Article pubs.acs.org/JAFC
Hepatoprotective Sesquiterpenes and Rutinosides from Murraya koenigii (L.) Spreng Qin-Ge Ma,† Yan-Gai Wang,‡ Wen-Min Liu,† Rong-Rui Wei,† Jian-Bo Yang,§ Ai-Guo Wang,§ Teng-Fei Ji,§ Jin Tian,∥ and Ya-Lun Su*,§ †
College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyan 473061, People’s Republic of China Department of Pharmacy, Xuanwu Hospital of Capital Medical University, Beijing 100053, People’s Republic of China § 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 ∥ College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: Three new sesquiterpenes (1−3) and two new rutinosides (4 and 5) along with 17 known compounds (6−22) were isolated from the leaves of Murraya koenigii (L.) Spreng. The new compounds were elucidated as (3R,5S,6R)-3,5,6trihydroxy-1,1,5-trimethylcyclohexyl-8-butyn-9-one (1), (8E,9R)-ethyl-7-(3S,5R,6S)-3,6-dihydroxy-1,1,5-trimethylcyclohexyl-9hydroxybut-8-enoate (2), (3R)-3-O-β-D-glucoside-6′-D-apiose-β-ionone (3), 4-O-β-D-rutinosyl-3-methoxyphenyl-1-propanone (4), and 1-O-β-D-rutinosyl-2(R)-ethyl-1-pentanol (5) based on their spectroscopic data. Compounds 1, 4, 5, 18, and 21 (10 μM) exhibited moderate hepatoprotective activities. KEYWORDS: Murraya koenigii, sesquiterpene, rutinoside, hepatoprotective activity
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spectrometer, a Nicolet 5700 FTIR microscope spectrometer, and a Hitachi UV-240 spectrophotometer, respectively.11 Nuclear magnetic resonance (NMR) spectra were recorded on Varian Mercury-300 and Inova-500 spectrometers, with tetramethylsilane (TMS) as an internal standard. The high-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired on an Agilent 1100 series LC/ MSD Trap SL mass spectrometer. Reversed-phase high-performance liquid chromatography (HPLC) was performed on a Shimadzu LC6AD instrument using a SPD-20A detector and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography was carried out on Sephadex LH-20 (Amersham Pharmacia, Sweden), silica gel H, 100−200 and 200−300 mesh (Qingdao Marine Chemical, Inc., Qingdao, China). Thin-layer chromatography (TLC) was performed on pre-coated silica gel GF254 plates, and the spots were visualized under UV light (254 or 356 nm) or spraying with 10% H2SO4 in 95% EtOH, followed by heating.12 Plant Material. The leaves of M. koenigii, family Rutaceae, were collected from Xishuangbanna, Yunnan, China, in August 201010 and authenticated by Prof. Lin Ma (Institute of Materia Medica, Beijing, China). A voucher specimen (ID-S-2436) has been deposited in the Herbarium of Institute of Materia Medica, Beijing, China. Extraction and Isolation. The air-dried leaves of M. koenigii (22.50 kg) were extracted 3 times with 95% EtOH (3 × 20 L) heating under reflux to give 2.10 g of crude extract. The combined extracts were successively partitioned with CHCl3, EtOAc, and n-butanol to yield three fractions: CHCl3-soluble fraction (33.50 g), EtOAc-soluble fraction (24.90 g), and n-butanol-soluble fraction (221.20 g). The n-butanol-soluble fraction was subjected to the macroporous adsorbent resin (Diaion-101) column, which were eluted with 20, 40, 60, and 95% ethanol to obtain four fractions: A (25.50 g), B (30.60 g),
INTRODUCTION Murraya koenigii L. (Rutaceae) is well-known as curry leaf, which has been widely cultivated as a spice in tropical and subtropical countries. The leaf of M. koenigii has been used for hundreds of years in southeast Asia as a traditional medicine and functional food to prevent and treat various diseases.1 Different kinds of compounds, including carbazole alkaloids,2 volatile oils,3 and other compounds, had been isolated from the leaves of M. koenigii.4 These compounds exhibited various bioactivities, such as hypoglycemic and hypolipemic,5 antidiarrheal,6 antioxidant and radical-scavenging,7 and antimicrobial activities.8 A preliminary screening assay exhibited that the n-butanolsoluble fraction from the leaves of M. koenigii inhibited hepatoprotective activities, which prompted us to further investigate its constituents. As a result, 22 compounds (Figure 1), including three new sesquiterpenes (1−3) and two new rutinosides (4 and 5) along with 17 known compounds (6− 22), were isolated from the active fraction of M. koenigii. Their structures (Figure 1) were elucidated by extensive analysis of their spectroscopic data. Meanwhile, we evaluated their hepatoprotective activities against D-galactosamine-induced WB-F344 cell damage and found that compounds 1, 4, 5, 18, and 21 (10 μM) showed moderate hepatoprotective activities.
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MATERIALS AND METHODS
General Experimental Procedures. The optical rotations were measured on a PerkinElmer 241 polarimeter at 20 °C.9 Melting points were recorded on a XT5B microscopic melting point apparatus (Beijing Tech Electro-Optical Instrument Factory, China), which was uncorrected.10 The circular dichroism (CD), infrared (IR), and ultraviolet (UV) spectra were measured with a JASCO J-815 CD © 2014 American Chemical Society
Received: Revised: Accepted: Published: 4145
January 29, 2014 April 21, 2014 April 21, 2014 April 21, 2014 dx.doi.org/10.1021/jf5005034 | J. Agric. Food Chem. 2014, 62, 4145−4151
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Figure 1. Structures of compounds 1−22. 2932.7, 1670.8, 1381.7, 1044.6 cm−1. 1H NMR (CD3COCD3, 300 MHz) and 13C NMR (CD3COCD3, 125 MHz) data: see Table 1. (8E,9R)-Ethyl-7-(3S,5R,6S)-3,6-dihydroxy-1,1,5-trimethylcyclohexyl-9-hydroxybut-8-enoate (2). White amorphous power. [α]20 D −5.49 (c 0.30, MeOH). mp: 157.4−156.9 °C. HRESIMS: m/z 309.1648 (calcd for C15H26O5Na, 309.1672). UV (MeOH) λmax: 203 nm. CD (MeOH): 222 (Δε, −7.50) nm; IR νmax: 3392.5, 2967.2, 2933.0, 1737.5, 1368.3, 1045.2 cm−1. 1H NMR (CD3COCD3, 300 MHz) and 13 C NMR (CD3COCD3, 125 MHz) data: see Table 1. (3R)-3-O-β-D-Glucoside-6′-D-apiose-β-ionone (3). White amorphous power. [α]20 D +1.50 (c 0.67, MeOH). mp: 187.7−188.5 °C. HRESIMS: m/z 525.2323 (calcd for C24H38O11Na, 525.2306). UV (MeOH) λmax: 203, 221, 291 nm. CD (MeOH): 276 (Δε, +0.64), 322 (Δε, −0.49) nm. IR νmax: 3318.6, 2925.0, 1667.0, 1373.2, 1072.3 cm−1. 1 H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 2. 4-O-β-D-Rutinosyl-3-methoxyphenyl-1-propanone (4). White amorphous power. [α]20 D −2.45 (c 0.50, MeOH). mp: 194.1−195.5 °C. HRESIMS: m/z 511.1797 (calcd for C22H32O12Na, 511.1786). UV (MeOH) λmax: 203, 222, 266, 301 nm. IR νmax: 3399.3, 1671.8, 1592.9, 1512.1 cm−1. 1H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 3.
C (12.80 g), and D (5.40 g). The A part was purified on a Sephadex LH-20 column with a gradient system of MeOH/H2O to give two subfractions: A-a and A-b. The separation of A-b (6.40 g) by mediumpressure liquid chromatography (MPLC) (10−30% MeOH−H2O) and preparative HPLC (detection at 205 nm, 7 mL/min),9 successively, yielded compounds 4 (9.00 mg), 5 (7.00 mg), and 22 (7.00 mg). Similarly, the B part was chromatographed over Sephadex LH-20 eluted with MeOH to give two sub-fractions: B-a and B-b. The separation of B-a (3.65 g) by MPLC (15−40% MeOH−H2O) and preparative HPLC (detection at 205 nm, 7 mL/min), successively, yielded compounds 3 (10.00 mg), 13 (4.00 mg), 14 (10.31 mg), and 16 (11.76 mg). The separation of B-b (12.50 g) by MPLC (20−50% MeOH−H2O) and preparative HPLC (detection at 205 nm, 7 mL/ min), successively, yielded compounds 1 (6.51 mg), 2 (7.40 mg), 6 (3.10 mg), 7 (3.30 mg), 8 (4.70 mg), 9 (9.53 mg), 10 (12.30 mg), 11 (3.44 mg), 12 (6.63 mg), 15 (16.23 mg), 17 (18.43 mg), 18 (10.60 mg), 19 (5.43 mg), 20 (6.10 mg), and 21 (12.31 mg). (3R,5S,6R)-3,5,6-Trihydroxy-1,1,5-trimethylcyclohexyl-8-butyn-9one (1). White amorphous power. [α]20 D −2.48 (c 0.08, MeOH). Melting point (mp): 153.4−154.9 °C. HRESIMS: m/z 263.1264 (calcd for C13H20O4Na, 263.1254). UV (MeOH) λmax: 201, 264 nm. CD (MeOH): 314 (Δε, +0.33), 263 (Δε, −2.04) nm. IR νmax: 3454.9, 4146
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Table 1. 1H NMR (300 MHz, CD3COCD3), 13C NMR (125 MHz, CD3COCD3), and HMBC Correlations of Compounds 1 and 2 1 δH 1 2eq 2ax 3 4eq 4ax 5 6 7 8 9 10 11 12 13 14 15
1.56 1.53 4.08 1.91 1.47
2.32 1.31 0.79 0.96
(m) (m) (m) (m) (m)
(s) (s) (s) (s)
δC
2 δH
HMBC (1H−13C)
39.5 46.8 46.8 63.6 45.8 45.8 57.7 93.0 75.8 107.6 191.6 16.4 22.5 26.7 26.3
C-1, C-3, C-4, C-6 C-1, C-3, C-4, C-6
1.66 1.22 3.77 1.66 1.22 1.95
C-2, C-3, C-6 C-2, C-3, C-6
δC
(m) (m) (m) (m) (m) (m)
5.83 (d, 16.5) 5.81 (dd, 16.5, 4.8) 4.68 (d, 4.8) C-8 4.16 1.22 0.97 0.84 0.79
(dd, 14.1, 6.6) (t, 14.1, 6.6) (s) (s) (d, 6.9)
HMBC (1H−13C)
40.2 46.1 46.1 66.5 40.1 40.1 34.9 77.6 137.7 128.2 72.5 173.8 61.4 14.5 25.7 24.9 16.4
C-1, C-3, C-4, C-6 C-1, C-3, C-4, C-6 C-2, C-3, C-5, C-6 C-2, C-3, C-5, C-6 C-1, 15-CH3 C-6, C-8, C-9 C-7, C-9, C-10 C-10 C-10
Table 2. 1H NMR (300 MHz, CD3OD), 13C NMR (125 MHz, CD3OD), and HMBC Correlations of Compound 3 3 δH 1 2eq 2ax 3 4eq 4ax 5 6 7 8 9 10 11 12
2.00 1.58 4.11 2.58 2.20
(m) (m) (m) (m) (m)
7.37 (d, 16.2) 6.18 (d, 16.2) 2.33 (s) 1.17 (s) 1.14 (s)
δC 37.8 47.4 47.4 72.9 40.5 40.5 134.0 137.1 144.4 133.1 201.2 27.2 28.8 30.5
3 δH
HMBC (1H−13C) C-1, C-3, C-1, C-3, C-1′ C-2, C-3, C-2, C-3,
13 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″a 4″b 5″
C-4, C-6 C-4, C-6 C-6 C-6
C-1, C-5, C-8, C-9 C-6, C-9, C-10 C-9 C-1 C-1
1.82 4.46 3.43 3.18 3.37 3.32 4.00 3.63 5.02 3.79 3.26 3.97 3.91 3.59
(s) (d, 7.8) (m) (m) (m) (m) (m) (m) (d, 39) (m) (m) (m) (m) (s)
δC
HMBC (1H−13C)
21.8 102.7 76.8 75.1 78.0 71.7 68.6 68.6 110.8 75.0 80.5 78.0 78.0 65.7
C-5 C-3, C-2′ C-4′ C-4′, C-5′ C-3′, C-5′ C-2′, C-3′ C-5′ C-5′ C-2″, C-3″, C-6′ C-3″, C-4″, C-5″ C-4″ C-2″, C-5″ C-2″, C-5″ C-2″, C-3″, C-4″
calculated as inhibition (%) = [(OD(sample) − OD(control))/(OD(normal) − OD(control))] × 100.16 Statistical Analysis. Compounds 1−22 were bioassayed with the bicyclol (hepatoprotective activity drug) as the positive control for their hepatoprotective activities against D-galactosamine-induced toxicity in WB-F344 cells.14 The results of pharmacological activities are shown in Table 4, with the inhibition (%) of compounds 1, 4, 5, 18, and 21 based on the computing formula with values of 43.7, 54.3, 45.1, 27.0, and 49.5, respectively. Furthermore, all of the values were expressed as means ± standard deviation (SD) of three experiments. The significance of unpaired observations between normal or control and tested samples was determined by Student’s t test.16 Differences were considered significant at p < 0.05.15 Therefore, compounds 1, 4, 5, 18, and 21 exhibited moderate hepatoprotective activities.
