Laboratory of Biomedicine-Pharmaceutics of China, Department of Pharmacology, Harbin Medical University, Harbin 150086, PR China
6
a r t i c l e
i n f o
a b s t r a c t
9 13 10 11 12
Article history: Received 27 September 2014 Accepted in revised form 21 November 2014 Available online xxxx
20 21 22 23
Keywords: Nardosinane-type sesquiterpenoids Nardostachys chinensis Batal Myocardial injury
24
1. Introduction
25 26
Nardostachys chinensis Batal, belonging to the genus Nardostachys (Valerianaceae), is a perennial herbal plant distributed in the Himalayan mountain areas [1]. Its underground part is used as a traditional Chinese medicine for the treatment of stomachic and sedative effects [2]. Phytochemical studies on this plant led to the isolation of a series of sesquiterpenoids, including guaiane, aristolane and nardosinane-type compounds [3–6], most of which possessed various bioactivities, such as antinociceptive [5], antimalarial [6], and cytotoxic activities [7]. Our preliminary study on N. chinensis discovered an aristolane-chalcone derivative and a nor-aristolane sesquiterpenoid [8], as well as several new sesquiterpenes [9,10]. In this paper, we mainly reported the isolation and structural elucidation of four new nardosinanetype sesquiterpenoids nardosinanone F–I (1–2, 4–5), along with eight known ones (3, 6–12) (Fig. 1). As far as we know, nardosinane-type sesquiterpene is a rare class of sesquiterpenes, which has only been found in soft corals, liverworts and Nardostachys genus plants [3,11–14]. Compounds 1 and 2 are another two new nardosinane sesquiterpenes bearing a rare
P
8
Q5 14 15 16 17 18 19
33 34 35 36 37 38 39 40 41 42 43 44
R
R
O
31 32
N C
29 30
U
27 28
E
C
T
E
D
Four new nardosinane-type sesquiterpenoids nardosinanone F–I (1–2, 4–5), along with eight known sesquiterpenoids (3, 6–12) were isolated from the underground parts of Nardostachys chinensis Batal. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compounds 1 and 2 were new nardosinane sesquiterpenoids processing a rare 4,11-epoxy group in nature. In addition, compounds 1, 5–7, 11 and 12 showed protective effects on neonatal rat cardiomyocyte injury induced by hydrogen peroxide. © 2014 Published by Elsevier B.V.
⁎ Corresponding author. Tel.: +86 20 85220785; fax: +86 20 85221559. E-mail address: [email protected] (Y. Dai).
4,11-epoxy group in nature. Up to date, there is only one new nardosinane-type sesquiterpene with such structural feature reported from the underground parts of N. chinensis [3]. The plausible biogenetic pathway was also proposed. Moreover, compounds 1, 2, 4–7, 11 and 12 were evaluated for their protective effects on neonatal rat cardiomyocyte injury induced by hydrogen peroxide.
45 46
2. Experimental
52
2.1. General experimental procedures
53
Optical rotations were measured on a Jasco P-1020 polarimeter. HR-ESI-MS spectra were acquired using a Waters Synapt G2 mass spectrometer. The NMR spectra were measured with Bruker AV300 and 400 spectrometers in CDCl3. Diaion HP-20 (Mitsubishi-Chemical, Japan), silica gel (100– 200 mesh, 200–300 mesh, Qingdao Marine Chemical Ltd., China), octadecylsilanized (ODS) silica gel (50 μm; YMC Ltd., Japan) and Sephadex LH-20 (50 μm; Amersham Pharmacia Biotech, Sweden) were used for open column chromatography (CC). Preparative HPLC was performed on a Shimadzu LC6AD system equipped with a UV detector, using a Cosmosil C-18 column (20 × 250 mm, 5 μm; Nacalai Tesque Inc., Japan; detector set at 210 nm and 280 nm) with a flow rate of
54
http://dx.doi.org/10.1016/j.fitote.2014.11.020 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
47 48 49 50 51
55 56 57 58 59 60 61 62 63 64 Q6 65 66
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2.2. Plant material
72 73
76
The plant material was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. and identified as N. chinensis by Dr. Qingcun Tian. A voucher specimen (20091020NC) was deposited at the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, China.
