Note pubs.acs.org/jnp
Iridal-Type Triterpenoids with Neuroprotective Activities from Iris tectorum Chun-Lei Zhang, Yan Wang, Yan-Fei Liu, Gang Ni, Dong Liang, Huan Luo, Xiu-Yun Song, Wan-Qing Zhang, Ruo-Yun Chen, Nai-Hong Chen, and De-Quan Yu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *
ABSTRACT: Six novel iridal-type triterpenoids with a previously unreported 3,6-dihydro-2H-pyran moiety, named spirioiridotectals A−F (1−6), were isolated from the ethanol extract of the rhizomes of Iris tectorum. Their structures were elucidated on the basis of extensive spectroscopic analysis. Furthermore, in in vitro bioactivity assays, compounds 1, 2, and 6 exhibited neuroprotective activities against serumdeprivation-induced PC12 cell damage.
and 5.11, a hydroxymethine signal at δH 5.02, three hydroxymethylene signals at δH 4.08, 4.07, 4.04, 4.01, 3.57, and 3.47, and four methyl signals at δH 1.85, 1.68, 1.61, and 1.17, suggesting that this compound is an iridal derivative. Comparison of the 1H and 13C NMR data of 1 with those of 28-deacetylbelamcandal led to the assignment of the same spiro-bicyclic unit for both compounds.13 The 13C NMR spectra revealed the presence of four double bonds, of which two were assignable to the terminal isoprene unit and the α,βunsaturated aldehyde group. HMBC correlations observed from H-14 at δH 2.45 to δC 138.4 and 122.3 pointed to the location of one double bond at C-15/C-16. An oxymethine proton at δH 5.02, which is in a bis-allylic position as judged from its chemical shift, showed cross-peaks with the two olefinic protons at δH 5.78 and 5.55 (H-16) in the 1H−1H COSY spectrum, suggesting the remaining double bond to be located between C-18 and C-19. The locations of the three hydroxymethylene groups were determined by analysis of the HMBC correlations as shown in Figure 1. The aldehyde group, four double bonds, and the spiro-bicyclic unit accounted for seven degrees of unsaturation; therefore there should be one additional ring to represent its unsaturation level. The presence of a 3,6-dihydro-2H-pyran ring was established by the HMBC correlations observed from H-26 to C-17 and from H-17 to C26. The configuration of the typical six-membered iridal ring system has been determined unequivocally by X-ray analysis and chemical degradation.14,15 From biosynthetic considerations, the absolute configurations of C-6, C-10, and C-11 should be 6R,10S,11S. The Me-27 signal displayed NOESY correlations with H-14 and H-26 (Figure 2), suggesting the
Iris tectorum Maxim. (Iridaceae) is a perennial herb, indigenous to mainland China. As a traditional Chinese medicine with heat-relieving and detoxifying actions, it has been used commonly for the treatment of sore throats.1 Numerous isoflavonoids and iridal-type triterpenoids have been isolated from this plant.2−4 Iridal-type triterpenoids are recognized generally as characteristic metabolites of the plant family Iridaceae.5,6 These substances show a broad range of biological activities including cytotoxicity,7,8 ichthyotoxicity,9 antiplasmodial effects,10 and PKC activation.11,12 As part of a research program to search for bioactive compounds from medicinal plants, six novel iridals (1−6) were isolated from the ethanol extract of the rhizomes of I. tectorum. These structurally related compounds represent the first examples of iridals with an unusual 3,6-dihydro-2H-pyran moiety. Compounds 1, 2, and 6 exhibited neuroprotective activities against serum-deprivationinduced PC12 cell damage. To the best of our knowledge, this is the first report to study the neuroprotective effects of iridals. Herein, we report the isolation, structure elucidation, and biological activities of these compounds. The EtOH-soluble extract from the air-dried rhizomes of I. tectorum was suspended in water and partitioned successively with EtOAc and n-BuOH. The EtOAc fraction was subjected to column chromatography on silica gel, Sephadex LH-20, and reversed-phase C18 silica gel, as well as preparative HPLC to afford six novel iridals, spirioiridotectals A−F (1−6). Spirioiridotectal A (1) gave a molecular formula of C30H46O6, as established by HRESIMS at m/z 525.3206 [M + Na]+, indicating eight degrees of unsaturation. The UV (254 nm) and the IR (1707, 1610 cm−1) absorptions indicated the presence of an α,β-unsaturated aldehyde group. The 1H NMR spectrum exhibited a characteristic singlet due to an aldehyde group at δH 10.21, three olefinic proton signals at δH 5.78, 5.55, © 2014 American Chemical Society and American Society of Pharmacognosy
Received: November 11, 2013 Published: January 16, 2014 411
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Figure 1. Selected HMBC (→) and 1H−1H COSY (−) correlations for 1.