1-O-β-D-Rutinosyl-2(R)-ethyl-1-pentanol (5). White amorphous power. [α]20 D −1.55 (c 0.50, MeOH). mp: 143.3−145.6 °C. HRESIMS: m/z 447.2214 (calcd for C19H36O10Na, 447.2201). UV (MeOH) λmax: 203 nm. CD (MeOH): 242 (Δε, −0.04) nm. IR νmax: 3384.6, 2928.8, 1362.8 cm−1. 1H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 3. Hepatoprotective Assay. We evaluated the hepatoprotective activities of compounds 1−22 (Figure 1), which were tested against Dgalactosamine-induced toxicity in WB-F344 cells using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method.8 The WB-F344 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3% fetal calf serum, 100 units/mL penicillin, and 100 units/mL streptomycin in 5% CO2 and incubated at 37 °C, which were placed in a 96-well microplate and precultured for 24 h.13 After this, the cultured cells were measured for cytotoxic effects, which were exposed to 40 mM D-galactosamine after 24 h. At last, the medium was replaced with the surum-free medium (0.5 mg/mL MTT) for 3.5 h of incubation. After removal of the medium and the addition of DMSO (150 μL/well) into the microplate, the formazan crystals were redissolved.13 The optical density (OD) was measured by a microplate reader at a wavelength of 492 nm, and the inhibition was
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RESULTS AND DISCUSSION Structure Elucidation of New Compounds. Compound 1 was obtained as a white amorphous power. Its molecular formula was deduced as C13H20O4 from the analysis of its HRESIMS at m/z 263.1264 (calcd for C13H20O4Na, 263.1254) corresponding to four degrees of unsaturation. The UV spectrum showed the adamantane-type sesquiterpene absorp4147
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Table 3. 1H NMR (300 MHz, CD3OD), 13C NMR (125 MHz, CD3OD), and HMBC Correlations of Compounds 4 and 5 4 δH 1 2 3a 3b 4a 4b 5 6a 6b 7 8 9 3-OCHH3 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″ 5″ 6″
7.61 (d, 1.8)
7.22 (d, 8.4) 7.70 (dd, 8.4, 1.8) 7.70 (dd, 8.4, 1.8) 3.07 1.20 3.93 5.03 3.52 3.49 3.39 3.61 4.04 3.62 4.69 3.83 3.71 3.39 3.69 1.19
(dd, 14.4, 7.2) (t, 7.2, 3.6) (s) (d, 7.2) (m) (m) (m) (m) (m) (m) (d, 1.8) (m) (m) (m) (m) (d, 7.2)
5
δC 131.4 114.9 149.3 149.3 150.7 150.7 110.9 122.3 122.3 200.6 30.9 7.4 55.2 100.4 73.3 76.5 70.0 75.7 66.4 66.4 100.7 70.8 70.9 72.6 68.4 16.5
C-4, C-6, C-7
C-1, C-3, C-4 C-4, C-5, C-7 C-4, C-5, C-7
normal control bicyclol 1 4 5 18 21
C-3′, C-4, C-5′
C-2″, C-3″, C-6′
cell survival rate (percentage of normal)
inhibition (percentage of control)
± ± ± ± ± ± ± ±
25.9 43.7 54.3 45.1 27.0 49.5
100.0 45.5 59.6 69.3 75.1 70.1 60.2 72.5
3.4 6.9 7.1b 7.1b 9.0b 7.2b 0.2b 8.5b
δC
4.06 1.60 2.18 1.16 2.09 1.17 0.87 1.70 1.19 0.89
(d, 2.1) (m) (m) (m) (m) (m) (s) (m) (m) (s)
83.8 45.8 36.7 36.7 27.3 27.3 19.3 28.7 28.7 13.9
4.25 3.40 3.37 3.60 3.83 3.95 3.54 5.02 3.30 3.93 3.13 3.58 0.85
(d, 7.5) (s) (m) (m) (m) (m) (m) (d, 1.8) (m) (m) (m) (m) (m)
102.9 76.6 78.1 71.0 77.6 67.9 67.9 110.4 71.7 74.7 74.9 65.8 20.2
HMBC (1H−13C) C-1′ C-4 C-2, C-6 C-2, C-6
C-2, C-3 C-2, C-3
C-7, C-9
C-1, C-3′, C-5′
C-3″, C-6′
the correlations of H-2/C-1, C-3, C-4, and C-6; H-4/C-2, C-3, and C-6; and 10-CH3/C-8 (Figure 2) in the heteronuclear multiple-bond correlation (HMBC) spectrum, and its relative configuration was determined by the correlations of H-3/5-OH and 3-OH/13-CH3 (Figure 2) in the nuclear Overhauser effect spectrometry (NOESY) spectrum. The absolute configuration of compound 1 was determined by the CD method. Because the Mo2(OAc)4-induced CD method has been successfully used to determine the absolute configuration of dozens of organic compounds with a 1,2-diol substructure,19 we study the Mo2(OAc)4-induced CD spectrum of compound 1, which contained a 5,6-diol unit. A positive Cotton effect was observed at 314 nm in the CD spectrum, and the absolute configurations of C-5 and C-6 were determined as 5S and 6R, respectively, by applying the spiral rule.19 In addition, according to the correlations of the NOESY spectrum, the absolute configuration of C-3 was deduced as 3R. Thus, compound 1 was identified as (3R,5S,6R)-3,5,6-trihydroxy1,1,5-trimethylcyclohexyl-8-butyn-9-one. Compound 2 was isolated as a white amorphous power. The molecular formula was deduced as C15H26O5 from HRESIMS at m/z 309.1648 (calcd for C15H26O5Na, 309.1672), indicating three degrees of unsaturation. Its UV spectrum showed absorbance at λmax 203 nm, and the IR spectrum indicated the presence of hydroxy (3392.5 cm−1), carbonyl (1737.5 cm−1), and methyl (1368.3 cm−1) functionalities. The UV and IR spectrum data of compound 2 were similar to those of compound 1. Therefore, compound 2 was concluded to be an adamantane-type sesquiterpene.17 The 1H NMR spectrum of compound 2 showed the presence of a multiplet at δH 3.77 (1H, m, H-3) and three methyl group singlets at δH 0.97 (3H, s), 0.84 (3H, s), and 0.79 (3H, s)
Table 4. Hepatoprotective Effects of Selective Compounds (10 μM) against D-Galactosamine-Induced Toxicity in WBF344 Cellsa compound
δH
HMBC (1H−13C)
Results were expressed as means ± SD (n = 3; for normal and control, n = 6). Bicyclol was used as a positive control (10 μM). bp < 0.05.
a
tions at λmax 201 and 264 nm.17 Its IR spectrum indicated the presence of hydroxy (3454.9 cm−1), carbonyl (1670.8 cm−1), and methyl (1381.7 cm−1) functionalities. The 1H NMR spectrum of compound 1 showed the presence of a multiplet at δH 4.08 (1H, m, H-3) and three methyl group singlets at δH 1.31 (3H, s), 0.96 (3H, s), and 0.79 (3H, s) (Table 1). These spectral characteristics coupled with the biosynthetic pathway (biogenetic isoprene rule) indicated the presence of the adamantane-type sesquiterpene18 skeleton in compound 1. The 13C NMR spectrum (Table 1) showed 13 carbon signals. Besides one carbonyl (δC 191.6), four methyl carbons, and two sp-hybridized carbons, the remaining six sp3hybridized carbons were attributed to the cyclohexane. The planar structure of compound 1 was established on the basis of 4148
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Figure 2. Key HMBC (H → C) and NOESY correlations of compounds 1−5.