77
2.3. Extraction and isolation
78
The air-dried underground parts (5 kg) of N. chinensis were chopped and refluxed twice with 60% EtOH for 2 h each time. The crude extract (850 g) was subjected to a macroporous resin HP-20 column chromatography (10 × 100 cm), eluted with water and aqueous ethanol in gradient (0:100; 10:90; 30:70; 50:50; 70:30; 95:5). The 70% EtOH–H2O eluate (171.7 g) was fractionated by silica gel CC (10 × 48 cm) eluted with CHCl3– MeOH (100:0 → 0:100) to afford twelve fractions (Fr. 1–12). Fr. 3 (CHCl3–MeOH 99:1 eluate, 53.4 g) was further chromatographed over silica gel (5.5 × 48 cm) using a gradient elution of petroleum ether–EtOAc to yield nine subfractions (Fr. 3.1–3.9). Fr. 3.8 (petroleum ether–EtOAc 10:1 eluate, 9.1 g) was then subjected to an opening ODS CC (3.5 × 18 cm) eluted with MeOH–H2O (50% → 100%) to afford ten fine-fractions (Fr. 3.8.1–3.8.10). Fr. 3.8.2 (60% MeOH–H2O eluate, 410.6 mg) was purified by preparative ODS HPLC with 45% MeOH–H2O to yield compound 1 (7.9 mg, tR = 18 min) and compound 3 (3.8 mg, tR = 16 min). Fr. 3.8.3 (60% MeOH–H2O eluate, 876.1 mg) was subjected to a Sephadex LH-20 CC (2 × 120 cm; CHCl3–MeOH 7:3) followed by preparative ODS HPLC with 55% MeOH–H2O to afford compounds 4 (36.9 mg, tR = 27 min) and 5 (73.1 mg, tR = 31 min). Fr. 5 (CHCl3–MeOH 99:1 eluate, 37.4 g) was chromatographed over silica gel (8 × 44 cm) using a gradient elution of petroleum ether–EtOAc to yield six subfractions (Fr. 5.1–5.6). Fr. 5.1 (petroleum ether–EtOAc 8:2 eluate, 6.3 g) was further
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103
C
E R
R
O
83 84
C
81 82
N
79 80
U
74 75
D
71
subjected to an opening ODS CC (3.5 × 18 cm) eluted with MeOH–H2O in gradient to give eight subfractions (Fr. 5.1.1– 5.1.8). Fr. 5.1.1 (40% MeOH–H2O eluate, 209 mg) was separated by preparative ODS HPLC with 45% MeOH–H2O to yield compound 7 (17.6 mg, tR = 30 min). Fr. 5.1.2 (50% MeOH– H2O eluate, 3.7 g) was further separated to an opening ODS CC (3.5 × 18 cm) eluted with 50% MeOH–H2O to give compound 9 (135 mg). Fr. 5.1.4 (60% MeOH–H2O eluate, 108 mg) was chromatographed on preparative ODS HPLC with 20% MeCN– H2O to afford compound 8 (10.7 mg, tR = 24 min), compound 11 (10.0 mg, tR = 45 min) and compound 12 (5.4 mg, tR = 49 min). Fr. 5.2 (petroleum ether–EtOAc 8:2 eluate, 3.1 g) was purified using Sephadex LH-20 CC (2 × 120 cm; CHCl3–MeOH 7:3) to afford compound 6 (2.9 g). Fr. 5.3 (petroleum ether– EtOAc 7:3 eluate, 10.6 g) was subjected to an opening ODS CC (3.5 × 30 cm) eluted with MeOH–H2O in gradient to give ten fractions (Fr. 5.3.1–5.3.10). Fr. 5.3.2 (eluted with 45% MeOH– H2O, 2.38 g) was chromatographed on Sephadex LH-20 (2 × 120 cm; 80% MeOH–H2O) to afford compound 10 (2.08 g) and Fr. 5.3.2.1 (291 mg), followed by preparative ODS HPLC with 40% MeOH–H2O to yield compound 2 (3.0 mg, tR = 20 min). Nardosinanone F (1): yellow, oil; [α]26 D −114.6 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 221 (4.02) nm, 258 (4.02) nm, 323 (4.02) nm; IR (KBr) νmax 3381, 2985, 2937, 1713 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 253.1200 [M + Na]+ (calcd for C15H18O2Na 253.1204). Nardosinanone G (2): yellow, oil; [α]26 D −73.4 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 206 (4.12) nm, 256 (3.05) nm; IR (KBr) νmax 3385, 2986, 2933, 1710 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 247.1335 [M + H]+ (calcd for C15H19O3 247.1334). Nardosinanone H (4): yellow, oil; [α]26 D −343.0 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 229 (4.03) nm, 265 (3.09) nm; IR (KBr) νmax 2989, 1667 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 231.1389 [M + H]+ (calcd for C15H19O2 231.1385). Nardosinanone I (5): yellow, oil; [α]26 D −253.8 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 247 (3.01) nm; IR (KBr) νmax 2985, 1662 cm−1; 1H and 13C NMR: see Table 1; HR-ESI-MS (positive ion mode): m/z 233.1536 [M + H]+ (calcd for C15H21O2 233.1542).