further upfield shift (Δδ 4.5) of C-6 and a downfield shift (Δδ 3.8) of C-8 in the 13C NMR spectrum as compared with the corresponding signals of 1, were observed. This indicated that 2 is a geometrical isomer of 1 at the α,β-unsaturated aldehyde moiety. The conclusion was confirmed by NOESY correlations between the aldehyde hydrogen signal and H-6 and between the vinyl methyl and one H-8. Spirioiridotectal C (3) was assigned the molecular formula C30H46O5, on the basis of HRESIMS (m/z 509.3211 [M + Na]+). The 1H NMR data of 3 were found to be close to those of 1 except for the presence of signals for a vinyl methyl group and the absence of any hydroxymethylene signals. The vinyl methyl signal at δH 1.75 displayed HMBC correlations with C18 and C-19. Taking the molecular weight, 16 mass units lower than that of 1, into consideration, the hydroxy group at C-29 in 1 was shown to be absent in 3. Thus, the structure of spirioiridotectal C was characterized as depicted. Spirioiridotectal D (4) gave the same molecular formula, C30H46O5, as 3 from its HRESIMS (m/z 509.3245 [M + Na]+). The NMR data were essentially the same as those of 3. In the 1 H NMR spectrum, the H-6 signal and one H-8 proton signal were shifted to a lower field and a higher field, respectively, as compared with the corresponding signals of 3. Furthermore, differences were observed in the chemical shifts of the C-6 and C-8 resonances in the 13C NMR spectrum of these substances. The above data suggested that 4 is a geometrical isomer of 3 at the α,β-unsaturated aldehyde moiety. NOESY correlations between the aldehyde hydrogen signal and H-6 and between the vinyl methyl and one H-8 in the NOESY spectrum further supported this conclusion. Spirioiridotectal E (5) was assigned an identical molecular formula (C30H46O5) to that of 3 from its HRESIMS (m/z 509.3245 [M + Na]+). The 1H NMR data of 5 were almost
Figure 2. Selected NOE (↔) correlations for 1.
configurations of 14S and 26R, respectively.13 A NOESY correlation between H-18 and H-26 indicated the 17S configuration. NOESY correlations between the aldehyde hydrogen signal and one H-8 and between the vinyl methyl and H-6 suggested the geometric configuration of the 2,7double bond as E. The geometry of the 15,16-double bond was deduced as E from the NOESY cross-peak between H-18 and H-29. Therefore, the structure of spirioiridotectal A was elucidated as shown. Spirioiridotectal B (2) exhibited the same molecular formula, C30H46O6, as 1, as established by HRESIMS at m/z 525.3210 [M + Na]+. The NMR data of 2 were very similar to those of 1. However, a downfield shift (Δδ 0.55) of the H-6 signal and an upfield shift (Δδ 0.50) of one H-8 signal in the 1H NMR spectrum relative to the corresponding signals of 1, and a 412
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Table 1. 1H NMR Spectroscopic Data for Compounds 1−6 in CDCl3 (600 MHz) position 1 3
1
14 16 17 18 20 21
2.27, m
2.27, m
22 24 25 26 27 28