C-8, and C-9; H-8/C-6, C-9, and C-10; 10-CH3/C-9; H-1′/C3; and H-1″/C-6′ (Figure 2) and the NOESY correlations of H-2/H-3 and H-3/H-4. The absolute configuration of C-3 was determined by the CD method. First, compound 3 was hydrolyzed22 to afford the aglycone 3a. The positive Cotton effect at 276 nm and the negative Cotton effect at 322 nm were observed in the CD spectrum of compound 3a. According to the spiral rule and the literature,23 the absolute configuration of C-3 was determined as 3R. Therefore, compound 3 was identified as (3R)-3-O-β-Dglucoside-6′-D-apiose-β-ionone. Compound 4 was obtained as a white amorphous power. Its molecular formula was established as C22H32O12 on the basis of HRESIMS at m/z 511.1797 (calcd for C22H32O12Na, 511.1786) corresponding to four degrees of unsaturation. The UV spectrum showed absorptions at λmax 203, 222, 266, and 301 nm. Its IR spectrum displayed the presence of hydroxy (3399.3 cm−1) and benzene ring (1671.8, 1592.9, and 1512.1 cm−1) functionalities. In the 1H NMR spectrum of compound 4, an ABX system was shown at δH 7.61 (1H, d, J = 1.8 Hz, H-2), 7.22 (1H, d, J = 8.4 Hz, H-5), and 7.70 (1H, dd, J = 8.4, 1.8 Hz, H-6) in the aromatic field. The anomeric signals at δH 5.03 (1H, d, J = 7.2 Hz, H-1′) and 4.69 (1H, d, J = 1.8 Hz, H-1″) suggested the existence of a rutinosyl moiety24 in compound 4 according to the 1H and 13C NMR spectra (Table 3). Additionally, a quartet at δH 3.07 (2H, dd, J = 14.4 and 7.2 Hz, H-8) and a triplet at δH 1.20 (1H, t, J = 7.2 and 3.6 Hz, H-9) were displayed in the 1H NMR spectrum, which suggested the presence of the −CH2CH3 fragment in compound 4. The structure of compound 4 was determined by the HMBC correlations of H-2/C-4, C-6, and C-7; H-5/C-1, C-3, and C-4; H-6/C-4, C-5, and C-7; H-8/C-7 and C-9; H-1′/C-3′, C-4, and C-5′; and H1″/C-2″ and C-6′ (Figure 2). Consequently, compound 4 was identified as 4-O-β-D-rutinosyl-3-methoxyphenyl-1-propanone. Compound 5 was isolated as a white amorphous power. Its molecular formula was deduced as C19H36O10 from HRESIMS data at m/z 447.2214 (calcd for C19H36O10Na, 447.2201), implying two degrees of unsaturation. The UV spectrum showed absorption at λmax 203 nm, and the IR spectrum indicated the presence of hydroxy (3384.6 cm−1) and methyl (2928.8 and 1362.8 cm−1) functionalities.
(Table 1). The above data suggested that compound 2 possessed the adamantane-type sesquiterpene skeleton.20 A trans-double bond at δH 5.83 (1H, d, J = 16.5 Hz, H-7) and 5.81 (1H, d, J = 16.5 Hz, H-8) and a −CH2CH3 fragment at δH 4.16 (2H, dd, J = 14.1 and 6.6 Hz) and 1.22 (3H, t, J = 14.1 and 6.6 Hz) were observed in the 1H NMR spectrum of compound 2. The 13C NMR spectrum (Table 1) showed 15 carbon signals consisting of one carbonyl (δC 173.8), two trans-double bond carbons, six saturated carbons of the cyclohexane, etc. Moreover, the structure of compound 2 was determined by the HMBC correlations of H-2/C-1, C-3, C-4, and C-6; H-4/ C-2, C-3, C-5, and C-6; H-5/C-1 and 15-CH3; H-7/C-6, C-8, and C-9; H-8/C-7, C-9, and C-10; and H-9/C-10 (Figure 2) and the NOESY correlations of H-3/H-5 and H-5/15-CH3. The absolute configurations of C-3, C-5, and C-6 were determined as 3S, 5R, and 6S, respectively, by biosynthetic considerations and the literature.20 In addition, the absolute configuration of C-9 was determined as 9R on the basis of a negative Cotton effect at 222 nm in the CD spectrum.20 Hence, compound 2 was identified as (8E,9R)-ethyl-7-(3S,5R,6S)-3,6dihydroxy-1,1,5-trimethylcyclohexyl-9-hydroxybut-8-enoate. Compound 3 was obtained as a white amorphous power. Its molecular formula was deduced as C24H38O11 based on HRESIMS at m/z 525.2323 (calcd for C 24 H 38 O 11 Na, 525.2306), indicating six degrees of unsaturation. The UV spectrum showed absorptions at λmax 203, 221, and 291 nm. Its IR spectrum displayed absorptions of hydroxy (3318.6 cm−1), carbonyl (1667.0 cm−1), and methyl (1373.2 cm−1) functionalities. The 1H NMR spectrum of compound 3 showed a multiplet at δH 4.11 (1H, m, H-3) and three methyl groups singlet at δH 1.17 (3H, s), 1.14 (3H, s), and 1.82 (3H, s), which revealed the typical characteristic of the adamantane-type sesquiterpene skeleton.20 In the aromatic field, the signals at δH 7.37 (1H, d, J = 16.2 Hz, H-7) and 6.18 (1H, d, J = 16.2 Hz, H-8) suggested the existence of a trans-double bond. Moreover, two anomeric protons resonated at δH 4.46 (1H, d, J = 7.8 Hz, H-1′) and 5.02 (1H, d, J = 3.9 Hz, H-1″) indicated the presence of glucosyl and apiosyl moieties21 in compound 3 according to the data of 1H and 13C NMR spectra (Table 2). The structure of compound 3 was established on the HMBC correlations of H-2/C-1, C-3, C4, and C-6; H-3/C-1′; H-4/C-2, C-3, and C-6; H-7/C-1, C-5, 4149
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In comparison of the 1H NMR data of compounds 5 and 4, compound 5 also had two typical doublets at δH 4.25 (1H, d, J = 7.5 Hz, H-1′) and 5.02 (1H, d, J = 1.8 Hz, H-1″) in the 1H NMR spectrum and a rutinosyl24 was concluded in compound 5 by the 1H and 13C NMR spectra (Table 3). Moreover, some typical signals at δH 4.06 (2H, d, J = 2.1 Hz, H-1), 1.60 (1H, m, H-2), 0.87 (3H, s, 5-CH3), and 0.89 (3H, s, 7-CH3) were observed in the 1H NMR spectrum, which concluded the 2ethyl-2-pentanol fragment in compound 5 according to correlations of H-2/C-4; H-3/C-2 and C-6; and H-6/C-2 and C-3 in the HMBC spectrum. Finally, the planar configuration of compound 5 was determined by the HMBC correlations of H-1/C-1′; H-1′/C-1, C-3′, and C-5′; and H-1″/ C-3″ and C-6′ (Figure 2). Acid hydrolysis of compound 5 afforded the aglycone 5a,25 and the 2R chirality of 5a was deduced from the optical value [α]D20 −3.20 (c 0.10, MeOH) by comparing to ref 26. Therefore, compound 5 was identified as 1-O-β-D-rutinosyl2(R)-ethyl-1-pentanol. In addition, 17 known compounds were isolated and identified as (R)-(−)-dehydrovomifoliol (6),27 blumenol C (7),28 blumenol A (8),29 icariside B1(9),30 icariside B1 aglycone (10),31 (3S,5R,6R,8E)-3,5,6-trihydroxy-1,1,5-trimethylcyclohexyl-8-buten-9-one (11),32 (4R)-4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-methanol (12),33 (8E)-3(R)-O-β-Dglucopyranosyloxy-1,5,5-trimethyl-7-cyclohexen-8-buten-7-one (13),34 3β-glucopyranosyloxy-β-ionone (14),35 3β-hydroxy5α,6α-epoxy-7-megastigmen-9-one (15),36 3-O-β-D-glucopyranosyloxy-5α,6α-epoxy-7-megastigmen-9-one (16),37 loliolide (17),38 (−)-epiloliolide (18),39 (3R,6R,7E)-3-hydroxy-α-ionone (19),38 5,6-dihydrovomifoliol (20),40 (3S,9R)-9-hydroxy-7butynyl-1,1,5-trimethyl-5-cyclohexen-3-ol (21),41 and 8-phenylethyl-O-β-D-rutinoside (22)24 by comparison of their physical and spectroscopic data to those reported in the references. Among them, compounds 6−21 belonged to sesquiterpenes and compound 22 belonged to rutinosides. Acid Hydrolysis of Compounds 3−5. Compounds 3−5 (4 mg) were each treated in 5% HCl (0.5 mL) and heated at 90 °C for 2 h.42 After cooling, each reaction mixture was extracted with EtOAc, and the aqueous layer was neutralized with 0.1 M NaOH. Therefore, glucose and apiose were obtained from compound 3, which was detected by TLC with authentic sugars. Similarly, we obtained glucose and rhamnose from compounds 4 and 5. The types of these sugars were identified by the TLC method.43 Under the condition of 14:6:1 CHCl3/ MeOH/H2O (Kieselgel 60 F254 plate), the Rf values of glucose, apiose, and rhamnose were 0.13, 0.31, and 0.24, respectively. Meanwhile, the Rf values (6:4:3 n-BuOH/pyridine/H2O, Cellulose 60 F plate) of glucose, apiose, and rhamnose were 0.37, 0.55, and 0.47, respectively. Hepatoprotective Activities. Compounds 1−22 were evaluated for their hepatoprotective activities against Dgalactosamine-induced WB-F344 cell damage. Among of them, compounds 1, 4, 5, 18, and 21 (10 μM) exhibited moderate hepatoprotective activities (Table 4).
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Article
AUTHOR INFORMATION
Corresponding Author
*Telephone/Fax: +86-10-6316-5327. E-mail: [email protected]. cn. Funding
This project was supported by the National Science and Technology Project of China (2012ZX09301002-002)-PCSIRT (IRT1007).10 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors express their appreciation to the Department of Instrumental Analysis of Peking Union Medical College and Chinese Academy of Medical Sciences for the NMR, IR, UV, HRESIMS, and CD measurements.