T
69 70
8.0 mL/min. Thin-layer chromatography (TLC) was performed on silica gel GF254 plates (Yantai Chemical Inst., China), and spots were visualized by spraying with concentrated sulfuric acid–vanillin solution followed by heating.
E
67 68
P
Fig. 1. Chemical structures of compounds 1–12.
R O
O
F
2
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
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J.-B. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx t1:1 Q1 t1:2
3
Table 1 1 H NMR and 13C NMR data for compounds 1 and 2 (400/100 MHz, in CDCl3) and 4 and 5 (300/75 MHz, in CDCl3).
t1:3
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
t1:4 t1:5
1 2
126.0 124.2
6.92 (d, 5.7) 6.23 (dd, 9.4, 5.7)
127.3 197.0
6.44 (s)
128.9 199.4
6.67 (s)
136.9 26.0
t1:6
3
135.7
5.92 (d, 9.4)
42.3
2.32 (m)
26.4
t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15
4 5 6 7 8 9 10 11 12
80.2 49.0 57.5 145.1 131.4 187.6 137.7 79.0 27.3
2.32 (m)
32.3 40.8 52.2 148.3 130.8 187.9 140.4 57.4 55.5
6.90 (dd, 6.2, 2.8) 2.26 (m) 2.16 (m) 1.42 (m) 1.55 (m) 1.99 (m)
t1:16 t1:17 t1:18
13 14 15
31.9 20.6 24.3
4
45.8
2.44 (d, 17.5) 2.76 (d, 17.5)
0.87 (s)
84.7 52.0 57.1 146.6 130.5 189.0 154.6 81.3 27.9
0.94 (s)
34.8 42.0 55.0 150.9 128.7 186.8 157.0 141.1 118.7
1.44 (s) 0.97 (s) 1.36 (s)
32.2 20.5 24.9
1.43 (s) 1.27 (s) 1.35 (s)
18.7 19.9 15.4
154 155
E
152 153 Q9
R
150 151 Q8
3. Results and discussion
157
The extract was subjected to column chromatography to yield four new nardosinane-type sesquiterpenoids nardosinanone F–I (1–2, 4–5), as well as eight known compounds, including nardonoxide (3) [3], nardosinonediol (6), kanshone E (7), isonardosmone (8) [17], kanshone A (9) [18], desoxo-narchinolA (10) [7], narchinol B (11) [19], and narchinol A (12) [20]. Nardosinanone F (1) was obtained as a yellow oil with [α]26 D −114.6 (c 0.50, MeOH). HR-ESI-MS of 1 displayed the pseudomolecular ion peak at m/z 253.1200 [M + Na]+ (calcd for C15H18O2Na, 253.1204) corresponding to the molecular formula C15H18O2 with seven degrees of unsaturation. The 13C
164 165 166 167
O
N C
162 163
U
160 161
R
156
158 159
D
Primary culture of neonatal rat cardiomyocytes was prepared from 1 to 3-day-old Wistar rats by trypsin according to the method described in Lu's report [15,16]. Firstly, 1 μM of different compounds was used to evaluate protective effects on neonatal rat cardiomyocytes from H2O2 injury (100 μM), and salvianolic acid B was used as the positive control. Then the effective compounds were further studied using the dose dependent effect with different concentrations (0.01 μM, 0.1 μM, 1 μM).