5.11, t (7.2) 1.68, s 10.21, s 4.32, d (3.6) 1.17, s 4.08, d (13.2) 4.04, d (13.2) 4.07, d (13.2) 4.01, d (13.2) 1.61, s
5.12, 1.70, 1.81, 4.33, 1.20, 4.08, 4.05, 4.09, 4.01, 1.62,
4 5 6 8 9 12 13
29 30
s m m m m m d (10.2) brd (13.8) brtd (13.8, 3.0) m m m m m m m d (3.0) brd (9.0) d (9.0) m
2
1.85, 3.57, 3.47, 1.37, 1.86, 1.81, 3.07, 3.20, 2.69, 1.77, 1.64, 1.64, 1.42, 1.96, 1.65, 2.45, 5.55, 5.02, 5.78, 2.11,
10.20, s 3.59, m 3.46, m 1.35, m 1.75, m 1.72, m 3.62, d (10.2) 2.70, td (14.4, 6.4) 2.49, brd (14.4) 1.77, m 1.63, m 1.52, m 1.42, m 1.96, m 1.63, m 2.47, m 5.56, d (3.6) 5.01, brd (9.0) 5.76, d (9.0) 2.06, m
t (7.2) s s d (3.0) s d (13.2) d (13.2) d (13.2) d (13.2) s
3
4
5
6
1.84, s 3.53, m
10.12, s 3.46, t (6.0)
1.82, brs 3.53, m
10.21, s 3.53, t (6.6)
1.39, 1.93, 1.63, 3.10, 3.21, 2.71, 1.77, 1.62, 1.43,
1.36, 1.94, 1.63, 3.10, 3.21, 2.70, 1.76, 1.62, 1.41,
m m, m d (10.2) d (13.8), td (13.8, 2.4) m m m
1.93, m 1.66, m 2.38, m 5.56, d (3.0) 4.98, brd (8.4) 5.47, d (8.4) 2.32, m 2.02, m 2.14, m 2.06, m 5.12, t (6.0) 1.70, s 10.22, s 4.30, d (3.0) 1.18, s 4.09, d (13.2) 4.05, d (13.2) 1.77, s
1.31, m 2.02, m 1.79, m 3.56, d (10.2) 2.67, td (13.8, 4.8), 2.45, brd (13.8) 1.69 m 1.58 m 1.45, m 1.34, m 1.89, m 1.55, m 2.40, m 5.50, d (4.2) 4.91, brd (9.0) 5.43, d (9.0) 2.24, m 1.97, m 2.10, m 1.98, m 5.01, t (6.0) 1.65, s 1.76, s 4.30, d (3.0) 1.14, s 4.02, d (13.2), 3.90, d (13.2) 1.71, s
2.10, 1.62, 2.36, 5.59, 4.97, 5.45, 2.04,
m m m d (3.6) dt (9.0, 2.4) d (9.0) m
1.36, 1.95, 1.63, 3.63, 2.70, 2.50, 1.75, 1.62, 1.53, 1.39, 1.95, 1.62, 2.42, 5.59, 4.97, 5.46, 2.04,
1.62, s
1.57, s
m m m d (10.2) brd (13.8) td (13.8, 3.6) m m m
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superimposable with those of 3. Differences were observed in the chemical shifts of the C-20 and C-29 resonances between the 13C NMR spectra of these compounds. A NOESY correlation between H-18 and H-20 suggested that 5 is a geometrical isomer of 3 with respect to the 18,19-double bond. Spirioiridotectal F (6) was deduced to have the same molecular formula of C30H46O5 as 5 from its HRESIMS (m/z 509.3230 [M + Na]+). The 1H and 13C NMR spectra of 6 were closely comparable to those of 5. Significant differences between the 1H NMR spectra of these compounds were observed in the chemical shifts of H-6 and one of the H-8 proton signals, which in 6 were at a lower field and an upper field, respectively, when compared with the corresponding signals in 5. The differences of these signals for 6 and 5 were comparable with those observed for 2 and 1, and 4 and 3. Therefore, compound 6 was concluded to be a geometrical isomer of 5 at the α,β-unsaturated aldehyde moiety. Analysis of the 13C NMR and NOESY spectra also supported the conclusion. In an in vitro assay, as summarized in Table 3, compounds 1, 2, and 6 exhibited moderate neuroprotective activities against serum-deprivation-induced PC12 cell damage at a 10 μM concentration.