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REFERENCES
(1) Zhou, L. X.; Zheng, H. C.; Yang, C. R. The research progress on plants of Murraya. J. Pharm. Pract. 1997, 15, 214−219. (2) Anupam, N.; Suvra, M.; Avijit, B.; Julie, B. Review on chemistry and pharmacology of Murraya koenigii Spreng (Rutaceae). J. Chem. Pharm. Res. 2010, 2, 286−299. (3) Rana, V. S.; Juyal, J. P.; Rashmi; Amparo Blazquez, M. Chemical constituents of the volatile oil of Murraya koenigii leaves. Int. J. Aromather. 2004, 14, 23−25. (4) Luo, Q.; Wang, S. M.; Lu, Q.; Luo, J.; Cheng, Y. X. Identification of compounds from the water soluble extract of Cinnamomum cassia barks and their inhibitory effects against high-glucose-induced mesangial cells. Molecules 2013, 18, 10930−10943. (5) Dineshkumar, B.; Analava, M.; Manjunatha, M. Antidiabetic and hypolipidemic effects of mahanimbine (carbazole alkaloid) from Murraya koenigii (rutaceae) leaves. Int. J. Phytomed. 2010, 2, 22−30. (6) Suvra, M.; Anupam, N.; Manoj, K.; Samir, K. B.; Ashes, D.; Upadhyay, S. N.; Singh, R. K.; Avijit, B.; Julie, B. Antidiarhoeal activity of carbazole alkaloids from Murraya koenigii Spreng (Rutaceae) seeds. Fitoterapia 2010, 81, 72−74. (7) Jagan Mohan Rao, L.; Ramalakshmi, K.; Borse, B. B.; Baghavan, B. Antioxidant and radical-scavenging carbazole alkaloids from the oleoresin of curry leaf (Murraya koenigii Spreng). Food Chem. 2007, 100, 742−747. (8) Rahman, M. M.; Gray, A. I. A benzoisofuranone derivative and carbazole alkaloids from Murraya koenigii and their antimicrobial activities. Phytochemistry 2005, 66, 1601−1606. (9) Liu, H.; Li, C. J.; Yang, J. Z.; Ning, N.; Si, Y. K.; Li, L.; Chen, N. H.; Zhao, Q.; Zhang, D. M. Carbazole alkaloids from the stems of Clausena lansium. J. Nat. Prod. 2012, 75, 677−682. (10) Ma, Q. G.; Tian, J.; Yang, J. B.; Wang, A. G.; Ji, T. F.; Wang, Y. G.; Su, Y. L. Bioactive carbazole alkaloids from Murraya koenigii (L.) Spreng. Fitoterapia 2013, 87, 1−6. (11) Wu, X. F.; Hu, Y. C.; Gao, S.; Yu, S. S.; Pei, Y. H.; Tang, W. Z.; Huang, X. Z. Two new compounds from the roots of Lysidice rhodostegia. J. Asian Nat. Prod. Res. 2007, 9, 471−477. (12) Yu, L.; Yang, J. Z.; Chen, X. G.; Shi, J. G.; Zhang, D. M. Cytotoxic triterpenoid glycosides from the roots of Gordonia chrysandra. J. Nat. Prod. 2009, 72, 866−870. (13) Hsiao, P. C.; Liaw, C. C.; Hwang, S. Y.; Cheng, H. L.; Zhang, L. J.; Shen, C. C.; Hsu, F. L.; Kuo, Y. H. Antiproliferative and hypoglycemic cucurbitane-type glycosides from the fruits of Momordica charantia. J. Agric. Food Chem. 2013, 61, 2979−2986. (14) Liu, Y. F.; Liang, D.; Luo, H.; Hao, Z. Y.; Wang, Y.; Zhang, C. L.; Zhang, Q. J.; Chen, R. Y.; Yu, D. Q. Hepatoprotective iridoid glycosides from the roots of Rehmannia glutinosa. J. Nat. Prod. 2012, 75, 1625−1631. (15) Lin, M. H.; Liu, H. K.; Huang, W. J.; Huang, C. C.; Wu, T. H.; Hsu, F. L. Evaluation of the potential hypoglycemic and β-cell
ASSOCIATED CONTENT
* Supporting Information S
Additional spectra of compounds 1−5. This material is available free of charge via the Internet at http://pubs.acs.org. 4150
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activity of glucosylated carotenoid metabolites from Cydonia vulgaris fruits. J. Agric. Food Chem. 2006, 54, 9592−9597. (36) Cheng, J. T.; He, J.; Li, X. Y.; Wu, X. D.; Shao, L. D.; Dong, L. B.; Deng, X.; Gao, X.; Peng, L. Y.; Cheng, X.; Li, Y.; Zhao, Q. S. Three new sucrose fatty acid esters from Equisetum hiemale L. Helv. Chim. Acta 2012, 95, 1158−1163. (37) Yamamoto, M.; Akita, T.; Koyama, Y.; Sueyoshi, E.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Takashima, A.; Aramoto, M.; Takeda, Y. Euodionosides A−G: Megastigmane glucosides from leaves of Euodia meliaefolia. Phytochemistry 2008, 69, 1586−1596. (38) Machida, K.; Kikuchi, M. Norisoprenoids from Viburnum dilatatum. Phytochemistry 1996, 41, 1333−1336. (39) Borkosky, S.; Valdes, D. A.; Bardon, A.; Diaz, J. G.; Herz, W. Sesquiterpene lactones and other constituents of Eirmocephala megaphylla and Cyrtocymura cincta. Phytochemistry 1996, 42, 1637− 1639. (40) Gonzalez, A. G.; Guillermo, J. A.; Ravelo, A. G.; Jimenez, I. A.; Gupta, M. P. 4,5-Dihydroblumenol A, a new nor-isoprenoid from Perrottetia multiflora. J. Nat. Prod. 1994, 57, 400−402. (41) Skouroumounis, G. K.; Sefton, M. A. Acid-catalyzed hydrolysis of alcohols and their β-D-glucopyranosides. J. Agric. Food Chem. 2000, 48, 2033−2039. (42) Chang, J.; Case, R. Phenolic glycosides and ionone glycoside from the stem of Sargentodoxa cuneata. Phytochemistry 2005, 66, 2752−2758. (43) He, J.; Shen, Y.; Jiang, J. S.; Yang, Y. N.; Feng, Z. M.; Zhang, P. C.; Yuan, S. P.; Hou, Q. New polyacetylene glucosides from the florets of Carthamus tinctorius and their weak anti-inflammatory activities. Carbohydr. Res. 2011, 346, 1903−1908.
protective constituents isolated from corni fructus to tackle insulindependent diabetes mellitus. J. Agric. Food Chem. 2011, 59, 7743− 7751. (16) Li, Y.; Zhang, D. M.; Li, J. B.; Yu, S. S.; Li, Y.; Luo, Y. M. Hepatoprotective sesquiterpene glycosides from Sarcandra glabra. J. Nat. Prod. 2006, 69, 616−620. (17) Park, J. H.; Lee, D. G.; Yeon, S. W.; Kwon, H. S.; Shin, D. J.; Park, H. S.; Kim, Y. S.; Bang, M. H.; Baek, N. I. Isolation of megastigmane sesquiterpenes from the silkworm (Bombyx mori L.) droppings and their promotion activity on HO-1 and SIRT1. Arch. Pharm. Res. 2011, 34, 533−542. (18) Zhang, Z.; Zhang, W.; Ji, Y. P.; Zhao, Y.; Wang, C. G.; Hu, J. F. Gynostemosides A−E, megastigmane glycosides from Gynostemma pentaphyllum. Phytochemistry 2010, 71, 693−700. (19) Liu, J.; Du, D.; Si, Y. K.; Lv, H. J.; Wu, X. F.; Li, Y.; Liu, Y. Y.; Yu, S. S. Application of dimolybdenum reagent Mo2(OAc)4 for determination of the absolute configurations of vic-diols. Chin. J. Org. Chem. 2010, 30, 1270−1278. (20) Cirilo, P.; Juan, T. Absolute structures of two new C13norisoprenoids from Apollonias barbujana. J. Nat. Prod. 1996, 59, 69− 72. (21) Jun, C.; Ryan, C. Phenolic glycosides and ionone glycoside from the stem of Sargentodoxa cuneata. Phytochemistry 2005, 66, 2752− 2758. (22) Liu, C. M.; Li, B.; Shen, Y. H.; Zhang, W. D. Heterocyclic compounds and aromatic diglycosides from Bretschneidera sinensis. J. Nat. Prod. 2010, 73, 1582−1585. (23) Frederick, K.; Chang, A. N. Synthesis of (3S)-and (3R)-3hydroxy-bionone and their transformation into (3S)-and (3R)-βcryptoxanthin. Synthesis 2011, 3, 509−516. (24) Umehara, K.; Hattori, I.; Miyase, T.; Ueno, A.; Hara, S.; Kageyama, C. Studies on the constituents of leaves of Citrus unshiu Marcov. Chem. Pharm. Bull. 1988, 36, 5004−5008. (25) Erika, M.; Koichi, M.; Masao, K. Chemical constituents of Hypericum erectum Thunb. J. Nat. Med. 2008, 62, 467−469. (26) Herbert, C. B.; Ramachandra, G. N.; Raman, K. B.; Chongsuh, P.; Bakthan, S. Chiral synthesis via organoboranes. 4. Synthetic utility of boronic esters of essentially 100% optical purity, synthesis of homologated boronicacids and esters of very high enantiomeric purities. J. Org. Chem. 1985, 50, 5586−5592. (27) Mori, K. Synthesis of the optically active dehydrovomifoliol, synthetic proof of the absolute configuration of (+)-abscisic acid. Tetrahedron Lett. 1973, 28, 2635−2638. (28) Matsunami, K.; Otsuka, H.; Takeda, Y. Structural revisions of blumenol C glucoside and byzantionoside B. Chem. Pharm. Bull. 2010, 58, 438−441. (29) Kuo, P. C.; Yang, M. L.; Hwang, T. L.; Lai, Y. Y.; Li, Y. C.; Thang, T. D.; Wu, T. S. Anti-inflammatory diterpenoids from Croton tonkinensis. J. Nat. Prod. 2013, 76, 230−236. (30) Luecha, P.; Umehara, K.; Miyase, T.; Noguchi, H. Antiestrogenic constituents of the thai medicinal plants Capparis flavicans and Vitex glabrata. J. Nat. Prod. 2009, 72, 1954−1959. (31) Li, S. M.; Yang, X. W.; Shen, Y. H.; Feng, L.; Wang, Y. H.; Zeng, H. W.; Liu, X. H.; Tian, J. M.; Shi, Y. N.; Long, C. L.; Zhang, W. D. Chemical constituents of Aeschynanthus bracteatus and their weak antiinflammatory activities. Phytochemistry 2008, 69, 2200−2204. (32) Tan, S. T.; Wilkins, A. L.; Holland, P. T.; Mcghie, T. K. Extractives from New Zealand honeys. 3. Unifloral thyme and willow honey constituents. J. Agric. Food Chem. 1990, 38, 1833−1838. (33) Ruettimann, A.; Mayer, H. Synthesis of optically active natural carotenoids and structurally related natural products, v. synthesis of (3R,3′R)-, (3S,3′S)- and (3R,3′S;meso)-zeaxanthin by asymmetric hydroboration, a new approach to optically active carotenoid building units. Helv. Chim. Acta 1980, 63, 1456−1462. (34) Baumes, R. L.; Aubert, C. C.; Gunata, Z. Y.; Moor, W. D.; Bayonove, C. L. Structures of two C13-norisoprenoid glucosidic precursors of wine flavor. J. Essent. Oil Res. 1994, 6, 587−599. (35) Fiorentino, A.; Dabrosca, B.; Pacifico, S.; Mastellone, C.; Piscopo, V.; Monaco, P. Spectroscopic identification and antioxidant 4151
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Hepatoprotective Sesquiterpenes and Rutinosides from Murraya koenigii (L.) Spreng Qin-Ge Ma,† Yan-Gai Wang,‡ Wen-Min Liu,† Rong-Rui Wei,† Jian-Bo Yang,§ Ai-Guo Wang,§ Teng-Fei Ji,§ Jin Tian,∥ and Ya-Lun Su*,§ †
College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyan 473061, People’s Republic of China Department of Pharmacy, Xuanwu Hospital of Capital Medical University, Beijing 100053, People’s Republic of China § 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 ∥ College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: Three new sesquiterpenes (1−3) and two new rutinosides (4 and 5) along with 17 known compounds (6−22) were isolated from the leaves of Murraya koenigii (L.) Spreng. The new compounds were elucidated as (3R,5S,6R)-3,5,6trihydroxy-1,1,5-trimethylcyclohexyl-8-butyn-9-one (1), (8E,9R)-ethyl-7-(3S,5R,6S)-3,6-dihydroxy-1,1,5-trimethylcyclohexyl-9hydroxybut-8-enoate (2), (3R)-3-O-β-D-glucoside-6′-D-apiose-β-ionone (3), 4-O-β-D-rutinosyl-3-methoxyphenyl-1-propanone (4), and 1-O-β-D-rutinosyl-2(R)-ethyl-1-pentanol (5) based on their spectroscopic data. Compounds 1, 4, 5, 18, and 21 (10 μM) exhibited moderate hepatoprotective activities. KEYWORDS: Murraya koenigii, sesquiterpene, rutinoside, hepatoprotective activity