1 2 3 15
4
O
O
O 9
10 5
8 6
O
O
O 1H-1H
7
11
14
O
17.7 22.3 16.6
12
2.82 (d, 4.8) 2.68 (d, 4.8) 1.07 (s) 1.01 (s) 0.93 (d, 6.6)
E
147
4.98 (br s) 4.96 (br s) 1.48 (s) 1.21 (s) 0.99 (d, 6.2)
2.14 (d, 6.3) 6.88 (dd, 10.1, 6.3) 6.28 (d, 10.1)
NMR and DEPT-135 spectra revealed 15 carbon signals, attributable to four methyl, six methine (five olefinic), and five quaternary carbons (one carbonyl, two oxygenated, and one olefinic). In the 1H–1H COSY spectrum (Fig. 2), two sets of spin coupling systems suggested the presence of the following structural units: C-1–C-2–C-3 and C-6–C-7–C-8. Moreover, the key HMBC correlations of H3-14/C-4, C-5, C-6 and C-10; H315/C-3 and C-4; H-1/C-9; H-7/C-9; and H3-12/C-6 and C-11 permitted the establishment of a nardosinane-type skeleton bearing the carbonyl group at C-9. The chemical shifts of C-4 (δC 80.2) and C-11 (δC 79.0) indicated that each of C-4 and C-11 owned a hydroxyl group. Taking into account the molecular formula, which showed two oxygens in the structure, the gross structure of 1 was deduced as a novel nardosinane sesquiterpene bearing a rare 4,11-epoxy group. In the NOESY spectrum, the correlations from H3-14 to H-6/H3-15 demonstrated that H3-14, H3-15 and H-6 are located in the same orientation (Fig. 3). The nardosinane sesquiterpene is a rather rare type of sesquiterpenes in nature; they are considered to derive from an aristolane precursor. (+)-Aristolone occurs in liverworts and sponges, while the (−)-aristolone is characteristic of terrestrial plants and soft corals [9,21]. Compound 1 had the same rotation sign (−114.6) as (−)-aristolone, and demonstrated that they belonged to the same enantiomeric series. Thus, the absolute configuration of 1 was assigned as 4S, 5R, 6R. Nardosinanone G (2) was obtained as a yellow oil with [α]26 D −73.4 (c 0.50, MeOH). It possessed the molecular formula of
C
2.4. Culture of neonatal rat cardiomyocytes and the assay for cell viability
3.25 (d, 6.5) 6.88 (dd, 10.1, 6.5) 6.30 (d, 10.1)
P
3.07 (d, 6.2) 6.87 (dd, 10.0, 6.2) 6.33 (d, 10.0)
T
2.93 (d, 6.3) 6.81 (dd, 10.1, 6.3) 6.31 (d, 10.1)
145 146
148 149
5
F
2
R O O
1
O
O
COSY
HMBC
13
1
2
4
5
Fig. 2. Key HMBC and 1H–1H COSY correlations of compounds 1–2 and 4–5.
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
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O
F
4
210
P
D
E
T
C
208 209
E
206 207
configuration determined by NOESY data, an absolute configuration of 4S, 5R, 6R was assigned to 2. Nardosinanone H (4) was obtained as a yellow oil with [α]26 D −343.0 (c 0.50, MeOH). Its molecular formula C15H18O2 was deduced by HR-ESI-MS, with seven degrees of unsaturation. The 13C NMR and DEPT-135 spectra revealed 15 carbons, including three methyl, two methylene (one olefinic), five methine (three olefinic), and five quaternary carbons (two carbonyl and two olefinic). The remaining two degrees of unsaturation required 4 to be a bicyclic sesquiterpene containing two terminal double bonds. The 1H–1H COSY showed two structural units: C-3–C-4–C-15 and C-6–C-7–C-8 (Fig. 2). Additionally, the HMBC correlations of H3-14/C-4, C-5, C-6 and C-10; H-1/C-3 and C-9; H-4/C-2; H-7/C-9; and H3-13/C-6, C-11 and C-12 permitted the establishment of the planar structure of 4 (Fig. 1). The NOESY correlations from H3-14 to
O
O O deoxidation epoxidation O
O
7
[O]
O
OH
O
8
[-H2O] OH
[O]
O
[-H] O
OH
O
O
[-H2O]
O
OH
9
10
1 [H] [-H2O]
O
cleavage
OH
OH
epoxidation
[-H2O] [O]
O
O
O
O
4
5
O
O
R
204 205
R
202 203
O
200 201
C
198 199
C15H18O3, which was determined by HREIMS at m/z 247.1335 [M + H]+ (calcd for C15H19O3 247.1334), with seven degrees of unsaturation. 1D- and 2D-NMR data analyses demonstrated that 2 was a nardosinane skeleton bearing two carbonyl groups at C-2 and C-9 (Fig. 2). The comparative analysis of 1H- and 13C NMR data (Table 1) of 1 and 2 revealed that two olefinic carbons at δC 124.2, 135.7 in 1 were replaced by one carbonyl carbon at δC 197.0 and one methylene carbon at δC 45.8 in 2. This assumption was further confirmed by the HMBC correlations H3-15/C-3 and H-3/C-2. NOESY correlations from H3-14 to H-6 and H3-15 indicated that 2 had the same relative configuration as 1. In the CD spectrum of 2, the negative Cotton effects at 343 nm (Fig. S2-8, Supplementary data) confirmed the 6R configuration in 2 according to octane rule of cyclohexanone [22] (the exciton coupling of n–π* transition in α, β-unsaturated ketone moiety of cyclohexenone). Considering the relative
N
196 197
U
195
R O
Fig. 3. Key NOESY correlations of compounds 1, 4 and 5.