m m m, d (10.2) td (14.4, 4.8) brd (14.4) m m m m m m m d (3.6) brd (8.4) d (8.4) m
2.10, m
2.10, m
5.09, t (6.6) 1.67, s 10.21, s 4.29, d (3.0) 1.16, s 4.09, d (13.2) 4.05, d (13.2) 1.75, brs
5.10, 1.68, 1.82, 4.31, 1.19, 4.10, 4.05, 1.75,
1.60, s
1.61, s
t (7.2) s s d (3.6) s d (13.2) d (13.2) s
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter, and UV spectra with a JASCO V-650 spectrophotometer. IR spectra were recorded on a Nicolet 5700 spectrometer by an FT-IR microscope transmission method. NMR measurements were performed on VNS-600 spectrometers in CDCl3. HRESIMS were obtained using an Agilent 1100 series LC/MSD ion-trap mass spectrometer. Preparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD20A detector and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Silica gel (200−300 mesh, Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China), Sephadex LH-20 (GE), and ODS (50 μm, YMC, Japan) were used for column chromatography. TLC was carried out with GF254 plates (Qingdao Marine Chemical Factory). Plant Material. The two-year-old rhizomes of I. tectorum were purchased in November 2011 from an herbal medicine market in Chengdu, Sichuan Province, People’s Republic of China. The plant material was authenticated by Prof. Lin Ma, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (ID-S-2469) is deposited at the Herbarium of the Department of Medicinal Plants, the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing. Extraction and Isolation. The air-dried, powdered rhizomes of I. tectorum (20 kg) were extracted exhaustively with 95% EtOH under reflux (3 × 100 L). The extracts were combined and concentrated under a vacuum to give a residue (4 kg), which was suspended in water and partitioned successively with EtOAc and n-BuOH. The EtOAcsoluble fraction (2 kg) was subjected to passage over a silica gel
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Table 2. 13C NMR Spectroscopic Data for Compounds 1−6 in CDCl3 (150 MHz) position
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
11.7 132.2 63.0 32.1 30.3 47.1 164.0 20.3 39.5 74.2 59.0 35.2 30.0 41.4 138.4 122.3 69.2 122.6 142.3 27.4 28.4 123.5 132.5 25.7 191.1 74.0 28.1 65.5 65.7 17.7
191.0 132.5 63.0 31.9 30.0 42.6 162.5 24.1 38.6 74.1 58.6 35.7 29.9 41.4 138.4 122.2 69.3 122.9 142.3 27.4 28.4 123.5 132.7 25.7 11.0 74.1 28.6 65.7 66.0 17.7
11.8 132.2 63.1 32.1 30.2 47.0 163.7 20.2 39.5 74.2 58.9 35.4 29.8 41.7 137.7 122.8 69.6 122.4 139.5 32.3 26.8 123.7 132.4 25.7 191.1 73.9 28.1 65.7 23.6 17.7
191.2 132.1 62.8 31.8 29.5 42.6 163.1 24.0 38.4 73.9 58.5 35.7 29.8 41.5 137.7 122.5 69.5 122.5 139.1 32.2 26.7 123.5 132.5 25.6 10.9 73.8 28.3 65.4 23.4 17.6
11.7 132.4 63.0 31.9 30.1 46.9 163.8 20.2 39.5 74.2 58.8 35.4 29.6 41.8 137.5 122.6 69.8 121.9 139.7 39.7 26.4 123.7 131.8 25.4 191.1 73.9 28.1 65.6 16.5 17.7
191.1 132.8 62.9 31.8 30.0 42.5 162.3 24.0 38.6 74.1 58.5 35.9 29.4 41.8 137.6 122.6 69.8 121.8 139.7 39.7 26.4 123.8 131.8 25.3 11.0 74.0 28.5 65.7 16.5 17.7
H2O, 5 mL/min) to obtain 5 (14 mg, tR 72.2 min) and 6 (5.5 mg, tR 58.4 min). Spirioiridotectal A (1): glassy solid; [α]20D +156.2 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 254 (4.32) nm; IR νmax 3398, 2927, 2868, 1707, 1651, 1610, 1450, 1378 cm−1; 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) spectra, see Tables 1 and 2; (+)-HRESIMS m/z 525.3206 [M + Na]+ (calcd for C30H46O6Na, 525.3187). Spirioiridotectal B (2): glassy solid; [α]20D +114.3 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 254 (4.44) nm; IR νmax 3373, 2928, 2868, 1711, 1648, 1609, 1452, 1377 cm−1; 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) spectra, see Tables 1 and 2; (+)-HRESIMS m/z 525.3210 [M + Na]+ (calcd for C30H46O6Na, 525.3187). Spirioiridotectal C (3): glassy solid; [α]20D +125.1 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 254 (4.34) nm; IR νmax 3386, 2929, 2870, 1710, 1651, 1610, 1449, 1378 cm−1; 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) spectra, see Tables 1 and 2; (+)-HRESIMS m/z 509.3211 [M + Na]+ (calcd for C30H46O5Na, 509.3237). Spirioiridotectal