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spectrometer, a Nicolet 5700 FTIR microscope spectrometer, and a Hitachi UV-240 spectrophotometer, respectively.11 Nuclear magnetic resonance (NMR) spectra were recorded on Varian Mercury-300 and Inova-500 spectrometers, with tetramethylsilane (TMS) as an internal standard. The high-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired on an Agilent 1100 series LC/ MSD Trap SL mass spectrometer. Reversed-phase high-performance liquid chromatography (HPLC) was performed on a Shimadzu LC6AD instrument using a SPD-20A detector and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography was carried out on Sephadex LH-20 (Amersham Pharmacia, Sweden), silica gel H, 100−200 and 200−300 mesh (Qingdao Marine Chemical, Inc., Qingdao, China). Thin-layer chromatography (TLC) was performed on pre-coated silica gel GF254 plates, and the spots were visualized under UV light (254 or 356 nm) or spraying with 10% H2SO4 in 95% EtOH, followed by heating.12 Plant Material. The leaves of M. koenigii, family Rutaceae, were collected from Xishuangbanna, Yunnan, China, in August 201010 and authenticated by Prof. Lin Ma (Institute of Materia Medica, Beijing, China). A voucher specimen (ID-S-2436) has been deposited in the Herbarium of Institute of Materia Medica, Beijing, China. Extraction and Isolation. The air-dried leaves of M. koenigii (22.50 kg) were extracted 3 times with 95% EtOH (3 × 20 L) heating under reflux to give 2.10 g of crude extract. The combined extracts were successively partitioned with CHCl3, EtOAc, and n-butanol to yield three fractions: CHCl3-soluble fraction (33.50 g), EtOAc-soluble fraction (24.90 g), and n-butanol-soluble fraction (221.20 g). The n-butanol-soluble fraction was subjected to the macroporous adsorbent resin (Diaion-101) column, which were eluted with 20, 40, 60, and 95% ethanol to obtain four fractions: A (25.50 g), B (30.60 g),
INTRODUCTION Murraya koenigii L. (Rutaceae) is well-known as curry leaf, which has been widely cultivated as a spice in tropical and subtropical countries. The leaf of M. koenigii has been used for hundreds of years in southeast Asia as a traditional medicine and functional food to prevent and treat various diseases.1 Different kinds of compounds, including carbazole alkaloids,2 volatile oils,3 and other compounds, had been isolated from the leaves of M. koenigii.4 These compounds exhibited various bioactivities, such as hypoglycemic and hypolipemic,5 antidiarrheal,6 antioxidant and radical-scavenging,7 and antimicrobial activities.8 A preliminary screening assay exhibited that the n-butanolsoluble fraction from the leaves of M. koenigii inhibited hepatoprotective activities, which prompted us to further investigate its constituents. As a result, 22 compounds (Figure 1), including three new sesquiterpenes (1−3) and two new rutinosides (4 and 5) along with 17 known compounds (6− 22), were isolated from the active fraction of M. koenigii. Their structures (Figure 1) were elucidated by extensive analysis of their spectroscopic data. Meanwhile, we evaluated their hepatoprotective activities against D-galactosamine-induced WB-F344 cell damage and found that compounds 1, 4, 5, 18, and 21 (10 μM) showed moderate hepatoprotective activities.
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MATERIALS AND METHODS
General Experimental Procedures. The optical rotations were measured on a PerkinElmer 241 polarimeter at 20 °C.9 Melting points were recorded on a XT5B microscopic melting point apparatus (Beijing Tech Electro-Optical Instrument Factory, China), which was uncorrected.10 The circular dichroism (CD), infrared (IR), and ultraviolet (UV) spectra were measured with a JASCO J-815 CD © 2014 American Chemical Society
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Figure 1. Structures of compounds 1−22. 2932.7, 1670.8, 1381.7, 1044.6 cm−1. 1H NMR (CD3COCD3, 300 MHz) and 13C NMR (CD3COCD3, 125 MHz) data: see Table 1. (8E,9R)-Ethyl-7-(3S,5R,6S)-3,6-dihydroxy-1,1,5-trimethylcyclohexyl-9-hydroxybut-8-enoate (2). White amorphous power. [α]20 D −5.49 (c 0.30, MeOH). mp: 157.4−156.9 °C. HRESIMS: m/z 309.1648 (calcd for C15H26O5Na, 309.1672). UV (MeOH) λmax: 203 nm. CD (MeOH): 222 (Δε, −7.50) nm; IR νmax: 3392.5, 2967.2, 2933.0, 1737.5, 1368.3, 1045.2 cm−1. 1H NMR (CD3COCD3, 300 MHz) and 13 C NMR (CD3COCD3, 125 MHz) data: see Table 1. (3R)-3-O-β-D-Glucoside-6′-D-apiose-β-ionone (3). White amorphous power. [α]20 D +1.50 (c 0.67, MeOH). mp: 187.7−188.5 °C. HRESIMS: m/z 525.2323 (calcd for C24H38O11Na, 525.2306). UV (MeOH) λmax: 203, 221, 291 nm. CD (MeOH): 276 (Δε, +0.64), 322 (Δε, −0.49) nm. IR νmax: 3318.6, 2925.0, 1667.0, 1373.2, 1072.3 cm−1. 1 H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 2. 4-O-β-D-Rutinosyl-3-methoxyphenyl-1-propanone (4). White amorphous power. [α]20 D −2.45 (c 0.50, MeOH). mp: 194.1−195.5 °C. HRESIMS: m/z 511.1797 (calcd for C22H32O12Na, 511.1786). UV (MeOH) λmax: 203, 222, 266, 301 nm. IR νmax: 3399.3, 1671.8, 1592.9, 1512.1 cm−1. 1H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 3.
C (12.80 g), and D (5.40 g). The A part was purified on a Sephadex LH-20 column with a gradient system of MeOH/H2O to give two subfractions: A-a and A-b. The separation of A-b (6.40 g) by mediumpressure liquid chromatography (MPLC) (10−30% MeOH−H2O) and preparative HPLC (detection at 205 nm, 7 mL/min),9 successively, yielded compounds 4 (9.00 mg), 5 (7.00 mg), and 22 (7.00 mg). Similarly, the B part was chromatographed over Sephadex LH-20 eluted with MeOH to give two sub-fractions: B-a and B-b. The separation of B-a (3.65 g) by MPLC (15−40% MeOH−H2O) and preparative HPLC (detection at 205 nm, 7 mL/min), successively, yielded compounds 3 (10.00 mg), 13 (4.00 mg), 14 (10.31 mg), and 16 (11.76 mg). The separation of B-b (12.50 g) by MPLC (20−50% MeOH−H2O) and preparative HPLC (detection at 205 nm, 7 mL/ min), successively, yielded compounds 1 (6.51 mg), 2 (7.40 mg), 6 (3.10 mg), 7 (3.30 mg), 8 (4.70 mg), 9 (9.53 mg), 10 (12.30 mg), 11 (3.44 mg), 12 (6.63 mg), 15 (16.23 mg), 17 (18.43 mg), 18 (10.60 mg), 19 (5.43 mg), 20 (6.10 mg), and 21 (12.31 mg). (3R,5S,6R)-3,5,6-Trihydroxy-1,1,5-trimethylcyclohexyl-8-butyn-9one (1). White amorphous power. [α]20 D −2.48 (c 0.08, MeOH). Melting point (mp): 153.4−154.9 °C. HRESIMS: m/z 263.1264 (calcd for C13H20O4Na, 263.1254). UV (MeOH) λmax: 201, 264 nm. CD (MeOH): 314 (Δε, +0.33), 263 (Δε, −2.04) nm. IR νmax: 3454.9, 4146
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Table 1. 1H NMR (300 MHz, CD3COCD3), 13C NMR (125 MHz, CD3COCD3), and HMBC Correlations of Compounds 1 and 2 1 δH 1 2eq 2ax 3 4eq 4ax 5 6 7 8 9 10 11 12 13 14 15
1.56 1.53 4.08 1.91 1.47
2.32 1.31 0.79 0.96
(m) (m) (m) (m) (m)
(s) (s) (s) (s)
δC
2 δH
HMBC (1H−13C)
39.5 46.8 46.8 63.6 45.8 45.8 57.7 93.0 75.8 107.6 191.6 16.4 22.5 26.7 26.3
C-1, C-3, C-4, C-6 C-1, C-3, C-4, C-6
1.66 1.22 3.77 1.66 1.22 1.95
C-2, C-3, C-6 C-2, C-3, C-6
δC
(m) (m) (m) (m) (m) (m)
5.83 (d, 16.5) 5.81 (dd, 16.5, 4.8) 4.68 (d, 4.8) C-8 4.16 1.22 0.97 0.84 0.79
(dd, 14.1, 6.6) (t, 14.1, 6.6) (s) (s) (d, 6.9)
HMBC (1H−13C)
40.2 46.1 46.1 66.5 40.1 40.1 34.9 77.6 137.7 128.2 72.5 173.8 61.4 14.5 25.7 24.9 16.4
C-1, C-3, C-4, C-6 C-1, C-3, C-4, C-6 C-2, C-3, C-5, C-6 C-2, C-3, C-5, C-6 C-1, 15-CH3 C-6, C-8, C-9 C-7, C-9, C-10 C-10 C-10
Table 2. 1H NMR (300 MHz, CD3OD), 13C NMR (125 MHz, CD3OD), and HMBC Correlations of Compound 3 3 δH 1 2eq 2ax 3 4eq 4ax 5 6 7 8 9 10 11 12
2.00 1.58 4.11 2.58 2.20
(m) (m) (m) (m) (m)
7.37 (d, 16.2) 6.18 (d, 16.2) 2.33 (s) 1.17 (s) 1.14 (s)
δC 37.8 47.4 47.4 72.9 40.5 40.5 134.0 137.1 144.4 133.1 201.2 27.2 28.8 30.5
3 δH
HMBC (1H−13C) C-1, C-3, C-1, C-3, C-1′ C-2, C-3, C-2, C-3,
13 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″a 4″b 5″
C-4, C-6 C-4, C-6 C-6 C-6
C-1, C-5, C-8, C-9 C-6, C-9, C-10 C-9 C-1 C-1
1.82 4.46 3.43 3.18 3.37 3.32 4.00 3.63 5.02 3.79 3.26 3.97 3.91 3.59
(s) (d, 7.8) (m) (m) (m) (m) (m) (m) (d, 39) (m) (m) (m) (m) (s)
δC
HMBC (1H−13C)
21.8 102.7 76.8 75.1 78.0 71.7 68.6 68.6 110.8 75.0 80.5 78.0 78.0 65.7
C-5 C-3, C-2′ C-4′ C-4′, C-5′ C-3′, C-5′ C-2′, C-3′ C-5′ C-5′ C-2″, C-3″, C-6′ C-3″, C-4″, C-5″ C-4″ C-2″, C-5″ C-2″, C-5″ C-2″, C-3″, C-4″
calculated as inhibition (%) = [(OD(sample) − OD(control))/(OD(normal) − OD(control))] × 100.16 Statistical Analysis. Compounds 1−22 were bioassayed with the bicyclol (hepatoprotective activity drug) as the positive control for their hepatoprotective activities against D-galactosamine-induced toxicity in WB-F344 cells.14 The results of pharmacological activities are shown in Table 4, with the inhibition (%) of compounds 1, 4, 5, 18, and 21 based on the computing formula with values of 43.7, 54.3, 45.1, 27.0, and 49.5, respectively. Furthermore, all of the values were expressed as means ± standard deviation (SD) of three experiments. The significance of unpaired observations between normal or control and tested samples was determined by Student’s t test.16 Differences were considered significant at p < 0.05.15 Therefore, compounds 1, 4, 5, 18, and 21 exhibited moderate hepatoprotective activities.