nardosinonediol (6)
3
2
[O] O
O O
HO
[-H] OH
OH 11
12 Fig. 4. Possible biosynthetic pathway of compounds 1–12.
Please cite this article as: Zhang J-B, et al, Nardosinane-type sesquiterpenoids of Nardostachys chinensis Batal, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.020
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B
120
120
0.01µM 0.1µM 1µM
80
*
*
*
*
*
*
*
60
40
viability (%)
100 80
* *
* * * *
*
*
*
*
*
* *
* *
*
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Conflict of interest
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The authors declare no conflict of interest.
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This research was financially supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” of China (Grant No. 2011ZX09201201-28), the National Program on Key Basic Research Project of China (973 Program Grant No. 2012CB518606), and the Program of Introducing Talents of Discipline to Universities (B13038).
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Appendix A. Supplementary data
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The HRESI-MS, 1D NMR, and 2D NMR for compounds 1–2 and 4–5 are available in Supplementary data. Supplementary data to this article can be found online at http://dx.doi.org/10. 1016/j.fitote.2014.11.020.
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References
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Acknowledgments
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H3-15/H-6 and from H3-13 to H-4 demonstrated the relative configuration as shown in Fig. 3. Taking the biosynthesis of natural products into account, aristolane sesquiterpenes (the putative biosynthetic precursor of nardosinane) isolated from this plant bear an R-configuration methyl group at C-4 [5,6,8,23]. Thus, the absolute configuration of 4 was determined as 4R, 5R, 6S. Nardosinanone I (5), obtained as a yellow oil with [α]26 D −253.8 (c 0.50, MeOH), was assigned with the molecular formula C15H20O2 according to its HR-ESI-MS at m/z 233.1536 [M + H]+ (calcd for C15H21O2, 233.1542), indicating six degrees of unsaturation. The 1H and 13C NMR data of 5 were very similar to those of kanshone A (9) [18], except that one oxygenated quaternary carbon (δC 57.4, C-11) and one methylene (δC 55.5; C-12) in 5 replaced one hydroxy group and one methyl in 9, indicating a three-membered epoxide ring at C-11 and C-12. Meanwhile, 2D-NMR correlations suggested the deduction and established the planar structure of 5 as shown in Fig. 2. The NOESY correlations from H3-14 to H3-15/H-6 and from H3-13 to H-4 indicated the relative configuration. 5 had the same rotation sign (−253.8) as 1; it could be concluded that the absolute configuration of 5 was assigned as 4R, 5R, 6R, 11S. In a previous phytochemistry investigation, an epoxy dienone (38 in reference) had been synthesized [24], however the NMR spectral data were not available. In the present study, we carried out detailed NMR data analysis and confirmed its structure as nardosinanone I. The plausible biogenetic pathway for 1–12 was proposed as shown in Fig. 4. The aristolane-type sesquiterpene precursor, nardosinonediol (6), would be transformed into 1–12 through a series of biochemical reactions including oxidation, dehydration, and cleavage as well as epoxidation reactions. Eight compounds (1, 2, 4–7, 11, 12) were evaluated for their protective effects on neonatal rat cardiomyocyte injury induced by hydrogen peroxide (H2O2) with salvianolic acid B as the positive control. Compounds 1, 5, 6, 7, 11 and 12 had protective effects on H2O2 induced myocardial injury (p b 0.05, vs H2O2 group) (Fig. 5). Compounds 1, 5, 11 and 12 can dosedependently protect neonatal rat cardiomyocytes from H2O2 injury from 0.01 to 1 μM (p b 0.05, vs H2O2 group) (Fig. 5).
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Fig. 5. Effect of compounds 1, 2, 4–7, 11, and 12 on neonatal rat cardiomyocyte injury induced by H2O2.
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