D (4): glassy solid; [α]20D +165.3 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 254 (4.33) nm; IR νmax 3403, 2927, 2870, 1711, 1651, 1610, 1450, 1378 cm−1; 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) spectra, see Tables 1 and 2; (+)-HRESIMS m/z 509.3245 [M + Na]+ (calcd for C30H46O5Na, 509.3237). Spirioiridotectal E (5): glassy solid; [α]20D +164.1 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 252 (4.35) nm; IR νmax 3400, 2927, 2871, 1711, 1651, 1610, 1452, 1378 cm−1; 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) spectra, see Tables 1 and 2; (+)-HRESIMS m/z 509.3245 [M + Na]+ (calcd for C30H50O5Na, 509.3237). Spirioiridotectal F (6): glassy solid; [α]20D +144.2 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 254 (4.25) nm; IR νmax 3425, 2926, 2874, 1709, 1654, 1610, 1451, 1377 cm−1; 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) spectra, see Tables 1 and 2; (+)-HRESIMS m/z 509.3230 [M + Na]+ (calcd for C30H46O5Na, 509.3237). Neuroprotective Assay (ref 16). The potential neuroprotective effects of the compounds against serum-deprivation-induced PC12 cell damage were assayed with the MTT method in vitro, using nerve growth factor (NGF) as a positive control. PC12 cells at a density of 5 × 103 cells per well in 96-well plates were suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum and 5% horse serum. The plates were incubated at 37 °C in a humidified, 5% CO2 atmosphere for 24 h. Then, the cells were cultured with or without test compounds (10 μM) in DMEM without serum. After incubation for 48 h, 10 μL of MTT (5 mg/mL) was added and maintained for another 4 h, and then the medium was carefully removed. The formazan crystals were dissolved in dimethyl sulfoxide, and absorbance was determined at 550 nm using an Ultramark microplate reader. The survival rate of PC12 cells was evaluated.
Table 3. Effects of Compounds 1, 2, and 6 at 10 μM on the Survival Rate of PC12 Cells Injured by Serum Deprivation (SD)a group normal SD SD + NGFe SD + 1 SD + 2 SD + 6
OD value 0.60 0.25 0.38 0.31 0.32 0.39
± ± ± ± ± ±
0.02 0.02b 0.02d 0.05c 0.04d 0.04d
survival rate (%) 100 41.4 63.5 50.9 54.1 63.8
± ± ± ± ± ±
3.7 3.8b 5.4d 8.7c 6.3d 5.8d
a
The OD value is the absorbance at 550 nm. The survival rate is OD% (normal = 100%). bp < 0.001 vs normal. cp < 0.01. dp < 0.001 vs SD group; one-way ANOVA was used (n = 6). ePositive control substance.
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ASSOCIATED CONTENT
S Supporting Information *
column and eluted with petroleum ether−acetone (20:1, 3:1, 1:1, and 0:1). The fraction (100 g) that eluted with 3:1 petroleum ether− acetone was chromatographed further on a reversed-phase C18 silica gel column (80 × 6 cm), eluted with 50%, 60%, 70%, 80%, 90%, and 100% MeOH in H2O, to afford eight fractions (F1−F8). Fraction 2 (5 g) was purified using Sephadex LH-20 (CHCl3−MeOH, 1:1), followed by preparative HPLC (35% MeCN in H2O, 5 mL/min), to afford 1 (12 mg, tR 76.2 min) and 2 (5 mg, tR 69.8 min). Fraction 6 (4 g) was fractionated over silica gel (CH2Cl2−MeOH, 20:1) and purified by preparative HPLC (44% MeCN in H2O, 5 mL/min) to yield 3 (14 mg, tR 85.4 min) and 4 (34 mg, tR 78.4 min). Fraction 4 (12 g) was submitted to a silica gel column and eluted with CH2Cl2−MeOH (25:1−15:1) to produce four subfractions (F4a−F4d). Of these subfractions, F4b was purified by preparative HPLC (45% MeCN in
Copies of IR, UV, NMR, and mass spectra for compounds 1−6. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86-10-63165224. Fax: +86-10-63017757. E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 414
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Note
ACKNOWLEDGMENTS This research was supported by the National Mega-project for Innovative Drugs (No. 2012ZX09301002-002) and the State Key Laboratory of Bioactive Substance and Function of Natural Medicines (No. GTZC201201), Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.
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REFERENCES
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