1-O-β-D-Rutinosyl-2(R)-ethyl-1-pentanol (5). White amorphous power. [α]20 D −1.55 (c 0.50, MeOH). mp: 143.3−145.6 °C. HRESIMS: m/z 447.2214 (calcd for C19H36O10Na, 447.2201). UV (MeOH) λmax: 203 nm. CD (MeOH): 242 (Δε, −0.04) nm. IR νmax: 3384.6, 2928.8, 1362.8 cm−1. 1H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD, 125 MHz) data: see Table 3. Hepatoprotective Assay. We evaluated the hepatoprotective activities of compounds 1−22 (Figure 1), which were tested against Dgalactosamine-induced toxicity in WB-F344 cells using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method.8 The WB-F344 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3% fetal calf serum, 100 units/mL penicillin, and 100 units/mL streptomycin in 5% CO2 and incubated at 37 °C, which were placed in a 96-well microplate and precultured for 24 h.13 After this, the cultured cells were measured for cytotoxic effects, which were exposed to 40 mM D-galactosamine after 24 h. At last, the medium was replaced with the surum-free medium (0.5 mg/mL MTT) for 3.5 h of incubation. After removal of the medium and the addition of DMSO (150 μL/well) into the microplate, the formazan crystals were redissolved.13 The optical density (OD) was measured by a microplate reader at a wavelength of 492 nm, and the inhibition was
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RESULTS AND DISCUSSION Structure Elucidation of New Compounds. Compound 1 was obtained as a white amorphous power. Its molecular formula was deduced as C13H20O4 from the analysis of its HRESIMS at m/z 263.1264 (calcd for C13H20O4Na, 263.1254) corresponding to four degrees of unsaturation. The UV spectrum showed the adamantane-type sesquiterpene absorp4147
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Table 3. 1H NMR (300 MHz, CD3OD), 13C NMR (125 MHz, CD3OD), and HMBC Correlations of Compounds 4 and 5 4 δH 1 2 3a 3b 4a 4b 5 6a 6b 7 8 9 3-OCHH3 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″ 5″ 6″
7.61 (d, 1.8)
7.22 (d, 8.4) 7.70 (dd, 8.4, 1.8) 7.70 (dd, 8.4, 1.8) 3.07 1.20 3.93 5.03 3.52 3.49 3.39 3.61 4.04 3.62 4.69 3.83 3.71 3.39 3.69 1.19
(dd, 14.4, 7.2) (t, 7.2, 3.6) (s) (d, 7.2) (m) (m) (m) (m) (m) (m) (d, 1.8) (m) (m) (m) (m) (d, 7.2)
5
δC 131.4 114.9 149.3 149.3 150.7 150.7 110.9 122.3 122.3 200.6 30.9 7.4 55.2 100.4 73.3 76.5 70.0 75.7 66.4 66.4 100.7 70.8 70.9 72.6 68.4 16.5
C-4, C-6, C-7
C-1, C-3, C-4 C-4, C-5, C-7 C-4, C-5, C-7
normal control bicyclol 1 4 5 18 21
C-3′, C-4, C-5′
C-2″, C-3″, C-6′
cell survival rate (percentage of normal)
inhibition (percentage of control)
± ± ± ± ± ± ± ±
25.9 43.7 54.3 45.1 27.0 49.5
100.0 45.5 59.6 69.3 75.1 70.1 60.2 72.5
3.4 6.9 7.1b 7.1b 9.0b 7.2b 0.2b 8.5b
δC
4.06 1.60 2.18 1.16 2.09 1.17 0.87 1.70 1.19 0.89
(d, 2.1) (m) (m) (m) (m) (m) (s) (m) (m) (s)
83.8 45.8 36.7 36.7 27.3 27.3 19.3 28.7 28.7 13.9
4.25 3.40 3.37 3.60 3.83 3.95 3.54 5.02 3.30 3.93 3.13 3.58 0.85
(d, 7.5) (s) (m) (m) (m) (m) (m) (d, 1.8) (m) (m) (m) (m) (m)
102.9 76.6 78.1 71.0 77.6 67.9 67.9 110.4 71.7 74.7 74.9 65.8 20.2
HMBC (1H−13C) C-1′ C-4 C-2, C-6 C-2, C-6
C-2, C-3 C-2, C-3
C-7, C-9
C-1, C-3′, C-5′
C-3″, C-6′
the correlations of H-2/C-1, C-3, C-4, and C-6; H-4/C-2, C-3, and C-6; and 10-CH3/C-8 (Figure 2) in the heteronuclear multiple-bond correlation (HMBC) spectrum, and its relative configuration was determined by the correlations of H-3/5-OH and 3-OH/13-CH3 (Figure 2) in the nuclear Overhauser effect spectrometry (NOESY) spectrum. The absolute configuration of compound 1 was determined by the CD method. Because the Mo2(OAc)4-induced CD method has been successfully used to determine the absolute configuration of dozens of organic compounds with a 1,2-diol substructure,19 we study the Mo2(OAc)4-induced CD spectrum of compound 1, which contained a 5,6-diol unit. A positive Cotton effect was observed at 314 nm in the CD spectrum, and the absolute configurations of C-5 and C-6 were determined as 5S and 6R, respectively, by applying the spiral rule.19 In addition, according to the correlations of the NOESY spectrum, the absolute configuration of C-3 was deduced as 3R. Thus, compound 1 was identified as (3R,5S,6R)-3,5,6-trihydroxy1,1,5-trimethylcyclohexyl-8-butyn-9-one. Compound 2 was isolated as a white amorphous power. The molecular formula was deduced as C15H26O5 from HRESIMS at m/z 309.1648 (calcd for C15H26O5Na, 309.1672), indicating three degrees of unsaturation. Its UV spectrum showed absorbance at λmax 203 nm, and the IR spectrum indicated the presence of hydroxy (3392.5 cm−1), carbonyl (1737.5 cm−1), and methyl (1368.3 cm−1) functionalities. The UV and IR spectrum data of compound 2 were similar to those of compound 1. Therefore, compound 2 was concluded to be an adamantane-type sesquiterpene.17 The 1H NMR spectrum of compound 2 showed the presence of a multiplet at δH 3.77 (1H, m, H-3) and three methyl group singlets at δH 0.97 (3H, s), 0.84 (3H, s), and 0.79 (3H, s)
Table 4. Hepatoprotective Effects of Selective Compounds (10 μM) against D-Galactosamine-Induced Toxicity in WBF344 Cellsa compound
δH
HMBC (1H−13C)
Results were expressed as means ± SD (n = 3; for normal and control, n = 6). Bicyclol was used as a positive control (10 μM). bp < 0.05.
a
tions at λmax 201 and 264 nm.17 Its IR spectrum indicated the presence of hydroxy (3454.9 cm−1), carbonyl (1670.8 cm−1), and methyl (1381.7 cm−1) functionalities. The 1H NMR spectrum of compound 1 showed the presence of a multiplet at δH 4.08 (1H, m, H-3) and three methyl group singlets at δH 1.31 (3H, s), 0.96 (3H, s), and 0.79 (3H, s) (Table 1). These spectral characteristics coupled with the biosynthetic pathway (biogenetic isoprene rule) indicated the presence of the adamantane-type sesquiterpene18 skeleton in compound 1. The 13C NMR spectrum (Table 1) showed 13 carbon signals. Besides one carbonyl (δC 191.6), four methyl carbons, and two sp-hybridized carbons, the remaining six sp3hybridized carbons were attributed to the cyclohexane. The planar structure of compound 1 was established on the basis of 4148
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Figure 2. Key HMBC (H → C) and NOESY correlations of compounds 1−5.
C-8, and C-9; H-8/C-6, C-9, and C-10; 10-CH3/C-9; H-1′/C3; and H-1″/C-6′ (Figure 2) and the NOESY correlations of H-2/H-3 and H-3/H-4. The absolute configuration of C-3 was determined by the CD method. First, compound 3 was hydrolyzed22 to afford the aglycone 3a. The positive Cotton effect at 276 nm and the negative Cotton effect at 322 nm were observed in the CD spectrum of compound 3a. According to the spiral rule and the literature,23 the absolute configuration of C-3 was determined as 3R. Therefore, compound 3 was identified as (3R)-3-O-β-Dglucoside-6′-D-apiose-β-ionone. Compound 4 was obtained as a white amorphous power. Its molecular formula was established as C22H32O12 on the basis of HRESIMS at m/z 511.1797 (calcd for C22H32O12Na, 511.1786) corresponding to four degrees of unsaturation. The UV spectrum showed absorptions at λmax 203, 222, 266, and 301 nm. Its IR spectrum displayed the presence of hydroxy (3399.3 cm−1) and benzene ring (1671.8, 1592.9, and 1512.1 cm−1) functionalities. In the 1H NMR spectrum of compound 4, an ABX system was shown at δH 7.61 (1H, d, J = 1.8 Hz, H-2), 7.22 (1H, d, J = 8.4 Hz, H-5), and 7.70 (1H, dd, J = 8.4, 1.8 Hz, H-6) in the aromatic field. The anomeric signals at δH 5.03 (1H, d, J = 7.2 Hz, H-1′) and 4.69 (1H, d, J = 1.8 Hz, H-1″) suggested the existence of a rutinosyl moiety24 in compound 4 according to the 1H and 13C NMR spectra (Table 3). Additionally, a quartet at δH 3.07 (2H, dd, J = 14.4 and 7.2 Hz, H-8) and a triplet at δH 1.20 (1H, t, J = 7.2 and 3.6 Hz, H-9) were displayed in the 1H NMR spectrum, which suggested the presence of the −CH2CH3 fragment in compound 4. The structure of compound 4 was determined by the HMBC correlations of H-2/C-4, C-6, and C-7; H-5/C-1, C-3, and C-4; H-6/C-4, C-5, and C-7; H-8/C-7 and C-9; H-1′/C-3′, C-4, and C-5′; and H1″/C-2″ and C-6′ (Figure 2). Consequently, compound 4 was identified as 4-O-β-D-rutinosyl-3-methoxyphenyl-1-propanone. Compound 5 was isolated as a white amorphous power. Its molecular formula was deduced as C19H36O10 from HRESIMS data at m/z 447.2214 (calcd for C19H36O10Na, 447.2201), implying two degrees of unsaturation. The UV spectrum showed absorption at λmax 203 nm, and the IR spectrum indicated the presence of hydroxy (3384.6 cm−1) and methyl (2928.8 and 1362.8 cm−1) functionalities.
(Table 1). The above data suggested that compound 2 possessed the adamantane-type sesquiterpene skeleton.20 A trans-double bond at δH 5.83 (1H, d, J = 16.5 Hz, H-7) and 5.81 (1H, d, J = 16.5 Hz, H-8) and a −CH2CH3 fragment at δH 4.16 (2H, dd, J = 14.1 and 6.6 Hz) and 1.22 (3H, t, J = 14.1 and 6.6 Hz) were observed in the 1H NMR spectrum of compound 2. The 13C NMR spectrum (Table 1) showed 15 carbon signals consisting of one carbonyl (δC 173.8), two trans-double bond carbons, six saturated carbons of the cyclohexane, etc. Moreover, the structure of compound 2 was determined by the HMBC correlations of H-2/C-1, C-3, C-4, and C-6; H-4/ C-2, C-3, C-5, and C-6; H-5/C-1 and 15-CH3; H-7/C-6, C-8, and C-9; H-8/C-7, C-9, and C-10; and H-9/C-10 (Figure 2) and the NOESY correlations of H-3/H-5 and H-5/15-CH3. The absolute configurations of C-3, C-5, and C-6 were determined as 3S, 5R, and 6S, respectively, by biosynthetic considerations and the literature.20 In addition, the absolute configuration of C-9 was determined as 9R on the basis of a negative Cotton effect at 222 nm in the CD spectrum.20 Hence, compound 2 was identified as (8E,9R)-ethyl-7-(3S,5R,6S)-3,6dihydroxy-1,1,5-trimethylcyclohexyl-9-hydroxybut-8-enoate. Compound 3 was obtained as a white amorphous power. Its molecular formula was deduced as C24H38O11 based on HRESIMS at m/z 525.2323 (calcd for C 24 H 38 O 11 Na, 525.2306), indicating six degrees of unsaturation. The UV spectrum showed absorptions at λmax 203, 221, and 291 nm. Its IR spectrum displayed absorptions of hydroxy (3318.6 cm−1), carbonyl (1667.0 cm−1), and methyl (1373.2 cm−1) functionalities. The 1H NMR spectrum of compound 3 showed a multiplet at δH 4.11 (1H, m, H-3) and three methyl groups singlet at δH 1.17 (3H, s), 1.14 (3H, s), and 1.82 (3H, s), which revealed the typical characteristic of the adamantane-type sesquiterpene skeleton.20 In the aromatic field, the signals at δH 7.37 (1H, d, J = 16.2 Hz, H-7) and 6.18 (1H, d, J = 16.2 Hz, H-8) suggested the existence of a trans-double bond. Moreover, two anomeric protons resonated at δH 4.46 (1H, d, J = 7.8 Hz, H-1′) and 5.02 (1H, d, J = 3.9 Hz, H-1″) indicated the presence of glucosyl and apiosyl moieties21 in compound 3 according to the data of 1H and 13C NMR spectra (Table 2). The structure of compound 3 was established on the HMBC correlations of H-2/C-1, C-3, C4, and C-6; H-3/C-1′; H-4/C-2, C-3, and C-6; H-7/C-1, C-5, 4149
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In comparison of the 1H NMR data of compounds 5 and 4, compound 5 also had two typical doublets at δH 4.25 (1H, d, J = 7.5 Hz, H-1′) and 5.02 (1H, d, J = 1.8 Hz, H-1″) in the 1H NMR spectrum and a rutinosyl24 was concluded in compound 5 by the 1H and 13C NMR spectra (Table 3). Moreover, some typical signals at δH 4.06 (2H, d, J = 2.1 Hz, H-1), 1.60 (1H, m, H-2), 0.87 (3H, s, 5-CH3), and 0.89 (3H, s, 7-CH3) were observed in the 1H NMR spectrum, which concluded the 2ethyl-2-pentanol fragment in compound 5 according to correlations of H-2/C-4; H-3/C-2 and C-6; and H-6/C-2 and C-3 in the HMBC spectrum. Finally, the planar configuration of compound 5 was determined by the HMBC correlations of H-1/C-1′; H-1′/C-1, C-3′, and C-5′; and H-1″/ C-3″ and C-6′ (Figure 2). Acid hydrolysis of compound 5 afforded the aglycone 5a,25 and the 2R chirality of 5a was deduced from the optical value [α]D20 −3.20 (c 0.10, MeOH) by comparing to ref 26. Therefore, compound 5 was identified as 1-O-β-D-rutinosyl2(R)-ethyl-1-pentanol. In addition, 17 known compounds were isolated and identified as (R)-(−)-dehydrovomifoliol (6),27 blumenol C (7),28 blumenol A (8),29 icariside B1(9),30 icariside B1 aglycone (10),31 (3S,5R,6R,8E)-3,5,6-trihydroxy-1,1,5-trimethylcyclohexyl-8-buten-9-one (11),32 (4R)-4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-methanol (12),33 (8E)-3(R)-O-β-Dglucopyranosyloxy-1,5,5-trimethyl-7-cyclohexen-8-buten-7-one (13),34 3β-glucopyranosyloxy-β-ionone (14),35 3β-hydroxy5α,6α-epoxy-7-megastigmen-9-one (15),36 3-O-β-D-glucopyranosyloxy-5α,6α-epoxy-7-megastigmen-9-one (16),37 loliolide (17),38 (−)-epiloliolide (18),39 (3R,6R,7E)-3-hydroxy-α-ionone (19),38 5,6-dihydrovomifoliol (20),40 (3S,9R)-9-hydroxy-7butynyl-1,1,5-trimethyl-5-cyclohexen-3-ol (21),41 and 8-phenylethyl-O-β-D-rutinoside (22)24 by comparison of their physical and spectroscopic data to those reported in the references. Among them, compounds 6−21 belonged to sesquiterpenes and compound 22 belonged to rutinosides. Acid Hydrolysis of Compounds 3−5. Compounds 3−5 (4 mg) were each treated in 5% HCl (0.5 mL) and heated at 90 °C for 2 h.42 After cooling, each reaction mixture was extracted with EtOAc, and the aqueous layer was neutralized with 0.1 M NaOH. Therefore, glucose and apiose were obtained from compound 3, which was detected by TLC with authentic sugars. Similarly, we obtained glucose and rhamnose from compounds 4 and 5. The types of these sugars were identified by the TLC method.43 Under the condition of 14:6:1 CHCl3/ MeOH/H2O (Kieselgel 60 F254 plate), the Rf values of glucose, apiose, and rhamnose were 0.13, 0.31, and 0.24, respectively. Meanwhile, the Rf values (6:4:3 n-BuOH/pyridine/H2O, Cellulose 60 F plate) of glucose, apiose, and rhamnose were 0.37, 0.55, and 0.47, respectively. Hepatoprotective Activities. Compounds 1−22 were evaluated for their hepatoprotective activities against Dgalactosamine-induced WB-F344 cell damage. Among of them, compounds 1, 4, 5, 18, and 21 (10 μM) exhibited moderate hepatoprotective activities (Table 4).
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Article
AUTHOR INFORMATION
Corresponding Author
*Telephone/Fax: +86-10-6316-5327. E-mail: [email protected]. cn. Funding
This project was supported by the National Science and Technology Project of China (2012ZX09301002-002)-PCSIRT (IRT1007).10 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors express their appreciation to the Department of Instrumental Analysis of Peking Union Medical College and Chinese Academy of Medical Sciences for the NMR, IR, UV, HRESIMS, and CD measurements.
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REFERENCES
(1) Zhou, L. X.; Zheng, H. C.; Yang, C. R. The research progress on plants of Murraya. J. Pharm. Pract. 1997, 15, 214−219. (2) Anupam, N.; Suvra, M.; Avijit, B.; Julie, B. Review on chemistry and pharmacology of Murraya koenigii Spreng (Rutaceae). J. Chem. Pharm. Res. 2010, 2, 286−299. (3) Rana, V. S.; Juyal, J. P.; Rashmi; Amparo Blazquez, M. Chemical constituents of the volatile oil of Murraya koenigii leaves. Int. J. Aromather. 2004, 14, 23−25. (4) Luo, Q.; Wang, S. M.; Lu, Q.; Luo, J.; Cheng, Y. X. Identification of compounds from the water soluble extract of Cinnamomum cassia barks and their inhibitory effects against high-glucose-induced mesangial cells. Molecules 2013, 18, 10930−10943. (5) Dineshkumar, B.; Analava, M.; Manjunatha, M. Antidiabetic and hypolipidemic effects of mahanimbine (carbazole alkaloid) from Murraya koenigii (rutaceae) leaves. Int. J. Phytomed. 2010, 2, 22−30. (6) Suvra, M.; Anupam, N.; Manoj, K.; Samir, K. B.; Ashes, D.; Upadhyay, S. N.; Singh, R. K.; Avijit, B.; Julie, B. Antidiarhoeal activity of carbazole alkaloids from Murraya koenigii Spreng (Rutaceae) seeds. Fitoterapia 2010, 81, 72−74. (7) Jagan Mohan Rao, L.; Ramalakshmi, K.; Borse, B. B.; Baghavan, B. Antioxidant and radical-scavenging carbazole alkaloids from the oleoresin of curry leaf (Murraya koenigii Spreng). Food Chem. 2007, 100, 742−747. (8) Rahman, M. M.; Gray, A. I. A benzoisofuranone derivative and carbazole alkaloids from Murraya koenigii and their antimicrobial activities. Phytochemistry 2005, 66, 1601−1606. (9) Liu, H.; Li, C. J.; Yang, J. Z.; Ning, N.; Si, Y. K.; Li, L.; Chen, N. H.; Zhao, Q.; Zhang, D. M. Carbazole alkaloids from the stems of Clausena lansium. J. Nat. Prod. 2012, 75, 677−682. (10) Ma, Q. G.; Tian, J.; Yang, J. B.; Wang, A. G.; Ji, T. F.; Wang, Y. G.; Su, Y. L. Bioactive carbazole alkaloids from Murraya koenigii (L.) Spreng. Fitoterapia 2013, 87, 1−6. (11) Wu, X. F.; Hu, Y. C.; Gao, S.; Yu, S. S.; Pei, Y. H.; Tang, W. Z.; Huang, X. Z. Two new compounds from the roots of Lysidice rhodostegia. J. Asian Nat. Prod. Res. 2007, 9, 471−477. (12) Yu, L.; Yang, J. Z.; Chen, X. G.; Shi, J. G.; Zhang, D. M. Cytotoxic triterpenoid glycosides from the roots of Gordonia chrysandra. J. Nat. Prod. 2009, 72, 866−870. (13) Hsiao, P. C.; Liaw, C. C.; Hwang, S. Y.; Cheng, H. L.; Zhang, L. J.; Shen, C. C.; Hsu, F. L.; Kuo, Y. H. Antiproliferative and hypoglycemic cucurbitane-type glycosides from the fruits of Momordica charantia. J. Agric. Food Chem. 2013, 61, 2979−2986. (14) Liu, Y. F.; Liang, D.; Luo, H.; Hao, Z. Y.; Wang, Y.; Zhang, C. L.; Zhang, Q. J.; Chen, R. Y.; Yu, D. Q. Hepatoprotective iridoid glycosides from the roots of Rehmannia glutinosa. J. Nat. Prod. 2012, 75, 1625−1631. (15) Lin, M. H.; Liu, H. K.; Huang, W. J.; Huang, C. C.; Wu, T. H.; Hsu, F. L. Evaluation of the potential hypoglycemic and β-cell
ASSOCIATED CONTENT
* Supporting Information S
Additional spectra of compounds 1−5. This material is available free of charge via the Internet at http://pubs.acs.org. 4150
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activity of glucosylated carotenoid metabolites from Cydonia vulgaris fruits. J. Agric. Food Chem. 2006, 54, 9592−9597. (36) Cheng, J. T.; He, J.; Li, X. Y.; Wu, X. D.; Shao, L. D.; Dong, L. B.; Deng, X.; Gao, X.; Peng, L. Y.; Cheng, X.; Li, Y.; Zhao, Q. S. Three new sucrose fatty acid esters from Equisetum hiemale L. Helv. Chim. Acta 2012, 95, 1158−1163. (37) Yamamoto, M.; Akita, T.; Koyama, Y.; Sueyoshi, E.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Takashima, A.; Aramoto, M.; Takeda, Y. Euodionosides A−G: Megastigmane glucosides from leaves of Euodia meliaefolia. Phytochemistry 2008, 69, 1586−1596. (38) Machida, K.; Kikuchi, M. Norisoprenoids from Viburnum dilatatum. Phytochemistry 1996, 41, 1333−1336. (39) Borkosky, S.; Valdes, D. A.; Bardon, A.; Diaz, J. G.; Herz, W. Sesquiterpene lactones and other constituents of Eirmocephala megaphylla and Cyrtocymura cincta. Phytochemistry 1996, 42, 1637− 1639. (40) Gonzalez, A. G.; Guillermo, J. A.; Ravelo, A. G.; Jimenez, I. A.; Gupta, M. P. 4,5-Dihydroblumenol A, a new nor-isoprenoid from Perrottetia multiflora. J. Nat. Prod. 1994, 57, 400−402. (41) Skouroumounis, G. K.; Sefton, M. A. Acid-catalyzed hydrolysis of alcohols and their β-D-glucopyranosides. J. Agric. Food Chem. 2000, 48, 2033−2039. (42) Chang, J.; Case, R. Phenolic glycosides and ionone glycoside from the stem of Sargentodoxa cuneata. Phytochemistry 2005, 66, 2752−2758. (43) He, J.; Shen, Y.; Jiang, J. S.; Yang, Y. N.; Feng, Z. M.; Zhang, P. C.; Yuan, S. P.; Hou, Q. New polyacetylene glucosides from the florets of Carthamus tinctorius and their weak anti-inflammatory activities. Carbohydr. Res. 2011, 346, 1903−1908.
protective constituents isolated from corni fructus to tackle insulindependent diabetes mellitus. J. Agric. Food Chem. 2011, 59, 7743− 7751. (16) Li, Y.; Zhang, D. M.; Li, J. B.; Yu, S. S.; Li, Y.; Luo, Y. M. Hepatoprotective sesquiterpene glycosides from Sarcandra glabra. J. Nat. Prod. 2006, 69, 616−620. (17) Park, J. H.; Lee, D. G.; Yeon, S. W.; Kwon, H. S.; Shin, D. J.; Park, H. S.; Kim, Y. S.; Bang, M. H.; Baek, N. I. Isolation of megastigmane sesquiterpenes from the silkworm (Bombyx mori L.) droppings and their promotion activity on HO-1 and SIRT1. Arch. Pharm. Res. 2011, 34, 533−542. (18) Zhang, Z.; Zhang, W.; Ji, Y. P.; Zhao, Y.; Wang, C. G.; Hu, J. F. Gynostemosides A−E, megastigmane glycosides from Gynostemma pentaphyllum. Phytochemistry 2010, 71, 693−700. (19) Liu, J.; Du, D.; Si, Y. K.; Lv, H. J.; Wu, X. F.; Li, Y.; Liu, Y. Y.; Yu, S. S. Application of dimolybdenum reagent Mo2(OAc)4 for determination of the absolute configurations of vic-diols. Chin. J. Org. Chem. 2010, 30, 1270−1278. (20) Cirilo, P.; Juan, T. Absolute structures of two new C13norisoprenoids from Apollonias barbujana. J. Nat. Prod. 1996, 59, 69− 72. (21) Jun, C.; Ryan, C. Phenolic glycosides and ionone glycoside from the stem of Sargentodoxa cuneata. Phytochemistry 2005, 66, 2752− 2758. (22) Liu, C. M.; Li, B.; Shen, Y. H.; Zhang, W. D. Heterocyclic compounds and aromatic diglycosides from Bretschneidera sinensis. J. Nat. Prod. 2010, 73, 1582−1585. (23) Frederick, K.; Chang, A. N. Synthesis of (3S)-and (3R)-3hydroxy-bionone and their transformation into (3S)-and (3R)-βcryptoxanthin. Synthesis 2011, 3, 509−516. (24) Umehara, K.; Hattori, I.; Miyase, T.; Ueno, A.; Hara, S.; Kageyama, C. Studies on the constituents of leaves of Citrus unshiu Marcov. Chem. Pharm. Bull. 1988, 36, 5004−5008. (25) Erika, M.; Koichi, M.; Masao, K. Chemical constituents of Hypericum erectum Thunb. J. Nat. Med. 2008, 62, 467−469. (26) Herbert, C. B.; Ramachandra, G. N.; Raman, K. B.; Chongsuh, P.; Bakthan, S. Chiral synthesis via organoboranes. 4. Synthetic utility of boronic esters of essentially 100% optical purity, synthesis of homologated boronicacids and esters of very high enantiomeric purities. J. Org. Chem. 1985, 50, 5586−5592. (27) Mori, K. Synthesis of the optically active dehydrovomifoliol, synthetic proof of the absolute configuration of (+)-abscisic acid. Tetrahedron Lett. 1973, 28, 2635−2638. (28) Matsunami, K.; Otsuka, H.; Takeda, Y. Structural revisions of blumenol C glucoside and byzantionoside B. Chem. Pharm. Bull. 2010, 58, 438−441. (29) Kuo, P. C.; Yang, M. L.; Hwang, T. L.; Lai, Y. Y.; Li, Y. C.; Thang, T. D.; Wu, T. S. Anti-inflammatory diterpenoids from Croton tonkinensis. J. Nat. Prod. 2013, 76, 230−236. (30) Luecha, P.; Umehara, K.; Miyase, T.; Noguchi, H. Antiestrogenic constituents of the thai medicinal plants Capparis flavicans and Vitex glabrata. J. Nat. Prod. 2009, 72, 1954−1959. (31) Li, S. M.; Yang, X. W.; Shen, Y. H.; Feng, L.; Wang, Y. H.; Zeng, H. W.; Liu, X. H.; Tian, J. M.; Shi, Y. N.; Long, C. L.; Zhang, W. D. Chemical constituents of Aeschynanthus bracteatus and their weak antiinflammatory activities. Phytochemistry 2008, 69, 2200−2204. (32) Tan, S. T.; Wilkins, A. L.; Holland, P. T.; Mcghie, T. K. Extractives from New Zealand honeys. 3. Unifloral thyme and willow honey constituents. J. Agric. Food Chem. 1990, 38, 1833−1838. (33) Ruettimann, A.; Mayer, H. Synthesis of optically active natural carotenoids and structurally related natural products, v. synthesis of (3R,3′R)-, (3S,3′S)- and (3R,3′S;meso)-zeaxanthin by asymmetric hydroboration, a new approach to optically active carotenoid building units. Helv. Chim. Acta 1980, 63, 1456−1462. (34) Baumes, R. L.; Aubert, C. C.; Gunata, Z. Y.; Moor, W. D.; Bayonove, C. L. Structures of two C13-norisoprenoid glucosidic precursors of wine flavor. J. Essent. Oil Res. 1994, 6, 587−599. (35) Fiorentino, A.; Dabrosca, B.; Pacifico, S.; Mastellone, C.; Piscopo, V.; Monaco, P. Spectroscopic identification and antioxidant 4151
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